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Stem cell therapies in orthopaedics: How far are we?
Chelsea Bahney, PhD Assistant Professor UCSF, Department of Orthopaedic Surgery
WHAT STILL NEEDS TO BE OVERCOME
② How do stem cells promote repair?
① Which “stem” cells to use?
③ How to delivery stem cells?
NOT ALL “STEM CELLS” ARE THE SAME!
Which stem cells to deliver depends on what you want them to do!
TOTIPOTENT stem cells can differenSate into all embryonic and extraembryonic cells (placenta)
PLURIOTENT stem cells can differenSate into all adult cell types but not extraembryonic cells Ø Inner cell mass of the blastocyst (EMBRYONIC STEM CELL) Ø Trophoblast stem cells (forms placenta)
MULTIPOTENT stem cells can differenSate into closely related cell types Ø Germ layer cells: endoderm, mesoderm, ectoderm Ø Tissue specific stem cells: blood, skin, neural, mesenchymal
OLIGOPOTENT stem cells can differenSate into only a few cells Ø Lymphoid, hair follicle (bulge), and endothelial stem cells
UNIPOTENT cells can produce only one cell type, their own but have the property of self renewal which disSnguishes them from non-‐stem cells Ø Satellite cells (muscle)
ADULT STEM CELLS
MacArthur BD et al. Systems biology of stem cell fate and cellular reprogramming. Nature Reviews Molecular Cell Biology 10, 672-‐681. (2009)
HOW CAN YOU CONTROL STEM CELL DIFFERENTIATION
FACTORS INFLUENCING DIFFERENTIATION
ü BioacSve Factors ü Mechanical Forces
ü Extracellular Matrix Microenvironment (shape, porosity, sSffness, ect)
EMBRYONIC STEM CELLS
OPPORTUNITIES
ü In vitro method to study embryonic development ü Model of human diseases (rare diseases: fibrodysplasia ossificans progressiva, FOP)
ü Can differenSate into all cell types à Sssue regeneraSon
CHALLENGES
ü Ethical consideraSons/debate over use ü Only a few “lines” are available for research and they
have different behaviors ü Mechanism for differenSaSon not always known ü Difficult to expand in culture & maintain pluripotency ü Teratoma formaSon1,2
(1) Wakitani, S. et al. Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint. Rheumatology (Oxford) 42, 162-‐165 (2003). (2) Nakajima, M., et al. In vivo mechanical condiSon plays an important role for appearance of carSlage Sssue in ES cell transplanted joint. J Orthop Res 26, 10-‐17 (2008).
MULTIPOTENT (ADULT) STEM CELLS
ADVANTAGES
ü Tissue repair and regeneraSon ü Established differenSaSon protocols
ü Accessible in stem cell niches
ü In vitro expansion
ü Immunoprivilaged1
David Scadden. Nature 441, 1075-‐1079 (29) June 2006)
Stem Cell
Stromal/Support Cell
CHALLENGES
ü ReplicaSng mechanical properSes of orthopaedic Sssues
ü Age related decrease in stem cell number2 ü RecreaSng the stem cell niche ü Different niche sources à variable results and differenSaSon protocols
(1) Schu, S et al. Immunogenicity of allogeneic mesenchymal stem cells. J Cell Mol Med 2011. (2) Caplan AI & Boyan BD. Endochondral bone formaDon: the lineage cascade. In Bone, vol 8, HallB (ed.). CRC Press: Boca Raton, FL, 1994; 1–46.
SKELETAL STEM CELL NICHES
Grassel & Ahmed. Frontiers in Bioscience 12, 4946-4956, September 1 (2007)
① BONE MARROW
② SYNOVIUM
③ ADIPOSE TISSUE
④ PERIOSTIUM
⑤ PERICYTES
MULTILINEAGE DIFFERENTIATION
Adipocytes Bone (Osteocytes) Chondrocytes Stromal Cells
Tendon/Ligament
DifferenDaDon potenDal & protocol not idenDcal for all the different stem cell niches…are these cells idenDcal??
