MUSCULOSKELETAL CENTER OFMUSCULOSKELETAL CENTER OF EXCELLENCE

STEM CELLS: OPPORTUNITIES AND STEM CELLS: OPPORTUNITIES AND CHALLENGESCHALLENGES

• Center for Stem Cell Biology & Regenerative Medicine• UMass Memorial Musculoskeletal Center of ExcellenceUMass Memorial Musculoskeletal Center of Excellence

STEM CELLS: OPPORTUNITIES AND CHALLENGES

• Biology of stem cells and applications for treatingBiology of stem cells and applications for treatinghuman disease

• UMASS initiatives in developing stem ll b d thcell-based therapy

STEM CELLS: OPPORTUNITIES AND CHALLENGES (Cont’d)

• Biology of stem cells and applications for treatingBiology of stem cells and applications for treatinghuman disease

• UMASS initiatives in developing stem cell-based ththerapy

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

• What is a Stem Cell?

• What are different types of stem cells?• What are different types of stem cells?

• What is the promise of stem cell research?

S C ll Wh h ?Stem Cells: What are they?

D l f H E bDevelopment of a Human Embryo

Totipotent Pluripotent Lineage Commitment

Single-cellembryo

3-dayembryo

5-7dayembryo

4-weekembryo

6-weekembryoembryo embryo embryo embryo embryo

Isolation of Embryonic Stem Cells

Properties of Embryonic Stem CellsProperties of Embryonic Stem Cells

Isolated from an early stage embryo

Exhibit normal karyotypean early stage embryo

Express Markers Pluripotent

Th P i f S C ll R hThe Promise of Stem Cell Research

Why Stem CellsWhy Stem Cells…

Ti li f S C ll R hTimeline of Stem Cell Research

1956First successful bone marrow transplant

1981Embryonic

1998Human embryonic stem cells isolated at Univ. Wisconsin

Embryonicstem cells are isolated from

blmouse blastocysts

2007iPS cells first developed

T f S C llTypes of Stem Cells

• Adult Stem Cells

• Embryonic Stem Cells (hESC)

• Somatic Cell Nuclear Transfer (SCNT) Stem Cells

• Induced Pluripotent Stem (iPS) Cells

Ad l S C llAdult Stem Cells

Where have adult stem cells been found?

Ad l S C ll ( ’d)Adult Stem Cells (cont’d)

Derivation Method Isolation from adult tissues.

AdvantagesSuccessful treatments demonstrated.

Stem cells can be matched to patient

Limitations

Cells not found in all tissues.

Produce a limited number of cell types.

Difficult to identify, isolate and grow.

Current UsesResearch.

Current UsesSome in clinical use (e.g. bone marrow)

Ethical Concerns None to date.

Lineage Committed Stem Cells S B F iSupport Bone Formation

(Development and Tissue Regeneration)

OCHistoneFibronectinCollagenc-Fos/c-JunTGFβ1

APBSPCollagenFra2/JunB

OCOPCollagenase

BAXp53

ProliferationMatrix

Maturation Mineralization

Osteopontin p53c-FosMsx2

Self RenewalLineage

Commitment

BMPTGFβ

BMPTGFβ

Vitamin DTGFβ

GlucocorticoidVitamin D

BMP

Stem Cell Osteoproginator Pre-Osteoblast Osteocyte

E b i S C llEmbryonic Stem Cells

Human Embryonic Stem Cells (hES Cells)

H ES ll l

Human Embryonic Stem Cells (hES Cells)

Human ES cell colony on mouse fibroblast

feedersHuman Blastocyst

feeders

Inner Cell Mass (ICM)

hES C ll ( t’d)hES Cells (cont’d)

Derivation MethodRemoval of cells from Inner Cell Mass (ICM) of blastocyst embryo from IVF.

Differentiate into all somatic cell typesAdvantages

Differentiate into all somatic cell types.

Excess of IVF embryos exist.

Limited number of lines available for federally funded

Limitationsresearch.

Risk of tumors (teratomas) from transplanting undifferentiated cells

Current Uses

Research.

Currently no clinical trials for therapeutic use.

In development for drug screening and toxicity testing

Ethical ConcernsDestruction of embryo.

Donor consent requiredDonor consent required.

D i ti f i d d Pl i t t St C llDerivation of induced Pluripotent Stem Cells

Induced Pluripotent Stem CellsInduced Pluripotent Stem Cells

Reprogramming & Correcting Genetic Defect

mm

ing

rogr

amRe

pr

iPS CellsiPS Cells

Derivation MethodReprogramming of somatic cells by introduction of specific regulatory factor genes.p g y g

Advantages“ES cell – like” characteristics.

Stem cells can be matched to patientStem cells can be matched to patient.

Unknown if cells can differentiate into all cell types.

Risk of tumors (teratomas) from transplantingLimitations Risk of tumors (teratomas) from transplanting undifferentiated cells and from expression of introduced genes.

