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Burn-Derived Stem Cells - A Promising New Cell Source For Skin Regeneration
Cellules souches issues de peau brûlée
- Une source prometteuse de cellules pour la régénération de la peau
by
Reinhard Dolp
A thesis submitted in conformity with the requirements for the degree of Master of Science
Institute of Medical Science University of Toronto\
ii
Burn-Derived Stem Cells - A Promising New Cell Source For Skin Regeneration
Reinhard Dolp
Doctor of Medicine
Institute of Medical Science University of Toronto
2017
Abstract
Introduction: Burns affect millions of people worldwide. Available wound coverage materials
are insufficient due to a lack of cells. Mesenchymal stem cells (MSCs) promote wound healing
but are limited by lack of availability. We hypothesize that burned skin contains functioning MSCs
(burn derived MSCs; BD-MSCs) that promote wound healing.
Methods: BD-MSCs were compared to umbilical cord MSCs in terms of key biological
characteristics. Then, skin-scaffolds were cellularized with BD-MSCs, applied onto excisional
porcine wounds and observed over 30d.
Results: We found no difference between BD- and UC-MSCs in mitochondrial function,
proliferation, colony formation, cell cycle stage distribution, reactive oxygen species, and MHC
I/II expression. BD-MSCs are safe and improved wound healing in mice and pigs.
Conclusion: Burned skin contains healthy MSCs that promote wound healing. Key biological
functions are not altered by burn trauma. Further studies are needed to evaluate the role of BD-
MSCs in wound healing.
iii
Acknowledgments
First and foremost, I would like to thank my supervisor Professor Dr. Marc Jeschke and Dr.
Amini-Nik for welcoming me into the team and enabling me to achieve my dream to become an
academic clinician in Canada.
Next, I would like to thank my committee members Prof. Post and Prof. Morshead for their
valuable feedback.
I am very grateful for the people that I have worked with - in particular, the fantastic lab-
technicians Alexa Parousis, Andrea-Kaye Datu, and Nazihah Bakhtyar.
Finally, I would like to thank my family - Robert, Monika, and Maximilian Dolp - as well as my
partner Jonathan Ausman.
iv
Contributors
The following people/organizations have been instrumental in the collection of materials and
data for this thesis:
1. The obstetrical and gynecological department of Sunnybrook, Dr. Herer, and Dr. Zaltz,
for the collection of umbilical cords.
2. The Ross Tilley Burn Center with all their members and patients for providing the burned
skin.
3. The Integra LifeScience Corporation for providing Integra®.
I want to thank Toronto Hydro for their generous donations that enabled our research.
v
Table of Contents
Acknowledgments.......................................................................................................................... iii
Contributors ................................................................................................................................... iv
Table of Contents ............................................................................................................................ v
List of Tables ................................................................................................................................. ix
List of Figures ................................................................................................................................. x
List of Abbreviations .................................................................................................................... xii
Chapter 1 Introduction .................................................................................................................... 1
Introduction ............................................................................................................................. 1
1.1 Burn injury ...................................................................................................................... 1
1.1.1 Definition and current treatment ............................................................................. 1
1.1.2 Challenges in modern burn care ............................................................................. 4
1.2 Skin substitutes ............................................................................................................. 12
1.3 Physiology of wound healing and its meaning for cell therapy .................................... 16
1.4 The role of stem cells in skin regeneration and wound healing .................................... 22
1.4.1 Overview - classifications and characteristics of stem cells ................................ 22
1.4.2 Mesenchymal stem cells as wound therapy .......................................................... 31
1.5 Preliminary data ............................................................................................................ 34
Chapter 2 Rationale, Hypothesis, and Aim ................................................................................... 37
Rationale, Hypothesis, and Aim ........................................................................................... 37
2.1 Rationale ....................................................................................................................... 37
2.2 Hypothesis..................................................................................................................... 37
vi
2.3 Aim ............................................................................................................................... 38
Chapter 3 Material and Methods................................................................................................... 39
Material and Methods ........................................................................................................... 39
3.1 Stem Cell extraction and culturing ............................................................................... 39
3.1.1 Conventional Stem Cell Extraction Method ......................................................... 41
3.1.2 Enzymatic Stem Cell Extraction Method ............................................................. 41
3.1.3 Cell culturing ........................................................................................................ 42
3.1.4 Quantification of cell yield ................................................................................... 42
3.2 Determination of mesenchymal stem cell character ..................................................... 43
3.2.1 Flow Cytometry for stem cell surface markers ..................................................... 43
3.2.2 Differentiation Assay ............................................................................................ 44
3.3 Determination of key biological characteristics ........................................................... 45
3.3.1 Population doubling time ...................................................................................... 45
3.3.2 Colony forming assay ........................................................................................... 46
3.3.3 Proliferation via bromodeoxyuridine (BrdU) staining .......................................... 46
3.3.4 Cell cycle analysis................................................................................................. 47
3.3.5 Reactive oxygen species (ROS) expression .......................................................... 47
3.3.6 Apoptosis .............................................................................................................. 48
3.3.7 Glycolytic and Mitochondrial function ................................................................. 49
3.4 Tumorigenicity .............................................................................................................. 50
3.4.1 In Vitro .................................................................................................................. 50
3.4.2 In Vivo .................................................................................................................. 51
3.5 Immunogenicity and -reactivity .................................................................................... 52
vii
3.5.1 Flow cytometry for major histocompatibility complex (MHC) I and II and toll-
like receptor (TLR) 4 ............................................................................................................ 52
3.5.2 QPCR for toll-like receptor (TLR) 1-10 ............................................................... 53
3.5.3 Secretion Profile.................................................................................................... 56
3.6 Stem cell integration into Integra® ............................................................................... 57
3.7 Porcine Model ............................................................................................................... 58
3.7.1 Experimental overview ......................................................................................... 58
3.7.2 Animals and Housing ............................................................................................ 60
3.7.3 Surgical wounds .................................................................................................... 60
3.7.4 Anesthesia and pain control .................................................................................. 61
3.7.5 Cellularization of skin scaffolds ........................................................................... 62
3.7.6 Wound dressings ................................................................................................... 63
3.7.7 Endpoints .............................................................................................................. 65
3.7.8 Wound Assessment ............................................................................................... 65
3.7.9 Safety Assessment ................................................................................................ 69
3.8 Statistical Analysis and graphical Representation ........................................................ 70
Chapter 4 Results .......................................................................................................................... 71
Results ................................................................................................................................... 71
4.1 Optimization of the current protocol for extraction of BD-MSCs ................................ 71
4.2 Characterization of key biological functions/characteristics of BD-MSCs .................. 73
4.2.1 Cells extracted from burned skin are mesenchymal stem cells ............................ 73
4.2.2 Burn does not cause cell dysfunction.................................................................... 77
4.3 Determination of potential risks for later clinical usage ............................................... 82
4.3.1 BD-MSCs are not tumorigenic ............................................................................. 82
viii
4.3.2 BD-MSCs display a low expression of MHC I, II, and TLR-4 ............................ 85
4.3.3 BD-MSCs differ in their cytokine and growth factor secretion profile from UC-
MSCs 89
4.4 Incorporation of BD-MSCs into synthetic skin substitutes .......................................... 91
4.5 Evaluation of BD-MSCs safety in a swine model ........................................................ 94
4.6 Evaluation of BD-MSCs' wound healing capacity in a swine model ........................... 98
4.6.1 Wound treatment with cellularized skin scaffolds improved epithelialization ..... 98
4.6.2 BD-MSC wound treatment does not affect epidermal thickness ........................ 103
4.6.3 BD-MSCs does not affect collagen deposition ................................................... 105
Chapter 5 Discussion .................................................................................................................. 107
Discussion ........................................................................................................................... 107
5.1 Cell extraction and delivery method ........................................................................... 107
5.2 Porcine wound healing model ..................................................................................... 110
5.3 Existence of viable and functioning mesenchymal stem cells in severely burned skin
111
5.4 Safety of BD-MSCs .................................................................................................... 116
5.5 BD-MSCs and inflammation ...................................................................................... 118
5.6 Effect on wound healing ............................................................................................. 120
Chapter 6 Future directions ......................................................................................................... 128
Chapter 7 Conclusion .................................................................................................................. 130
References ................................................................................................................................... 131
ix
List of Tables
Table 1. Classification of burns based on their depth. .................................................................... 3
Table 2. Primers for toll-like receptor 1-10. ................................................................................. 55
Table 3. Overview of pigs used. ................................................................................................... 59
Table 4. Safety data of BD-MSC wound treatment in porcine study ........................................... 96
x
List of Figures
Figure 1. Schematic overview of the different skin layers and the skin grafts. .............................. 6
Figure 2. Schematic overview of early excision and the usage of Integra®. ................................. 8
Figure 3. Schematic overview of scar types. ............................................................................... 11
Figure 4. Classification of skin substitutes. .................................................................................. 14
Figure 5. Phases of wound healing. .............................................................................................. 18
Figure 6. Overview of stem cells based on their source and their potency. ................................. 23
Figure 7. Relationship between stem cell potency and differentiation. ........................................ 25
Figure 8. Overview of adult stem cells. ........................................................................................ 30
Figure 9. Burned skin contains viable cells. ................................................................................. 36
Figure 10. Stem cell extraction methods....................................................................................... 40
Figure 11. Pig wounds and dressing layers. .................................................................................. 64
Figure 12. Overview of important time points.............................................................................. 66
Figure 13. The enzymatic stem cell extraction method is superior to conventional stem cell
extraction method.......................................................................................................................... 72
Figure 14. Burn derived dermal cells show MSC surface markers. ............................................. 74
Figure 15. Extracted cells from burned skin can differentiate into mesenchymal tissues. ........... 76
Figure 16. BD-MSCs are comparable to UC-MSCs in key biological characteristics. ................ 79
Figure 17. BD-MSCs are comparable to UC-MSCs in key biological characteristics. ................ 81
Figure 18. BD-MSCs are not tumorigenic. ................................................................................... 84
xi
Figure 19. BD-MSCs have a low immunogenicity and -reactivity comparable to UC-MSCs. .... 86
Figure 20. BD-MSCs do not differ in the expression of TLRs from UC-MSCs. ........................ 88
Figure 21. Cytokine, chemokine and growth factor expression profile of BD-MSCs differ from
UC-MSCs. ..................................................................................................................................... 90
Figure 22. Cellularization of Integra®. ......................................................................................... 93
Figure 23. The optical appearance of the scars at day 30. ............................................................ 97
Figure 24. BD-MSCs delivered via Integra improved epithelialization in vivo. ........................ 100
Figure 25. BD-MSCs delivered via PG-1 improved epithelialization in vivo. ........................... 102
Figure 26. BD-MSCs do not affect epidermal thickness. ........................................................... 104
Figure 27. In vitro cellularized skin scaffolds do not improve collagen deposition. .................. 106
Figure 28. BD-MSCs improve wound healing in mice. ............................................................. 121
Figure 29. BD-MSCs shorten proliferative phase of wound healing. ......................................... 122
Figure 30. Burn derived mesenchymal stem cells are integrated into the granulation tissue. .... 125
xii
List of Abbreviations
Ab/Am Antibiotic-antimycotic solution
BD Burn-derived
BrdU Bromodeoxyuridine
DMEM Dulbecco's Modified Eagle Medium
ECAR Extracellular acidification rate
Fb Fibroblast
FBS Fetal bovine serum
FCCP Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone
FGF Fibroblast growth factor
IFN Interferon
IL Interleukin
IP Interferon gamma-induced protein
MDC Macrophage-derived chemokine
MHC Major histocompatibility complex
MIP Macrophage inflammatory protein
MSC Bcl-2-related protein A1
OCR Oxygen consumption rate
P Passage
SC Stem cell
TBSA Total body surface area
TLR Toll-like receptor
UC Umbilical cord
1
Chapter 1
Introduction
Introduction
1.1 Burn injury
Burns are one of the most devastating traumata a patient can suffer from and continue to be a
severe public health issue (1). They are the fourth most common type of trauma and affect
more than two million people annually in the United States alone with a global incidence rate
of 1.1 per 100,000 population, causing approximately 330,000 death per year globally - a
number that is even believed to be grossly underestimated (1-4). After the initial treatment in
a highly specialized burn unit, such as the Ross Tilley Burn Center in Toronto, burn survivors
need a complex and costly interdisciplinary aftercare due to inevitable complications such as
debilitating scarring and contractures (5,6). This literature review highlights the immense
need for advances in burn treatment.
1.1.1 Definition and current treatment
Burn injury is defined by the American Burn Association (ABA) as an " injury to the skin or
other organic tissue primarily caused by thermal or another acute trauma”. It occurs when
some or all the cells in the skin or other tissues are destroyed by hot liquids (scalds), hot
solids (contact burns), or flames (flame burns). Injuries to the skin or other organic tissues
due to radiation, radioactivity, electricity, friction, or contact with chemicals are also
identified as burns (7,8)."
2
It is important for the treatment and outcome prediction to determine the exact extent of the
burn in its horizontal (total body surface area, TBSA; calculated as a percentage of the
whole-body surface area, TBSA%) and vertical axis (depth). The nomenclature to describe
the depth of the burn has changed over time (9,10) - see table 1:
3
Table 1. Classification of burns based on their depth.
Current Nomenclature
Former Nomenclature Depth Of burn
1. First degree burns
1. First degree burns
1. Confined to the epidermis
2. Partial-thickness or
dermal burns
a. Superficial partial-
thickness burns
b. Deep partial-
thickness burn
2. Second degree burns
a. Second degree a
b. Second degree b
2. Reaches into dermis
a. confined to the
upper (papillary)
dermis
b. extends to the deep
(reticular) dermis
3. Full-thickness burns 3. Third degree burns 3. Reaches through the
dermis into the
subcutaneous fat
4. Fourth degree burns 4. Fourth degree burns 4. Affects deep tissues
underneath skin and
subcutaneous fat
Content modified from Shahrokhi S. Burn Care E-Book. 1st ed. Shahriar Shahrokhi Books
4
First degree burns are treated with analgesics and polysporin cream. These burns heal without
scarring or pigmentation irregularities, and therefore are not considered in the calculation of
the TBSA%. Superficial dermal burns are very painful and usually heal spontaneously within
three weeks without leaving functional or optical impairments.
Deep dermal burns are less painful, but lead to (hypertrophic) scarring and functional
impairment if untreated (see below " Challenges in modern burn care"). Therefore, they are
treated like full-thickness burns and require mandatory excision of the burned skin followed
by grafting (see below) (11). This should be done preferably within the first days after burn
("early excision and grafting"), but no consensus exists about the exact timing (9). The main
rationale behind this practice is that by removing necrotic skin, which is an ideal environment
for infections, severe infectious complications such as sepsis, multi-organ failure and death
can be reduced (10). The removed skin is considered medical waste and discarded.
1.1.2 Challenges in modern burn care
A 2016 published European study emphasized wound treatment as the biggest challenge in
modern burn care, next to the management of inhalation injuries and the volume resuscitation
(12).
After burn injury, early excision of the burned skin followed by rapid wound coverage is
essential in deep-partial, full thickness and fourth degree burns not only for the survival of
the patient, but also to reduce debilitating hypertrophic scarring (13). Every tissue that was
damaged by the thermal stimulus is completely removed and requires replacement by a graft
(autologous, heterogeneous, synthetic, or xenograft) promptly to protect the wound from
5
dehydration, protein loss, and infection. After decades of research and various options,
autologous skin grafting is still considered the gold standard for wound coverage after
removal of the burned skin (14).
Human skin consists of epidermis, dermis and hypodermis. Epidermis (mainly keratinocytes)
is the outer layer and functions as a physical and chemical barrier. The dermis lays
underneath the epidermis and is composed of a majority of fibroblasts and collagen, which
gives the skin strength and flexibility. This layer also contains several important skin
appendices such as eccrine and apocrine glands or nerve endings. The furthest away from the
skin surface is the hypodermis - a fat layer that contains the major skin vessels and nerves
(9). Depending on the wound depth, the location of the injury or the pre-treatment of the
wound, skin grafts can be obtained in differing thicknesses’ - split-thickness or full-thickness
grafts. Figure 1 gives a schematic overview of the skin layers and the different depths of skin
grafts.
6
Figure 1. Schematic overview of the different skin layers and skin grafts.
Skin consists of Epidermis (outer layer), Dermis and Hypodermis. Classification of skin
grafts is based on their thickness, resp. the skin layers they contain: Full-thickness skin
grafts consist of the full epidermis and dermis, whereas split-thickness grafts also contain
epidermis but only part of the dermis.
7
Autologous skin grafts are harvested with a special knife - the dermatome. This procedure is
invasive, creating a new wound in a healthy skin area, and bears itself the risk for wound
complications such as pain and delayed healing (14,15). Unfortunately, the larger the burn in
terms of TBSA%, the less healthy skin is left for autologous skin grafting. Harvested skin
grafts can be expanded via meshing, but this expansion is limited and often the high
expansions rates of 1:4 or 1:6 claimed by the meshing device companies cannot be achieved
(14). This calls for alternative/synthetic wound coverage materials that need to have a high
availability and cost-effectiveness (16).
The most commonly used synthetic wound coverage material is Integra® (INTEGRA®
Dermal Regeneration Template, Integra LifeSciences Corporation, USA) consisting of
bovine collagen to facilitate cell ingrowth with an optional second layer of silicone on top for
better mechanical properties and wound coverage (17). This product was developed in the
1980s by Burke and Yannas and is considered a breakthrough in burn management (18-22).
While the wound is protected from dehydration, protein loss, and infection, dermal cells such
as fibroblasts can grow into the material and fill out the wound defect (see figure 2). Once
the dermal cells completely fill out the regeneration template, the silicone layer can be peeled
off and replaced by a thin split-thickness graft. This immediate wound coverage provides
enough time to stabilize the patient, but also reduces the depth of the autograft needed.
8
Figure 2. Schematic overview of early excision and the usage of Integra®.
After removal of the burned tissue, Integra® is placed onto the wound with the bovine
collagen layer directly connecting to the wound bed. Over time, cells such as fibroblasts
and monocytes migrate into the bovine collagen and form the granulation tissue. Once
this tissue fills out the entire bovine collagen, the upper layer of Integra® (silicone) can
be replaced by a thin split-thickness skin graft.