SKELETAL LINEAGE Adipocytes
Bone (Osteocytes) Chondrocytes Stromal Cells
Tendon/Ligament
MESODERMAL TISSUES Skeletal muscle Smooth muscle Cardiac muscle Endothelial cells
TRANSGERMAL TISSUES Neuralectoderm
HematopoieSc Lineage Endoderm
Skin
MSC
BONE MARROW PERIOSTEUM SYNOVIUM WHITE FAT
DIFFERENTIATION POTENTIAL OF MESENCHYMAL STEM CELLS
Elaine Fuchs. Nature 445, 834-‐842 (22 February 2007)
Spradling A, et al. Nature 414, 98-‐104 (1 November 2001)
SKIN STEM CELL NICHES
EPITHELIAL STEM CELLS
GUT STEM CELL NICHES
INTESTINAL STEM CELLS
0.0
20.0
40.0
60.0
80.0
100.0
120.0
Newborn Teen 30 50 80
# MSCs p
er M
arrow Cells
AGE
1/ 10,0000
1/ 100,000
1/ 2,000,0000
1/ 250,000
1/ 400,000
BAD NEWS……NUMBER OF MSCS DECLINES WITH AGE
Ø These cells are rare, need proper technique to obtain and need to expand significantly for therapeuSc benefit
Caplan AI & Boyan BD. Endochondral bone formaDon: the lineage cascade. In Bone, vol 8, HallB (ed.). CRC Press: Boca Raton, FL, 1994; 1–46.
GOOD NEWS……MSCS ARE IMMUNOPRIVILEGED (WE THINK!)
Immunoprivileged = Does not illicit grau versus host response
Immunosupressive = AcSvely downregulate inflammaSon
Normanton et al: hwp://journals.plos.org/plosone/arScle?id=10.1371/journal.pone.0106673
ü Stem cell banking ü Universal donors
WHAT STILL NEEDS TO BE OVERCOME
② How do stem cells promote repair?
① Which “stem” cells to use?
③ How to delivery stem cells?
MSCs Trophic Factors
SSmulate Tissue Repair
DifferenSaSon to CarSlage & Bone
Tissue Engineering
Modulate Healing & Reduce Scarring
AnS-‐inflamatory Cytokines
Paracrine stimulation
Build a new tissue
THERAPEUTIC POTENTIAL OF SKELETAL STEM CELLS (MSCS)?
Modified from: Caplan AI & Dennis JE. Mesenchymal Stem Cells as Trophic Mediators, Journal of Cellular Biochemistry. Vol 98, Issue 5, Aug 2006
PARACRINE FACTORS FROM MSCS
BIOACTIVE FACTORS1,2
ü transforming growth factor beta (TGF-‐β) ü stem cell factor (SCF) ü insulin-‐like growth factor (IGF) ü epidermal growth factor (EGF) ü granulocyte/macrophage colony sSmulaSng factors (G/M-‐CSF)
ANTI-‐INFLAMMATORY MOLECULES
ü interleukin-‐103 ü nitric oxide4 ü prostaglandins5
REFERENCES (1) Haynesworth, S. E. et al. J Cell Physiol 166, 585-‐592 (1996). (2) Caplan, A. I. J Pathol 217. (3) Bartholomew, A. et al. Exp Hematol
30, 42-‐48 (2002). (4) Sato, K. et al. Blood 109, 228-‐234 (2007). (5) Aggarwal, S. & Piwenger, M. F. Blood 105, 1815-‐1822 (2005). (6) Murphy JM et al. Stem cell therapy in a caprine model of OA. ArthriSs Rheum. 2003 Dec;48(12):3464-‐74.
CONTROLLING MACROPHAGE POLARIZATION
Laskin DL. Chem. Res. Toxicol., 2009, 22 (8). DOI: 10.1021/tx900086v
Stem Cells Aging Diabetes RA, COPD Obesity
IMPROVING HEALING RESPONSE WITH MSCS
hwp://www.osiris.com/technology/#msc-‐primer
WHAT STILL NEEDS TO BE OVERCOME
② How do stem cells promote repair?
① Which “stem” cells to use?
③ How to delivery stem cells?
