Stem Cells and Regenerative Medicine

Geoffrey C. Gurtner, MD, FACSStanford University School of Medicine

Lucile Salter Packard Children’s Hospital

Human Response to InjuryHuman Response to Injury

Regeneration

Injury

Repair with Scar

Human Response to Injury Human Response to Injury

Injury

Regeneration

Repair with Scar

Regenerative Medicine

Is Regeneration Possible?Insights from Developmental Biology

Regenerative MedicineRegenerative Medicine

STEM CELLS

TISSUE ENGINEERING

REGENERATIVE MEDICINE

Nanosciences/devices

Developmental biology

Material Science

Bioengineering

Genomics

What is a Stem Cell?What is a Stem Cell?

1. Cells that can be grown indefinitely (self renewal)

2. Can be differentiated into different specialized cells

Stem vs. Progenitor CellStem vs. Progenitor Cell1. Stem Cell

• Asymmetric Division to produce itself (self renewal)

• Differentiate into specialized daughter cells

2. Progenitor Cell• Cannot self renew• Differentiate into

specialized daughter cells

http://www.nih.gov/news/stemcell/scireport.htm

Stem Cells: From Embryo or AdultStem Cells: From Embryo or AdultTotipotent: stem cells capable of giving rise to an intact organism

Pluripotent: stem cells capable of giving rise to cells of all (3) germ layers

Multipotent: stem cells capable of giving rise to cells that comprise a germ layer or organ

http://www.nih.gov/news/stemcell/scireport.htm

Different Stem Cell Types

? Immunogenic, pluripotent

Pluripotent

Immunogenic, ? Pluripotent vs

multipotent

Immunogenic,multipotent

Embryonic Stem Cells

Embryonic Stem CellsEmbryonic Stem Cells

•Derived from inner cell mass of blastocyst

•Pluripotent

•Can differentiate into all adult cell types

Human Embryonic Stem CellsHuman Embryonic Stem Cells

• Human ESCs can differentiate into myocardium

• Human ESCs can produce insulin secreting endocrine cells

Eur J Cardiothorac Surg. 2006 Jan;29(1):50-5

Nat Biotechnol. 2006 Oct 19

Human Embryonic Stem CellsHuman Embryonic Stem Cells• 22 NIH-registered human embryonic stem cells

available• No current human trials with embryonic stem

cells

• Primary current enthusiasm is to use as disease models for diseases such as ALS

Blastocyst Trophoblast cells removed

Inner Cell Mass Human ESCs

HF Chen et al. Hum Reprod. 2006 Oct 27

California Institute for Regenerative Medicine (CIRM)

• Proposition 71 established "California Institute for Regenerative Medicine" to regulate stem cell research and provide funding through grants and loans.

• Established constitutional right to conduct stem cell research; prohibited funding of human reproductive cloning research.

• Authorized issuance of general obligation bonds to finance Institute activities up to $3 billion subject to annual limit of $350 million.

Problems with Embryonic Stem CellsProblems with Embryonic Stem Cells

• Cells are immunogenic

• Difficult to control and differentiate in vivo, possible tumorgenicity

• NIH approved lines are maintained on murine feeder cells for necessary ex vivo expansion

• Composition of growth media, factors, conditions for culture unclear

• Ethical and religious

““EmbryonicEmbryonic--likelike”” Stem CellsStem Cells

• Amniotic sac derived stem cells contain oct-4 and nanog like ES cells

• Cells not immortal, but can be differentiated (Stemnion, Inc.)

• Similar claims made for placenta derived mesenchymal cells (Stemcell Pharma, Inc.) and cord blood

• Unknown immunogenicity

Embryonic Stem Cells

• We know all the genes that are upregulated in embryonic stem cells

• What happens if we turn on these genes in normal (non-stem cells) using gene therapy?

• This approach is called epigenetic reprogramming of somatic cells

InducedInduced Pluripotent Stem (iPS) Pluripotent Stem (iPS) CellsCells

• 22 stem cell genes added to skin fibroblasts using retroviruses

• Results in embryonic stem cell-like cells

• Teratomas are formed in vivo

• In vitro differentiation into all 3 germ layers

Takahashi and Yamanaka. Cell 126, 2006.

