Stem cells

Tissue dynamics-Histogenesis-Formation

Cellular fate processesCell communications

-Microenvironment

Tissue engineering applications-Bone marrow transplantation-Skin and vascular grafts-Pancreas-Cartilage and chondrocytes

Fundamental challenges of tissue engineering

Stem Cells- Undifferentiated cells with potential to differentiate into mature cells of many lineages

Embryonic stem cellsAdult Cord blood

Image of cell line SA02, humanembryonic stem (hES) cell colonyon a mouse embryonic fibroblast(MEF) feeder layer.

Stem CellsFacts about Stem Cells- Undifferentiated cells with potential to differentiate into mature cells of many lineages- Self-renewal: ability to go through numerous cycles of cell division while maintaining

the undifferentiated state- Unlimited potency - capacity to differentiate into any mature cell type

Pluripotent, embryonic stem cells originate as innermass cells within a blastocyst. The stem cells canbecome any tissue in the body, excluding a placenta.Only the morula's cells are totipotent, able to becomeall tissues and a placenta.

Examples of stem cell systems: mesenchymal stem cells (MSCs)

Stem Cells and Tissue Engineering- Stem cells build tissues.- Stem cells do age: early stems cells that can divide without differentiation(fetal liver and umbilical cord)

- Isolation of stem cells for scientific and clinical purposes: difficult because of rarity

Stem Cell Aging- Telomerases, DNA Stability, and Natural Cell Senescence- Telomeres: noncoding repeating sequences

on ends of chromosomes- Telomerase: DNA polymerase that can rebuild

telomeres-By lengthening telomeres, aging might slow andin exchange increase vulnerability to cancer.Weinstein and Ciszek, Experimental Gerontology, 2002, 615

Telomeres

•at ends of all chromosomes (not bacterial DNA circular) •roles in chromosome replication and maintenance (1) replication for replicating the ends of linear chromosomes (2) maintenance proposed to provide each cell with a replication counting mechanism that helps prevent unlimited proliferation •each cell division shortens telomere 50–100 nucleotides •DNA 100s to 1,000s repeats of a simple-sequence containing clusters of G residues (humans AGGGTT) •Telomerase enzyme maintains length

Reading material:

Mechanisms of Stem Cell Self-RenewalHe, Nakada, and Morrison, Annual Review of Cell and Developmental Biology 377-406, 2009

A. Tzatsos and N. Bardeesy, “Ink4a/Arf Regulation by let-7b and Hmga2:A Genetic Pathway Governing Stem Cell Aging,” Cell Stem Cell, 469, 2008

“As Hmga2 is turned off in ageing stem cells by increasing let-7bexpression, this allows Ink4a and Arfto be expressed,” explains Morrison. “The take-home message of the paper is that we have identified an entire pathway of genes that change in expression with age in stem cells.”

Bmi-1 is known as a tumor promotor, and genes that it repress - Ink4a/Arfare tumor suppressors. Hmga2 is anti-aging, but is a tumor promotor. So, aging of adult stem cell system actually protects us from cancer.

Bianco et al., “Stem cells in tissue engineering,”Nature 414, 118-121 (2001)

a, Skin autografts are produced by culturing keratinocytes (which may be sorted for p63, the recently described, epidermal stem cell marker) under appropriate conditions not only to generate an epidermal sheet, but also to maintain the stem cell population (holoclones). The epidermal sheet is then placed on top of a dermal substitute comprising devitalized dermis or bioengineered dermal substitutes seeded with dermal fibroblasts. Such two-dimensional composites, generated ex vivo, completely regenerate full-thickness wounds.

b, Bone regeneration requires ex vivo expansion of marrow-derived skeletal stem cells and their attachment to three-dimensional scaffolds, such as particles of a hydroxyapatite/tricalcium phosphate ceramic. This composite can be transplanted into segmental defects and will subsequently regenerate an appropriate three-dimensional structure in vivo.

