stem cells, lineage development, plasticity, & regeneration functional histology dean tang,...

Stem Cells, Lineage Development, Plasticity, & Regeneration

Functional HistologyDean Tang, SPRD, MDACCAug. 31, 2009

Flatworm(planarian)

Newt MRL mice

Stem cell development

Terminal differentiation

Death (PCD)

Senescence

Stem cells

Progenitors/Precursor cells

1. Characteristics & Definition

2. Types: germ stem cells (GSC), embryonic SC (ESC), and somatic (adult) SC (SSC). Adult SCs in many cases are called adult progenitor or precursor cells.

3. (Adult) SC Identification: multiple approaches

4. SC Niche: location, location, location!

5. Plasticity, Reprogramming, & iPS Cells

6. SCs & Tissue Regeneration

7. SCs & Cancer

Stem Cell Biology

-Rare -Are generally small- Normally localized in a ‘protected’ environment called NICHE, where they only occasionally divide - But they possess HIGH PROLIFERATIVE POTENTIAL and can give rise to large clones of progeny in vitro or in vivo following injury or appropriate stimulation- Possess the ability to SELF-RENEW (i.e, asymmetric or symmetric cell division)- Can generate (i.e., DIFFERENTIATE into) one or multiple or all cell types (uni-, oligo-, multi-, pluri-, or toti-potent)

Stem Cells

SC

Committed cells

SC Self-renewal, Proliferation, and Differentiation

SC Development: Self-renewal, proliferation, differentiation

LT-SC ST-SC Lateprogenitors

Differen-tiated cells

Differen-tiating cells

Earlyprogenitors

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Pro

life

rati

onDifferentiation

Transformationtargets

Self-renewal

Niche Commitment Differentiation







7. SCs & Cancer

Stem Cell Biology

•Mouse ESCs were generated early 1980s by Evans and Martin •mES cells are cultured on mouse fibroblast feeders (irradiated or mitomycin C-treated) together with LIF.mES cells are widely used in gene targeting•Human ES (hES) cells were first created by Jim Thomson (Uni. Wisconsin) in 1998 •hES cells were initially cultured also on mouse fibroblast feeders but without LIF. Now they can be maintained in defined medium with high bFGF (100 ng/ml), activin, and some other factors

Embryonic Stem Cells (ESCs)

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) How can hES cells be derived?

16-cell morula

Primitive ectoderm Trophectoderm

Primitive Endoderm

A. Nagy

ES cells

A. Nagy

TS cells A. Nagy

A. Nagy

hAPlacZ GFP wildtype

A. Nagy

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heart pancreas testis

liver brain kidney

A. Nagy

GFP positive ES cell contribution to the brain

A. Nagy

Tetraploid

ES cells

A. Nagy

ES cell <-> tetraploid embryo aggregation

A. Nagy

•Derived from umbilical cord •Primarily blood stem cells•Also contain mesenchymal stem cells that can differentiate into bone, cartilage, heart muscle, brain, liver tissue etc.*CB-SC could be stimulated to differentiate into neuron, endothelial cell, and insulin-producing cells

Cord Blood Stem Cells (CB-SC)

Germline Stem Cells (GSC)

Other ‘embryonic’ SCs







7. SCs & Cancer

Stem Cell Biology

How to identify and characterize (adult) stem cells?

1. Marker analysis2. Clonal/clonogenic assays3. Side population (SP): BCRP or ABCG24. Label-retaining cells (LRC)5. Aldefluor assay (Aldh1 expression)6. Cell size-based enrichment7. Genetic marking

Passegué, Emmanuelle et al. (2003) Proc. Natl. Acad. Sci. USA 100, 11842-11849

Hematopoietic and progenitor cell lineages

(~1:5,000 or 0.02%;lifetime self-renewal)

(~1:1,000 or 0.1%;self-renewal for 8 wks)

(No self-renewal)

(Nestin)

(GFAP)

(Pax6)(A2B5)

(NG2)(MBP)

(NeuM)

(Mash-1)

(PDGFR)

Sue Fischer

CLONAL vs CLONOGENIC ASSAYS

Clonal

*Plate cells at clonal density(50-100 cells/wellin 6-well plateor 10-cm dishor T25 flask)

*Plate single cellsinto 96-well plates(or using flow sorting)- limiting dilution

Holoclone Mero- or paraclone

a. Cloning efficiency (CE; %)b. Clonal size (cell number/clone)c. Clonal development (tracking)d. Clone types

A clone: a two-dimensional structure

Plating efficiency

Prolif. potential

Clonogenic

‘In-gel’ assays(plate cells at low density)

‘On-gel’ assays(plate at low density)

a. Efficiency (%)b. Colony/sphere size (cell number)c. Colony/sphere development (tracking)d. Immunostaining/tumor exp.