Confirmation and RefinementTransduction with only four genes: Oct3/4, Klf4, Sox2, c-Myc (Okita, et al. Nature 448(19), 2007) or…Activation of endogenous Oct4 or Nanoggene (Wernig, et al. Nature 448(19), 2007)Results in iPS cells whose DNA methylation pattern, gene expression, and chromatin state are identical to ES cells

.

Further Characterization of iPS CellsFurther Characterization of iPS Cells

• Proliferation similar to ES cells, with slightly longer doubling times

• Transplantation into nude mice resulted in teratomas with all three germ layers

Okita, et al. Nature 448(19), 2007.

Reprogrammed Fibroblasts Yield Viable Chimeras

• Can function as pluripotent stem cells

• GFP labeling shows high contribution to pup development

Wernig, et al. Nature 448(19), 2007.

The Promise• Reprogrammed iPS cells are nearly

indistinguishable from ES cells

• iPS cells develop into a wide variety of tissues, just like embryonic stem cells

• iPS cells contribute to the germ line and yield viable transgenic animals

• Can normal fibroblasts transduced with a handful of genes replace embryonic stem cells?

The Reality

Okita, et al., report that 20% of iPS cell-derived mice developed cancer, probably from Myc activation

Retroviral vectors have previously caused leukemia in human gene therapy trials

Thus clinical application is a ways off, but at least we are beginning to understand “stem-ness”

Adult Bone Marrow Stem CellsAdult Bone Marrow Stem Cells

Adult Bone Marrow Derived Adult Bone Marrow Derived Stem CellsStem Cells

• Hematopoietic Stem Cells (HSCs)

• Bone Marrow Derived Mesenchymal Stem Cells (BM-MSCs)

• Endothelial Progenitor Cells (EPCs)

• Cord Blood Derived Stem Cells

Hematopoietic stem cellsHematopoietic stem cells

1. Portion of the bone marrow that forms RBCs, WBCs, etc.

2. Unlike other putative stem cells, discrete markers have been identified (SCA1+, lin-, and c-Kit+, Flk2+/Thy1.1lo ) allowing a pure population of these cells to be isolated and studied

3. True stem cells, able to self-renew and differentiate

Reya T, Morrison SJ, Clarke MF, Weissman IL., Nature 2001;414:105-111

Established Clinical ApplicationsEstablished Clinical Applications

• 40 year track record of success• HSC harvested from bone marrow or

cytokine mobilized peripheral blood• Treatment of leukemia, blood

dyscrasias, mucopolysaccharidoses and cancer

Other Proposed Applications of Hematopoietic Stem Cells

• HSCs have been purported to differentiate into myocardium, endothelium, small intestine, skeletal muscle, lung, and liver

• Most of these reports are now being attributed to cell fusion• Cell fusion is being explored for therapeutic purposes

PNAS 2006;103;13156-13161

Nature Medicine 2004: 10, 744 - 748

HSC to endothelial cell

HSC to hepatocyte

PNAS 2006;103;6321-6325

HSC to intestinal epithelium

Bone Marrow Derived Mesenchymal Stem Cells

• No widely accepted Markers

• Exist within the lin-, CD45-, Sca-1+ fraction of the bone marrow

• Ability to mobilize from bone marrow and home to injured tissue remains controversial

http://www.nih.gov/news/stemcell/scireport.htm

Characterization of Bone MarrowCharacterization of Bone Marrow

HSC:Lin-Sca+Ckit+CD34+ (human)CD45+Thy1.1+

Shizuru, et al, Ann Rev Med, 2005

MSC:Lin-CD14-CD45-CD29+CD90+CD95+CD166+

Wang, et al, Stem Cells, 2005

Bone Marrow Mesenchymal Stem CellTherapy of Myocardial InfarctionTherapy of Myocardial Infarction

