干細胞癌癥描述課件

1、Flatworm (planarian)NewtMRL miceNature. 2012 Sep 27;489(7417):561-5. doi: 10.1038/nature11499. Skin shedding and tissue regeneration in African spiny mice (Acomys).Seifert AW, Kiama SG, Seifert MG, Goheen JR, Palmer TM, Maden M.Flatworm (planarian)NewtMRL miStem Cell DevelopmentAdult stem/progenitor

2、sfunctional maturationPCD (apoptosis)DifferentiationDeathSenescenceESCschronological agingStem Cell DevelopmentAdult steStem Cells & CancerThree 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 t

3、umors.Large numbers of established tumor cells have to be injected to re-initiate an orthotopic tumor in mice.Key reviews:Reya T et al. Stem cells, cancer, and cancer stem cells. Nature 414, 105-111, 2001.Dick JE. Stem cell concepts renew cancer research. Blood 112: 4793-4807, 2008.Visvader JE, and

4、Lindeman GJ. Cancer Stem Cells: Current Status and Evolving Complexicities. Cell Stem Cell 10: 717-728, 2012.4. Tang DG. Understanding cancer stem cell heterogeneity and plasticity. Cell Res, 22(3):457-472, 2012. 5. Magee JA, Piskounova E, & Morrison SJ. Cancer Stem Cells: Impact, Heterogeneity, and

5、 Uncertainty. Cancer Cell 21: 283-296, 2012.(Dean Tang, Basic Concepts of Tumor Biology, Oct 31, 2012)Stem Cells & CancerThree tumor1. Characteristics & Definition2. SC Identification3. SC Niche & Plasticity4. SCs & Cancer5. Cancer Stem Cells (CSCs)Stem Cells & Cancer1. Characteristics & DefinitioRa

6、re 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 S

7、ELF-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 CellsStem CellsCommitted progenitor cellssymmetric SC renewalasymmetric celldivision (ACD)symmetric SC commitment (d

8、ifferentiation)SCTang, Cell Res. 2012Committed progenitor cellssymmLT-SCST-SCLateprogenitorsDifferen-tiated cellsDifferen-tiating cellsEarlyprogenitorsProliferationDifferentiationTransformationprobabilitySelf-renewalNicheCommitmentDifferentiationExpansionTang, Cell Res. 2012Cell lineage development:

9、 Self-renewal, proliferation, & differentiationLT-SCST-SCLateDifferen-DiffereMouse 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)

10、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)Embryonic Stem Ce

11、lls (ESCs)Leftover or dead-end IVF embryos (PGD) How can hES cells be derived?How can hES cells be derived?16-cell morula16-cell morulaPrimitive ectodermTrophectodermPrimitive EndodermA. NagyPrimitive ectodermTrophectoderES cellsA. NagyES cellsA. NagyTS cellsA. NagyTS cellsA. NagyA. NagyA. NagyA. Na

12、gyA. NagyA. NagyA. NagyheartpancreastestisliverbrainkidneyA. Nagyheartpancreastestisliverbraink干細胞癌癥描述課件Derived from umbilical cord Primarily blood stem cellsAlso contain mesenchymal stem cells that can differentiate into bone, cartilage, heart muscle, brain, liver tissue etc.*CB-SC could be stimula

13、ted to differentiate into neuron, endothelial cell, and insulin-producing cellsCord Blood Stem Cells (CB-SC)Germline Stem Cells (GSC)Other embryonic SCsCord Blood Stem Cells (CB-SC)G1. Characteristics & Definition2. SC Identification3. SC Niche & Plasticity4. SCs & Cancer5. Cancer Stem Cells (CSCs)S

14、tem Cells & Cancer1. Characteristics & DefinitioFunctional Assays of Stem Cells(Candidate) Stem CellsStem Cells in situ(Xeno)TransplantationLineage tracingFunctional Assays of Stem CellHow to identify and characterize (adult) stem cells?Marker analysisLabel-retaining cells (LRC): Pulse-chase exper.C

15、lonal/clonogenic assaysFunctional analysis: Side population (SP) assayFunctional analysis: Aldefluor assayCell size-based enrichmentGenetic marking & lineage tracing How to identify and characteriPassegu, Emmanuelle et al. (2003) Proc. Natl. Acad. Sci. USA 100, 11842-11849Hematopoietic stem/progenit

16、or 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)Lin-Sca1+ckit+CD150+CD48- (20%-50% such mouse BM cells are SCs)Till JE & McCulloch EA. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res 14,

17、 213-222, 1961. Lin-CD34+CD38-CD45RA-Thy1+RholoCD49f+(Notta F.Dick JE. Science 333, 218-221, 2011)Passegu, Emmanuelle et al. (2(Nestin)(GFAP)(Pax6)(A2B5)(NG2)(MBP)(NeuM)(Mash-1)(PDGFRa)(Nestin)(GFAP)(Pax6)(A2B5)(NG2Sue FischerSue FischerHow to identify and characterize (adult) stem cells?Marker anal