Stem cells in tissue engineeringTissue Dynamics

- Tissue histogenesis (normal steady-state function): formation of different tissues from undifferentiated cells

- Tissue formation- Tissue repair

Tissue Histogenesis- Turnover rate- Cell replication- Cell differentiation- Cell motility- Cell apoptosis- Cell adhesion

Tissue histogenesis (embryogenesis)

Histogenesis in highly prolific tissues

Bone Marrow and Blood Cell Formation

Histogenesis in highly prolific tissues

The Villi in the Small Intestine

The Villi in the Small Intestine

Microvilli show electron dense plaques (open arrow) at their apices.

Histogenesis in highly prolific tissues

Skin

Tissue Formation

Tissue Repair

Organization of Tissues into Functional Subunits- 11 major organ systems- organs: functional subunits

(100 um)- tissue- cells (10 um)

Cellular Fate Processes

- Cell Differentiation- Cell Motion- Cell Replication- Interacting cellular fate processes determine overall tissue dynamics.

Cellular Communications

How Do Cells Communicate?- Cells secrete soluble signals (cyto-, and chemokines).- Cells touch each other and communicate via direct cell-cell contact.- Cells make proteins that alter the chemical microenvironment (ECM).

Soluble Growth Factors

Direct Cell-Cell Contact

Cell-Matrix Interactions

Tissue Microenvironment

Cellular Function in Vivo: Tissue Microenvironment and Communication withOther Organs

Cellularity- Cell density: typically 100-500 x 106 cells/cm3. Less than 1000 cells perfunctional unit- Cartilage ~ 1 cell/(100 um)3

Dynamics

Size and Geometry

Bone anatomy

Cellular Therapies- Cell differentiation- Cell division (mitosis, meiosis)- Cell migration (motility)- Cell communication- Cell death (apoptosis, necrosis)

Human Cells as Therapeutic Agents- Blood transfusion- Bone marrow transplantation (BMT)- Hematopoietic stem cell transplants- Allogeneic- Autologous- Xenogenic- Syngenetic- Reconstitution of tissues ex vivo- Tissue engineering

Tissue Engineering

Image from http://www.the-scientist.com/2006/9/1/35/1/

Tissue engineering timeline

E. S. Place et al., “Complexity in biomaterials for tissue engineering,”Nature Materials 8, 457 - 470 (2009)

Incidence of Organ and Tissue Deficiencies*

BoneJoint replacement 558,000Bone graft 275,000Internal fixation 480,000Facial reconstruction 30,000

CartilagePatella 319,400Meniscus 250,000Arthritis (Knee) 149,900Arthritis (Hip) 219,300Small Joints 179,000

Tendon 33,000Ligament 90,000

SkinBurns, Sores, 3,650,000Ulcers 1,100,000

Heart 754,000Blood Vessels 606,000Liver 205,000Pancreas 728,000

Blood transfusions 18,000,000Dental 10,000,000

* From Langer and Vacanti, 1993

In vitro Tissue Culture

Biodegradable Polymer Scaffold

CellsOsteoblastsChondrocytesHepatocytesEnterocytesUrothelial Cells

In Vivo Implantation

NewBoneCartilageLiverIntestineUreter

fibroblast nih3t3 - cancer cell line, image from http://mydaisydaze.blogspot.com

Allogeneic transplants: skin, cornea, heart, liver, kidney, bone marrow, bone- subject to immune rejection

Autologous transplants: bone marrow, cartilage, bone, blood- no immune rejection

Xenogenic transplants: porcine heart valves, pig liver (temporary), pig neuralcells (Parkinson’s disease)- shortage of organs for clinical (allogeneic) transplantation- 60% of patients awaiting replacement organs die on the waiting list.- problems: immune rejection, xenozoonosis (potential for infectious diseaseto spread from the donor animal, porcine endogenous retroviruses: PERVs),ethical issues (pigs, cows vs. primates), religious beliefs

Syngenetic: isograft between twins, in practice, usually covered withimmunosuppressants in case they are not 100% genetically identical

Tissue Engineering

Bone Marrow Transplantation (BMT)1

Red bone marrow is the site of all hematopoiesis in adults.