• BM MSC injected intra-arterially increase vasculogenesis in mycardium

• BM MSC increase perfusion and viability of myocardium

Nature Medicine. 2001 Apr;7(4):430-6

Circulation. 2002 Oct 8;106(15):1913-8

““SideSide”” Population Marrow Cells Population Marrow Cells for Wound Healingfor Wound Healing

• Topical application accelerated wound healing in diabetic mice

• Side population defined as c-kit+, lin-, sca-1+, CD 34-

• Orgill et al, PRS, in press

Cord Blood Stem CellsCord Blood Stem Cells

• Obtained from blood retrieved from the umbilical cord and placenta at the time of birth

• Isolated by Fluorescence Activated Cell Sorting (FACS)

• Infused via an IV and find their way to the bone marrow, “stem cell homing”

Established Clinical Applications Cord Blood Stem Cells

Autologous Transplant• To date over 6,000 cord

blood transplants have been performed

• In the US ½ of all pediatric marrow transplants from unrelated donors come from cord blood

• In Japan, this is true for adults as well

Dana-Farber Cancer Institute

Allogenic Transplant

Johns Hopkins

Other Proposed Uses of Umbilical Cord Stem Cells

Medical Hypothesis 2006

Circulation 114 (Suppl.1) 125-131, 2006

“Overall, the results of these studies of a combined total of 376 patients do not promote the use of intracoronary infusions of autologous bone marrow to improve ventricular function”

NEJM September 21,2006

The only established clinical The only established clinical application for hematopoietic application for hematopoietic stem cells or cord blood stem stem cells or cord blood stem

cells is in the treatment of blood cells is in the treatment of blood disorders and cancerand cancer

Adult Tissue Derived Stem CellsAdult Tissue Derived Stem Cells

Adult Tissue Specific Stem CellsEctoderm

epithelial stem cellEndoderm

intestinal crypt cell

Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature 414, 98-104, 2001

Adult Tissue Specific Stem CellsAdult Tissue Specific Stem Cells

• Neural Stem Cells

• Adipose Derived Mesenchymal Stem Cells (AdipoMSCs)

• Skin Stem Cells

Neural Stem CellsNeural Stem Cells• Subventricular zone along lateral ventricles of

human brain have greatest density of NSCs

Sanai N. N England J Med. 2005 Aug 25;353(8):811-22

Neural Stem CellsNeural Stem Cells

• Progenitor cells that can differentiate to neurons or glial cells

• Neural stem cells have also been implicated in development of gliomas

• Bone morphogenetic proteins (BMPs), EGF and FGF-2 implicated in neural stem cell differentiation

Sanai N. N England J Med. 2005 Aug 25;353(8):811-22

NeurogenesisNeurogenesis• Progenitor neural stem cells can potentially

mature into neurons and support axonal growth• Potential application in neurodenerative

disorders ie. Alzheimer’s

Steindler DA. Glia. 2006 Dec;54(8):815-25

Proposed Clinical Application: Proposed Clinical Application: Spinal Cord InjurySpinal Cord Injury

•The adult central nervous system has a limited capacity to regenerate after injury

•Neurogenesis occurs at sites of injury but is very inefficient

•No human trials yet due to concerns regarding source and safety of neural stem cell transplants Journal of Neuroscience21(23):9334-9344, 2001.

Adipose Derived Mesenchymal Stem Cells (AdipoMSCs)

Suction-assisted lipectomy(elective cosmetic surgery patients)

Raw lipoaspirate

Extensive washingproteolytic digestion

(collagenase)

Collection ofcells = PLA

In Vitro Differentiation of AdipoMSCs

OsteogenicMyogenic

AdipogenicNeurogenicChondrogenic

Proposed Clinical Application:Proposed Clinical Application:Bone RegenerationBone Regeneration

Bioengineered Scaffolds

Regeneration

AdipoMSCs

AdipoMSCs Form Bone in Modified Standard Media

Von Kossa

at 28 days

Alkaline Phosphatase

at 7 days

Control Media

ODM

D E F

A B C

ODM + RA + BMP-2

Nature Biotechnology May, 2004

Case ReportCase ReportAdipose derived stem cells harvested and injected into calvarial defect (Lendeckel S et al. Journal of Cranio-

Maxillofacial Surgery 2004: 32, 370–373.)

