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Stem Cells & Cancer

Stem Cells & Cancer. Three tumor biology puzzles: Most tumors are of a clonal origin but tumor cells are heterogeneous. It is very difficult to establish stable tumor cell lines from tumors. Large numbers of established tumor cells have to be injected to re-initiate

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Stem Cells & Cancer

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  1. Stem Cells & Cancer Three tumor biology puzzles: Most tumors are of a clonal origin but tumor cells are heterogeneous. It is very difficult to establish stable tumor cell lines from tumors. Large numbers of established tumor cells have to be injected to re-initiate an orthotopic tumor in mice. Recent key reviews: 1. Reya T et al. Stem cells, cancer, and cancer stem cells. Nature 414, 105-111, 2001. 2. Visvader JE, and Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 8: 755-768, 2008. 3. Dick JE. Stem cell concepts renew cancer research. Blood 112: 4793-4807, 2008. 4. Shackleton M, Quintana E, Fearon ER, and Morrison SJ. (2009). Heterogeneity in cancer: cancer stem cells versus clonal evolution.Cell 138, 822-829, 2009. (Dean Tang, Basic Concepts of Tumor Biology, Sept. 22, 2010)

  2. Stem Cells & Cancer 1. Characteristics & Definition 2. Types & (Adult) SC Identification 3. SC Niche & Plasticity 4. SCs & Cancer 5. Cancer Stem Cells (CSCs)

  3. Flatworm (planarian) Newt MRL mice

  4. Stem cell development Terminal differentiation Progenitors/ Precursor cells Senescence Death (PCD) Stem cells

  5. Stem Cells • Rare • 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).

  6. SC Self-renewal, Proliferation, and Differentiation SC Committed cells

  7. SC Development: Self-renewal, proliferation, differentiation Differentiation Transformation targets Self-renewal ? LT-SC ST-SC Late progenitors Differen- tiating cells Differen- tiated cells Early progenitors Proliferation Niche Commitment Differentiation

  8. Embryonic Stem Cells (ESCs) • 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.

  9. How can hES cells be derived? • Leftover or dead-end IVF embryos (PGD)

  10. 16-cell morula

  11. Primitive ectoderm Trophectoderm Primitive Endoderm A. Nagy

  12. ES cells A. Nagy

  13. TS cells A. Nagy

  14. A. Nagy

  15. A. Nagy

  16. A. Nagy

  17. heart pancreas testis liver brain kidney A. Nagy

  18. Other ‘embryonic’ SCs Germline Stem Cells (GSC) Cord Blood Stem Cells (CB-SC) • 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

  19. How to identify and characterize (adult) stem cells? Marker analysis Clonal/clonogenic assays Side population (SP): BCRP or ABCG2 Label-retaining cells (LRC) Aldefluor assay (Aldh1 expression) Cell size-based enrichment Genetic marking

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

  21. (Nestin) (PDGFR) (Mash-1) (NeuM) (Pax6) (A2B5) (GFAP) (MBP) (NG2)

  22. Sue Fischer

  23. How to identify and characterize (adult) stem cells? Marker analysis Clonal/clonogenic assays Side population (SP): BCRP or ABCG2 Label-retaining cells (LRC) Aldefluor assay (Aldh1 expression) Cell size-based enrichment Genetic marking

  24. Anchorage- independ. survival Plating efficiency Clonogenic ‘In-gel’ assays (plate cells at low density) Prolif. potential Prolif. Clonal *Plate cells at clonal density (50-100 cells/well in 6-well plate or 10-cm dish or T25 flask) ‘On-gel’ assays (plate at low density) *Plate single cells into 96-well plates (or using flow sorting) - limiting dilution Gels: Agar Agarose Methylcellulose Matrigel Poly-HEMA fibroblasts Spheres (sphere-formation assays) Colonies (colony-formation assays) Holoclone Mero- or paraclone A colony/sphere: a 3-D structure Efficiency (%) Colony/sphere size (cell number) Colony/sphere development (tracking) Immunostaining/tumor exp. CLONAL vs CLONOGENIC ASSAYS A clone: a two-dimensional structure Cloning efficiency (CE; %) Clonal size (cell number/clone) Clonal development (tracking) Clone types

  25. Mixing Experiments to Demonstrate the Clonality of Clones/Spheres DU145:DU145 GFP (1:1) Clonal Assay phase DU145:DU145 GFP (1:1) MC GFP DU145 RFP:DU145 GFP (1:1) MC

  26. How to identify and characterize (adult) stem cells? Marker analysis Clonal/clonogenic assays Side population (SP): BCRP or ABCG2 Label-retaining cells (LRC) Aldefluor assay (Aldh1 expression) Cell size-based enrichment Genetic marking

  27. Identification of HSC by SP Zhou et al., Nature Med 7, 1028, 2001

  28. How to identify and characterize (adult) stem cells? Marker analysis Clonal/clonogenic assays Side population (SP): BCRP or ABCG2 Label-retaining cells (LRC) Aldefluor assay (Aldh1 expression) Cell size-based enrichment Genetic marking

  29. LRCs in the Bulge ARE Stem Cells Tumbar et al., Science 303, 359-363, 2004; Fuchs et al., Cell 116, 769, 2004 Fuchs E: The tortoise and the hair: Slow-cycling cells in the stem cell race. Cell 137, 811-819, 2009.