18、ysisLabel-retaining cells (LRC): Pulse-chase exper.Clonal/clonogenic assaysFunctional analysis: Side population (SP) assayFunctional analysis: Aldefluor assayCell size-based enrichmentGenetic marking & lineage tracing How to identify and characteriTill JE & McCulloch EA. A direct measurement of the

19、radiation sensitivity of normal mouse bone marrow cells. Radiat. Res 14, 213-222, 1961. The earliest report in which putative stem cells were identified by their ability to retain labeled radionucleotides for long period of timeCotsarelis G, Cheng SZ, Dong G, Sun TT & Lavker RM. Existence of slow-cy

20、cling libmal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells. Cell 57, 201-209, 1989.Cotsarelis G, Sun TT, & Lavker RM. Label-retaining cell reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair

21、 cycle, and skin carcinogenesis. Cell 61, 1329-1337, 1990.Till JE & McCulloch EA. A direLRCs in the Bulge & BM ARE Stem CellsTumbar T et al., Defining the epithelial stem cell niche in skin. Science 303, 359-363, 2004. Blanpain, C., et al., Self-renewal, multipotency, and the existence of two cell p

22、opulations in an epithelial stem cell niche. Cell 118, 635-648, 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.Fuchs E & Horsley V. Ferreting out stem cells from their niches. Nat Cell Biology 13: 513-518, 2

23、011.Wilson A et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 135, 1118-1129, 2008.Foudi A et al. Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells. Nat. Biotechnol.27, 84-90, 2009.LRCs in the Bulge

24、 & BM ARE SteNot All Stem Cells Are Slow-CyclingFuchs E & Horsley V. Ferreting out stem cells from their niches. Nat Cell Biology 13: 513-518, 2011.Li L & Clevers H. Co-existence of quiescent and active adult stem cells in mammals. 327, 542-545, 2010. and even for the ones that do, approximately onl

25、y 5-6 divisions of the label-retaining stem cell or its progeny can be monitored after a pulse-chase beforethe label has diluted out to the point where it can no longer be traced.Not All Slow-Cycling Cells Are Stem CellsNot All Stem Cells Are Slow-CyHow to identify and characterize (adult) stem cell

26、s?Marker analysisLabel-retaining cells (LRC): Pulse-chase exper.Clonal/clonogenic assaysFunctional analysis: Side population (SP) assaysFunctional analysis: Aldefluor assayCell size-based enrichmentGenetic marking & lineage tracing How to identify and characteriERheinwald JG & Green H. Serial cultiv

27、ation of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell 6, 331-343, 1975.Sun TTCell 9, 511-521, 1976Nature 269, 489-493, 1977Cell 14, 469-476, 1978Fuchs ECell 19, 1033-1042, 1980Cell 25, 617-625, 1981Barrandon YPNAS 82, 5390-4, 1985Cell 50, 1131-1137, 1

28、987Rice RHCell 11, 417-422, 1977Cell 18, 681-694, 1979Watt FMJCB 90, 738-742, 1981ERheinwald JG & Green H. SeriCLONAL 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)- lim

29、iting dilutionHolocloneMero- or paracloneCloning efficiency (CE; %)Clonal size (cell number/clone)Clonal development (tracking)Clone typesA clone: a two-dimensional structurePlating efficiencyProlif. potential ClonogenicIn-gel assays(plate cells at low density)On-gel assays(plate at low density)Effi

30、ciency (%)Colony/sphere size (cell number)Colony/sphere development (tracking)Immunostaining/tumor exp.A colony/sphere: a 3-D structureColonies(colony-formationassays)Anchorage-independ.survivalProlif. Spheres(sphere-formation assays)Gels: Agar Agarose Methylcellulose Matrigel Poly-HEMA fibroblastsC

31、LONAL vs CLONOGENIC ASSAYS Mixing Experiments to Demonstrate the Clonality of Clones/SpheresDU145 RFP:DU145 GFP (1:1) MCDU145:DU145 GFP (1:1) MCphaseGFPDU145:DU145 GFP (1:1) Clonal AssayPastrana E, Silva-Vargas V, and Doetsch F.Eyes wide open: A critical review of sphere-formation as an assay of ste

32、m cells.Cell Stem Cell 8, 486-498, 2011Mixing Experiments to DemonstHow to identify and characterize (adult) stem cells?Marker analysisLabel-retaining cells (LRC): Pulse-chase exper.Clonal/clonogenic assaysFunctional analysis: Side population (SP) assaysFunctional analysis: Aldefluor assayCell size-

33、based enrichmentGenetic marking & lineage tracing How to identify and characteriGoodell MA et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 183, 1797-1806, 1996.Golebiewska A, Brons NH, Bjerkvig R, and Niclou SP. Critical apprai