Pre Operative

Post Operative

Proposed Clinical Application for Proposed Clinical Application for AdipoMSCs: Soft Tissue AugmentationAdipoMSCs: Soft Tissue Augmentation

• Historical basis in fat grafting which has been performed in plastic surgery for over a hundred years

• Enormous interest in identifying the ideal soft tissue “filler” for cosmetic and reconstructive applications

• Ideal filler would be integrated, vascularized and non-immunogenic

QuestionsQuestions1. What are we injecting?

Adipocytes, Fibroblasts, Smooth Muscle Cells, Perivascular Cells, Endothelial Cells, Stem Cells

2. What happens after we inject it?

3. What impact does this have on tumorigenesis in the breast?

?

Adipose Tissue Contains Multipotent Cells

• Limiting Dilution Assay and Cloning Rings used to isolate 500 cell clones

• 7 of 500 clones (1.4%) demonstrated in vitrodifferentiation along 3 mesodermal lineages• Osteogenic• Adipogenic• Chondrogenic

PBS WashCollagenase Digest

Culture ofAdherent Cells

ADIPOGENESIS OSTEOGENESIS CHONDROGENESIS

Alcian Blue StainOil Red O Stain Alizarin Red Stain

Different Processing:Different Processing:LABORATORY ISOLATION: 1. Collagenase digestion2. Red cell lysis3. Centrifugation4. Plating and Culture

1. CLINICAL ISOLATION: 2. Lipoaspirate undergoes

centrifugation 3. Separate into oil, adipose,

and red cell layers

Fat grafting performed by current techniques is not predominantly stem

cell transfer

Survival of Implanted CellsSurvival of Implanted CellsAngiogenesis• Diffusion is unable to sustain

cells at a distance of greater then 2mm from a blood vessel

• Recent advances in fat grafting have probably increased cell viability

• Still unclear what the optimal injection volume per pass is to maximize cell viability

• Any non viable cells can result in calcification

Commercialization of Adipose “Stem Cell Isolation” for Fat Transfer

The Celution™ System makes cells available in about one hour and these cells should be enriched for MSCs

Duality of Stem CellsDuality of Stem Cells

Tissue Regeneration Tumorigenesis andCancer Stem Cells

In Animal Models, Mesenchymal Stem Cells Increase Breast Cancer Metastases

The authors show that human breast cancer cell lines become metastatic when in physical contact with mouse mesenchymal stem cells.

Using subcutaneous xenografts of human and mouse cells combined, Karnoub et al. show increased metastases to the lung while control grafts having human or mouse cells alone did not result in metastasis.

Even without stem cells, tissue damage to leads Even without stem cells, tissue damage to leads to the release of tumorigenic factorsto the release of tumorigenic factors

• Published techniques delivering 0.2 to 0.02 cc per “pass” require up to 13,900 “passes” to deliver the average volume (278cc) over an 4-6 hour period.

• Fat grafting is traumatic and results in the deposition of a disorganized mixture with mechanical damage, inflammation, and necrosis.

• Fibroblasts activated by surgical trauma and infiltrating immune cells secrete cytokines that have been shown to potentiate tumor growth including TGF-β, FGF-2 , and PDGF (Sieweke, Science, 1990; Blobe, N. Engl. J.Med. 2000; Skobe, Proc. Natl Acad. Sci. 1998).

• Specific to breast tissue, Lochter (J. Cell Biol. 2000) demonstrate inflammation specific activation of matrix proteases are involved in breast cancer progression.