Recent evidence shows that one pluripotential hematopoitic stem cell in bone marrow produces all types of blood cells.• Hematopoitic stem cells divide at a slow rate to produce more pluripotential stem cells and differentiated cells.

• Differentiated cells produce different blood cell types.

Pluripotential stem cells first form committed myeloid stem cells and committed lymphoid stem cells.• Myeloid—produce separate progenitor cells for erythrocytes, eosinophils, basophils, megakaryocytes, and monocytes/neutrophils.

• Lymphoid—some seed thymus (T-lymphocytes), others stay in bone marrow (B-lymphocytes)—both then seed other lymphoid tissue.

Bone Marrow Transplantation (BMT)- BMT replaces diseased, non-functioning bone marrow with healthy functioning bone marrow (for

conditions such as leukemia, aplastic anemia, and sickle cell anemia).- Replaces the bone marrow and restore its normal function after high doses of chemotherapy or

radiation are given to treat a malignancy. This process is often called "rescue" (for diseases such as lymphoma, neuroblastoma, and breast cancer).

- Replaces bone marrow with genetically healthy functioning bone marrow to prevent further damage from a genetic disease process (such as Hurler's syndrome, and adrenoleukodystrophydisorder).

- Some of the diseases that have been treated with bone marrow transplant include the following: leukemia, lymphomas, some solid tumors (i.e., neuroblastoma, rhabdomyosarcoma, brain tumors), aplastic anemia, immune deficiencies (severe combined immunodeficiency disorder, Wiskott-Aldrich syndrome), sickle cell disease, thalassemia, Blackfan-Diamond anemia, metabolic/storage diseases (i.e., Hurler's syndrome, adrenoleukodystrophy disorder), cancers of the breast, ovaries, and kidneys

1

- Types of BMT• Allogeneic (leukemia)• Autologous (lymphoma, and breast and testicular cancer): the donor can be a patrent, identical twin, or an unrelated donor with the genetically matched marrow

• Umbilical cord blood transplant

Skin and Vascular Grafts-Skin grafts and transplants help reduce infection and fluid loss (which burn patients with extensive skin loss are especially susceptible to), but transplants carry several risks, including bleeding, infection, and rejection of the graft.

-Dermis (fibroblasts) and epidermis (keratinocytes)-Good ex vivo cultivation-Transplanted dermal fibroblasts nonimmunogenic

2

Trancyte, derived from human foreskins (Advanced Tissue Sciences, Inc. CA, USA)

Biobrane (Dow Hickam /Bertek Pharmaceuticals, TX, USA)

Dermagraft

http://www.hpnonline.com/inside/2008-03/0803-IC-WoundCare.html

1. 임시 피부 대체재(1) Biobrane (Dow Hickam/Bertek Pharmaceuticals, TX, USA)Biobrane은 얇은 실리콘 막이 붙어 있는 나이론 망사에 돼지의 폴리펩타이드를 코팅한 제품으로 2도 이하의 화상 (partial-thickness burn)에 효과적이다. 기존의 silver sulfadiazine에 비해 훨씬 짧은 드레싱 교환 주기와 진통제 사용량 감소 효과를 볼 수 있다.

(2) Transcyte (Advanced Tissue Sciences, Inc. CA, USA)Transcyte은 Biobrane보다 한 단계 발전된 제품으로 돼지의 콜라겐을 코팅한 나이론 망사에 newborn human fibroblast cell을 배양시켜서 이를 반투과성의 실리콘 막에 붙여 화상 부위에 적용하는 것이다. 여기서 핵심은 나이론 망사 안의 fibroblast가 성장하면서, 인간 피부 콜라겐, 기질 단백질 그리고 성장 인자를 분비하여 화상부위의 회복을 촉진하는 것이다. 사용되는 막이 반투과성이 있어서 액체와 가스의 교환이 가능하고 또한 이 제품의 물리적인 특성이 상당한 유연성이 있기 때문에 특히 얼굴 화상처럼 곡면이 심한경우 효과적이다. 2도 이하의 화상에 효과적이다.