A colony/sphere: a 3-D structure

Colonies(colony-formationassays)

Anchorage-independ.survival

Prolif.

Spheres(sphere-formation assays)

Gels: Agar Agarose Methylcellulose Matrigel Poly-HEMA fibroblasts

Mixing Experiments to Demonstrate the Clonality of Clones/Spheres

DU145 RFP:DU145 GFP (1:1) MC

DU145:DU145 GFP (1:1) MC

phas

eG

FP

DU145:DU145 GFP (1:1) Clonal Assay

Identification of HSC by SP

Zhou et al., Nature Med 7, 1028, 2001

LRCs in the Bulge ARE Stem Cells

Tumbar et al., Science 303, 359-363, 2004; Fuchs et al., Cell 116, 769, 2004Fuchs E: The tortoise and the hair: Slow-cycling cells in the stem cell race.

Cell 137, 811-819, 2009.







7. SCs & Cancer

Stem Cell Biology

Stem Cell Niche

The most important function of a stem cell niche is to keep the stem cells quiescent and from differentiating and simultaneously maintain their “stemness” (i.e., the repertoire of gene expression profiles characteristic of stem cells).

Stem Cell Niche in Hair Follicles: The Bulge

Moore KA & Lemischka IR. Science 311, 1880-1885, 2006

Bulge Stem Cells

Tumbar et al., Science 303, 359-363, 2004; Fuchs et al., Cell 116, 769, 2004

Stem Cell Niche in Small Intestine: The Crypt


Stem Cell Niche in BM: The Osteoblast Niche


Stem Cell Niche

In Drosophila, the GSC niches can be experimentally “emptied” - but the niches still persist and can even “recruit” SSC into the niches and maintain their SC properties for some time, although the SSCs never become GSC (Kai and Spradling, PNAS 100, 4633, 2003).

Sato T et al., Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262-265, 2009.







7. SCs & Cancer

Stem Cell Biology

Stem cell lineage

Differentiatedcells

Death (PCD)

Senescence

Stem cells


Other cell(s)

Adult Stem Cell Plasticity

Plasticity: the ability of SCs to regenerate and trans-differentiate into (many) other cell types (the cell type-specific programming of apparently committed primary progenitors is not irrevocably fixed, but may be radically re-specified in response to a single

transcriptional regulator. Heyworth C et al., EMBO J. 21, 3770-3781, 2002).

**Transdifferentiation vs Dedifferenitation: Transdifferentiation refers to adult stem cells directly differentiating into other cell lineages of cells; de-differentiation refers to somatic stem/progenitor cells first reverting back to a more primitive state then differentiating into a specific cell type.

Blelloch R. Nature 455, 604-605, 2008

*First report: Long-term cultured adult brain (stem) cells can reconstitute the whole blood in lethally irradiated mice (Bjornson et al., Science 283, 534-537, 1999).

*Cells from skeletal muscle have hematopoietic potential (Jackson et al., PNAS 96, 14482-14486, 1999) and can also “differentiate” into many other cell types (Qu-Petersen, Z, et al., JCB 157, 851-864, 2002).

*CNS “SCs” can “differentiate” into muscle cells (Clarke et al., Science 288, 1660-1663, 2000; Galli et al., Nat. Neurosci 3, 986-991, 2000; Tsai and McKay, J. Neurosci 20, 3725-3735, 2000).

*Vice versa, “SCs” from blood or bone marrow can “transdifferentiate” into muscle (Ferrari et al., Science 279, 1528-1530, 1998; Gussoni et al., Nature 401, 390-394, 1999), hepatocytes (Petersen et al., Science 284, 1168-1170, 1999; Lagasse et al., Nat Med 6, 1229-1234, 2000), cardiac myocytes (Orlic et al., Nature 410, 701-705, 2001), or neural cells (Mezey et al., Science 290, 1779-1782, 2000; Brazelton et al., Science 290, 1775-1779, 2000).