AdipoMSCs for Soft Tissue AdipoMSCs for Soft Tissue AugmentationAugmentation

Plastic and Reconstructive Surgery, Supplement 4, 2006

Other Proposed Applications of Other Proposed Applications of AdipoMSCsAdipoMSCs

• To increase vascularity in cardiovascular and wound healing applications

• As substitutes for neural progenitor cells • Advantages are ease of harvest and

excellent yields

Skin Stem Cells and Skin Stem Cells and Wound HealingWound Healing

*modified from Singer & Clark, NEJM, 1997

Endogenous Sources of Stem/Progenitor Cells During Wound Healing

Blood Borne,(EPC, HSC)

Not a new ideaNot a new idea……

Epidermal Multipotent Stem CellsEpidermal Multipotent Stem CellsMultipotent epidermal stem cells reside in the bulge (Bu) zone of hair follicle (HF)

Blanpain C, Lowry WE, Geoghegan A, Polak L, Fuchs E. Cell, Vol. 118, 635–648, September 3, 2004,

Multipotent epidermal stem cells Multipotent epidermal stem cells can differentiate into:can differentiate into:

1. epidermis2. sebaceous gland 3. hair follicles

Transplanted Bulge Hair Regeneration

Blanpain C, Lowry WE, Geoghegan A, Polak L, Fuchs E. Cell, Vol. 118, 635–648, September 3, 2004,

Sebaceous Gland Sebaceous Gland RegenerationRegeneration

• Blimp1+ cells responsible for sebaceous gland regeneration and controls the size, activity, and number of sebocytes Blimp1+

SG

Fuchs E et al. Cell. 2006 Aug 11;126(3):597-609

Epidermal Stem Cells and Epidermal Stem Cells and Wound HealingWound Healing

• Bulge epidermal stem cells migrate to healing wounds and are involved in re-epithelialization after injury

• Are eventually eliminated as wound heals

Cotsarelis G et al. Nature Medicine. 2005 Dec;11(12):1351-4

Hair Follicle Regeneration During Wound Healing

Ito, et al. Nature 447(17), 2007.

• De novo hair regeneration occurs after wound closure in mice

• Histology shows that regenerating hair follicles mimic stages of normal embryonic hair development

Regenerated Hair Originates from Interfollicular Stem Cells

Genetic lineage analysis using transgenic mice:

1) Tg(Krtl-15-cre/PGR)22Cot;R26R LacZ + only in bulge cells (arrow)

Only TRANSIENT presence of bulge cells in regenerated hair follicles

2) Krtl-15-CrePR*LacZ + in 70% bulge and 50% non-bulge epidermal cells (red arrowhead)

Hair follicles are chimeric, indicating multiple progenitor cells are involved

Hair Follicle Development Requires Wnt Protein

KRT17

• Regenerated follicles parallel embryonic follicle development at molecular level:

• KRT17, alkaline phosphatase, Lef1, Wnt10b, Shh Alkaline

phosphatase

• Secreted DKK1, a Wnt inhibitor, blocks follicular neogenesis

• Overexpression of secreted Wnt7aresults in significant follicular neogenesis

The Bottom Line• New hair follicles can be formed following

wound formation in mice.

• Wnt signaling plays an important role in hair follicle neogenesis.

• It may be possible to regrow hair follicles in humans using local trauma (dermabrasion) and wnt signaling.

Skin Regeneration Using Autologous Stem Cells

Nature 414, 98-104, 2001

Unanswered Questions and Unanswered Questions and Obstacles Obstacles

Do stem cells incorporate into Do stem cells incorporate into tissues or do they function as tissues or do they function as cytokine/growth factor delivery cytokine/growth factor delivery

vehicles?vehicles?

The answer to this question is critical The answer to this question is critical for the problem of immunogenicityfor the problem of immunogenicity

1. With investigation, most stem cells and their progeny have been found to be immunogenic

2. Thus will need HLA matching and/or immunosuppression if they are permanent cell replacement

3. However if they are not permanent this will be less of an obstacle

www.osiris.com

Therapeutic Cloning to Avoid Therapeutic Cloning to Avoid ImmunogenicityImmunogenicity

http://stemcells.nih.gov/

Secondary Questions

1.1. If these are functioning as cytokine/growth If these are functioning as cytokine/growth factor delivery systems, is there an factor delivery systems, is there an advantage for stem cell based therapy over advantage for stem cell based therapy over differentiated cell based therapy?differentiated cell based therapy?