Current Uses Research.

Ethical Concerns None to date.

Comparative Properties of Stem CellsComparative Properties of Stem Cells

• Embryonic Stem Cells: Indefinite growth; Potential to differentiate into any cell typeto differentiate into any cell type

• Lineage-Specific Stem Cells: Limited growth;• Lineage-Specific Stem Cells: Limited growth; Potential to differentiate into defined cell types

• Cancer Stem Cells: Indefinite growth; Cannot differentiate into a defined cell typedifferentiate into a defined cell type

Why so many types of stem cells?

hi l C C ll/ i il bili

Because none are perfect.

Ethical Concerns

Immune Rejection Concerns

CancerConcerns

Cell/Tissue Types

Availability

hESC Yes Yes Yes All Many lines

SCNT ES cells Yes Yes Yes All? No

Adult Stem Cells No No No Limited

Many(limited types)

iPS Cells No No Yes All? Many

UMASS Initiatives in Developing Stem Cell-Based Therapy

A Collaboration of:

• UMASS Center for Stem Cell Biology & Regenerative MedicineRegenerative Medicine

• UMASS Memorial Musculoskeletal• UMASS Memorial Musculoskeletal Center for Excellence

UMASS Contributions to two Rate-LimitinggParameters of Stem Cell Control

• Mechanisms that regulate rapid and infinite• Mechanisms that regulate rapid and infinite proliferation of stem cells

• Mechanisms that establish and maintain lineage commitment following differentiationcommitment following differentiation

Genetic and Epigenetic RegulationGenetic and Epigenetic Regulation

• Genetic Control: DNA-encoded Regulatory Information.

Epigenetic Control: Regulatory Information for cell structure, function and specificity NOT encoded in DNA that IS transmitted to progeny cells during cell division.

Genetic and Epigenetic RegulationGenetic and Epigenetic Regulation

• Genetic Control

Genetic and Epigenetic RegulationGenetic and Epigenetic Regulation

• Epigenetic Control Histone CodeHistone Code

Genetic and Epigenetic RegulationGenetic and Epigenetic Regulation

• Epigenetic Control Regulatory Factor “Architectural Signature” Regulatory Factor Architectural Signature

Transcriptional Suppression

HDACHDAC YAPYAP

Basal Basal –– Suppressed (Differentiated)Suppressed (Differentiated)

HLHHLH** RUNXRUNX C/EBPC/EBP

APAP 11 DLXDLXAcAcAcAcRUNXRUNXAPAP--11

HDACHDAC YAPYAPYAPYAPRUNXRUNX

mRNAmRNASite ASite A Site BSite B Site CSite C

OCOC--BoxBoxTATATATA

C/EBPC/EBPEE--BoxBox

VDREVDRE

RUNXRUNXAPAP--11 DLXDLXRUNXRUNXAPAP 11RUNXRUNX

Maximal Expression

H ATH AT H LHH LH TFIIBTFIIB TAF’STAF’SV DR/RX RV DR/RX R HATHATAcAcA cA cAA

V itam in D V itam in D -- Enhanced (D ifferentiated)Enhanced (D ifferentiated)

A cA cAcAc

p

H ATH AT H LHH LH**

TFIIBTFIIBR N A R N A PolPol IIII

TAF S

TFIIDTFIIDRU NXRU NXm R N Am R N A

S ite AS ite A S ite BS ite B S ite CS ite CO CO C --Bo xBo x

TATATATAC /EBPC /EBP

EE --BoxBoxVD REVD RE

RU NXRU NX C /EBPC /EBPA PA P --11 D LXD LXRU NXRU NX

A PA P --11AcAcAcAcA cA cA cA c A cA c A cA c

M h i th t t l id d i fi it• Mechanisms that control rapid and infinite proliferation of stem cells

• Mechanisms that establish and maintain lineage it t f ll i diff ti ticommitment following differentiation

The Mammalian Cell CycleThe Mammalian Cell Cycle

An Abbreviated Cell Cycle in Human yEmbryonic Stem Cells

Dynamics of Combinatorial Organizationy gand Assembly of Regulatory Machinery in

Nuclear Microenvironments

RNA P l II M di t d R l t M h i f C ll C l (Hi t ) GRNA Pol-II Mediated Regulatory Mechanisms for Cell Cycle (Histone) Gene

Expression:

G1 G1/S S G2

• Mechanisms that control rapid and infinite proliferation of stem cells

• Mechanisms that establish and maintain lineage commitment following differentiation

Lineage Specific Retention of RegulatoryLineage-Specific Retention of RegulatoryMachinery for Cell Fate Determination

During MitosisDuring Mitosis

• Post-mitotic gene expression requires restoration of nuclear organization and assembly of regulatorynuclear organization and assembly of regulatory complexes.