9
The main limitation of this product is that it is an acellular scaffold that completely depends
on the regeneration abilities and ingrowth of the patient’s own skin cells. Additionally, it still
requires a thin split-thickness autograft after the removal of the upper silicone layer once the
cell ingrowth into the bovine collagen (5,23).
After Hayflick and Morehead showed that the serial cultivation of human fibroblasts is
possible in 1961 (24), Rheinwald and Green achieved the same for keratinocytes in 1975 (25)
and hopes were high that lost skin due to burn injury could be replaced quickly and easily in
the near future, instead of waiting for the patient's own cells to regrow (23). This hope was
dashed in the following decades and to date, we still don't have satisfactory replacement
material resembling the human skin (26-29). This may be because the human skin is an
extremely complex structure consisting of multiple different cells originating from all three
germ layers (30), but also because "complex and costly products will not find commercial
applications" (31-33).
The herculean task for new therapies in burn wound management is, not only to accelerate
and improve wound healing, but also to prevent excessive tissue formation in the form of
hypertrophic scarring. Every burn wound that takes more than three weeks to heal (deep
partial thickness burns, full thickness burns, and fourth degree burns) leads to severe scarring
and needs to be treated with excision and grafting (34,35). Therefore, it is important to
determine the burn depth precisely: An underestimation leaves deep wounds untreated
increasing the risk for non-healing and scar formation, while an overestimation creates
unnecessary wounds (30). Even though, surgical techniques have improved over time,
leading to more functional and optimal outcomes (36), debilitation scarring remains one of
10
the most serious and costly long term complications tremendously reducing the quality of life
(37).
Since only fetal skin can heal completely without scarring, scar formation is a normal
physiological consequence of adult wound (9). However, excessive scar formation needs to
be prevented and treated because it not only causes a massive optical impairment, it also
restricts the movement of the patient and can lead to complete joint immobility. Two
different entities in excessive scarring exist: hypertrophic scars and keloids. If the excessive
tissue formation is confined to the area of the former wound (vertical axis), it is denominated
hypertrophic scar, however, if it exceeds this area it is referred to as a keloid (vertical and
horizontal axis) - see figure 3.
11
Figure 3. Schematic overview of scar types.
Excessive scarring can be categorized as hypertrophic scarring or keloid formation.
A scar is considered hypertrophic if excessive tissue is formed along the vertical
axis. If tissue also exceeds the horizontal border of the original injury, the scar is
considered a keloid.
12
While intact skin collagen secretion and break-down are balanced (37), burns cause a
disequilibrium resulting in excessive collagen deposition (9), which is the primary culprit for
hypertrophic scarring and keloid formation (38-41). The incidence of their occurrence is as
high as 90% after burn injury (42). While both have minor different microscopical
characteristics, such as collagen organization or dominant collagen type (38), both lead to
itching, pain, optical impairments such as pigmentation irregularities, and contractures that
can result in joint fusions and massive functional impairment (9). In addition, the differences
between those two entities seem to be more of academic interest than of clinical relevance, as
both are treated with the same techniques/drugs (42). A variety of treatment modalities are
available, from ablative laser therapy to cortisol injections, however, they are not satisfactory
due to high recurrence and treatment failure rates (15,43). Additionally, scar removal
treatments are not risk free (44), and in the case of scar surgery, this means another invasive
procedure for the patient.
New wound treatments for burn patients should consider both aspects - they need to improve
wound healing by replacing or stimulating dermal and epidermal cell proliferation, all the
while preventing over-stimulation to avoid excessive scarring.
1.2 Skin substitutes
A multitude of different approaches have been undertaken in the last decades to manufacture
and improve skin substitutes. Various attempts exist to classify the growing number of skin
substitutes, while trying to take into account the steadily increasing materials available, and
their different physical and biological properties (14,45). The most basic way to classify
them is based on the material used (i.e. synthetic or biological), or based on whether they are
13
cell-free or cell-containing (46,47). While cell-free materials such as Integra® are aimed to
promote autologous healing, the cell containing substitutes ideally provide an immediate
functioning skin replacement. Figure 4 depicts a more differentiated way of categorizing
skin substitutes, broadcasting their immense diversity (based on the proposal of the Plastic
Surgery Service of Hospital das Clínicas of the School of Medicine of Universidade de São
Paulo) (44,48,49).
14
Figure 4. Classification of skin substitutes.
Skin substitutes can be classified by the origin of their main material, their durability, the
skin layer they are used to substitute, and the cells that were integrated into them.
Graphic based on the proposal of the Plastic Surgery Service of Hospital das Clínicas of
the School of Medicine of Universidade de São Paulo.
15
The larger the burn is in terms of TBSA% the higher the need for cell-containing materials. It
is safe to say that patients who lose up to 95% of their skin surface do not have the
physiological resources to regrow their own skin and therefore require skin substitutes that
contain cells to immediately replace the lost ones. This high demand for cells can potentially
be met by differentiated cells or by stem cells, whether autologous or allogenic (50,51).
DIFFERENTIATED CELLS: Even though composite skin substitutes with incorporated
differentiated skin cells (allogenic neonatal foreskin keratinocytes and fibroblasts) are already
commercially available, such as Apligraf® (Organogenesis, Inc, Canton, Massachusetts) or
Orcel® (Forticell Bioscience, Inc, New York, New York) , allogenic differentiated cells do
not persist in the wound, nor do they contain a full representation of all skin cells such as
Langerhans cells or Melanocytes and therefore do not replace lost skin sufficiently
(44,48,50,51). The benefits of those products are that they supply the healing wound with
extracellular matrix proteins and wound healing promoting factors such as cytokines from
both epidermal and dermal cells (52).
Autologous differentiated cells, especially keratinocytes, cannot yet be cultured in the amount
and time required to treat burn patients (53,54). Various products using cultured epithelial
autografts (CEA) have been tested or are still undergoing clinical testing. Their limitations
include slow culturing rates of cells, high costs, durability, and vulnerability of the grafted
cells. Due to those limitations, it is unclear if they will have a future in burn care (44). In
addition, skin is a highly complex tissue consisting of multiple cells and structures. A true
skin substitute with differentiated cells is required to contain not only keratinocytes and
fibroblasts, but also more complex structures such as sweat glands and hair follicles, essential
for normal skin function (55). Many attempts and approaches have been undertaken to
16
achieve this, however, these approaches are at an experimental stage or have not shown any
clinical relevance (51,56-58).
STEM CELLS: The thought advantage of stem cells for skin regeneration is their ability to
differentiate into various skin cell types, simplifying the process of producing an "off-shelf"
skin by narrowing the materials down to two main components: stem cells and a scaffold
(59,60). Stem cells have been shown to have highly beneficial paracrine effects that promote
wound healing, including the secretion of tissue growth factor (TGF) and epidermal growth
factor (EGF) (61). Treatment of burn and non-burn wounds with different types of stem cells
has already shown very promising results in animal models and humans (see chapter "The
role of stem cells in skin regeneration and wound healing"). However, it is yet to be
determined how to incorporate stem cells into skin scaffolds, which skin scaffold is the most
feasible for stem cell therapy, and most importantly, which stem cell source offers the most
capable stem cells for skin regeneration in terms of promotion of wound healing and
prevention of excessive scarring.
1.3 Physiology of wound healing and its meaning for cell therapy
The local application of skin substitutes with or without incorporated cells onto the burn
wound will interfere with the body’s own wound healing, therefore it is important to
understand the physiology of wound healing and its challenges and possible opportunities for
cellular therapies. Burns differ from other skin injuries in terms of systemic complications,
such hypermetabolism and systemic inflammation, nevertheless wound healing always
follows the same principles outlined below (23).
17
Physiological wound healing consists of four phases (62): hemostasis, inflammation,
proliferation, and remodeling phase (see figure 5). Those phases are not strictly distinct from
each other and can occur simultaneously depending on the severity, the type of burn and the
patient's individual wound healing resources.
18
Figure 5. Phases of wound healing.
Wound healing occurs in four consecutive and sometimes simultaneous phases:
hemostasis, inflammation, proliferation, and remodeling.
19
Thermal injury leads to a destruction of skin vessels and a vasoconstriction followed by a
thrombus formation (hemostasis phase). Usually two to three days later, granulocytes and
monocytes will migrate to the wounded area, marking the beginning of the inflammatory
phase. Fibroblasts are stimulated and secrete various extracellular matrix (ECM) proteins
such as collagen and elastin (proliferation phase). The now formed tissue is called
granulation tissue. This phase can last up to one month depending on the wound size. Lastly -
in the remodelling phase - the earlier secreted ECM material will become more organized.
Simultaneously to the proliferation of fibroblasts, epidermal repair begins with the migration
of keratinocytes from the wound edges towards the wound center (49).
Since the hemostasis phase is mainly a direct correlate to the tissue trauma and occurs within
minutes (63,64), the inflammatory phase is the first phase that offers a real therapeutic
opportunity. In addition, the practice of the gold standard "early excision and grafting" in full
and deep-partial thickness burns (65) takes place in this specific time period of wound
healing (52,66). The physiological meaning of the inflammatory phase is the degradation of
necrotic tissue, the prevention of wound infections and the induction of further wound repair
via cytokine and chemokine expression by immune cells (mainly neutrophils and monocytes)
(67).
It is unclear to what extent this inflammation shifts from aiding wound healing to impeding
it. On the one hand, this initial inflammatory response to the burn tissue damage is essential
for wound healing: the immune cells (mainly neutrophils and monocytes) activate fibroblasts
and keratinocytes, respectively, and their progenitors inducing wound closure and re-
epithelialization (proliferation phase) (68). Animals that lack these vital immune cells, for
20
example macrophage-depleted mice, display great deficiencies in all phases of wound
healing, resulting in delayed wound closure, decreased angiogenesis, collagen synthesis,
granulation tissue formation, and reductions in important growth factors such as VEGF and
TGF-beta.
On the other hand, the same inflammation is closely linked to the formation of hypertrophic
scars and keloids (69) - one of the major long-term complications in burn survivors leading
not only to high psychological distress but also to severe physical impairments. In addition,
the vasodilatation caused by the inflammation results in tissue edema that further
compromises wound healing (70-72).
Since inflammation is a double-edged sword with an essential meaning for wound healing by
initiating and regulating it, it will be a great challenge of every new therapy to find the right
balance between pro- and anti-inflammatory effects.
It is known that bone-marrow derived stem cells have the capacity to translocate from the
bone-marrow to injured tissue and improve its regeneration. Rea at al. claim that over half of
the fibroblast population in the granulation tissue and a small percentage of keratinocytes
have their origin in the bone marrow, indicating the great importance of stem cell migration
and differentiation in the healing of burn wounds (73). This confirmed previous studies in
excisional wounds as well as in healthy skin- that bone marrow stem cells are an important
part of skin regeneration and maintenance. Burn patients that lose a major part of their body
surface, thus require highly functioning bone-marrow derived stem cells to aid in skin
regeneration. Interestingly, a lung fibrosis model also linked bone marrow stem cells to scar
formation (74-76). Unfortunately, severe illness and infections are known to suppress bone
marrow, first noticed historically as anemia in critically ill patients. This data would mean
21
that the more bone marrow is suppressed, the iller a burn patient is, and the higher is his need
for external sources of stem cells.
The last and longest phase in wound healing is the remodeling phase. Most cellular
components of the earlier formed granulation tissue such as macrophages and myofibroblasts
undergo apoptosis, protein syntheses, decreases in cell proliferation, and reorganization of
secreted collagen (77). While inflammatory cells play an essential role in initiating wound
healing and the proliferation phase, they also seem to play a key role in the remodeling phase
by directing ECM breakdown, production, and remodeling. A T-helper cell type 2 (Th2)
dominant environment is suspected to cause hypertrophic scarring in burn patients, by
inducing ECM production and reducing its breakdown. Macrophages and lymphocytes have
an extensive cross talk and stimulate each other (78). Interestingly, T-lymphocytes (adaptive
immune system) only seem to play an important role in larger or complex wounds such as
burns, in comparison to smaller, uncomplicated wounds that remain directed by granulocytes
and macrophages (innate immune system). The rearrangement and cross-linking of collagen
fibres as well as the substitution of the weaker collagen III with the stronger collagen I is an
essential part of the remodeling phase, and determines the strength of the newly formed tissue
(79). This shows that the balance between stimulation of (myo-) fibroblastic production and
its inhibition by cytokines from immune cells is of paramount importance for the properties
of the final scar. Therefore, an ideal wound therapy should carefully modulate inflammation
in order to reduce excessive collagen deposition and scarring.
Stem cells that have the ability to influence every phase of wound healing from the
inflammation to the remodeling phase - via paracrine mechanisms, ECM production or
22
differentiation into skin cells - are considered a promising new therapeutic approach
(76,77,80).
1.4 The role of stem cells in skin regeneration and wound healing
The discovery and continuously increasing understanding of stem cells and their abilities is
considered to be a milestone in regenerative medicine and offers the high hope of finding a
cure for various conditions from infertility to organ dysfunction. Despite a massive increase
in stem cell research and publications, this relatively new field of medical science is only at
its beginning and much more research is warranted to understand the clinical meaning and to
develop effective cell-based therapies using stem cells.
1.4.1 Overview - classifications and characteristics of stem cells
All stem cells have three unique defining characteristics: (1) the ability for self-renewal, (2)
their lack of specialization, and (3) the ability to differentiate into at least one specialized cell
type or lineage (potency) (81). The strength of those characteristics varies in different stem
cells, mainly due to their source. Figure 6 provides an orienting overview of important stem
cell sources, their names and their potency. Stem cells are usually categorized based on their
potency or source.
23
Figure 6. Overview of stem cells based on their source and their potency.
Stem cells can be categorized based on their general source: Embryonic, fetal/neonatal, and
adult stem cells. Further sub-classifications can be made using their exact tissue of origin.
Graphic based on Gargett C. Review article: stem cells in human reproduction. SAGE
Publications; 2007 Jul;14(5):405–24.
24
EMBRYONIC STEM CELLS: Stem cells harvested from an embryo at the time of formation
of the zygote (=fertilization of an oocyte by a sperm) to the 4-cell stage have the highest
potency - they are totipotent (figure 7 shows the different degrees of potencies and their
correlation with the differentiation of the stem cell, based on Wagers and Weissmann et al.
(82)).
25
Figure 7. Relationship between stem cell potency and differentiation.
The potency of a stem cell is negatively correlated to its differentiation. Graphic based on
Wagers AJ, Weissman IL. Plasticity of adult stem cells. Cell. 2004 Mar 5;116(5):639–48
26
This means that totipotent cells can give rise to an entire human organism, including the
placenta. If harvested at a later time point from the inner cell mass of the blastocyst, the stem
cells are less potent, referred to as pluripotent. They can develop into an entire human
organism, but not into a placenta (83). Embryonic stem cells can propagate indefinitely, a
characteristic that makes them highly interesting for regenerative medicine. It is known that
upon differentiation embryonic stem cells partially lose their favourable immunomodulatory
properties, making them susceptible to immunologic rejection when used as an allograft (84).
Embryonic stem cells have already displayed promising results in multiple fields of
regenerative medicine, ranging from the regeneration potential of neurons to the possible
reconstitution of skin (85,86). However, since extracting those cells means destroying or
damaging an embryo, a broad clinical application will be opposed by high ethical and moral
concerns. Klimanskaya et al. showed that human embryonic stem cells could potentially be
gained in the near future without interfering with embryonic development, however, further
research is warranted in this field (87). Apart from the ethical concerns, embryonic stem cells
have a risk of teratoma formations, a major obstacle for their clinical application (88). Tissue
that is created in vitro out of embryonic stem cells, needs to be completely depleted of any
remaining pluripotent cells before transplantation into the patients - a process that needs to be
refined in the future.
INDUCED PLURIPOTENT STEM CELLS: Recent advances in stem cell research made it
possible to create pluripotent stem cells out of differentiated adult cells (induced pluripotent
stem cells, iPSC). This technique was first developed in 2006 by Yamanaka et al. using
murine fibroblasts (89). The reprogramming was achieved by introducing genes for
transcription factors (via retroviral transduction) that are essential for maintaining
27
pluripotency in embryonic stem cells: octamer-binding transcription factor (Oct) 3/4, sex
determining region Y-box (Sox) 2, c- myelocytomatosis viral oncogene (Myc), and Kruppel-
like factor (Klf) 4. The possibility to create patient-specific autologous cells that are
pluripotent and have unlimited self-renewal is of great interest for regenerative medicine.
Another benefit of such approach is the elimination of immunologic rejection, since those
cells are autologous. Various researchers have shown the immense regenerative potential of
iPSCs. Satoshi et al. used iPSCs for neuronal regrowth in spinal cord injuries in mice (90)
while Shamis et al. found iPSC-derived fibroblasts to display an augmented production of
extracellular matrix - a finding that is highly important for wound healing. However, the
high safety concerns prohibit the clinical application of iPSCs in the near future (45) and the
first clinical trial using iPSCs was suspended recently, because of those concerns . Since
iPSCs are pluripotent cells, they also pose a risk of teratoma formation, similar to or even
higher than embryonic stem cells (91). The above described transduced transcription factors
are also expressed in malignant tumors further fueling safety concerns. In addition, the
changes at a molecular level that happen during reprogramming of an adult cell into an iPSC
are not well understood and require further research (91-93).
FETAL STEM CELLS: Fetal stem cells can be extracted from a variety of sources such as
the umbilical cord, the umbilical cord blood, fetal blood, the placenta, as well as from the
amniotic fluid or fetal organs such as the liver. Depending on their source, they are different
subtypes of fetal stem cells: The most commonly used ones in regenerative medicine are
hematopoietic stem cells (e.g. stem cells from umbilical cord blood) and MSCs (e.g. stem
cells from the umbilical cord), but is also possible to obtain endothelial (e.g. from the
placenta), epithelial (e.g. from the liver), and neural (e.g. from the brain) fetal stem cells (82).