INJECTING STEM CELLS – ISSUES AND CONSIDERATIONS
Ø InjecSon locaSon and technique mawers ü IP, IV or local ü Speed ü Carrier
MSC injecDons to cardiac Dssue: < 10% survive more than 24 hours < 1.4% remain at injured locaSon
MSC interarDcular injecDon to knees with surgically induced OA (goat)6: • protects carSlage and subchondral bone • minimal cell engraument
INJECTING STEM CELLS – IMPROVING OUTCOMES
2. Controlled and SLOW injecSon2
3. Directed cell therapy3 1. Hydrogel carrier to dissipate the sheer1
REFERENCES 1) Aguado BA, et al. Improving viability of stem cells during syringe need flow
through the design of hydrogel cell carriers. Tiss Eng A, Apr 2012. 2) Lam J, et al. Unpublished data. 3) Keen, TJ et al. MSCs: Delivery Routes and Engrabment, Cell-‐TargeDng Strategies,
and Immune ModulaDon. Stem Cells Int(2013) ArScle ID 732742, 13 pages
TISSUE ENGINEERING FOR ORTHOPAEDICS
Repair damaged or diseased Sssue with a regenerate that has metabolic and mechanical funcSon of naSve Sssue.
VinaSer, C., et al. 27, 307-‐314, (2009).
BONE
Ø DMB with bone marrow Ø SyntheSc allograus
CARTILAGE
Ø Microfracture Ø MACI
Ø Three dimensional scaffold Ø Cells Ø BioacSve factors
Very poor cell engraftment by injection
SMART SCAFFOLDS – THE NEXT GENERATION OF TISSUE ENGINEERING Tunable systems that try to re-‐engineer the desired Sssue by direcSng stem cells down the
“normal” developmental pathway
Cell Adhesion Domains “Biomime'c”
Physical ProperHes (porosity, mechanics, hydrophilicity)
Biodegradable Scaffold “Bioresponsive”
Growth Factors “Boiac've”
Pollock, J., and Healy, K.E., “BiomimeHc and Bio-‐responsive Materials in RegeneraHve Medicine: Intelligent Materials for Healing Living Tissues,” In Strategies in Regenera've Medicine, M. San'n (Ed.), Springer, 2009
DESIGN OF A CELL-‐MEDIATED BIORESPONSIVE HYDROGEL
+ Stem Cells
EnzymaScally Degradable Scaffold
CarSlage or Bone Matrix FormaSon& Matrix ElaboraSon
SCAFFOLD DEGRADATION
Light
Tissue Regenerate
Photoencapsulated stem cells in degradable scaffold
1Bahney CS, et al. FASEB Journal, February 3 2011. 2Burdickt JA. Biomacromoleucles 2005 3Kim & Healy. Biomacromolecules 2003. Wise. PLOS One 2010
CarSlage = MMP71, hyaluronan2 Bone = MMP-‐133, hyaluronan, collagen
GROWTH FACTOR DELIVERY IN SCAFFOLDS
BONE
ü BMP
ü VEGF
CARTILAGE
ü TGFβ
Place, E. S., Evans, N. D. & Stevens, M. M. Complexity in biomaterials for Sssue engineering. Nat Mater 8, 457-‐470, doi:nmat2441 [pii]10.1038/nmat2441 (2009).
HOW CLOSE ARE WE TO CLINICAL USE FOR ORTHOPAEDICS?
5 years!* *Caveat: I have been saying 5-‐years for 8 years L…..
STRIDES
Ø Controlled differenSaSon of MSCs to bone, carSlage, and adipose Sssues in vitro
Ø Tissue Engineering methods being developed to support orthopaedic regeneraSon (research phase and Phase I clinical trials)
Ø AnS-‐inflammatory funcSon can modulate immune response and reduce fibrosis (Phase II/III clinical trials)
Ø Trophic funcSon can sSmulate autologous Sssue repair (Phase II clinical trials)
HOW CLOSE ARE WE TO CLINICAL USE FOR ORTHOPAEDICS?
CONCERNS
Ø UNREGULATED/OFF-‐LABEL USE à DO NOT PAY OUT OF POCKET FOR THIS Ø Material source – regulaSon of stem cell integrity Ø Manufacturing consideraSons – storage, shipping, quality
Ø Limited long term safety & efficacy data
Ø Uncontrolled/non-‐specific Sssue differenSaSon in vivo (Phase II/III clinical trials)