*Bone marrow appears to contain two distinct SCs: the HSC and MSC. A single HSC could contribute to epithelia of multiple organs of endodermal and ectodermal origin (Krause et al., Cell 105 369-377, 2001). MSC, on the other hand, can adopt a wider range of fates (endothelial, liver, neural cells, and perhaps all cell types) (Pittenger et al., Science 284, 143-146, 1999;

Schwartz et al., JCI 109, 1291-1302, 2002; Jiang et al., Nature 418, 41-49, 2002).*Pluripotent “SCs” have also been isolated from skin that can “differentiate” into neural cells, epithelial

cells, and blood cells (Toma et al., Nat Cell Biol. 3, 778-784, 2001)*Highly purified adult rat hepatic oval “stem’ cells, which are capable of differentiating into hepatocytes

and bile duct epithelium, can “trans-differentiate” into pancreatic endocrine hormone-producing cells when cultured in a high glucose environment (Yang et al., PNAS 99, 8078-8083, 2002)

“Transdifferentiation” of Stem Cells: Exciting!!!

Adult SC Plasticity

Blau et al., Cell 105: 829-841, 2001

A. Dystrophin (green) and detection of Y-chromosome in the nucleiB. Detection of fumarylacetoacetate hydrolase (FAH) in the FAH-/- recipient mouse liverC. BM-derived gal+-heart muscle fibers in the recipient myocardial infarctionD. NeuN+, GFP+-neuron derived from the transplanted marked NSCs

*CNS stem cells rarely turn into blood (Morshead et al., Nat Med 8, 268-273, 2002).*Muscle-derived hematopoietic stem cells are hematopoietic in origin, i.e., CD45+

(McKinney-Freeman et al., PNAS 99, 1341-1346, 2002)*Cell fusion between co-cultured neurosphere-derived cells with ES cells (Yinh et al.,

Nature 416, 545-548, 2002) or between bone marrow cells and ES cells (Terada et al, Nature 416 542-545, 2002). The fused tetraploid cells inherit

the selectable markers for both cell types and the properties of ES cells, and can contribute to multiple somatic tissues.*Bone marrow-derived cells (BMDCs) turn to hepatocytes thru cell fusion rather than

transdifferentiation (Wang et al., Nature 422, 897, 2003; Vassilopoulos et al., Nature 422, 901, 2003).

*BMDCs stably fuse with Purkinje neurons to form heterokaryons (Weimann et al., Nature Cell Biol. 5, 959, 2003; Nature 425, 968, 2003).

*Single HSCs can integrate into myofibers and contribute to muscle regeneration through cell fusion (Camargo et al., Nat. Med. 9, 1520, 2003; Corbel et al., Nat. Med. 9, 1528, 2003).

Transdifferentiation of Stem Cells-------- Is it real?

Cell fusion underlies “transdifferentiation”

Medivinsky A and Smith, A. Nature 422, 823, 2003.

*Adult MSC turned into skeletal muscle fibers (De Bari et al., JCB, 160, 909-918, 2003).

*Muscle stem cells differentiate into hematopoietic lineages but retain myogenic potential (Nature Cell Biol. 5, 640, 2003).

Transdifferentiation of Stem Cells ----- It seems to be real!

*HSCs do not transdifferentiate into cardiac myocytes in myocardial infarcts (Nature 428, 664, 2004).

*HSCs adopt mature hematopoietic fates in ischemic myocardium (Nature 428, 668, 2004).

*BMDCs generate cardiomyocytes as a low frequency through cell fusion, but not transdifferentiation (Nature Med. 10, 494-501, 2004).

*Hematopoietic myelocytic cells are the major source of hepatocyte fusion partners(Camargo et al., JCI 113, 1266, 2004).

*Myelomonocytic cells are sufficient for therapeutic cell fusion in liver (Willenbring et al., Nature Med. 10, 744, 2004).

*Myelomonocytic cells are the cells that contribute to skeletal muscle regeneration(Doyonnas et al., PNAS 101, 13507, 2004).

Transdifferentiation of Stem Cells ----- It CAN’t be real!

*HSCs convert into liver cells within days without fusion (Jang et al., Nature Cell Biol. 6, 532-539, 2004).

*Epithelial cells can develop from BMDCs without cell fusion (Harris et al., Science 305, 90, 2004).

*NSCs can differentiate into endothelial cells without cell fusion (Wurmser et al.,Nature 430, 350, 2004).

Transdifferentiation of Stem Cells ----- Yes, it IS real!

Not only stem/progenitor cells but also terminally differentiated cells can fuse with other cells

Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle(Matsuura K et al., JCB 167, 351, 2004)

Pancreatic -cells: Interesting insulin-producing cells

*Insulin-producing -cells in adult mouse pancreas can self-duplicate during normal homeostasis as well as during injury (Dor et al., Nature 429, 41, 2004).