2. 영구 피부 대체재(1) Integra (integra Life Science Corp., NJ, USA)Integra는 두 개의 막으로 이루어져 있는데, 화상 부위와 직접 접촉되는 부위는 chondroitin-6-sulfate와 상어의 연골조직에서 추출한 glycoaminoglycan과 교차 결합된 소의 type-I 콜라겐으로 만들어진 섬유의 다공성 기질로 이루어져 있다. 이 다공성 기질 구조는환자의 fibroblast, macrophage, lymphocyte와 모세혈관들이 침투할 수 있는 판형을 제공하게 된다. 그리고 외부 쪽으로는 실리콘막으로 이루어져 있어서 외부 자극으로부터 상처를 보호하게 된다. 일단 새 피부 조직이 형성이 되면, 실리콘 막을 제거되고 그 위에 얇은 자가 피부 이식이 실시된다. 3도 화상 (full-thickness burn)에 사용이 가능하고 자가 피부 이식이 곤란한 환자들에게 새로운치료의 장을 열었다는데 큰 의미가 있다.

(2) Apligraf (Organogenesis, Inc., MA, USA and Novartis Pharmaceuticals Corp., NJ, USA)Apligraf는 제조 과정이 좀 특이하다. 즉 fibroblast를 소의 type-I 콜라겐과 섞은 뒤, 가열을 통해 fibroblast 기질을 만든다. 일 주일정도 자연스럽게 수축이 되도록 한 뒤, 신생아의 포피세포에서 채취한 keratinocyte를 첨가해 상피층을 만든다. 다시 몇 일을 기다린후, 이 혼합물을 공기에 노출시켜 keratinocyte가 분화를 하여 각질층을 만들게 한다. 이 시점에서 Apligraf는 cytokine이나 성장 인자 같은 기질 구성물질들을 분비하는 능력을 갖게 된다. Apligraf는 실제 피부와 많은 부분이 유사하다는 장점이 있어서 자가 피부이식과 병행 사용시 많은 효과를 보기는 하는데, 문제는 위에 언급한 제조 방식에서 볼 수 있듯이, 늘 신선한 상태로만 제조되기 때문에, 상온에서 보관 가능한 기간이 불과 5일이다.

(3) Dermagraft (Advanced Tissue Sciences, Inc., CA USA)Dermagraft는 생체 내에서 흡수가 가능한 polyglactin 망사에 신생아의 포피 세포에서 분리한 fibroblast를 심어 넣은 제품이다. 이fibroblast가 증식하며 피부 콜라겐, 성장 인자, glycosaminoglycan 그리고 fibronectin등을 분비하게 된다. 동종 피부 이식 (allograft)와 비교할 때 감염 문제나, 회복 속도에서 환자들로부터 높은 만족도를 받고 있다.

Pancreas / β-Islet Cells Beta cells producing Insulin and

Amylin (65-80% of the islet cells) Alpha cells releasing Glucagon (15-

20%) Delta cells producing Somatostatin

(3-10%) PP cells containing Pancreatic

polypeptide (1%) Epsilon cells containing ghrelin

3 Pancreas / β-Islet Cells Loss of ability to produce and secrete insulin for diabetic pancreas Insulin secreting β–islet cells lose properties in culture. Immune rejection causes allogeneic transplantation from the cadaver only temporary. Physical separation with a semipermeable membrane

3

Cartilage and Chondrocytes

- cartilage tissue: avascular, alymphatic, aneural- very low density chondrocytes are dispersed.- joints, rib cage, ear, nose, intervertebral discs- autologous chondrocyte implantation (ACI): Carticel, Genzyme (FDA approved)

Chondrosphere, co.don AG (http://www.codon.de)

4Cartilage Tissue Engineering

BEFORE cell seeding

AFTER 2 weeks in culture

A scaffold of biodegradeable material was fashioned into the shape of a 3 year old’s ear. Cartilage from a cow was then placed in the scaffold and then was implanted under the skin, but above the muscle of a nude mouse. A nude mouse was used because they have no immune system that would reject the cow cells. The mouse started to grow very thin blood vessels that were able to supply the cartilage with nutrients and the cartilage started to grow with the mouse. By the time the scaffold was dissolved, there was enough cartilage there to maintain the structure of the outer ear.