28
One single source can also contain more than one stem cell type such as the fetal blood,
which contains both mesenchymal and hematopoietic stem cells. Hematopoietic and MSCs
are multipotent, meaning they can develop into more than one cell type, however, their
differentiation potential is more limited than pluripotent stem cells. Hematopoietic stem cells
can only differentiate into cells of the hematopoietic lineages such as granulocytes or
lymphocytes (83). MSCs, on the other hand, can only differentiate into mesenchymal tissue
(mesenchyme is a specific part of the mesoderm - the second germ layer) such as cartilage,
adipose, and bone tissue. The ultimate differentiation of those cells is determined by various
intrinsic and extrinsic factors such as gene expression, microenvironment, and growth
factors.
Recent studies have shown that this seemingly pre-destined differentiation path is not as rigid
as it was believed to be. Gioretti et al. for example, reprogrammed human umbilical cord
stem cells to neuronal cells by inducing the ectopic expression of the transcription factor
Sox2 (84).
Fetal stem cells are considered a valuable component for regenerative medicine because they
are an intermediate between embryonic stem cells, which have the highest potency but also
the highest ethical concerns, and adult stem cells, which have the least ethical concerns but
also the lowest potency. In addition, fetal stem cells seem to have a favourable immune
profile making them a promising candidate for allogenic transplantation (85,86). Umbilical
cord blood transplantation is a well stablished clinical practice and has shown better
outcomes than the transplantation of adult bone marrow stem cells, especially in pediatric
patients. Various in vitro and in vivo studies on animals and humans already confirmed the
high regenerative capacities of fetal stem cells from muscle (88) to bone regeneration and
promotion of wound healing (89). Furthermore, fetal stem cells displayed superior
29
regenerative potential and seem to be better tolerated (90) when compared to adult stem
cells. However, the usage of fetal tissue in the form of stem cells is limited by availability and
ethical concerns.
ADULT STEM CELLS: Adult stem cells are undifferentiated cells with limited
differentiation and self-renewal capacity. They can be harvested from a variety of
differentiated tissues such as the liver or the gut (45). Since their differentiation potential is
mainly predetermined based on the germ layer they are derived from, they are often classified
based on their mesodermal (such as mesenchymal or cardiac stem cells), ectodermal (such as
neural stem cells or ocular stem cells), or endodermal (such as pulmonary epithelial stem
cells or gastrointestinal tract stem cells) origin. In some literature, fetal stem cells are also
considered adult stem cells. Figure 8 gives an overview of the different adult stem cells
classified by their germ layer-origin based on Körbling et al. (91). Mesodermal hematopoietic
stem cells seem to have a unique characteristic similar to pluripotent embryonic stem cells -
they can differentiate across germ layer boundaries into endodermal and ectodermal cells.
Moreover, the previous rigid understanding of tissue-specific differentiation of adult stem
cells, in general, is under question (91-93).
30
Figure 8. Overview of adult stem cells.
Adult stem cells are categorized based on the germ layer they are derived from: Endoderm,
mesoderm, and ectoderm. The germ layer origin determines the differentiation of the adult
stem cells. Graphic based on Körbling M, Estrov Z. Adult Stem Cells for Tissue Repair — A
New Therapeutic Concept? Massachusetts Medical Society; 2009 Oct. 7;349(6):570–82
31
The most commonly used adult stem cell source is bone marrow. Bone marrow contains
hematopoietic and MSCs, both of mesodermal origin (45). The use of this stem cell source is
already part of clinical practice since 1968 with the first bone marrow transplantation at the
University of Minnesota. It displayed extremely promising results in regenerative medicine
and even reached the stage of advanced clinical trials (94,95). Since adult stem cells include
cells from all three germ layers, their possible therapeutic application is vast with an
immense, steadily increasing number of publications in this field and needs to be discussed in
detail elsewhere. In summary, adult stem cells are a promising source for tissue regeneration
throughout the whole spectrum of somatic tissues, reaching from improving bone structure in
osteogenesis imperfecta patients via bone marrow transplantation (95) to improving cardiac
functions after infarction by intramyocardial stem cell implantation (96). Of all adult stem
cells, MSCs in particular seem to be an ideal candidate for cellular wound therapy, since they
are available in human skin and are known to be an essential part of physiological wound
healing while being considered safe if derived from an external source (97-101).
1.4.2 Mesenchymal stem cells as wound therapy
The human skin is a complex multicomposite tissue, containing different stem cell
populations in epidermis and dermis that play an important role in skin regeneration and
wound healing: Epidermal stem cells can regenerate a variety of epidermal structures such as
sebaceous and sweat glands, keratinocytes, and hair follicles (57,102-107). The dermis
contains MSCs (108-111) that are fibroblast-like and produce an extracellular matrix which is
responsible for skin strength and elasticity (49,110,112). In physiological wound healing,
MSCs - from the surrounding skin or the bone marrow - migrate towards the wound bed and
form the granulation tissue together with immune cells and fibroblasts, enabling epidermal
32
regeneration via paracrine mechanisms such as the secretion of epidermal growth factor
(97,98,113,114).
MSCs are multipotent fibroblast-like cells, also known as mesenchymal stromal cells. The
International Society for Cellular Therapy released 2006 minimal criteria to identify and
characterize them in vitro (115): (1) They must adhere to a plastic surface, (2) they must
express Cluster of Differentiation (CD) protein 105, 90, 73 while lacking CD 45, 34, and 14
or CD 11b, 79alpha, or CD 19 and HLA-DR, (3) they must differentiate into main
mesenchymal tissues - adipose tissue, cartilage, and bone tissue.
The initial mechanism of action through which MSCs are believed to contribute to wound
healing is their ability to differentiate into required cells for skin regeneration such as
fibroblasts and endothelial cells (114,116,117). When exposed to EGF or VEGF, for
example, MSCs could be differentiated towards epidermal and endothelial cells, respectively
(118). However, it is a very controversial finding that MSCs can differentiate across germ
layer borders. Nowadays the promotion of wound healing and skin regeneration is mainly
attributed to the various paracrine effects of the MSCs such as the secretion of vascular
endothelial growth factor (VEGF)-alpha, insulin-like growth factor (IGF)-1, epidermal
growth factor (EGF), and keratinocyte growth factor (46,47,119-121). They are also capable
of secreting essential ECM components such as collagen (122,123). Depending on the source
of MSCs, they display different characteristics. Amable et al. showed that umbilical cord
stem cells express more growth factors, while adipose-derived MSCs produce more ECM
components (123). The beneficial effect of MSCs in wound healing seems to extend beyond
their differentiation and paracrine potential. Some studies suggest that wound healing is
33
promoted even if the MSCs undergo apoptosis causing a release of immune modulating
factors such as TNF-stimulated gene 6 protein (TSG-6) (124,125).
MSCs have a complex interaction with the adaptive and innate immune system by cell-cell
direct contact as well as by paracrine mechanisms (126). They are shown to suppress the
proliferation and activation of T- and B-lymphocytes, antigen presenting cells, macrophages
and natural killer cells while upregulating regulatory T cells (127,128),(129-132). MSCs even
appear to be helpful in the treatment of systemic as well as local inflammation. This
mechanism is believed to be mainly due to prostaglandin E2 (PGE2) depended macrophage
reprogramming that reduces their (interleukin) IL-6 and TNFalpha secretion - the main
inflammatory cytokines - while upregulating the secretion of the anti-inflammatory IL-10
(133,134). This immunosuppressive mechanism also seems effective in the reduction of local
wound inflammation leading to an improvement in wound healing (135). However, the
interaction between MSCs and the immune system is not fully understood (52). The
reduction of inflammation is also believed to reduce scarring, as skin wounds heal without
scarring in embryos that have no developed immune system (136). Compared with syngeneic
MSC injection into surgical wounds in mice, allogenic MSCs were equally efficient in the
promotion of wound healing and did not show a higher amount of inflammation or
immunologic rejection (132). Additionally, MSCs also seem to have antimicrobial properties
by the secretion of the peptide LL-37 (137).
The promising effects of MSCs in skin regeneration obtained in vitro and in animals, has
been reproduced in humans (138-140). Zhiyong et al. for example were able to successfully
transplant sweat glands created out of bone marrow-derived MSCs into burn patients (139).
34
A combination of autologous fibroblasts and MSCs aided in wound healing in non-healing
diabetic foot ulcers (140).
However, to date, there is no ideal source for MSCs for skin regeneration. Allogenic sources
always have the risk of Graft-versus-Host immune reactions (see above) and might require
immune suppression - fatal for burn patients in which sepsis and infections are recognized as
one of the most dangerous and potentially lethal morbidities (141). Therefore, autologous
sources are preferred, since they do not bear the risk of immunologic rejection, however,
bone marrow derived stem cells that showed promising results require an invasive procedure
facilitating osteomyelitis which is not acceptable for the severely ill burn patients that have a
high risk of infection (141). In addition, the cell number that can be gained with a bone
marrow aspiration is very limited (142). The extraction of MSCs out of the patient’s own
healthy skin is also not an option, since this would create another wound, or - because in the
case of larger burns - there is not enough unburned skin left. This shows a great need for an
autologous source of MSCs, providing a high number of cells without the need for invasive
procedures.
1.5 Preliminary data
Burned skin needs to be excised in every patient to reduce the risk of wound infections and
decrease mortality. We made the groundbreaking discovery that this severely burned skin -
which is considered dead tissue and discarded as medical waste - contains viable cells (see
figure 9). Those cells showed a high proliferation and good attachment to the plastic surface
of the cell culture flask, fulfilling one of the definition criteria for MSCs.
35
The finding that burned skin might contain MSCs would be revolutionary for regenerative
medicine and for the treatment of burn patients, since it would meet the demand for a new
stem cell source that is not limited by ethical concerns, additional invasive procedures, and
might even bear the potential of autologous stem cell transplantation overcoming adverse
immunological effects.
36
9. Burned skin contains viable cells.
Viable cells could be extracted out of severely burned skin and cultured. The dotted line
shows the outline of a cell attached to the plastic surface of a cell culture flask 1 week after
extraction from burned skin.
37
Chapter 2
Rationale, Hypothesis, and Aim
Rationale, Hypothesis, and Aim
2.1 Rationale
Burn injury is one of the most severe traumata a human can suffer resulting in a high personal
and socioeconomic burden. Despite impressive advances in the last decades, to date, there is
no replacement for the lost skin after burn injury, leaving a great need for new therapeutic
options in this area. Those options ideally improve not only wound healing but are also able
to prevent debilitating scarring. MSCs from various sources such as bone marrow and
umbilical cord have been shown to fulfill those requirements: they accelerate wound healing
and reduce scarring in vitro and in vivo. Unfortunately, there is no ideal source of MSCs -
their extraction either requires an invasive procedure, goes along with high ethical concerns,
has a low cell yield or bears the risk of immunologic rejection. For the first time, our lab has
shown that burned skin, which must be excised in every burn patient and is considered
medical waste, contains viable cells with characteristics of MSCs. Those cells need to be
fully classified and systematically examined in terms of their biological functionality and
their wound healing capacity.
2.2 Hypothesis
Cells extracted from burned skin are healthy, fully functioning mesenchymal stem cells -
burn-derived mesenchymal stem cells (BD-MSCs) - that promote wound healing.
38
2.3 Aim
The aim of this thesis is to characterize the cells extracted from the burned skin and to
evaluate their future role for regenerative medicine and wound healing. To achieve this, the
following sub-aims were established:
1) Optimizing the current protocol for extraction of BD-MSCs to obtain a higher yield of stem
cells
2) Characterization of key biological functions/characteristics of BD-MSCs
3) Determination of potential risks for later clinical usage
4) Incorporation of BD-MSCs into synthetic skin substitutes
5) Evaluation of BD-MSCs safety well as wound healing capacity in a swine model
39
Chapter 3
Material and Methods
Material and Methods
3.1 Stem Cell extraction and culturing
The study was ethically approved by the Sunnybrook Research Institute Research Ethics
Board (REB) ethics committee: REB # 017-2011. Cells were extracted from either skin or
umbilical cord tissue that was stored in Dulbecco's Modified Eagle Medium (Gibco™
DMEM, Thermo Fischer Scientific, Canada) enriched with 1% antibiotic-antimycotic
solution (Ab/Am; Gibco® Antibiotic-Antimycotic, Thermo Fischer Scientific, Canada) for a
maximum period of 24 hours in the fridge at 4°C. Before the extraction, the tissue was
washed with 70% ethanol for 30 seconds followed by 1 % Phosphate-buffered saline (PBS;
Wisent Inc., Canada) with 1% Ab/Am and PBS with 2% Ab/Am for 1 minute each. Stem
cells were either extracted via an enzymatic cell extraction method or via the conventional
cell extraction method - see figure 10.
All umbilical cords were donated by our obstetrical department after consent from the
patients were obtained and collected within 6h after birth. Burned skin was excised at the
Ross Tilley Burn center and reached the lab within 3h after surgery. For each experiment we
used cells derived from at least three different tissue donors. Premature neonates, septic
births, and septic/infected burn patients as well as tattooed skin samples were excluded. The
tissue itself as well as the extracted cells were not solely used for this project. Cells from one
donor were used in various experiments. Selection was only based on passage numbers since
the samples were de-identified during the experiments.
40
Figure 10. Stem cell extraction methods.
(A) Human burned skin or (B) Human umbilical cords were subjected to either (C) the
enzymatic extraction method or (D) the conventional cell extraction method.
41
3.1.1 Conventional Stem Cell Extraction Method
For the conventional cell extraction method, the tissue was cut in 0.5cm2 squares, placed in a
cell culture dish and submersed with DMEM medium enriched with 10% FBS (Gibco™ fetal
bovine serum, Life Technologies Corporation, USA) and 1% Ab/Am. Scratches were made
into the dermal surface of the tissue to enhance the number of isolated cells. Outgrowth of
MSCs from the tissue pieces was visible under the light microscope and was controlled daily.
Once cell outgrowth took place, the tissue pieces were removed. When the cells reached 90%
confluency they were split and seeded into cell culture flasks.
3.1.2 Enzymatic Stem Cell Extraction Method
For the enzymatic cell extraction method, the tissue was minced with a scalpel and then
incubated in an enzymatic cocktail consisting out of human collagenase 1 (Worthington
Biochemical Corporation, USA), dispase (Life Technologies Corporation, USA), trypsin
(Life Technologies Corporation, USA), DMEM medium with 1% Ab/Am. After 60min
incubation under constant rotation at 37.5°C, the cell-enzyme mix was diluted with PBS
50:50 and filtered through a 10μm cell strainer (Falcon® 10µm Cell Strainer, Corning, USA).
The filtered cell-enzyme mix was centrifuged at 1000rpm for 10min and - after discarding of
the supernatant - plated in cell culture flasks with 7ml of DMEM-medium enriched with
1%Ab/Am and 10%FBS.
42
3.1.3 Cell culturing
The standard culture medium consisted out of Dulbecco's Modified Eagle Medium, 10%
FBS, and 1% antibiotic-antimycotic solution. Media changes took place every second or third
day. For all experiments, only cells that were extracted with the enzymatic cell extraction
method were used.
All cells were cultured in 75cm2 cell culture flasks and split when reaching a confluency of
90%. To split the cells, we first removed the cell culture medium, then washed the cells with
PBS, followed by incubation with trypsin (5ml/flask, incubation time 5-6min in the tissue
incubator). The cell-trypsin mix was then diluted with 5ml cell culture medium to stop the
enzymatic reaction of trypsin. After centrifugation (5min, 600rpm) and removal of the
supernatant, the cells were resuspended in 7ml of cell culture medium and plated in fresh cell
culture flasks.
3.1.4 Quantification of cell yield
One cm2 of each burned skin and five cm of umbilical cord, respectively, was subjected to
the enzymatic or the conventional extraction method and placed in a cell culture flask each.
Cell culture flasks were evaluated after 24 under a light microscope: We assessed the
number of adherent cells as described below.
The outline of the cell culture flask was drawn on a see-through plastic foil. The so created
shape was divided into 0.5 x 0.5cm squares and according gridlines were created. The plastic
foil with the grid lines was attached to the bottom of the flask while assessing it under the
43
light microscope. Five of those 0.5 x 0.5cm squares were randomly chosen for cell counting
and used consistently for all flasks.
We recorded the attached cells for each of those five squares using a manual hand counter.
Since one flask has 300 squares, we multiplied each cell count by 300 to extrapolate the total
number of cells in the culture flask based on one counted 0.5 x 0.5cm square. An average was
created out of those five values and was reported as the average number of cells per flask
after 24h extracted from 1cm2 of burned skin.
3.2 Determination of mesenchymal stem cell character
3.2.1 Flow Cytometry for stem cell surface markers
Cells from three different umbilical cords and three different burn patients were extracted via
the enzymatic method. Cells had either a passage number of one (equal to 1-1.5 weeks in
culture) or a passage number of three to four (equal to 3-4 weeks in culture). After reaching a
confluency of 90%, they were trypsinized, resuspended in flow buffer consisting out of
Hank's Balanced Salt Solution (HBSS; Wisent Inc., Canada) and 1% bovine serum albumin
(WISENT Inc., Canada). They were incubated with the conjugated antibodies (ratio 1:100) -
CD105 (eBioscience, Thermo Fisher Scientific, Canada), CD 90 (Thermo Fisher Scientific,
Canada), CD73 (Thermo Fisher Scientific, Canada), and CD34 (Invitrogen, Thermo Fisher
Scientific, Canada) - for 30min on ice in the dark. After washing, cells were analyzed with
the BDTM LSR II flow cytometer (BD Biosciences, Canada) using the BD FACSDIVA™
SOFTWARE (BD Biosciences, Canada). The graphical and statistical analysis was done in
FlowJo™ v10 for MAC and Prism 5 for Mac OS X.
44
3.2.2 Differentiation Assay
Cells were incubated in the specific differentiation media for 10 days, followed by fixation
and staining. We used an n=3 for each cell line i.e. cells came from 3 different patients.
Experiments were conducted in triplicates per patient. Cells had a maximum passage number
of 4 which equals a culture time of maximum 4 weeks after extraction.