*In vivo reprogramming of adult pancreatic exocrine cells to bcells using 3 TFs (Ngn3, Pdx1, and Mafa), suggesting aparadign for directing cell reprogramming without reversion to a pluripotent cell state (Zhou et al., Nature455, 627-632, 2008).

*In response to injury, a population of pancreatic progenitorscan generate glucagon-expressing alpha cells that thentransdifferentiate (with ectopic expression of Pax4) intobeta cells (Collmbat et al, Cell 138, 449-462, 2009).

Cell-cycle re-entry and de-differentiation*Dedifferentiation is a genetically regulated process that may ensure a return path to the undifferentiated state when necessary (Katoh et al., PNAS 101, 7005, 2004).*Regeneration of male GSC by spermatogonial dedifferentiation in vivo (Brawley and Matunis,

Science 304, 1331, 2004).*Many ‘post-mitotic’ cells such as hepatocytes, endothelial cells, and Schwann cells have long

been known to retain proliferative (progenitor) potential.*Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors

(Nature 449, 473-477, 2007).*During Salamander limb regeneration, complete de-differentiation to a pluripotent state is not required – Progenitor cells in the blastema keep a memory of their tissue origin (Nature 460, 60-65, 2009).*Evidence for cardiomyocyte renewal in humans (Bergmann O et al., Science 324, 98-102, 2009). (Cardiomyocytes turn over at an estimated rate of ~1% per year at age 20, declining to 0.4% per year at age 75. At age 50, 55% of human cardiomyocytes remain from birth while 45% were generated afterward. Over the first decade of life, cardiomyocytes often undergo a final round of DNA synthesis and nuclear division without cell division, resulting in ~25% of human cardiomyocytes being binucleated. Most adult cardiomyocytes are polyploid as they undergo DNA synthesis without nuclear division).*Neuregulin 1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury

(Bersell et al., Cell, 138, 257-270, 2009).

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Induced Pluripotent Cells (iPS cells)(Infection of somatic cells with 2-4 factors: Sox2, Oct4, Klf-4, Myc)

Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006 Aug 25;126(4):663-76. Takahashi K, Okita K, Nakagawa M, Yamanaka S. Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc. 2007;2(12):3081-9.Maherali N, ……., Hochedlinger K. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell. 2007 Jun 7;1(1):55-70.Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 2007 Jul 19;448(7151):313-7. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from

adult human fibroblasts by defined factors. Cell. 2007 Nov 30;131(5):861-72. Yu J, ……, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007 Dec 21;318(5858):1917-20. Hanna J, …. Jaenisch R. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin.

Science. 2007 Dec 21;318(5858):1920-3. Nakagawa M, …… Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol. 2008 Jan;26(1):101-6.Park IH, …….. Daley GQ. Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 2008 Jan 10;451(7175):141-6. Brambrink T…..Jaenisch R. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells.Cell Stem Cell. 2008 Feb 7;2(2):151-9.Lowry WE……Plath K. Generation of human induced pluripotent stem cells from dermal fibroblasts. PNAS. 2008 Feb 26;105(8):2883-8.Wernig M…..Jaenisch R. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. PNAS 2008 Apr 15;105(15):5856-61.Hanna J……Jaenisch R. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell. 2008 Apr 18;133(2):250-64.

Induced Pluripotent Cells (iPS cells)(Infection of somatic cells with 2-4 factors: Sox2, Oct4, Klf-4, Myc)

Stadtfeld M, Brennand K, Hochedlinger K. Reprogramming of pancreatic beta cells into induced pluripotent stem cells. Curr Biol. 2008 Jun 24;18(12):890-4.Eminli S, Utikal JS, Arnold K, Jaenisch R, Hochedlinger K. Reprogramming of Neural Progenitor Cells into iPS Cells in the Absence of Exogenous Sox2 Expression. Stem Cells. 2008 Jul 17. [Epub ahead of print]Narazaki G…..Yamashita JK. Directed and systematic differentiation of cardiovascular cells from mouse induced pluripotent stem cells. Circulation. 2008 Jul 29;118(5):498-506. Epub 2008 Jul 14.Mauritz C, ……Martin U. Generation of functional murine cardiac myocytes from induced pluripotent stem cells. Circulation. 2008 Jul 29;118(5):507-17.Kim JB, Zaehres H, ….. Schöler HR. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature. 2008 Jul 31;454(7204):646-50. Aoi T, ….. Yamanaka S. Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science. 2008 Aug 1;321(5889):699-702. Epub 2008 Feb 14.Mali P, Ye Z, Hommond HH, Yu X, Lin J, Chen G, Zou J, Cheng L. Improved efficiency and pace of generating induced pluripotent stem cells from human adult and fetal fibroblasts. Stem Cells. 2008 Aug;26(8):1998-2005.Dimos JT, ….. Eggan K. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science. 2008 Aug 29;321(5893):1218-21.Park IH, …… Daley GQ. Disease-Specific Induced Pluripotent Stem Cells. Cell. 2008 Sep 5;134(5):877-86.