  30. How to identify and characterize (adult) stem cells? Marker analysis Clonal/clonogenic assays Side population (SP): BCRP or ABCG2 Label-retaining cells (LRC) Aldefluor assay (Aldh1 expression) Cell size-based enrichment Genetic marking

  31. 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).

  32. Stem Cell Niche in Hair Follicles: The Bulge Moore KA & Lemischka IR. Science 311, 1880-1885, 2006

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

  34. Stem Cell Niche in Small Intestine: The Crypt Moore KA & Lemischka IR. Science 311, 1880-1885, 2006

  35. Stem Cell Niches in BM Moore KA & Lemischka IR. Science 311, 1880-1885, 2006 Naveiras O et al., Bone-marrow adipocytes as negative regulators of the hematopoietic microenvironment. Nature 460, 259, 2009. Mendez-Ferrer, S et al., Mesenchymal and hematopoietic stem cells form a unique bone marrow niche. Nature 466, 829-834, 2010.

  36. Stem cell lineage Differentiated cells Progenitors/ Precursor cells Other cell(s) Senescence Death (PCD) Stem cells

  37. 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.

  38. Blelloch R. Nature 455, 604-605, 2008

  39. “Transdifferentiation” of Stem Cells: Exciting!!! *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)

  40. Cell fusion as one mechanism of trans-differentiation: 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)

  41. De-differentiation: Cell-cycle re-entry *Many ‘post-mitotic’ cells such as hepatocytes, endothelial cells, and Schwann cells have long been known to retain proliferative (progenitor) potential. *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). *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). *Epigenetic reversion of post-implantation epiblast to pluripotent embryonic cells (Nature, 461, 1292-1295, 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.) *Neuregulin 1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury (Bersell et al., Cell, 138, 257-270, 2009). *MafB/c-Maf deficiency enables self-renewal of differentiated functional macrophages (Aziz A, et al., Science 326, 867-871, 2009).

  42. Pancreatic b-cells: Interesting insulin-producing cells *Insulin-producing b-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 b cells using 3 TFs (Ngn3, Pdx1, and Mafa), suggesting a paradign for directing cell reprogramming without reversion to a pluripotent cell state (Zhou et al., Nature 455, 627-632, 2008). *In response to injury, a population of pancreatic progenitors can generate glucagon-expressing alpha cells that then transdifferentiate (with ectopic expression of Pax4) into beta cells (Collmbat et al, Cell 138, 449-462, 2009). *Conversion of adult pancreatic a-cells to b-cells after extreme b-cell loss (Nature 464, 1149-1154, 2010).

  43. CELL 126, 652-655, 2006 NUCLEAR REPROGRAMMING

  44. 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. 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 131, 861-872, 2007. 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, 318, 1920-1923, 2007). 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. Kobayashi T et al., Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 142, 787-799, 2010. *Up to now: >2000 PubMed publications on iPS cells! *Yamanaka S: Elite and stochastic models for induced pluripotent stem cell generation. Nature 460: 49-52, 2009. *Yamanaka S & Blau HM. Nuclear reprogramming to a pluripotent state by three approaches. Nature 465, 704, 2010

  45. Development and epigenetic (re)programming Hochedlinger 2009, Development 136, 509-523 Transcription factors and chromatin remodelers and key modulators of ‘potency’

  46. Nicholas CR & Kriegstein AR. Cell reprogramming gets direct. Nature 463, 1031-1032, 2010. Vierbuchen T et al., Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035-1041, 2010 (Ascl1, Brn2, and Myt1l). Ieda M, et al., Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell 142, 375-386, 2010 (Gata4, Mef2c, and Tbx5). Bonfanti P, et al., Microenvironmental reprogramming of thymic epithelial cells to skin multi- potent stem cells. Nature 466, 978-982, 2010. Bussard KM, et al., Reprogramming human cancer cells in the mouse mammary gland. Cancer Res 70, 6336-6343, 2010.

  47. Stem Cells & Tumorigenesis (Terminal) differentiation functional maturation Senescence Perinatal stem/ progenitors PCD Death Adult Stem/ Progenitor Cells ESCs Tumor cells

  48. Several fundamental tumor biology questions Why is it so difficult to establish tumor cell lines from established tumors or even metastases? 2. Why tens or hundreds of thousands of established tumor cells have to be injected to initiate an orthotopic tumor? 3. Tumors are clonal (i.e., all tumors were initially derived from ‘going bad’ of one cell) but why is the tumor itself heterogeneous?

  49. Tumorigenecities of Orthotopically Implanted Prostate Cancer Cells Cell type Cell# injected Incidence Latency (days) Du145 1,000 0/4 10,000 0/4 100,000 1/4 103 500,000 3/5 53, 53, 59 LAPC4 100 0/4 1,000 0/4 10,000 0/4 100,000 0/4 500,000 3/6 43, 43, 48 LAPC9 100 0/3 1,000 0/9 10,000 4/8 46, 53, 75, 75 100,000 6/9 32, 42, 42, 45, 62, 69 1,000,000 4/4 48, 56, 56, 69

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