Osteogenic differentiation: Cells were seeded in a 24-well plate. Differentiation Medium -
consisting of L-ascorbic acid-2-phosphate (Sigma-Aldrich, Canada), β-glycerophosphate
disodium salt hydrate (Sigma-Aldrich, Canada), Dexamethasone (Sigma-Aldrich, Canada),
Dulbecco's Modified Eagle Medium low-glucose media (Gibco™ DMEM, Thermo Fischer
Scientific, Canada), FBS (Gibco™ fetal bovine serum, Thermo Fischer Scientific, Canada),
and antibiotic-antimycotic solution (Gibco® Antibiotic-Antimycotic, Thermo Fischer
Scientific, Canada) - was added once the cells reached 90% confluency. Media change took
place every second day. After 10 days, cultured cells were fixed with 75% Ethanol and
stained with Alizarin Red S (Sigma-Aldrich, Canada). The positive stained area was
measured with Image J Version 1.51 for MAC.
Adipogenic Differentiation: Cells were seeded in a 24-well plate. Differentiation Medium -
consisting of 3-isobutyl-1-methylxanthine (Sigma-Aldrich, Canada), insulin (SAFC
Biosciences, USA), indomethacin (Sigma-Aldrich, Canada), Dexamethasone (Sigma-Aldrich,
Canada), Dulbecco's Modified Eagle Medium low-glucose media (Gibco™ DMEM, Thermo
Fischer Scientific, Canada), FBS (Gibco™ fetal bovine serum, Thermo Fischer Scientific,
Canada), and antibiotic-antimycotic solution (Gibco® Antibiotic-Antimycotic, Thermo
45
Fischer Scientific, Canada) - was added once the cells reached 90% confluency. Media
change took place every second day. After 10 days, cultured cells were fixed with 75%
Ethanol and stained with Oil Red O (Sigma-Aldrich, Canada).
Chondrogenic Differentiation: Cells were cultured in 15ml tubes (400.000 cells/tube).
Differentiation Medium - consisting of L-ascorbic acid-2-phosphate (Sigma-Aldrich,
Canada), Insulin-Transferrin-Selenium (Corning™ cellgro™ Insulin-Transferrin-Selenium,
Corning Incorporated, USA), Dexamethasone (Sigma-Aldrich, Canada), sodium pyruvate
Sigma Aldrich, Canada), TGF-β1, Dulbecco's Modified Eagle Medium low-glucose media
(Gibco™ DMEM, Thermo Fischer Scientific, Canada), FBS (Gibco™ fetal bovine serum,
Thermo Fischer Scientific, Canada), and antibiotic-antimycotic solution (Gibco® Antibiotic-
Antimycotic, Thermo Fischer Scientific, Canada) - was added after cells were centrifuged
with 1000rpm for 5min and the supernatant removed. After 10 days, cultured cells were fixed
with 4% paraformaldehyde (Electron Microscopy Sciences, USA) and stained with Alcian
Blue (Alcian Blue 8GX, Santa Cruz Biotechnology, Canada). The positive stained area was
measured with Image J Version 1.51 for MAC.
3.3 Determination of key biological characteristics
3.3.1 Population doubling time
Cells from three different umbilical cords and three different burn patients were extracted via
the enzymatic method. Cells with a passage number of 3-4 were seeded into 24-well culture
plates (100 cells per plate). Each biological sample was assessed in triplicates. Adhesive cells
46
were counted after 24 (Ni) and 48 hours (Nn). Population doubling time (PDT) was
calculated with the following formula: PDT=48h/((logNn)-(logNi)/log2).
For cells with the passage number of 1, we counted attached cells 24h and 48h after
enzymatic cell extraction in the cell culture flask as described above under "Quantification of
cell yield" and calculated the PDT with the same formula.
3.3.2 Colony forming assay
Cells from three different umbilical cords and three different burn patients were extracted via
the enzymatic method. Cells with a passage number of 3-4 were seeded into cell culture
dishes (100 cells per 60mmX15mm dish). Each biological sample was assessed in triplicates.
After 14 days, the cells were stained with 0.5% cresyl echt violet (Fisher Scientific, Thermo
Fisher, Canada) and colonies counted with a manual cell counter.
3.3.3 Proliferation via bromodeoxyuridine (BrdU) staining
All cells for staining were cultured in 8 chamber slides (Falcon™ Chambered Cell Culture
Slides, Fisher Scientific, Canada) until they reached a confluency of 80-90% before staining.
Cells from three different umbilical cords and three different burn patients were used. Each
biological sample was cultured and stained in doublets. BrdU (Cell Signaling Technology
Inc, Canada) was added to warm culture medium (1:200) and incubated with the cells for 12
hours. The cultured cells were then fixed in 4% paraformaldehyde (Electron Microscopy
Sciences, USA) followed by a permeabilization with 0.25% Triton X-100 (BioShop Canada
Inc., Canada) and an incubation in 1.5M hydrochloric acid (Sigma Aldrich, Canada).
47
Unspecific binding was prevented with 1% bovine serum albumin (WISENT Inc., Canada).
The samples were incubated with the primary antibody (BrdU (Bu20a) Mouse mAb #5292,
Cell Signaling Technology Inc, Canada) at 4 °C overnight, followed by a one-hour long
incubation at room temperature in the secondary antibody (Alexa Fluor® 488 dye, Thermo
Fischer Scientific, Canada). After staining the cell containing slides were mounted with
DAPI containing mounting medium (VECTASHIELD Antifade Mounting Medium with
DAPI, Vector Laboratories, USA).
3.3.4 Cell cycle analysis
Cells from three different umbilical cords and three different burn patients were extracted via
the enzymatic method. Cells had a passage number of three to four (equal to 3-4 weeks in
culture). We used the Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis (Abcam,
Canada). After reaching a confluency of 90%, cells were trypsinized, fixed in 75% ethanol,
and incubated with propidium iodide and RNase for 30min. DNA staining was analyzed with
the BDTM LSR II flow cytometer (BD Biosciences, Canada) using the BD FACSDIVA™
SOFTWARE (BD Biosciences, Canada). The graphical and statistical analysis was done in
FlowJo™ v10 for MAC and Prism 5 for Mac OS X. We used an n=3 for each cell line i.e.
cells came from 3 different patients. Cells had a maximum passage number of 4 equivalent to
a culture time of maximum 3 weeks after extraction.
3.3.5 Reactive oxygen species (ROS) expression
Cells from three different umbilical cords and three different burn patients were extracted via
the enzymatic method. For the 2′,7′-Dichlorofluorescin diacetate (DCFDA) staining, cells
were cultured in 96-well plates (Corning® 96 Well Flat Clear Bottom Black Polystyrene TC-
48
Treated Microplates, Corning Incorporated, USA) until they reached a 95% confluency. Each
biological sample was assessed in triplicates. Cells were incubated with 25μM 2′,7′-
Dichlorofluorescin diacetate (DCFDA) in PBS for 45min at 37 degrees Celsius. As positive
control, cells were additionally exposed to 0.1mM H2O2 (Laboratoire Atlas Inc, Canada) for
30min. Fluorescence intensity was measured with a plate reader (Synergy™ H4 Hybrid
Multi-Mode Microplate Reader, BioTek Instruments Inc., USA).
3.3.6 Apoptosis
Apoptosis was assessed via TdT-mediated dUTP Nick-End Labeling (Tunel) staining. Cells
at a passage number of 3-4 from three different umbilical cords and three different burn
patients were cultured in 8 chamber slides (Falcon™ Chambered Cell Culture Slides, Fisher
Scientific, Canada) until they reached a confluency of 80-90% before staining. We used the
DeadEnd™ Fluorometric TUNEL System-Kit (Promega Corporation, USA) to detect
fragmentation of DNA. The cultured cells were fixed in 4% paraformaldehyde (Electron
Microscopy Sciences, USA) followed by a permeabilization with 0.25% Triton X-100
(BioShop Canada Inc., Canada). After pH-equilibration with the equilibration buffer, the cells
were incubated for 60min in the dark at 37 degrees Celsius in the TdT reaction mix
containing recombinant deoxynucleotidyl dransferase (rTdT), fluorescein-12-dUTP and
equilibration buffer. The positive control was incubated with DNAse I before incubating in
the the TdT reaction mix. After staining the cell containing slides were mounted with DAPI
containing mounting medium (VECTASHIELD Antifade Mounting Medium with DAPI,
Vector Laboratories, USA).
49
3.3.7 Glycolytic and Mitochondrial function
The glycolytic and mitochondrial function was assessed with the Seahorse XFe24 (Seahorse
Bioscience, USA) using the Seahorse XF Glycolysis Stress Test Kit (Seahorse Bioscience
Inc., USA) and the Seahorse XF Cell Mito Stress Test Kit (Seahorse Bioscience, USA). Both
kits required additional XF24 cell culture plates, sensor cartridges and XF base medium from
the same company.
Cells at a passage number of 3-4 from three different umbilical cords and three different burn
patients were seeded in the XF24 cell culture plates (30.000 cells/well) and incubated for 12
hours in standard cell culture medium (DMEM, 10% FBS, 1% Ab/Am) at 37°C. Each
biological sample was analyzed in six replicates.
MITOCHONDRIAL FUNCTION: In short, the standard medium was washed off and
replaced by XF base medium supplemented with 25mM glucose, 2mM glutamine and 1mM
sodium pyruvate. Oligomycin, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone
(FCCP), rotenone, and antimycin A were loaded in the recommended dosages into the sensor
cartridge. Sensor cartridge and cell containing culture plate were inserted into the Seahorse
XFe24. Oxygen consumption rate (OCR) along with extracellular acidification rate (ECAR)
was measured at baseline and after sequential addition of oligomycin, FCCP, and rotenone &
antimycin A.
GLYCOLYTIC FUNCTION: Standard medium was washed off and replaced by XF base
medium supplemented 2mM glutamine. Glucose, oligomycin and 2-Deoxy-D-glucose (2-
DG) were loaded in the recommended dosages into the sensor cartridge. Simultaneously to
50
the mitochondrial stress kit, oxygen consumption rate (OCR) along with extracellular
acidification rate (ECAR) was measured at baseline and after the injection of the pre-loaded
substances.
DATA ANALYSIS: The measured data was analyzed using the supplied XF mito stress test
report generator and the XF glycolysis stress test report generator.
3.4 Tumorigenicity
3.4.1 In Vitro
Four different cell types were used: Human fibroblasts, human umbilical cord MSCs, human
BD-MSCs and highly aggressive breast cancer cells (231/LM 2-4). Preparation of the soft
agar colony formation followed an established protocol (143). In summary, 6-well cell
culture plates were filled with 2ml of 0.6% agarose gel. After solidification, the second layer
of agarose (0.3%: 1ml) containing 100,000cells per well was layered on top. Each cell type
was used in biological triplicates. To avoid cross-contamination, each cell line was placed in
separate 6 well-plates. In a third step, a feeding layer (0.3% agarose: 1ml) was poured on top
of the cell containing layer. Every 2-3 days an additional feeding layer was placed on top of
the old one. Cells were evaluated after 20 days under a light microscope and the number of
colonies counted.
51
3.4.2 In Vivo
ANIMALS AND HOUSING: We used homozygous male athymic nude mice (Crl:NU(NCr)-
Foxn1nu, Charles River Laboratories, USA) with an age of 4-6 weeks. This experiment was
reviewed by the ethics committee and approved (AUP #: 15-503). Animals were housed in
individual cages at room temperature and at a 12hr light-dark cycle with food and water ad
libitum at Sunnybrook Research Institute. Standard diet and animal care standard operation
procedures were obeyed. Mice were assessed daily using a standardized protocol elaborated
together with the animal staff. Pain medication was not needed.
CELL PREPARATION: Four different cell types were used: Human fibroblasts, human
umbilical cord MSCs, human BD-MSCs and highly aggressive breast cancer cells (231/LM
2-4). Cells were trypsinized, counted and resuspended in PBS at a concentration of 10,000
cells/µl. At the injection site, the 50µl of the cell/PBS-mix was combined with 50µl Matrigel
(Corning® Matrigel® Matrix, Corning, USA) and injected (see below)
CELL INJECTION: Anesthesia was done with Isoflurane (induction with 5% for 5-15sec,
maintenance 1-5% to effect). Each mouse received subcutaneous cell-injections (100µl of the
cell/Matrigel-mix containing 500,000 cells in total) at three different locations (1x
interscapular and 1x per flank). To avoid cross-contamination of the cell lines, 6 mice were
exclusively injected with MSCs only, while the remaining 3 mice were injected with
fibroblasts and cancer cells simultaneously: cancer cell injection sites were randomly
assigned to either flank or back with one injection per mice. The other two injection sites per
mice were used for fibroblast injections. Syringes containing the different cell lines were
52
prepared in a blinded manner and given a numeric code containing mouse number (1-6) and
injection site number (1-3). We used a 1ml syringe and a 16G 1/2 needle for cell application.
ENDPOINT: The endpoint of this study was the appearance of the first visible tumor mass at
any location. Humane endpoints were defined as therapy refractory distress of the animal,
and ulcerating tumors. One mouse showed therapy refractory respiratory distress in the
recovery phase of the narcosis and was euthanized. Euthanizing was done via cervical
dislocation.
TUMOR ASSESSMENT: Mice were evaluated for physiological parameters and pain daily
and bi-weekly for tumor masses. At reaching the primary endpoint of this study, pictures
were taken with an IPhone 6 and the visible tumor masses excised, fixed in Formaldehyde
and send for histological preparation.
3.5 Immunogenicity and -reactivity
3.5.1 Flow cytometry for major histocompatibility complex (MHC) I and
II and toll-like receptor (TLR) 4
Cells from three different umbilical cords and three different burn patients were used at a
passage number of 3-4. After fixing with 0.01% paraformaldehyde (Electron Microscopy
Sciences, USA), cells were incubated for 1hour at -4 degrees Celsius with the conjugated
antibodies - TLR-4 (eBioscience, Thermo Fisher Scientific Inc., Canada), MHC II
53
(eBioscience, Thermo Fisher Scientific Inc., Canada) and MHC I (eBioscience, Thermo
Fisher Scientific Inc., Canada) - together with flow buffer consisting of HBSS with 1%
bovine serum albumin. After washing, cells were analyzed with the BDTM LSR II flow
cytometer (BD Biosciences, Canada) using the BD FACSDIVA™ SOFTWARE (BD
Biosciences, Canada). The graphical and statistical analysis was done in FlowJo™ v10 for
MAC and Prism 5 for Mac OS X.
3.5.2 QPCR for toll-like receptor (TLR) 1-10
Cells from three different umbilical cords and three different burn patients were used at a
passage number of 3-4. Additionally, peripheral blood mononuclear cells from two different
patients were used as a positive control.
Total RNA was isolated using TRIzol reagent (Invitrogen, Canada) according to
manufacturer’s instructions. RNA concentration and quality were assessed using
spectrophotometry (Nanodrop 2000, Thermo Fischer Scientific Inc., Canada). We accepted
RNA with a 260/280 ratio >1.8.
The mRNA expression was quantified using StepONE Plus PCR System (Applied
Biosystems, California, USA) and Biorad SsoAdvanced Universal SYBR® Green Supermix
(Bio-Rad, California, USA) according to manufacturer’s directions. First-strand cDNA
synthesis from 2μg of total RNA was performed with random primers using High-Capacity
cDNA Reverse Transcription Kits (Applied Biosystems, USA) according to manufacturer’s
instructions. Forward and reverse primers were optimized to verify primer efficiency and
dissociation melt curves were analyzed for primer specificity. All samples were run in
duplicate, simultaneously with negative controls that contained no cDNA. Primers were
54
ordered from Life Technologies Inc., Canada. See table 2 for the full sequence of the
individual primers. Optimization was performed to determine a 1:20 dilution ratio of plasma
in nuclease free sterile water and 2ul of starting material was used per reaction. All samples
were run in duplicate, simultaneously with negative controls.
Transcript levels were normalized to GAPDH, and analyzed using the 2^-ΔΔCt
method. Statistical significance was calculated on Δct values. We chose GAPDH as a
housekeeping gene, since it is one of the most widely used reference genes in high impact
studies and considered a "classical" housekeeping gene. (144)
55
Table 2. Primers for toll-like receptor 1-10.
Target Forward Primer Reverse Primer
TLR 1 GCACCCCTACAAAAGGA
ATCTG
GGCAAAATGGAAGATGC
TAGTCA
TLR 2 CTGGTAGTTGTGGGTTG
AAGCA
GATTGGAGGATTCTTCCT
TGGA
TLR 3 TTAAAGAGTTTTCTCCAG
GGTGTTTT
AATGCTTGTGTTTGCTAA
TTCCAA
TLR 4 CCCCTTCTCAACCAAGA
ACC
ATTGTCTGGATTTCACAC
CTGGAT
TLR 5 TGCTAGGACAACGAGGA
TCATG
GAGGTTGCAGAAACGAT
AAAAGG
TLR 6 AGGCCCTGCCCATCTGT
AA
GCAATTGGCAGCAAATC
TAATTT
TLR 7 GCTATTGGGCCCATCTC
AAG
TCCACATTGGAAACACC
ATTTTT
TLR 8 TCAGTGTTAGGGAACAT
CAGCAA
AACATGTTTTCCTTTTTA
GTCTCCTTTC
TLR 9 GGGAGCTACTAGGCTGG
TATAAAAATC
GCTACAGGGAAGGATGC
TTCAC
TLR 10 TTTACTCTGGGACGACCT
TTTCC
ATAAGCCTTACCACCAA
AAGTCACA
56
3.5.3 Secretion Profile
For the detection of cytokines, chemokines, growth factors and immunomodulatory proteins
we used the HCYTOMAG-60K MILLIPLEX MAP Human Cytokine/Chemokine Magnetic
Bead Panel - Immunology Multiplex Assay (EMD Millipore Corporation, Germany). This kit
enables the detection of sCD40L, EGF, FGF-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF,
GRO, IFN-α2, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-
10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, MCP-1, MCP-3, MDC (CCL22),
MIP-1α, MIP-1β, PDGF-AB/BB, RANTES, TGF-α, TNF-α, TNF-β, VEGF, Eotaxin/CCL11,
and PDGF-AA. The basic principle of this kit is that the antigens of interest are bound to
color-coded magnetic beads on the one side, and to a fluorescent conjugate (Biotin-
Streptavidin) on the other side. The colour of the attached fluorescent bead is specific for the
antigen of interest, the emitted fluorescence from the Biotin-Streptavidin system is directly
proportionate to the amount of the bound antigen of interest.