• Two models: Degradation re-synthesis andDegradation, re synthesis, and

assembly/organization Retention and partitioningRetention and partitioning

Runx2: A Transcription FactorInvolved in Lineage Commitment and

Required for Osteogenesis in vivo

PluripotentPluripotentMesenchymalMesenchymal CellCell

Tendon CellsTendon CellsFibroblastsFibroblasts

??

PPARPPARγγ

MyoDMyoDMyotubesMyotubes

Runx2Runx2?

Runx2Runx2

Mature OsteoblastsMature OsteoblastsHypertrophicHypertrophic

ImmatureImmatureChondrocytesChondrocytes

AdipocytesAdipocytes

PPARPPARγγ

Runx2Runx2

Mature OsteoblastsMature OsteoblastsHypertrophicHypertrophicChondrocytesChondrocytes

Matrix ProteinsMatrix ProteinsMatrix ProteinsMatrix Proteins

Contributions of Runx Factors to Genetic and Epigenetic Control: Scaffolds for Spatial Organization of Promotor Regulatory Complexes (P/DNA and P/P)

Basal Basal –– Suppressed (Differentiated)Suppressed (Differentiated)Transcriptional Suppression

XHDACHDAC YAPYAP

pp ( )pp ( )

HLHHLH** RUNXRUNX C/EBPC/EBP

AP 1 DLXDLXAcAcAcAcRUNXRUNXAPAP 11

HDACHDAC YAPYAPYAPYAPRUNXRUNX

Transcriptional Suppression

y, 3

3: 3

84:7

491

282-

9445

X mRNAmRNASite ASite A Site BSite B Site CSite C

OCOC--BoxBoxTATATATA

C/EBPC/EBPEE--BoxBox

VDREVDRE

RUNXRUNXAPAP--11 DLXDLXRUNXRUNXAPAP--11RUNXRUNX

Bioc

hem

istr

yCe

ll Bi

ol, 1

9:Bi

ol C

hem

, 2

HLHHLH TFIIBTFIIB TAF’STAF’SVDR/RXRVDR/RXR HATHATAcAc

Vitamin D Vitamin D -- Enhanced (Differentiated)Enhanced (Differentiated)

AcAcAcAc

Maximal Expression

al (1

994)

B99

9) M

ol C

al (2

007)

J B

HATHAT HLHHLH**

TFIIBTFIIBRNA RNA PolPol IIII

TAF’STAF S

TFIIDTFIIDRUNXRUNXmRNAmRNA

Site ASite A Site BSite B Site CSite C TATATATAEE--BoxBox

RUNXRUNX C/EBPC/EBPAPAP--11 DLXDLXRUNXRUNX

APAP--11

HATHATAcAcAcAcAcAcAcAc AcAc AcAc

AcAcAc

onte

cino

et

aved

et a

l (1

utie

rrez

et a

Site ASite A Site BSite B Site CSite COCOC--BoxBox

TATATATAC/EBPC/EBP

EE BoxBoxVDREVDREM J a G

u

Re organization of Regulatory MachineryRe-organization of Regulatory MachineryDuring Mitosis

INTERPHASE MITOSIS

Young et al (2007) Nature, 445: 442

Runx2 Remains Stably Associated withRunx2 Remains Stably Associated withMitotic Chromosomes

Young et al (2007) PNAS, 104: 3189

Chromosomal Association of Runx2 is AllelicChromosomal Association of Runx2 is Allelic

Runx FociRunx Foci

Young et al (2007) Nature, 445: 442

A Multi Dimensional Approach to UnderstandA Multi-Dimensional Approach to Understand Stem Cell Biology and Therapeutic Potential

• Stem cells exhibit a shorter cell cycle: sustainedStem cells exhibit a shorter cell cycle: sustained growth and unrestricted availability.

• Cell fate, lineage commitment and maintenance require the combined contributions of genetic andrequire the combined contributions of genetic and epigenetic mechanisms in nuclear “microenvironments” during andmicroenvironments during and following cell division.

Challenges and Opportunities of g ppStem Cell Research

• Moral and ethical considerations

• Immuno compatibility

• Procedure-related compromises (e.g., insertionalmutagenesis, tumorigenesis)

• Retention of genetic basis for diseases

Success Stories with Stem CellsSuccess Stories with Stem Cells

• Bone Marrow Transplantation- Cancer

Hemotological Diseases- Hemotological Diseases

• Cardio-vascular Stem Cells

• Neurological Stem Cellsl- Spinal Cord Injury

- Adult Macular Degeneration- ALS- ALS

• Skeletal Reconstruction

Acknowledgements

Members of the UMASS Center for Stem Cell Biology &

Acknowledgements

Members of the UMASS Center for Stem Cell Biology & Regenerative Medicine

Members of the UMASS Memorial MusculoskeletalCenter of Excellence

Janet L. SteinJane B. LianJane B. Lian

Andre van WijnenKlaus A. Becker

Prachi GhuleMatthew Mandellive

Daniel YoungDaniel YoungSayyed Kaleem Zaidi