2. If they are incorporating, hohow can we control the differentiation of cells in vivo to produce a physiologic effectwhile preventing tumor creation?

How can we isolate and purify rare stem cell subsets in the absence of discrete markers?

In the case of EPCs what we know so far is:– Resident bone marrow cells– Respond to ischemic stimuli– Mobilize into the circulation– Differentiate into mature

endothelial cells in new blood vessels

What is an What is an ““EPCEPC””??1. Markers and

precise identification currently unclear

2. Nomenclature and identification in the literature inconsistent

3. Are circulating cells identical bone marrow resident cells?

4. Are one or many different cells involved?

Authors Marker Profile Origin Species

Asahara et al. Science (1997) CD34+/ KDR (VEGF-R2) Blood Mouse

Kalka and Asahara PNAS (2000) CD31/VE-Cadherin Blood MouseKDR/LDL-R/UAE-1 R

Kawamoto et al. Circ. (2001) CD31/UAE-1 binding Blood Human

Walter et al. Circ. (2002) Tie2? BM Mouse, RatHuman

Takahashi et al. Nat Med. (1999) CD31/Tie2/Sca1 BM Mouse

Dimmler et al. Circ. Res. (2001) CD34/KDR Blood Human

Rauscher et al. Circ. (2003) CD31/CD-45_ BM Mouse

Asahara and Isner Stem Cells (2004) Flk-1/AC133/CD34 BM? HumanTie2/c-Kit/Sca-1

Tie2/VE-cadherin BM MouseCD34/CD31

Chan and Verma Transl. Physiol. (2005) CD34/VE-cadherin Blood Human

Gurtner et al. Nat Med. (2004) CD34+/Flk1+/CD133+ Blood HumanCD11b-

Blood (2005) CD34+/Flk1+/sca-1+ Blood, BM Mouselin-/CD11b-

Embryonic Vasculogenesis

What is the What is the ““EPCEPC”” Lineage?Lineage?

Hemangioblast

HSC

EPC

Hemangioblast

HSC EPC

Hemangioblast

HSC EPC

Regulatory Issues: FDA Guidelines Regulatory Issues: FDA Guidelines for Stem Cell Therapyfor Stem Cell Therapy

N Engl J Med. 2006 Oct 19;355(16):1730-5

Can stem cells overcome a Can stem cells overcome a hostile environment to produce a hostile environment to produce a

physiologic effect? physiologic effect?

Stem Cell Mediated Regeneration: Importance of Seed and Soil

Seeds

Soil Growth

Seed and Soil in Stem Cell BiologySeed and Soil in Stem Cell Biology

Soil

Seed-stem cell-resident cell

Soil

Regenerative MedicineRegenerative Medicine

STEM CELLS

TISSUE ENGINEERING

REGENERATIVE MEDICINE

Nanosciences/devices

Developmental biology

Material Science

Bioengineering

Genomics

Tissue Engineered BladderTissue Engineered Bladder

Lancet 2006Biodegradable matrix (PLGA)

seeded with bladder smooth muscle cells from biopsy

Integration and Colonization by Integration and Colonization by Native UrotheliumNative Urothelium

Dermal SubstitutesDermal Substitutes

•Alloderm, MTF, Dermamatrix

•Acellular dermal matrix from cadavers

•Will also revascularize

Breast Contour AbnormalitiesBreast Contour Abnormalities

Implant Positioning and Implant Positioning and CoverageCoverage

Less inflammatory response in Less inflammatory response in implant apsulesimplant apsules

No Matrix Matrix

Prefabrication of Synthetic Matrix Prefabrication of Synthetic Matrix with a Vascular Pediclewith a Vascular Pedicle

Neumeister, PRS, 2007

ConclusionsConclusions

• Regenerative Medicine and Tissue Engineering are already here and will play an increasing role in clinical practice

• New products will combine regenerative cells and the proper environmental cues for maximal effect

• New advances in developmental biology may make personalized stem cells a reality in the next 5 years