Cells were cultured in 6-well plates until they reached a confluency of 90-95%, then they
were incubated for 48h in the fresh standard medium. A second group was incubated for 48h
in standard medium containing 1ug/ml lipopolysaccharide (LPS, Lipopolysaccharides from
Pseudomonas aeruginosa 10, Sigma Aldrich, Canada). Experimental design was strictly
following the Immunology Multiplex Assay protocol. In summary: The medium of the
different groups was filled in a 96-well plate and mixed with the magnetic beads, and the
fluorescent conjugate. All components that were not bound to the beads were washed off, and
the fluorescent conjugate was analyzed using the Luminex 100® Milliplex® Analyzer (EMD
Millipore Corporation, Germany).
57
Analyzing of the data was done with Microsoft Excel 2016 for MAC. We excluded secreted
proteins if all cells of one biological group were not in the detectable range of the machine.
This applied to GRO, MCP-3 MCP-1, IL1-b, Il-4, Il-5, Il-9, Il-10, and IL12-P70. To take in
account that the detection threshold is an arbitrary value that varies in different machines, we
took the lowest respectively the highest detectable threshold in case some samples were
below respectively above this threshold.
3.6 Stem cell integration into Integra®
MSCs from three different umbilical cords and three different burn patients were used at a
passage number of 3-4. 50,000 cells in 50µl standard cell culture medium were pipetted in
the middle of the bovine collagen part of 6mm Integra® punches. After 20min incubation at
37 degrees Celsius in a cell culture dish the seeding procedure was either repeated two more
times with or without additional spinning (max. 2000rpm for 5min) in-between the different
seedings to increase the seeding depth, or the cellularized Integra® was submersed in
standard cell culture medium right away and incubated for 12h in a cell culture incubator at
37 degrees Celsius and 5% CO.
For the evaluation of the seeding depth and the cell morphology, the cellularized Integra®
was washed three times in PBS, fixed in 4% paraformaldehyde for 30min, then stained with
ActinGreen (ActinGreen™ 488 ReadyProbes® Reagent, Thermo Fisher Scientific Inc.,
Canada) and mounted with DAPI containing mounting medium (VECTASHIELD Antifade
Mounting Medium with DAPI, Vector Laboratories, USA).
58
For the evaluation of the effect of three consecutive seedings, BD-MSCs were stained before
seeding with the Vybrant® Multicolor Cell-Labeling Kit (Thermo Fisher Scientifics Inc.,
Canada). Mounting was done without a DAPI containing mounting medium
(VECTASHIELD Antifade Mounting Medium, Vector Laboratories, USA).
3.7 Porcine Model
3.7.1 Experimental overview
In a pilot study, we compared wound healing in 4x4cm full-thickness excisional skin wounds
treated with either acellular skin scaffolds or with their cell-containing counterparts over a
total period of 30 days. As cells, we used BD-MSCs incorporated into the skin scaffolds in
different concentrations (500, 5000, or 50000 cells/cm2). We assessed for granulation tissue
formation, the speed of epithelialization, and epidermal thickness. In total, we used four pigs
- three pigs with Integra® and one pig with PG-1 (see table 3).
59
Table 3. Overview of the used pigs.
pig 1 pig 2 pig 3 pig 4
Acellular Skin Scaffold 3 3 2 2
BD-MSCs 500cells/cm2 3 3 0 0
5000cells/cm2 3 3 2 2
50.000cells/cm2 0 0 2 2
Skin Scaffold
Integra® Pullulan Integra® Integra®
60
Since the listed pigs were not exclusively used for this study, we had a variety of other
dressings simultaneously on those pigs. Assignment of the different wound dressing materials
was done via randomization without matching acellular (control) and cellular (treatment)
treated wounds.
3.7.2 Animals and Housing
We used male Yorkshire pigs with a weight of 20-30kg. This experiment was reviewed by
the ethics committee and approved (AUP #: 16-600). The pigs were housed in individual
pens at room temperature and at a 12hr light-dark cycle with food and water ad libitum at
Sunnybrook Research Institute. Feeding and daily care was performed by the in-house animal
staff and overseen by the assigned veterinarian. Standard diet and animal care standard
operation procedures were obeyed. All animals were fasted for at least 6 hours before surgery
and assessed daily using a standardized protocol elaborated together with the veterinarian.
Pain medication was adjusted accordingly.
3.7.3 Surgical wounds
After induction of anesthesia. Hair was removed via electrical shaving followed by chemical
depilation. The operation area was disinfected with povidone Iodine, skin excision in the
previously marked areas was done with a scalpel, the rest of the operation with a monopolar
diathermy knife that was also used for hemostasis.
Full-thickness excisional wounds were inflicted in all pigs, 4x4cm each, 12-16 per pig, 6-8 on
each side in two rows equidistant from the spine (see figure 11). The exact localization and
61
total number of the wounds were based on the anatomical characteristics of the pig. Wounds
were marked on the back of the animal before the operation.
After the excision, wounds were covered with skin substitutes, either Integra® or PG-1
(acellular or cellularized). Integra® was secured via metal staples that were removed at the
first dressing change. Stapling was not possible on PG-1 due to the physical properties of the
material.
All animals received a perioperative IM antibiotic administration with a cephalosporin
(Cefazolin 25mg/kg IV intraoperatively and Ceftiofur 5mg/kg IM pre- and post-op and on
arrival). In the case of wound site infection, antibiotics were given daily until the wound
infection resolved. Wound infection only occurred in pig number 1 and was resolved by day
14.
After surgery and bandaging, pigs could recover in a warm, well-ventilated recovery pen with
essential amounts of oxygen and under observation.
3.7.4 Anesthesia and pain control
ANESTHESIA: General anesthesia was induced by intramuscular injection of ketamine (1
mg/10 kg) or dexmedetomidine (0.03mg/kg) and maintained with isoflurane (to effect; 1-5%)
through endotracheal intubation in the initial operation. For the following dressing changes,
we used facial mask only.
62
PAIN CONTROL: Intraoperatively and during the experiment (beginning on the day of the
surgery until euthanizing at day 30), pigs received a basic dosage of tramadol (4-6mg/kg)
which was escalated when needed.
3.7.5 Cellularization of skin scaffolds
We used two different acellular skin scaffolds: bilayer Integra® as the most commonly used
and FDA-approved acellular skin scaffold in burn surgery and pullulan-gelatin first
generation hydrogel (PG-1) - a novel acellular skin scaffold recently developed by our
laboratory (145). The same process of cellularization was used for the two skin scaffolds.
Seeding was done in different concentrations (500, 5000, or 50000 cells/cm2; see table 3)
with a 16-channel multichannel pipet (VWR Signature™ Ergonomic high-performance
Multichannel Pipettor, VWR International, USA).
Cultured BD-MSCs were trypsinized, counted and resuspended in our standard cell culture
medium at a concentration of 500, 5000, or 50000 cells/30µl. Per channel, we used a total
amount of 30µl cell/media solution. Cells were pipetted for Integra® on top of the bovine
collagen, with the silicone side facing down on a sterile cell culture dish. PG-1 is a one-layer
scaffold and cells were pipetted on one side, while the other side was also facing down on a
sterile cell culture dish. Each scaffold was cellularized in a separate dish. After cell
application, scaffolds were incubated at 37 degrees Celsius at 5% CO for 15-20min, before
completely submersing them in medium and stored in a tissue incubator for 12 hours. Shortly
before surgery, the cellularized skin scaffolds were assessed under the microscope for
floating cells indicating cell death and/or failure to integrate. No floating cells could be
detected in either of the scaffolds, indicating a full cell integration. The cell culture medium
63
was washed off for 30min on a Rocker. Cellularized skin scaffolds were stored in PBS on ice
for a maximum of 2h until application onto the wounds.
3.7.6 Wound dressings
Dressing layers were as follows: The skin substitute was applied directly onto the wound bed,
followed by Jelonet® paraffin gauze, Polysporin® cream, wet gauze, by dry gauze, OpSite®
or Tegaderm® transparent film dressing, gauze rolls, and a lamb-tube compression jacket
(see figure 11). Dressing changes were done every 2-3 days under general anesthesia and
sterile conditions.
64
Figure 11. Pig wounds and dressing layers.
(A) Schema of the ideal wound distribution. (B) Pre-operative marked wound areas. (C)
Surgical full-thickness wound. (D) Integra® attached with metal staples. (E) Integra®
covered with Jelonet®. (F) Gauze layer on top of Integra® and Jelonet®, fixed with Opsite®.
(G) Dressing rolls. (H) Compression Jacket.
65
3.7.7 Endpoints
The primary endpoint was the closure of all wounds in one treatment i.e. the control group,
which was achieved in pig number one after 30days and therefore set as the standard length
of experiment for the following pigs.
Humane endpoints were therapy refractory wound infection, pain, and distress that cannot be
alleviated, persistent hemorrhage, self-mutilation, seizures, loss of consciousness, or
ambulatory difficulty.
Chemical euthanasia was performed by the animal staff under anesthesia using phenobarbital
IV.
Two pigs came bacteremia from the supplier and had to be euthanized and excluded from this
study.
3.7.8 Wound Assessment
3.7.8.1 Macroscopically
Photographs of the wounds together with a scale bar were taken intraoperatively with each
dressing change using an iPhone 6. Epithelialization was measured from day 13-14 (=first
dressing change after removal of the silicone layer) to day 22-24 (=first complete closure of a
wound by epithelialization). We measured from the hairline to the visible epithelialization
line. Measurements were done in Image J Version 1.51 for MAC. Figure 12 gives an
overview of the important time-points in the surgical wound group. For the number of
wounds per group and pig see table 3.
66
Figure 12. Overview of important time points.
67
Epithelialized Area: We measured total wound area and non-epithelialized area. The
epithelialized wound area was calculated as follows:
����� ���� ��� − ��� � �ℎ��� �� �ℎ�� ��
Epithelialization Speed: To take into account that due to different wound contractions and
different mechanical strains all wounds had a slightly different size, we calculated the
epithelialization speed between day 10 and day 23 as follows:
�� �ℎ�� �� �� ��� � ��^2 �� �� 23 ⁄ 13 ���
3.7.8.2 Microscopically
At the end of the experiment after 28-30 days, the swine was euthanized and the
wounds/scars were excised, fixed in formaldehyde and send for histological preparation. All
tissue for staining was embedded in paraffin, cut into 5μm thick slices and placed on standard
glass slides.
After Deparaffinization with Citrosol (CitriSolv Hybrid™, Decon Labs Inc., USA), the tissue
containing slides were incubated in Bouin's solution (Bouin's Fixative, Electron Microscopy
Sciences, USA) for 24 hours at room temperature. Staining was done as follows:
Hematoxylin (Harris Hematoxylin Solution, Sigma Aldrich, Canada), Bibrich Scarlet Acid
Fuchsin (Electron Microscopy Sciences, USA), followed by aniline blue (Electron
Microscopy Sciences, USA).
68
For the analysis of the below described criteria, pictures of the stained slides were taken
under a light microscope either with 20x magnification (collagen deposition and cellularity)
or with a 10x magnification (epidermal thickness). Imaging and evaluation was done in a
blinded manner by one person to keep consistency. Measurements and cell counting were
done in Image J Version 1.51 for MAC.
Collagen deposition: First, we measured the area covered by collagen per visual field (µm).
Light microscopy images of the trichrome stained wound sections were taken: one from the
wound center and one of the wound edge per wound (20x magnification). To gain a
standardized image for all wounds, images of the granulation tissue were taken 200µm below
the epithelial layer in a blinded manner. The blue area (=collagen) was measured
automatically in every picture by Image J Version 1.51 for MAC using following settings:
Filtering for blue (Hue values), Top slider 125, bottom slider 160, Saturation full spectrum
(0-255), Brightness full spectrum (0-255).
To account for a possible different collagen density per area, we also assessed the integrated
density. For that, Image J Version 1.51 for MAC calculated the mean blue density using the
same setting as above. The integrated density was calculated as follows: integrated density =
mean blue density x total blue area.
Cellularity: Cells per image were calculated manually in a blinded manner. We excluded
endothelial cells as well as erythrocytes to get a closer estimate for the number of fibroblasts
and immune cells present.
69
Epidermal Thickness: Epidermal thickness was assessed with Image J Version 1.51 for
MAC. We measured the thickest endothelial diameter in µm. We excluded the upper
acellular keratin layer from this measurement since this layer was destroyed resp. detached
during the histological preparation.
3.7.9 Safety assessment
Pigs were assessed for systemic and local wound site complications every 2-3 days with each
dressing change. Assessment and health scoring was done by our research group and
additionally by a blinded animal technician. No difference between the two scorings was
detected.
Local complications: We assessed for (1) arterial bleeding out of the wound site, (2) wound
infections, indicated by the commonly used criteria for wound infection - rubor, calor, dolor,
and pus, (3) detachment of the entire Integra, and (4) the formation of keloids or hypertrophic
scars. Keloids and hypertrophic scars were defined as outlined above in chapter 1.
Furthermore we assessed the optic appearance using the Manchester Scar Scale (146) - an
established clinical assessment tool for scar evaluation. This scale evaluates color, contour,
distortion, texture and "matt/shiny" surface of the scar tissue. Each item, except "matt/shiny"
surface, is scored from 1-4, with 1 being the best core. If the scar appears shiny it gets
additional 2 points, if it is matt it gets 1 point. Scars with the most unfavorable optics
therefore have a score of 18, scars with an optical appearance that resembles the original
tissue receive a score of 5.
Systemic complications: We used a standard health-scoring system consisting of 5 items
(pupil size and reactivity, activity, posture, food intake, and hydration). Each item was scored
70
from 1-5, with 5 indicating absence of problems. The scores of the 5 items were added to a
maximum score of 25, indicating a healthy, active and well-nourished pig. In addition, we
assessed for respiratory distress, fever and any signs of abnormal behavior that might indicate
systemic complications, such as shivering, and abnormal behavior towards humans or other
pigs.
3.8 Statistical Analysis and graphical Representation
Statistical analysis was done via Prism GraphPad Version 5.0a for Mac and Microsoft Excel
2016 for Mac. Two groups were compared with an unpaired t-test, more than two groups
with a one-way ANOVA with a post-hoc Tukey test. A p-value <0.05 was considered
statistical significant. All experiments were conducted with cells from a minimum number of
three different donors. Depending on the experiment, we used the cells from each donor in
duplicates or triplicates. The exact number of used biological samples and repeats is given in
the according Material and Methods section as well as in the figure legends.
All graphs are made with Prism GraphPad Version 5.0a for Mac and display mean ± SEM.
All graphics depict different cell lines resp. different animals, no biological or technical
replicates are shown.
71
Chapter 4
Results
Results
4.1 Optimization of the current protocol for extraction of BD-MSCs
Our preliminary finding show that burned skin contains viable cells. Those cells were
extracted with a conventional cell extraction method that is standard in our laboratory. Since
fast wound coverage is essential in burn patient management, we compared this extraction
method to an enzymatic extraction method known to achieve faster results in terms of usable
cells.
24 hours after enzymatic cell extraction out of burned skin 16140 ± 5418 cells attached to the
plastic surface of the cell culture flask (figure 13 A and D). No statistical difference could be
found in the cell yield between skins of the five different patients. In contrast, the
conventional cell extraction method did not lead to any attached cells after 24h (figure 13 B).
Visible cell growth out of the tissue pieces could be seen after 7 ± 4 days (conventional
method). The evaluated skins came from patients with deep partial- or full-thickness burns
with an average TBSA of 7 ± 0.8% caused by either flame or scalding (figure 13 C).
72
Figure 13. The enzymatic stem cell extraction method is superior to conventional
stem cell extraction method.
(A) The enzymatic method yielded 16140 ± 5418 attached cells per cm2 of processed burned skin after 24h, whereas (B) the conventional cell extraction method of the same skins did not result in any attached. (C) Characteristics of the used skins. (D) Light microscope image of attached cells 24h after enzymatic cell extraction (Arrows mark attached cells). TBSA = total body surface area.
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001;
**** P ≤ 0.0001. No stars represent P > 0.05.
73
4.2 Characterization of key biological functions/characteristics of
BD-MSCs
4.2.1 Cells extracted from burned skin are mesenchymal stem cells
We established that the enzymatic method is the fastest way to extract cells from burned skin.
In the next step, we aimed to characterize the nature of those cells using established and
defining MSCs markers/characteristics set by the International Society for Cellular Therapy.
After enzymatic cell extraction, significantly fewer cells from burned skin (burn derived, BD)
show a triple positivity for CD 73/90/105 while negative for CD34 when compared to
umbilical cord stem cells (66 ± 5% vs. 89 ± 5%; P=0.025). This relatively low percentage of
MSC-marker positive cells in BD group is significantly increased to 93 ± 2% (P=0.015) at a
passage number of 3-4 annihilating the difference between the UC and the BD group (figure
14 A and C). At a passage number of three to four, 93 ± 2% of the BD and 96 ± 2% of the
UC derived cells are positively identifiable as MSCs based on the used surface markers.
When the individual cell surface markers are compared on their own, only CD105 is
significantly lower in BD cells compared with UC cells (P=0.02) at a passage of 1 whereas
no difference can be found in a later passage. CD73 and CD90 are comparable between UC
and BD cells from passage 1 on (figure 14 B and C). In both groups, the rate of CD34
positive cells was less than 1%.
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Figure 14. Burn derived dermal cells show MSC surface markers.
(A) Most cells isolated from umbilical cords and burned skin were triple positive for CD37, 90, and 105 while negative for CD34. Note that umbilical cord cells only showed a significant higher amount of those cells (89 ± 5% vs. 66 ± 5%; P=0.025) at passage (P) 1. No difference between the cell lines was found at a later passage. (B) Skin derived cells showed less expression of CD105 in passage 1 (P=0.02). (C) Flow cytometry for CD73/90/105. Note that CD34 positive cells were gaited out before analyzing. (D) Flow cytometry of skin derived cells (passage 3-4) for CD105/73 and CD105/90. The upper panel shows unstained cells.