………………….

Raya A………Izpisua Belonte JC. Disease-corrected hematoppoietic progenitors from Fanconi anemia induced pluripotent cells. Nature July 2 60, 53-58, 2009.

*Up to now: ~700 PubMed publications on iPS cells!Yamanaka S: A fresh look at iPS cells. Cell 137, 13-17, 2009.Yamanaka S: Elite and stochastic models for induced pluripotent stem cell generation. Nature 460: 49-52, 2009.







7. SCs & Cancer

Stem Cell Biology

Degenerative Diseases

1. Alzheimer’s disease: wholesale death of serotonin-producing neurons involved in memory

2. Parkinson’s disease: death of dopamingergic neurons in the midbrain

3. Huntington’s disease: death of medium spiny neurons in the subcortical striatum

4. Amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease): damage of motor neurons in the wholeCNS especially spinal cord

5. Multiple sclerosis (MS): death of oligodendrocytes followed by death of neurons

6. Spinal cord injury: physical damage to neurons and their connections

7. Diabetes: death of insulin-producing -cells8. Myocardial infarction: acute death of cardiac myocytes

Sources for Cell therapy

*ES cells: differentiate into specified cell types and then transplant**Alternate ways to generate ES cells:

a) Generation of ES cells from embryos that cannot implant (i.e.,politically correct ES cells; Meissner A, and Jaenisch R. Nature 2006 439:212-5).

b) Generation of ES cells from single blastomeres (Chung Y, et al.Nature 2006 439:216-9; Klimanskaya I, et al. Nature. 2006 Aug 23).

*SCNT (somatic cell nuclear transfer) - derive ES cells (or disease-tailored ES cells) through nuclear reprogramming

*Nuclear reprogramming of somatic cells by fusion with hES cells (Science 309, 1369, 2005)

*iPS cells*Reprogramming by matrix elasticity (Cell 126, 677-689, 2006).*Rejuvenating old progenitors by young systematic environment

(Science 433, 760-764, 2005)*Adult SC: may possess certain plasticity; but limited proliferative and

differentiation capacities; origins often unclear

ES Cells Are Pluripotent/Totipotent

ES cells rescue heart defects

Chien et al., Science 306, 239, 2004Fraidenraich et al., Science 306, 247, 2004

Baker M: How to fix a broken heart? Nature 460, 18-19, 2009

Sources for Cell therapy

*ES cells: differentiate into specified cell types and then transplant**Alternate ways to generate ES cells:

a) Generation of ES cells from embryos that cannot implant (i.e.,politically correct ES cells; Meissner A, and Jaenisch R. Nature 2006 439:212-5).

b) Generation of ES cells from single blastomeres (Chung Y, et al.Nature 2006 439:216-9; Klimanskaya I, et al. Nature. 2006 Aug 23).

*SCNT (somatic cell nuclear transfer) - derive ES cells (or disease-tailored ES cells) through nuclear reprogramming

*Nuclear reprogramming of somatic cells by fusion with hES cells (Science 309, 1369, 2005)

*iPS cells*Reprogramming by matrix elasticity (Cell 126, 677-689, 2006).*Rejuvenating old progenitors by young systematic environment

(Science 433, 760-764, 2005)*Adult SC: may possess certain plasticity; but limited proliferative and

differentiation capacities; origins often unclear

Cloning for Cell Therapy







7. SCs & Cancer

Stem Cell Biology

Stem cell development

Terminal differentiation

Death (PCD)

Senescence

Stem cells


1. Every tumor contains stem-like cancer cells, which maintain tumor homeostasis, drive tumor progression, and probably mediate drug resistance and metastasis

2. Tumors are derived from normal stem cellsor their immediate progeny (progenitor cells)

An alternative explanation of tumor cell heterogeneity:The cancer stem cell (CSC) hypothesis

stem cells, lineage development, plasticity, & regeneration functional histology dean tang,...

Documents

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sc niche

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somatic adult sc ssc

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embryonic sc esc