It was never transplanted because, since it contained no human tissue, it would have been rejected by a human.

Mouse with a human ear?ca. 1995

Fundamental Questions- Clinically meaningful number of cells: 107 ~ 109

- Fundamental limitations•Hayflick limit: 30 ~ 50 doublings in culture with a primary cell•Noted in all organism cell types that have been fully differentiated•The only known way of circumventing the Hayflick limit is with the enzymetelomerase, which regenerates telomeres during DNA replication

•Exception to the Hayflick limit: stem cells, cancer cells

Rasnick, Auto-catalysed progression of aneuploidy explains the Hayflick limit of cultured cells, carcinogen-induced tumours in mice, and the age distribution of human cancer, Biochem. J. (2000) 348 (497–506)

Hayflick limit

Continuous epithelial turnover during ageing is thought to lead to telomere shortening. When coupled with somatic mutations inactivating retinoblastoma/INK4a/p53 checkpoints, the Hayflick limit (mortality stage 1 (M1) or replicative senescence) can be bypassed. Continuous proliferation beyond the Hayflick limit results in progressive telomere attrition and subsequent fusion–bridge–breakage cycles in cells with dysfunctional telomeres. This process culminates in aneuploidy and complex non-reciprocal translocations (NTRs), resulting in massive and rapid changes in gene dosage observed in early carcinogenesis. Telomerase reactivation in the carcinoma-in-situ stage leads to relative chromosomal stability, providing a genome in which additional gene-specific mutations arise that are critical for progression to invasive and metastatic stages.

DePinho, “The age of cancer,” Nature 408, 248-254 (2000)

Tissue Engineering Challenges

- Proper reconstitution of the microenvironment for the development of tissue function

- Scale-up to generate enough properly functioning microenvironments to be clinically meaningful

- Automation of the operation of a system to a clinically meaningful scale

- Implementation of the automated device in a clinical setting with all the cell-handling and preservation procedures that are required for the implementation of cellular therapies

Transplants made to order(http://www.the-scientist.com/2006/9/1/35/1/)

At the centimeter scale, tissue engineering should yield a mechanically stable construct of clinically relevant thickness, comparable to the thickness of the human myocardium (from millimeters to a centimeter). This requirement is hampered by the diffusional limitations of oxygen supply encountered in most tissue-culture vessels, coupled with the high metabolic demand of cardiomyocytes for oxygen.

At the millimeter scale, the tissue should consist of elongated myofibers aligned in parallel and capable of synchronous contractions, a requirement that is hampered by the lack of appropriate electromechanical stimulation in conventional cultures.

At the micrometer scale, cells in the engineered cardiac tissue must be coupled by functional gap junctions and be capable of electrical impulse propagation in order to prevent arrhythmia upon implantation.

At the nanometer scale, functional excitation-contraction machinery of individual cardiomyocytes needs to be established.

Tissue engineering in space

Freed et al., “Tissue engineering of cartilage in space,” PNAS 1997

Cartilage cells were seeded onto polymer scaffolds, and the resulting constructs were cultivated in rotatingbioreactors first for 3 months on Earth and then for 4 additional months either on the Mir Space Station (10-4–10-6 g) or on Earth (1 g).

Tissue engineering in space

Freed et al., “Tissue engineering of cartilage in space,” PNAS 1997

The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth.

Vocabulary

ApoptosisCellular therapiesChondrocytesDifferentiationExtracellular matrixFlow cytometryFunctional subunitsGraft vs. Host Disease (GVHD)HematopoiesisMesenchymal cellsMitosisSelf-renewalStem cellsAllogeneic, autologous, syngenetic, xenogeneic