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001; ****
P ≤ 0.0001. No stars represent P > 0.05. N=3 for each group; triplicates per biological
sample
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Cells extracted from burned skin via the enzymatic method were able to differentiate into
cartilage, adipose, and bone tissue after 10 days (figure 15). However, when compared to
umbilical cord-derived MSCs, burn derived cells showed a lower differentiation potential.
Adipogenic differentiation potential (measured as percentage of red-oil-o positive cells in
relation to the total amount of cells per visual field) and chondrogenic differentiation
potential (measured as alcian-blue positive area per visual field) was significantly lower
(P=0.001 and P=0.03, respectively), while osteogenic differentiation (measured as alizarin-
red positive area per visual field) only showed a clear signal of lower differentiation capacity
without reaching significance. Interestingly, only burned skin-derived cells showed highly
differentiated adipocytes (figure 15 G star).
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Figure 15. Extracted cells from burned skin can differentiate into mesenchymal tissues.
(A, D arrow) Cells extracted from burned skin differentiate after 10 days into adipocytes, (B, D arrow) osteoblasts, and (C, D arrow) chondrocytes. When compared to UC derived cells, BD derived cells show a lower differentiation potential. Differentiation into adipose tissue and cartilage is significantly lower (P=0.001 and P=0.03, respectively), osteogenic differentiation shows a clear signal without reaching significance. Only BD-MSCs showed highly differentiated adipocytes (star).
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001; ****
P ≤ 0.0001. No stars represent P > 0.05. N=3 for each group (=cells from 3 different
patients per group); triplicates per biological sample.
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Cells extracted from the burned skin attach to a plastic surface, show the characteristic
surface marker constellation for MSCs and can differentiate into key mesenchymal tissues -
fulfilling the identifying criteria for MSCs.
4.2.2 Burn does not cause cell dysfunction
In the previous experiments we showed that we can extract viable MSCs out of burned skin.
However in order to use those cells for skin regeneration, it is of paramount importance to
prove that burn trauma does not impair their biological functioning. In addition, we tested the
ROS content in these cells since ROS is known to be a highly damaging consequence of
tissue and cellular trauma. As a control, we used UC-MSCs, since they represent the
youngest and healthiest MSCs that can be gained without raising ethical concerns.
To assess whether thermal injury damages essential biological functions of the MSCs, BD-
MSCs were compared to UC-MSCs in terms of proliferation capacity and speed, colony
forming behavior, cell cycle phases, the expression of reactive oxygen species (ROS),
apoptosis, glycolytic capacity and mitochondrial function.
BD-MSCs display an average population doubling time (PDT) of 28 ± 5h which shows no
statistical difference to UC-MSCs with 36 ± 5h (figure 16 A). PDT for BD-MSCs was also
not significantly different between different passages (passage one and passage 3-4), that
previously showed a significant difference in the amount of CD 73/90/105 positive and CD34
negative cells (figure 16 B). In the colony forming assay, BD-MSCs were comparable to UC-
MSCs after 20 days: 29 ± 2 colonies vs. 30 ± 1 colonies formed after 14 days (figure 16 C).
Proliferation assessed via bromodeoxyuridine (BrdU) staining also showed no statistical
difference between BD-MSCs and UC-MSCs: 66 ± 13% vs. 76 ± 2% BrdU positive cells
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after 12 hours (figure 16 D and G). BD-MSCs and UC-MSCs showed a similar distribution
for cells in different cell cycle phases: 69 ± 2% vs. 61 ± 5% for G0/1 phase, 13.4 ± 2% vs.
13.1 ±2% for S phase, and 9.3 ± 1% vs. 12.8 ± 5% for G2/M phase (figure 16 E). No
statistical difference in the expression of ROS could be detected between the two cell lines:
11685 ± 2904 fluorescence intensity (fi) vs. 8871 ± 4675fi (figure 16 F). None of the cultured
cells showed apoptosis in the tunel staining (figure 16 H).
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Figure 16. BD-MSCs are comparable to UC-MSCs in key biological characteristics.
(A, B) Population doubling time (PDT). (C) Colonies formed after 14d. (D, G) Proliferation assessed via BrdU staining. (E) Cell cycle distribution. (F) Reactive oxygen species (ROS) expression. (H) Apoptosis assessed via tunnel staining.
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001; ****
P ≤ 0.0001. No stars represent a P > 0.05. N=3 for each group (=cells from 3 different
patients per group), triplicates per biological sample.
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Since burn injury does not seem to damage basic cell functioning, we assessed one of the
most essential cell mechanisms- energy gaining mechanisms in the form of ATP production
via anaerobic glycolysis and oxidative phosphorylation.
BD-MSCs did not differ from UC-MSCs in their glycolytic function: Glycolysis, glycolytic
reserve, and glycolytic capacity did not show a significant difference between the two cell
lines: 37.1 ± 6mpH/min vs. 35.7 ± 1mpH/min, 124.2 ± 6mpH/min vs. 132.9 ± 32mpH/min,
and 44.6 ± 6mpH/min vs. 39.7 ± 7mpH/min, respectively. (figure 17 A-D).
No statistical difference could be found between BD-MSCs and UC-MSCs in the measured
parameters for mitochondrial function (figure 17 E-H). However, BD-MSCs showed a signal
towards a higher oxidative metabolism, with a slightly higher basal respiration, maximal
respiration, and adenosine triphosphate (ATP) production: 27.3 ± 12pmol/min vs. 17.1 ±
8pmol/min, 39.1 ± 9pmol/min vs. 28.9 ± 12pmol/min, and 18.7 ± 17pmol/min vs. 4.2 ±
3pmol/min, respectively.
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Figure 17. BD-MSCs are comparable to UC-MSCs in key biological characteristics.
(A-D) Glycolytic function. (E-H) Mitochondrial function. ECAR = extracellular acidification rate, OCR = oxygen consumption rate.
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001; ****
P ≤ 0.0001. No stars represent P > 0.05. N=3 for each group (=cells from 3 different
patients per group), 6 replicates per biological sample.
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The thermal injury did not lead to a derangement of key biological cell functions. Although
coming from two different tissue sources, BD-MSCs seem to be comparable in basic cell
functions to healthy UC-MSCs.
4.3 Determination of potential risks for later clinical usage
After we showed that MSCs derived from burned skin are viable and not affected in their key
biological functions - making them a potential source for skin regeneration - we addressed
the most common safety concerns associated with cellular therapy: tumor formation and
adverse interactions with the host's immune system.
4.3.1 BD-MSCs are not tumorigenic
Tumor formation potential was assessed first in vitro. We continued to compare BD-MSCs
with UC-MSCs which are mainly considered safe. As a positive control, highly aggressive
breast cancer cells (231/LM2-4) were used. In addition, we added fibroblasts (Fb) as a
negative control since recent controversial studies hypothetically (see introduction and
discussion) attributed a minimal potential of tumor formation to MSCs. In soft agarose, BD-
MSCs did not lead to colony formation after 20 days (figure 18). From all the cell lines used,
only the cancer cells led to tumor colony formation: 38.8 ± 7 colonies after 20 days.
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After the negative results for BD-MSCs in the in vitro study, it was ethically justifiable to
confirm those results in vivo: When injected s.c. into athymic nude mice, BD-MSCs like UC-
MSCs and fibroblasts did not lead to a visible tumor formation after 20 days (figure 18). Only
231/LM2-4 cells caused macroscopical and microscopical tumor formation.
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Figure 18. BD-MSCs are not tumorigenic.
(A, C) BD-MSCs did not cause In vitro tumor formation in soft agarose in after 20d (arrow: tumor colony in the cancer cell group). (B, D) No tumor formation was observed within 20d in vivo after subcutaneous injection in athymic mice (arrow: visible tumor in the cancer cell group). (E, F) Trichrome stained tumor caused by cancer cells. Fb=fibroblasts, T=tumor, S=subcutis, D=dermis, E=epidermis.
N=3 (=cells from 3 different patients per group) in the UC- and BD-MSCs group, as well as
in the Fb group. Cancer cells were all derived from one patient. Each biological sample
was used as a triplicate in the in vitro model, and as a singlet in the in vivo experiment.
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4.3.2 BD-MSCs display a low expression of MHC I, II, and TLR-4
To be able to potentially predict immunologic rejection after transplantation, we determined
the expression of major histocompatibility complex (MHC) I and II molecules, one of the key
elements in transplant rejection.
Flow cytometry shows a very low number of cells expressing major histocompatibility
complex (MHC) I and II molecules in BD-MSCs: 0.98 ± 0.04% for MHC I and 0.07 ± 0.01%
for MHC II (figure 19 A). Compared with UC-MSCs (0.52 ± 0.09% for MHC I and 1.32 ±
0.6% for MHC II), no statistical difference could be found.
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Figure 19. BD-MSCs have a low immunogenicity and -reactivity comparable to UC-
MSCs.
(A) Less than 1% of BD-MSCs express major histocompatibility complex (MHC) I and II. No statistical difference to the expression profile of UC-MSCs could be found. (B) Toll-like receptor (TLR) 4 expression in BD-MSCs is 1% ± 0.03%, which shows no statistical difference to UC-MSCs. (C) Flow cytometry of BD-MSCs (passage 3-4) stained against MHC I, II, or TLR4. The upper panel shows the corresponding unstained cells.
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001; ****
P ≤ 0.0001. No stars represent a P > 0.05. N=3 for each group (=cells from 3 different
patients per group), triplicates per biological sample.
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To further assess if future of complications of BD-MSC treatment can be predicted, we
determined the expression of toll-like receptor (TLR) 4 on BD-MSCs. TLR-4 recognizes
lipopolysaccharide, a membrane element of gram negative bacteria commonly found on burn
patients, and is associated with massive scar formation (see discussion).
Toll-like receptor (TLR) 4 is expressed in only 1 ± 0.03% of BD-MSCs. However, this shows
a signal towards a higher TLR-4 expression compared with UC-MSCs that display 0.01 ±
0.01% positive cells (figure 19 B) but without reaching statistical significance.
This signal could be confirmed by qPCR (figure 20 A). The TLR-4 expression was less than
half compared to human fibroblasts in both cell lines: 0.4 ± 0.2-fold for BD-MSCs and 0.2 ±
0.2-fold for UC-MSCs.
Even though the function of the remaining human TLRs are not well understood, we tested
their expression on BD-MSCs to fully characterize our cells in this regard. No statistical
difference in the expression of TLR 1-10 could be found between UC-MSCs and BD-MSCs
(figure 20 B and C). Except for TLR3, TLR1-10 are less expressed in BD-MSCs and UC-
MSCs compared to peripheral blood mononuclear cells (PBMCs). TLR3 shows a higher
expression in UC-MSCs (1.6 ± 0.6-fold) and BD-MSCs (1.3 ± 0.3-fold) in relation to
PBMCs.
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Figure 20. BD-MSCs do not differ in the expression of TLRs from UC-MSCs.
(A) Quantitative PCR showed no difference between UC-MSCs and BD-MSCs in the expression of TLR-4. Expression was in both groups less than half of the expression by normal human fibroblasts. (E, D) TLR 1-10 expression did not show any significant difference between BD-MSCs and UC-MSCs.
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001;
**** P ≤ 0.0001. No stars represent a P > 0.05. N=3 for each group (=cells from 3
different patients per group), duplicates per biological sample.
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4.3.3 BD-MSCs differ in their cytokine and growth factor secretion profile
from UC-MSCs
To further address the concern of possible adverse immunologic effects of the BD-MSC
treatment, we measured cytokine expression of the BD-MSCs at baseline and under
lipopolysaccharide stimulation simulating an environment with gram negative bacteria,
which resembles the wound situation of burn patients more closely.
From the tested 34 cytokines, chemokines and growth factors, only 11 showed a statistical
difference between BD- and UC-MSCs at baseline (figure 21 A-D). UC-MSCs had a higher
expression in all significant factors: Il-1a (P=0.02), IL-6 (P=0.03), IL-17a (P=0.003), TNFa
(P=0.01), IFNa2 (P=0.047), IFN-Y (P=0.004), Il-2 (P=0.01), IL-7 (P=0.0004), FGF-2
(P=0.01), MDC (P=0.01), and MIP-1b (P=0.04). The difference between UC- and BD-MSC
expression was further reduced upon stimulation of the cells with LPS for 48h - only IL-1a
was significantly higher secreted by UC-MSCs (P=0.01). Interestingly, IP-10 that showed no
difference in the baseline secretion between the two different cell lines, was significantly
higher expressed in BD-MSCs after LPS stimulation compared to UC-MSCs (P=0.04).
When exposed to an inflammatory stimulus (LPS), BD-MSCs displayed a higher reactivity in
all parameters compared to UC-MSCs (figure 21 E-H), with Il-1a (P=0.04), IL-6 (P=0.02),
IL-17a (P=0.007), IP-10 (P=0.047), IL-7 (P=0.04), IL-8 (P=0.01; data not shown) and FGF-2
(P=0.046) reaching statistical significance.
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Figure 21. Cytokine, chemokine and growth factor expression profile of BD-MSCs
differ from UC-MSCs.
(A-D) BD-MSCs showed a statistical significant lower baseline expression in 11 out of 34 tested proteins. When stimulated with lipopolysaccharide (LPS) a significantly different expression between UC-MSCs and BD-MSCs could only be found in IL-1a (P=0.01) and IP-10 (0.04). (E-F) BD-MSCs showed significantly more secretory reactivity towards LPS stimulation.
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001; ****
P ≤ 0.0001. No stars represent a P > 0.05. N=3 for each group (=cells from 3 different
patients per group), duplicates per biological sample.
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4.4 Incorporation of BD-MSCs into synthetic skin substitutes
We have show so far that we can extract healthy and viable MSCs out of severely burned
skin and that those cells seem to be comparable in important safety parameters to the
clinically used UC-MSCs making in vivo experiments ethically and medically justifiable. To
add more clinical relevance and to make the application to a large wound bed possible, we
incorporated the MSCs in the following experiment in already existing acellular wound
coverage materials. We used Integra®, an FAD-approved and widely used material in burn
patients that would allow us - in case of success - to bring this therapy faster from bench to
bedside.
Integra® can be cellularized with MSCs in vitro prior to its application onto wounds. BD-
MSCs as well as UC-MSCs show attachment and a regular cell morphology indicative of
healthy cells 12h after seeding onto the bovine collagen part of Integra® (figure 22 A and B).
There was no morphological difference between UC-MSCs and BD-MSCs. No cell loss in
the form of floating cells was visible in the process of cellularization in either group,
indicating successful incorporation.
Pipetting the cells on top of the bovine collagen only led to an average sinking depth of 123 ±
21µm. Since Integra® has a thickness between 1 and 1.3mm, we tried to increase the
cellularization depth by centrifugation of the Integra® in between consecutive seedings.
First, we observed that when BD-MSCs were seeded in three consecutive steps, with a five-
minute centrifugation between each seeding, no distinct layers could be seen. For that
purpose we used yellow, red, and green live dye that was added to the cells 15min before
incorporation into Integra®. Cells of each seeding were present in the lowest as well as in
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the highest layer (first seeding: green cells, second seeding: red cells, third seeding: yellow
cells; figure 22 C and D).
Second, the cellularization depth could be increased max. by 13% through centrifugation:
123 ± 21µm vs. 180 ± 10µm (P=0.013; figure F and G). Further spinning could not increase
the seeding depth.
In summary, BD-MSCs could be incorporated into Integra® without cell loss. However, the
pipetting technique could only achieve a superficial cell incorporation with a max. depth of
under 200µm on the Integra® surface that will be in direct contact with the wound bed.
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Figure 22. Cellularization of Integra®.
(A) BD-MSCs seeded on Integra®. Stained after 12h with ActinGreen and DAPI. Horizontal view. (B) UC-MSCs seeded on Integra®. Stained after 12h with ActinGreen and DAPI. horizontal view. (C) Cellularization with three seeding: first green, second red, third yellow. Vertical view. (D) Cellularization with three seeding: first green, second red, third yellow. Horizontal view. (E, F) Seeding depth after 12 hours without spinning and (F, G) with spinning.
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001; ****
P ≤ 0.0001. No stars represent a P > 0.05. N=3 for each group (=cells from 3 different
patients per group).
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4.5 Evaluation of BD-MSCs safety in a swine model
With the ability to incorporate BD-MSCs into Integra®, we were now able to apply BD-MSC
therapy to large excisional porcine wounds with 4x4cm. The goal of this study was to assess
the safety of BD-MSC treatment in vivo on a porcine model, which is considered one of the
most accurate prediction models of future wound therapy (see discussion).
During the 30d experiment duration, none of the pigs showed systemic complication (Table
4). Every 2-3days we assessed respiratory distress, fever, signs of systemic infection, and
calculated a health score consisting of pupil size and reaction to light, activity, food intake,
posture, and hydration. This health score was also independently created by blinded, trained
animal technicians. No difference between our score and the independently created score
could be observed.
BD-MSC treatment did not lead to wound site complications (Table 4). There was no arterial
bleeding or detachment of Integra®. In the first pig on the sixth post-operative day, we
observed small bacterial infections on the entry sites of the metal staples that were used to
fixate the Integra® on the wound bed. These were only minor local infections and present
throughout all groups (control and BD-MSC treatment group). After removal of the staples
and local wound cleaning with 0.9% saline-solution, the infections resolved over the
following week. Signs of systemic infections were not present. The occurrence of wound site
infections could be completely prevented in the following pigs (pig2-4) with the removal of
the staples at the first dressing change at day three.
We further assessed the formation of keloids and hypertrophic scars as well as the optical
appearance (assessed with the Manchester Scar Scale) of the formed scars (Table 4 and figure
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27). BD-MSC treatment did not alter the optical appearance of the scar tissue and did not
cause excessive scarring in form of keloids and hypertrophic scarring.
BD-MSC wound treatment did not cause any adverse effects, neither systemically, nor
locally.