34、sal of the side population assayin stem cell and cancer stem cell research. Cell Stem Cell 8, 136-147, 2011.Goodell MA et al. Isolation anBcrp (ABCG2) is a major mediator of the SP phenotypeZhou et al., Nature Med 7, 1028, 2001Bcrp (ABCG2) is a major mediatHow to identify and characterize (adult) st

35、em cells?Marker analysisLabel-retaining cells (LRC): Pulse-chase exper.Clonal/clonogenic assaysFunctional analysis: Side population (SP) assaysFunctional analysis: Aldefluor assayCell size-based enrichmentGenetic marking & lineage tracing How to identify and characteriAlison MR, Guppy NJ, Lim SML, &

36、 Nicholson LJ. Finding cancer stem cells: Are aldehyde dehydrogenases fitfor purpose? J Pathol. 222, 335-344, 2010.Ma I & Allan AL. The role of human aldehyde dehydrogenase in normal and cancer stem cells. Stem Cell Rev and Report 7, 292-306, 2011.*ALDH1A1 and ALDH3A1: Seem to be the major isozymes

37、mediating the Aldefluor phenotype and are preferentially expressed in SC.*ALDH superfamily: 19 putatively functional genes in 11 families and 4 subfamilies. ALDH superfamily of NAD(P)+-dependent enzyme catalyzes oxidations of aldehydes to carboxylic acids.Kastan MB et al. Direct demonstration of ele

38、vated aldehyde dehydrogenase in human hematopoieticprogenitor cells. Blood 75, 1947-1960, 1990.Jones RJ et al., Assessment of aldehyde dehydrogenase in viable cells. Blood 85, 2742-46, 1995.Storms RW et al. Isolation of primitive human hematopoietic progenitors on the basis of aldehydedehydrogenase

39、activity. PNAS 96, 9118-9123, 1999.Alison MR, Guppy NJ, Lim SML, How to identify and characterize (adult) stem cells?Marker analysisLabel-retaining cells (LRC): Pulse-chase exper.Clonal/clonogenic assaysFunctional analysis: Side population (SP) assaysFunctional analysis: Aldefluor assayCell size-bas

40、ed enrichmentGenetic marking & lineage tracing How to identify and characteriFuchs E & Horsley V. Ferreting out stem cells from their niches. Nature Cell Biology 13: 513-518, 2011.Fuchs E & Horsley V. FerretingFuchs E & Horsley V. Ferreting out stem cells from their niches. Nature Cell Biology 13: 5

41、13-518, 2011.Liver J et al., Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.Nature 450, 56-62, 2007.Snippert HJ et al., Intestinal crypt homeostasis results from neutral competition between symmetrically dividingLrg stem cells. Cell 143, 134-144, 201

42、0.Fuchs E & Horsley V. Ferreting1. Characteristics & Definition2. SC Identification3. SC Niche & Plasticity4. SCs & Cancer5. Cancer Stem Cells (CSCs)Stem Cells & Cancer1. Characteristics & DefinitioStem Cell Niche in Hair Follicles: The BulgeMoore KA & Lemischka IR. Science 311, 1880-1885, 2006Stem

43、Cell Niche in Hair FollicBulge Stem CellsTumbar et al., Science 303, 359-363, 2004; Fuchs et al., Cell 116, 769, 2004Bulge Stem CellsTumbar et al.,Stem Cell Niche in Small Intestine: The CryptMoore KA & Lemischka IR. Science 311, 1880-1885, 2006Barker N et al., Cell Stem Cell 11: 452-460, 2012.Stem

44、Cell Niche in Small IntesStem Cell Niches in BMMoore KA & Lemischka IR. Science 311, 1880-1885, 2006Naveiras 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 u

45、nique bone marrow niche. Nature 466, 829-834, 2010.Stem Cell Niches in BMMoore KAStem cell lineageDifferentiatedcellsDeath (PCD)SenescenceStem cellsProgenitors/Precursor cellsOther differ.cell(s)Stem cell lineageDifferentiate*First report: Long-term cultured adult brain (stem) cells can reconstitute

46、 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).*C

47、NS “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,

48、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

49、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 (e

50、ndothelial, 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 ce

51、lls, 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

52、glucose environment (Yang et al., PNAS 99, 8078-8083, 2002)“Transdifferentiation” of Stem Cells*First report: Long-term cultuDe-differentiation: Cell-cycle re-entry*Many post-mitotic cells such as hepatocytes, endothelial cells, and Schwann cells have longbeen known to retain proliferative (progenit

53、or) 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).*Co

54、nversion 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 46

55、0, 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, decl

56、ining 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 cardiomyocyt

57、es 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).De-different

58、iation: Cell-cyclePancreatic 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 bcells usi

59、ng 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 thentransdiffer

60、entiate (with ectopic expression of Pax4) intobeta 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).Pancreatic b-cells: InterestinNUCLEAR REPROGRAMMINGCELL 126, 652-655, 2006干細胞癌癥描述課件 Induced Pl