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Table 4. Safety data of BD-MSC wound treatment in porcine study
Pig 1 Pig 2 Pig 3 Pig 4
Systemic Complications
Respiratory Distress1 no no no no
Fever1 no no no no
Systemic Infection1 no no no no
Health Score1,2,3
25/25 25/25 25/25 25/25
Local/Wound Site Complications
Arterial bleeding1 no no no no
Wound infection1 yes
4 no no no
Detachment of Integra®1 no no no no
Keloid/hypertrophic scar formation1 no no no no
1 assessed q2-3d
2 assessed items: (1) pupil size and reaction to light, (2) activity, (3) food intake, (4) posture, (5)
hydration; max. 5 points/item, overall max. 25 points/day 3 items were assessed q2-3d; values in the table are the MEAN of all assessed health score values
during the 30d period 4
small bacterial infection on staple entry points throughout all groups – resolved after removal of metal staples; no additional antibiotic therapy needed
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Figure 23. The optical appearance of the scars at day 30.
(A) Exemplary pictures of scars formed after 30d in the BD-MSC-treated group and the acellular control group. (B) No significant difference between the optical appearance between the two groups assessed via the Manchester Scare Scale.
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001;
**** P ≤ 0.0001. No stars represent a P > 0.05. N=3; 2-3 wounds/pig per treatment
group
Manchester Scar Scale
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4.6 Evaluation of BD-MSCs' wound healing capacity in a swine model
Since this study was designed to assess safety, it was lacking location matched acellular control
groups. All groups were randomly assigned to the excised wounds. Therefore, the following
evaluation of any therapeutic effects of the BD-MSC treatment can only be understood as
preliminary and with caution. To exclude any confounding effects caused by the carrier material
on wound healing, we also used PG-1 in addition to Integra® to deliver the BD-MSC treatment.
4.6.1 Wound treatment with cellularized skin scaffolds improved
epithelialization
Epithelialization was assessed between day 10 (removal of silicone layer in the Integra® group)
and day 23 (complete epithelialization of the first wound) in the Integra® treated animals.
This pilot study was aimed to establish a porcine model for wound healing at our Institution. For
this early study, we did not have location matched controls.
Since wounds of both groups were randomly distributed on the pig's back and not matched, we
first assessed the total amount of the epithelialized wound area (figure 23A). The epithelialized
area was significantly higher in the 5,000cells/cm2 treatment group at day 23 (P=0.040)
compared with the control group. Higher (50,000 cells/cm2) and lower cell concentrations (500
cells/cm2) did not show a statistical difference compared to the medium concentration treatment
group (data not shown).
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Last, we assessed the epithelialization speed as cm2/day in both groups. The medium
concentration treatment group showed a higher epithelialization speed compared to the control
group (P=0.038, figure 23C). Higher and lower cell concentrations did not show a statistical
difference compared to the medium concentration treatment group (data not shown).
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Figure 24. BD-MSCs delivered via Integra improved epithelialization in vivo.
(A) The total epithelialized area after 23 days was significantly higher in BD-MSC treated
wounds (P=0.040). (B) The overall epithelialization speed (cm2/day) assessed between day
10 and 23 was significantly higher in the BD-MSC group (P=0.038). (C) Representative wound images of the two groups on day 10, day 17, and day 23 (Blue arrow: start of wound edge; Black arrow: epithelialization boarder).
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001;
**** P ≤ 0.0001. No stars represent a P > 0.05. N=3; 2-3 wounds/pig per treatment
group
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Simultaneous to the wound coverage with (a)cellular Integra®, we started porcine wound
treatment on a separate pig with (a)cellular PG-1. Due to increased bleeding in the first week and
unfavorable mechanical properties of the material we decided to not continue with PG-1 on more
pigs.
Comparable to the results of the BD-MSC treatment delivered via Integra®, the cellularized PG-
1 skin scaffolds showed a sign of improved epithelialization in the form of total epithelialized
area and epithelialization speed (figure 24).
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Figure 25. BD-MSCs delivered via PG-1 improved epithelialization in vivo.
(A) The total epithelialized area after 23 days was higher in BD-MSC treated wounds. (D) The overall epithelialization speed (cm2/day) assessed between day 10 and 23 was higher in the BD-MSC group. (E) Representative wound images of the two groups on day 10, day 17, and day 23 (Blue arrow: start of wound edge; Black arrow: epithelialization boarder).
N=1; 2-3 wounds/pig per treatment group.
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4.6.2 BD-MSC wound treatment does not affect epidermal thickness
Epidermal thickness was measured at the wound edge and in the wound center in the Integra®-
pigs at the end of the observation period (d30) when >80% of all wounds were epithelialized. No
difference could be found in epidermal thickness between the treatment group and the control
group (figure 25).
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Figure 26. BD-MSCs do not affect epidermal thickness.
(A) No difference in epidermal thickness at day 30 (>90% of wounds epithelialized) between the BD-MSC group (5000 BD-MSC/cm2) and the acellular Integra group at the wound center, (B) as well as at the wound edge. (C) Representative trichrome stained sections from of the BD-MSC and the acellular group (arrows: epidermal thickness measured at the thickest point; E = epidermis, D = dermis).
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001; **** P
≤ 0.0001. No stars represent a P > 0.05. N=3; 2-3 wounds/pig per treatment group
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4.6.3 BD-MSCs does not affect collagen deposition
Wound treatment with cellularized Integra® (5,000 BD-MSCs/cm2) did not lead to a higher
collagen covered area or a higher collagen amount in the granulation tissue compared with
wounds treated with acellular Integra® (figure 26).
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Figure 27. In vitro cellularized skin scaffolds do not improve collagen deposition.
(A, B) After 30 days, no difference could be found between acellular and cellularized skin scaffolds in terms of collagen covered area and (C, D) amount of secreted collagen. (E) Trichrome stained granulation tissue of wound edge and center for both groups.
Statistical significance is indicated with stars: P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001;
**** P ≤ 0.0001. No stars represent a P > 0.05. N=3; 2-3 wounds/pig per treatment
group
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Chapter 5
Discussion
Discussion
5.1 Cell extraction and delivery method
The ideal cell extraction method yields a high number of cells after a short time without
imposing stress on the tissue that could alter cell metabolism and function. We compared two
different extraction methods - the conventional and the enzymatic method. The first one exposes
the tissue and its cells to the least stress possible since the only manipulation consists of cutting
the tissue with a scissor (147). However, this method only leads to a substantial number of cells
after 2 weeks and therefore is not usable for early cell-therapy in burn patients. The average time
for visible outgrow of MSCs from the tissue pieces was 7 ± 4 days, which is in accordance with
literature. De Bruyn et al. for example recorded an average time of 5 days for MSC outgrowth
out of Wharton's Jelly pieces (148). Taking into account that with the enzymatic method, almost
800.000 MSCs/cm2 burned skin could be harvested at the time of the first visible MSC
appearance in the conventional method, the enzymatic cell extraction should be the favored
method for early cell therapy in burn patients. The attachment of viable stem cells 24h after
enzymatic extraction was already recorded in literature for UC-MSCs and confirms our results
(149). It is known, however, that enzymatic cell extraction can cause damage to the cells and
even cell lysis (148), but the high yield of viable MSCs after a short time and the lack of an
equieffective extraction method outweighs these disadvantages.
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The ideal delivery method for local cell therapy needs to be precise in terms of its location and
the number of delivered cells, easy to handle for the surgical team and the cells need to remain
viable and in place after their application to the wound bed. We decided to use Integra® and
pullulan-gelatin first generation hydrogel (PG-1)(145) as the carrier for our stem cell application.
Our goal was a direct translational approach in this project to immediately evaluate a possible
clinical relevance. Our main focus was on Integra® - a FDA approved and clinically already
widely used material. Since its invention in the 1980s, Integra® is the most commonly used
artificial skin substitute in burn care and has proven to be highly beneficial in burn wound
management (9,10). It is classified as an acellular synthetic skin substitute and relies on the
patient’s own regenerative potential, providing a scaffold for cell ingrowth and a protection
against infections, dehydration and protein loss (9). We showed that BD-MSCs can be seeded
reliably onto Integra® and that those cells attach to the scaffold, stay in the place of the seeding,
are viable, and display a healthy cell-morphology. However, the structure of Integra® did not
allow us to completely cellularized the whole material - the maximal seeding depth that could be
reached was on average 180µm with centrifugation. Since centrifugation is not feasible for larger
skin scaffold pieces because of special limitations of the centrifuge, we cellularized only the skin
substitutes by applying the cells via pipetting and observed comparable results.
Considering that after skin injury the vascularization of Integra® takes up to 14 days (50) and
therefore the cells are initially nourished solely via diffusion from the wound bed (150), it is
unclear if cells in the upper layers would have been properly nourished. Therefore, we did not
see the low seeding depth as a limitation. A solution to overcome the low initial seeding depth
and the possible lack of nutrients in scaffold areas distant to the wound bed could be a serial
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injection of BD-MSCs through the upper silicone layer at different time points, e.g. with every
dressing change.
To not limit the results of our research to a singular brand product (Integra®) and to exclude
possible effects on wound healing used by the carrier material alone, we also explored the
application of BD-MSC cell therapy using an artificial skin substitute that was recently
developed by our laboratory called " pullulan-gelatin first generation hydrogel (PG-1)" (145).
PG-1 consists of the polysaccharide pullulan and gelatin and it was shown that different cell
types can be successfully incorporated into this material. Since this was published recently by
our lab, we did not repeat cell seeding and viability tests of cellularized PG-1. In a murine study,
PG-1 cellularized with differentiated fibroblasts and keratinocytes promoted wound healing and
reduced local inflammation. Displaying similar physical properties, we could apply the exact
same seeding and application protocol to PG-1 as we used for Integra® with the exception that
we could not staple PG-1 to the wound edges since it had no supporting silicone layer. The same
limitations concerning the seeding depth apply for this scaffold, however, due to the hydrogel
character of PG-1, a second seeding of BD-MSCs with a latency after grafting will not be
possible, since direct fluid injection into the hydrogel led to a destruction of the material.
The in vitro cellularized skin scaffolds had to be applied to the wound, which required manual
adjustment, stretching, in situ cutting and stapling. Physical stress is known to negatively affect
cell function and cell survival (151,152). An optimization of the application protocol that
minimizes this physical stress, e.g. cell application after fixation of the scaffolds to the wound
bed, would be ideal.
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5.2 Porcine wound healing model
Our preliminary data in a murine model already showed that local BD-MSC therapy leads to a
significant improvement of wound healing. However, the mouse as a model for skin regeneration
and wound healing is inferior to pigs (153): Small mammals such as mice and rabbits are only
frequently chosen first because of cost-effectiveness and uncomplicated handling, but the
difference in wound healing and skin anatomy compared to humans is substantial. Rodents heal
primarily by wound contraction and not by reepithelialisation like humans or pigs. In addition,
humans have an epidermal thickness of 50 to 120µm with a derma-epidermal ratio of 10:1 to
13:1, that can only be found in pigs (154). Apart from primates, which are only rarely used in
medical research because of massive ethical concerns (155), pigs show a great similarity to
humans in important aspects of their skin anatomy- amount of subdermal adipose tissue, dermal
papillary bodies (156-158), composition of the stratum corneum (159), biochemical properties
and distribution of porcine dermal collagen (160), vascularization (161,162). In addition, the
turnover time for keratinous protein fractions is similar in humans and swine (163). The pig also
seems to be comparable to humans in its physiology and functioning of major organs (164,165).
However, the porcine skin and wound healing also have some important differences. Porcine
skin is less elastic than human skin (166), and does not contain eccrine glands, instead, apocrine
glands can be found throughout the whole skin (154).
The close resemblance with human skin anatomy and wound healing physiology makes the pig
one of the best animal models to conduct wound healing studies (153). It is not only used to
understand the effect of stem cell therapy on wound healing (167,168), but it is also an excellent
model to specifically investigate treatments for burn wounds (169).
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5.3 Existence of viable and functioning mesenchymal stem cells in severely burned skin
It is known that the human skin contains MSCs (147,170). A 2012 published study by Vaculik et
al. was able to further subdivide those dermal MSCs based on specific surface markers such as
SSEA-4+ or CD271+ and showed that different subpopulations have different differentiation
potentials (170). However, there is no definitive single identifier for MSCs (170), it is rather a
combination of different surface markers, a specific differentiation profile and the ability to
adhere to a plastic surface as it was defined by the International Society for Cellular Therapy in
2006 (115). The problem, especially in MSC isolation from the dermis, is that they are very
similar to fibroblasts in morphology and surface markers (171). Therefore, a clear distinction
between the two cell lines is often difficult or not possible (172). Cells extracted from the burned
skin adhere to the plastic surface, show key surface markers of MSCs (positive for CD105, 90,
73, and negative for CD34), but are less capable of differentiating into the mesenchymal tissues
(cartilage, bone, adipose tissue) compared with umbilical cord MSCs. This could be due to the
fact that the cell population we extracted does contain terminally differentiated fibroblasts, thus
do not have the ability to differentiate into those tissues. Another reason for the lower
differentiation potential of the BD-MSCs is also the fact that the donor age is significantly higher
compared to umbilical cords that are used on the day of birth (173,174). The initial lower
percentage of cells positively identified as MSCs solely based on their surface marker expression
can be explained by the fact that the skin is more heterogeneous than the umbilical cord tissue,
and therefore a more heterogeneous cell population initially adheres to the plastic surface of the
culture flask (175). This is supported by the fact that with an increased culture time, the initial
heterogeneity is reduced to an almost complete population positive for the tested MSC surface
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markers. This, together with the positive differentiation assays for each tested patient sample,
makes it very likely that the extracted cells are indeed MSCs.
The extremely surprising novelty of this project is that we extract those MSCs out of the severely
burned skin, previously believed to be dead tissue and therefore discarded as medical waste. We
are the first ones to show that burned skin contains viable stem cells that can be used for
regenerative medicine. Before cell extraction, we removed attached muscle and adipose tissue to
ensure we only subject the burned skin to cell extraction. We did not find a difference in cell
yield between flame or scald thermal injuries, indicating that we have a small subpopulation of
extremely heat resistant stem cells in our skin. A higher heat tolerance of stem cells is not a new
concept; it can be used clinically before autologous stem cell transplantation in leukemic patients
to purge healthy stem cells from aberrant leukemic ones (176). However, this heat exposure is
not comparable to thermal injuries - it does not exceed 45 degrees and is used over a longer time
period such as 90-120min (177-179). The exact level of heat that BD-MSCs were exposed to is
unknown, but since we only used deep-partial or full-thickness burned skin, the thermal stimulus
exceeds 45 degrees Celsius by far. In our porcine model for example, we had to apply a 200-°C
hot metal block for 20 seconds to achieve a controlled full thickness burn. It is not known
whether stem cells can survive such intensive heat, or how this occurs, but it will be of great
importance for our understanding of stem cell biology and wound healing to investigate in detail
to what extent stem cells can survive extreme thermal stress, what the underlying mechanism is,
and if this is exclusive to MSCs or present in all stem cells. The fact that we could also extract
viable stem cells from porcine burned skin is of paramount importance, since this indicates that
whatever causes this cellular resistance is not exclusive to humans.
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Stem cells extracted out of burned skin are not only viable, but they also show no impairment in
key biological characteristics and functions when compared to umbilical cord MSCs. Intensive
heat exposure is known to cause severe damage to the cell integrity as well as to its functioning
(180,181). Fibroblasts exposed to 43°C showed an immediate increase in their oxidative stress
damage (181). By promoting oxidative defense mechanisms such as upregulation of glutathione
and mitochondrial superoxide dismutase, oxidative stress could be prevented. Increased ROS
production in MSCs is known to greatly affect their viability and functioning and therefore needs
to be minimized for their successful therapeutic application (182). Literature produced two
contradictory views on ROS in MSCs: On the one hand, it was shown that MSCs have a high
amount of glutathione (a strong antioxidant) and therefore have a great capacity to reduce ROS
(183). On the other hand, it was also suggested that MSCs have less capacities to cope with ROS,
for example keratinocytes (184). BD-MSCs did not show an increased reactive oxygen
production at a passage number of 3-4 compared to umbilical cord cells. It would be interesting
to elucidate in future studies if BD-MSCs at an earlier time point, e.g. right after extraction,
showed an increased ROS production or if those cells can prevent it right from the beginning
through a strong oxidative defense system. We used the enzymatic cell extraction method for our
study, since it was the only extraction method that yielded a substantial amount of MSCs after a
short time period. However, this method itself is highly stressful for the cells and lead to a high
amount of ROS production right after extraction in both the umbilical cord and the BD-MSC
group, with no difference between the group, overshadowing any possible effect of the initial
thermal injury (data not shown). The ability to reduce this initial high oxidative stress and reach
a normal cell function in terms of proliferation, cell cycle phase distribution and colony forming
behavior is beneficial for their application in regenerative medicine, since elevated ROS levels in
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the wound environment is believed to contribute to MSC loss after grafting (185-187). The fact
that BD-MSCs show no significant difference in their population doubling time and their
proliferation capacities compared to UC-MSCs is indicative that thermal injury does not lead to
oxidative cell damage, since this would cause impaired self-renewal and proliferation (188-191).
In recent years, studies have shown that cell exposure to moderate heat (heat shock) can also be
beneficial (192-196). The heat shock response leads to an increased activation of heat shock
transcription factors (HSF 1-5) resulting in a higher expression of various heat shock proteins
(Hsp) such as Hsp 70 or Hsp 90 (197-199). This response is not exclusive to thermal stimuli but
is also caused by a variety of different stressors such as starvation or hemodynamic stress (200).
Hsp plays an essential role in unstimulated stem cells and are closely related to their
differentiation and self-renewal potential (201), but they also seem to be highly protective
against thermal injuries, oxidative stress, infection and even sepsis (200,202). However, to reach
a protective effect, cells were preconditioned at mild heat to upregulate Hsp expression before
subjecting them to a severe damaging stimulus. Harder et. al for example could reduce iatrogenic
ischemic necrosis of a skin flap by preconditioning the skin with local moderate heat exposure
for 24h prior to the surgery (203). This particular mechanism was believed to be caused by
Hsp32, which has CO-like vasodilation effects. Various studies also showed that a
preconditioning of cells by up-regulation of Hsp improved resilience against tissue damage of
various kinds (193-196). However, this upregulation of the protective Hsp seems to occur with a
substantial delay of several hours and therefore is highly unlikely to be the cause of BD-MSC
resistance against thermal injury (204,205).
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BD-MSCs displayed proliferation, the number of colonies after 10d, and the cell cycle phase
distribution comparable to UC-MSCs. This is quite surprising, since UC-MSCs were shown to
be superior in their proliferation and colony forming capacities to MSCs from other cell sources
such as bone marrow or adipose tissue (206,207). The calculated population doubling time of
BD-MSCs of 28 ± 5h is almost identical with the reported PDT of 27 ± 3h for dermal MSCs
extracted from hair follicles of healthy skin (208). This data suggests that burn injury does not
affect the ability or speed of proliferation of the dermal MSCs at all. Future studies are warranted
to closely compare MSCs of unburned and of burned skin to elucidate this further.
The distribution of the mesenchymal stem cell population within the cell cycle phases seems to
be independent of their source of origin - with the majority of cells in the G0/G1 phase and only
a small percentage in a proliferative state (209,210). Based on previous literature showing that
severe stress such as heat or oxidative stress leads to cell cycle arrest, (211,212) we expected that
BD-MSCs had a different cell cycle phase distribution than UC-MSCs from undamaged tissue.
Because of the number of cells needed for cell cycle analysis via propodium iodine flow
cytometry, we could not assess cells at a passage number of 1. Therefore, we cannot comment if
there was a difference at an earlier stage closer to the burn.
BD-MSCs showed a comparable glycolytic function to UC-MSCs, but a clear signal towards a
higher oxidative phosphorylation. This is not surprising since Broderick et al. already showed in
2006 that heat increases mitochondrial respiration (213,214). The main energy gaining
mechanism in MSCs, however, is anaerobic glycolysis that is responsible for 97% of the ATP
production and that shows no difference between the two cell lines (210). This data shows that
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BD-MSCs did not only maintain their ability to produce rapid energy via anaerobic glycolysis,
but also gained a higher ability for oxidative phosphorylation.
This study shows for the first time that severely burned skin contains viable MSCs and that those
MSCs seem to be undamaged by the thermal injury and comparable to highly proliferative and
healthy umbilical cord stem cells in key biological functions.
5.4 Safety of BD-MSCs
The main safety concern in the usage of stem cells is their potential risk of tumor formation.
While this is a valid concern in pluripotent embryonic stem cells (213,215), MSCs independent
of the tissue they were extracted from are believed to be safe and therefore are widely used in
clinical trials (216). This belief was challenged in recent years by studies that showed MSCs can
indeed be tumorigenic in vivo (217). In some cases, long-term culturing of MSCs led to genetic
abnormalities further fueling the concern of malignant transformation (218). However, this is in
contrast to the majority of literature that could not detect a risk of malignancy (219).
Furthermore, the question was raised if the cells with the malignant potential are true MSCs or
the product of cross-contamination with cancer cells (220). We performed in vitro and in vivo
tumorigenic assays with BD-MSCs and could not detect a risk for tumor formation. In addition,
BD-MSC treatment did not cause any systemic or local adverse effects in swine nor in mice
(detailed description of the murine experiment of our laboratory below). To further confirm their
safety, and considering that they are derived from severely burned skin, it will be important to
assess whether the thermal stimulus lead to chromosomal or genetic abnormalities. However,
BD-MSCs did not show tumor formation in vitro and in vivo, they are identified as MSCs that
are already widely and successfully used in clinical trials without adverse effects, and we showed
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that BD-MSCs do not display altered cell function compared to UC-MSCs. Taken together, it is
justified to assume that BD-MSCs, like MSCs of other origins, will be safe for clinical usage.
Another concern in every cell therapy is the potential risk of immunologic rejection by the
recipient. The intended usage of BD-MSCs is as an autologous cell transplantation and therefore
does not contain this risk. However, we also examined BD-MSCs for a potential use as a
heterologous transplant. The extremely low expression of Major Histocompatibility Complex
(MHC) II (hypoimmunogeneicity) in various MSCs is known to be one of the main factors why
they are able to avoid T-cell recognition and immunologic rejection (123,221-225). In addition,
MSCs as an adjunctive therapy are also believed to aid in the reduction of organ transplant
rejection by secreting immunosuppressive proteins such as IL-10 (226). Depending on the source
of MSCs, the amount of expressed MHC can vary - bone marrow derived MSCs, for example,
were found to express slightly more MHC II than UC-MSCs (227). BD-MSCs showed a low
expression with less than 1% cells positive for MHC II and less than 1.5% positive for MHC I
comparable to UC-MSCs, indicating that they also could be used as allogenic transplants. In
addition, neither swine nor mice (see below) displayed signs of excessive local or systemic
inflammation under MSC wound-treatment.
Based on our results, BD-MSCs seem to be safe in vitro and in vivo in terms of tumor formation
and immunologic rejection. In addition, none of the treated pigs showed any adverse side effect -
neither locally when compared to the acellular control group - no tumor formation, no arterial
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bleeding, no hypertrophic scarring, and no keloid formation - nor systemically - no respiratory
distress, no fever, no signs of systemic infection, nor decreased well-being.
These confirm the safety of BD-MSCs as a wound treatment. The observation period of the pigs
was 30d, which does not allow us to definitively exclude possible long-term adverse effects such
as late keloid formation or hypertrophic scarring. However, the length of the observation period
was determined to be 30 days - a time frame where most of the wounds were completely closed
and epithelialized. No difference, microscopically or macroscopically, between the BD-MSC
treated wounds and the control group could be detected during wound healing and after complete
epithelialization, making the risk of a potential late-onset adverse effect highly unlikely to
negligible.
5.5 BD-MSCs and inflammation
Burn wounds, like all other large wounds, are heavily colonialized by bacteria within 48 hours
after the initial injury (228). It is of great importance to understand in what extent the applied
cells react to infectious stimuli.
Toll-like receptors (TLR) are expressed by various immune (such as peripheral blood
mononuclear cells) and non-immune cells (such as fibroblasts) and can recognize bacterial,
fungal and viral components (14). Of the 11 known human TLR, TLR 4, in particular, is of
importance since this receptor recognizes lipopolysaccharides (LPS) - a characteristic and
specific component of gram-negative bacteria such as pseudomonas aeruginosa (213,214), that is
amongst the most common and most lethal bacteria for burn patients (210). The exact meaning
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of TLR activation for wound healing, however, is unknown and contradictory. On the one hand
activation of TLR can lead to a secretion of proinflammatory cytokines (213,215) further fueling
the excessive inflammation caused by burn injury. On the other hand, it was shown that by
activating MSCs via TLR 3 and 4, their activation of the immunosuppressive regulatory T-cells
was increased (217), proinflammatory cytokines such as IL-1 were decreased while anti-
inflammatory ones such as IL-10 were increased (218). In addition, TLR also seems to play an
important role in proliferation and differentiation of MSCs. Xiaoqing, for example, showed that
the activation of TLR 4 increased proliferation and osteogenic differentiation in bone marrow
derived MSCs (219). Our study has shown that there is no statistical difference in the expression
profile of TLR in BD-MSCs compared to UC-MSCs. This suggests that BD-MSCs most likely
react in a comparable manner to UC-MSCs - which overall reduce wound inflammation and
enhance bacterial clearance (220) - when exposed to colonialized or infected wounds.
The suggested similar behavior of UC-MSCs and BD-MSCs in terms of infection and
inflammation, was further confirmed by the measurement of secreted essential cytokines,
chemokines, and growth factors. Of 34 assessed proteins, BD-MSCs only showed a significantly
different expression of 11 at baseline and 2 once stimulated by LPS, which takes the wound
colonialization into account. BD-MSCs seem to be slightly more reactive to LPS than UC-
MSCs, but in the end, only BD-MSCs showed a significantly higher expression of gamma
interferon (IFN-gamma)-inducible protein 10 (IP10) and interleukin (Il, hematopoietin) 1a. Both
cytokines are believed to have pro-inflammatory properties by chemo-attracting, recruiting, and
stimulating immune cells (123,221-225). Since IP10 was recently shown to be a potent inhibitor
of angiogenesis in vivo, the clinical relevance of its higher expression in BD-MSCs needs to be
further evaluated (226). On the other hand, IL-1a was also shown to have beneficial effects by
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improving the skin barrier and promoting epidermal lipid production (229). In vivo studies are
needed to determine the exact effect of those findings on wound healing.
The data of this study suggests that BD-MSCs will have a comparable reaction towards
colonialized or infected wounds than UC-MSCs and overall do not significantly differ in the
expression of cytokines, chemokines and growth factors, making them a promising candidate for
skin regeneration and comparable to the already successfully used UC-MSCs.
5.6 Effect on wound healing
Burn derived mesenchymal stem cell treatment did improve epithelialization in the form of
increasing the total epithelialized area and the epithelialization speed compared with the acellular
control group. The promotion of wound healing observed in pigs is in accordance with the effect
of BD-MSC treatment of punch wounds in mice, that also showed improved wound healing.
The murine study was conducted in our lab by Dr. Amini-Nik (Data not published yet). 6-8 week-
old immune-incompetent nude mice (Jackson Laboratories) received 4mm punch wounds and
were either treated with BD-MSCs incorporated into Matrigel (110.000 cells/wound) or acellular
Matrigel (n=5). Wound healing was observed over 12 days, followed by histological sampling at
day 12 (figure 27A,B). Mice treated with BD-MSCs showed a significantly smaller wound size
(figure 27C-E), a smaller keratinocyte layer (figure 27C, D, and F) and a shortened proliferative
phase of wound healing - measured via Ki67- and BrdU staining (figure 28).
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Figure 28. BD-MSCs improve wound healing in mice.
(A) Schematic of in vivo animal experiment. (B) Time course measurement of wounded skin shows a
faster healing in excisional biopsies which were treated with BD-MSCs compared with control group.
Note the arrows in days 8 and 12 post biopsies. (C) 4mm punch wounds 12 days after topical
treatment without cells shows a larger wound size (E) and more scarring (F) than wounds treated with
burn-derived stem cells (D-F).
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Figure 29. BD-MSCs shorten proliferative phase of wound healing.
(A-B) Ki67 staining of healed skin 12 days post wounding shows significantly less Ki67+ cells
in the healing bed of wounds treated with BD-MSCs. Arrows show Ki67+ cells. (C)
Quantification of the number of Ki67 positive cells. Error bar shows 95 confidence intervals of
the mean. (D-E) BrdU incorporation of healed skin 12 days post wounding shows significantly
less BrdU incorporation in the healing bed of wounds treated with BD-MSCs. Arrows show
cells which are incorporated with BrdU. (F) Quantification of the number of BrdU-positive cells.
Error bar shows 95 confidence intervals of the mean
123
Porcine and murine experiments show a beneficial effect of BD-MSCs on wound healing.
However, the porcine study is only a pilot study and did not have location matched controls. It is
essential to repeat this experiment in a higher number of pigs that contain exactly location-
matched control wounds to exclude different wound contraction and healing behavior of
different locations.
Epidermal thickness did not show a difference between the control and the BD-MSC treated
group in the porcine model, unlike the murine model. This could be due to the fact that wound
healing differs in varying species (with pigs closely resembling human wound healing; see
discussion above) and therefore BD-MSCs might affect the stages of wound healing differently.
However, at this early stage, no definitive conclusion can be drawn.
There are several variables that require investigation in the samples taken from this study and in
further studies with a higher power to understand the mechanism of BD-MSCs on wound
healing, but also to improve their effecting order and make them clinically relevant.
First, it is important to determine the best technique of applying BD-MSCs onto the wound bed.
In this pilot, we chose to pipette BD-MSCs onto the skin scaffolds before applying them onto the
wounds, because it is the fastest and most cost-effective method. However, during the
application of the (a)cellular skin scaffolds onto the wounds, they are exposed to substantial
physical strain - scaffolds have to be applied onto the wound bed, they have to be adjusted, cut
individually, stapled and pressed down. Considering that BD-MSCs only reached a seeding
depth of 130µm it is likely that they were damaged through the application process. As
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highlighted in detail in the introduction, physical/mechanical stress is a known and strong
contributor to cell death and cell dysfunction.
Second, it is of paramount importance to determine if cell death occurred, if BD-MSCs remained
in the wounds after 30d and if they were able to integrate. Dr. Amini-Nik has shown in the
immune-incompetent mice, a few number of human BD-MSCs remain in the newly formed
scar/granulation tissue after complete wound closure (day 12; see figure 27).
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Figure 30. Burn derived mesenchymal stem cells are integrated into the granulation
tissue.
HLA class 1ABC (EMR8-5) staining of healed skin 12 days post wounding shows
remaining BD-MSCs (Arrowheads: cells negative for HLA1; Arrows: cells positive for
HLA1)
126
However, this was done in severely immune compromised mice and it does not allow us to
conclude if BD-MSCs also remain in the granulation tissue in immune-competent animals.
Third, since we applied the BD-MSCs right after infliction of the wounds, it is possible that the
wound bed was not able to supply enough nutrition in the initial critical phase of wound healing
and therefore the MSCs could not reach their full potential. An in vitro study showed that
starving MSCs enter a G0/G1 phase of the cell cycle, rather than undergoing apoptosis right
away (227). However, a study published in the same year showed that starvation of the MSCs is
the main factor for inducing cell death after 48 hours (228). After re-gaining access to nutrition,
the BD-MSCs entered a more active phase of the cell cycle and could contribute to the wound
healing process, which is an explanation for the increased epithelialization in the treatment
group.
Furthermore, a detailed dose-effect study needs to be executed. We used 500, 5,000 and a
maximum amount of 50,000 cells/cm2 of the wound. With an initial cell yield of BD-MSCs of
around 1.000cells per cm2 burned skin 24 hours after extraction and a PDT of 28 ± 5h, those cell
numbers can be achieved after approximately 1, 5, and 8 days of culturing, respectively. Since
our goal was to deliver cellular therapy as soon as possible after burn injury, we did not want to
use higher cell numbers that would have required a longer culturing time. A solution for that
could be the usage of bioreactors that claim to achieve expansion factors of 100 to 1000 (14). In
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the above-mentioned follow-up study that is currently underway, we used cell numbers of up to
200,000 cells/cm2.
BD-MSCs did improve wound healing in mice and pigs. Further studies are warranted to
elucidate the exact mechanism behind that effect, to optimize cell application, and to show that
this data can be reproduced on a larger scale.
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Chapter 6
Future directions
We show for the first time that discarded, severely burned skin contains viable MSCs. These cells
can be extracted, expanded in vitro, and used as an adjunctive to already existing wound coverage
materials in a straightforward and cost-effective incorporation process. Their wound healing
potential was confirmed in two different animal models - mice and swine - without causing adverse
side effects. This is an exciting discovery for stem cell research as well as the burn community
since this could change the way we practice burn and wound care in the future.
Various challenges emerge from this important discovery: It is of paramount importance to
understand the mechanism behind the survival of MSCs in burned skin. It is not known what
might protect this group of cells from apoptosis and necrosis when exposed to heat far beyond
protein coagulation temperatures. It also needs to be answered if those surviving cells come from
a specific location of the dermis or epidermis, or if they are evenly distributed through the
excised burned skin. We plan to approach those questions with extracting MSCs from various
clear defined regions such as epidermal, dermal, and perivascular of unburned skin, expose them
to heat of different intensities and compare the surviving cells with the healthy skin-derived
MSCs.
The regenerative potential of BD-MSCs needs to be further explored. A comprehensive dose-
response study with location-matched control wounds should follow our initial pilot study to find
the optimal concentration of BD-MSCs to improve wound healing. In addition, different
129
application strategies of the BD-MSCs need to be evaluated such as spraying or injection into the
wound bed. We are currently conducting those experiments.
One of the most important questions that need to be answered is undoubtedly if BD-MSCs can
last and integrate into the newly formed tissue, and if that is the case, it needs to be evaluated if
the MSCs differentiate into skin cells such as fibroblasts and keratinocytes. We already started
investigating those issues by staining the porcine wound sections with anti-human nuclear
antigen antibody. In addition, we are completing the follow-up pig study where we changed the
mode of BD-MSC application to reduce physical stress on the cells to a minimum. Cells are now
injected into the skin scaffolds after their placement and fixation onto the wounds. Results of this
current study will elucidate further the effect of BD-MSCs on wound healing.
Now that we established the safety of our BD-MSC treatment and have shown favorable
outcomes in mice and swine, we meet the requirements to apply for a clinical trial to use our
gained knowledge in a direct translational approach. We intend to isolate BD-MSCs from burn
patients and graft them on the same patient in form of an autologous transplant.
Since the ideal treatment of severely burned patients ultimately requires the fast and complete
replacement of the lost skin, it also needs to be evaluated if BD-MSCs are a valuable adjunctive
to a cellular combination therapy in the form of compound skin replacements together with
differentiated skin cells such as fibroblasts and keratinocytes. Our vision for the future is the
production of autologous skin replacement by using patients’ own BD-MSCs.
130
Chapter 7
Conclusion
The discovery that burned skin contains viable MSCs that can be extracted and cultured reliably
and in significant quantities is of great importance for regenerative medicine and for burn care.
Burned skin as a stem cell source is free from ethical concerns, does not require additional
invasive procedures and even has the potential to serve as a source for autologous stem cell
transplantation.
We have shown that burn trauma did not adversely affect key biological characteristics making
BD-MSCs fully functioning, healthy stem cells comparable to UC-MSCs, that are known for
their beneficial wound healing properties. In addition, BD-MSCs appear to be safe: they did not
show tumor formation in vitro and in vivo or adverse effects in mice and pigs. Their predicted
interaction with the host immune system in case of transplantation is low, based on their low
MHC expression and on their cytokine expression profile that is similar to UC-MSC known to
evade immunologic rejection and to reduce inflammation.
We were able to integrate BD-MSCs into existing and novel acellular skin scaffolds without cell
loss or complications in an easy and cost-effective manner.
Human BD-MSCs showed improved wound healing in murine and in porcine surgical wounds,
in terms of wound healing speed and improved epithelialization.
Further studies are warranted to understand the mechanism of action of BD-MSCs as a wound
treatment and to optimize application and dosing.
131
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