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.Stem Cell DevelopmentAdult stem/progenitorsfunctional maturationPCD(apoptosis)Diffe
2、rentiationDeathSenescenceESCschronological agingStem Cells&CancerThree tumor biology puzzles:1.Most tumors are of a clonal origin but tumor cells are heterogeneous.2.It is very difficult to establish stable tumor cell lines from tumors.3.Large numbers of established tumor cells have to be injected t
3、o re-initiate an orthotopic tumor in mice.Key reviews:1.Reya T et al.Stem cells,cancer,and cancer stem cells.Nature 414,105-111,2001.2.Dick JE.Stem cell concepts renew cancer research.Blood 112:4793-4807,2008.3.Visvader JE,and Lindeman GJ.Cancer Stem Cells:Current Status and Evolving Complexicities.
4、Cell Stem Cell 10:717-728,2012.4.4.Tang DG.Understanding cancer stem cell heterogeneity and plasticity.Cell Res,22(3):457-472,2012.5.5.Magee JA,Piskounova E,&Morrison SJ.Cancer Stem Cells:Impact,Heterogeneity,6.and Uncertainty.Cancer Cell 21:283-296,2012.7.(Dean Tang,Basic Concepts of Tumor Biology,
5、Oct 31,2012)1.Characteristics&Definition2.SC Identification3.SC Niche&Plasticity4.SCs&Cancer5.Cancer Stem Cells(CSCs)Stem Cells&Cancer-Rare-Generally small-Normally localized in a protected environment called NICHE,where they only occasionally divide.-But they possess HIGH PROLIFERATIVE POTENTIAL an
6、d 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).S
7、tem CellsCommitted progenitor cellssymmetric SC renewalasymmetric celldivision(ACD)symmetric SC commitment (differentiation)SCTang,Cell Res.2012LT-SCST-SCLateprogenitorsDifferen-tiated cellsDifferen-tiating cellsEarlyprogenitorsProliferationDifferentiationTransformationprobabilitySelf-renewalNicheCo
8、mmitmentDifferentiationExpansionTang,Cell Res.2012Cell lineage development:Self-renewal,proliferation,&differentiationMouse 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
9、 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
10、Cells(ESCs)Leftover or dead-end IVF embryos(PGD)How can hES cells be derived?16-cell morulaPrimitive ectodermTrophectodermPrimitive EndodermA.NagyES cellsA.NagyTS cellsA.NagyA.NagyA.NagyA.NagyheartpancreastestisliverbrainkidneyA.NagyDerived from umbilical cord Primarily blood stem cellsAlso contain
11、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 cellsCord Blood Stem Cells(CB-SC)Germline Stem Cells(GSC)Other embryonic SCs1.Characteristics&Definitio
12、n2.SC Identification3.SC Niche&Plasticity4.SCs&Cancer5.Cancer Stem Cells(CSCs)Stem Cells&CancerFunctional Assays of Stem Cells(Candidate)Stem CellsStem Cells in situ(Xeno)TransplantationLineage tracingHow to identify and characterize(adult)stem cells?1.Marker analysis2.Label-retaining cells(LRC):Pul
13、se-chase exper.3.Clonal/clonogenic assays4.Functional analysis:Side population(SP)assay5.Functional analysis:Aldefluor assay6.Cell size-based enrichment7.Genetic marking&lineage tracing Passegu,Emmanuelle et al.(2003)Proc.Natl.Acad.Sci.USA 100,11842-11849Hematopoietic stem/progenitor cell lineages(1
14、: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 14,213-222,1961.Lin-CD34+CD3
15、8-CD45RA-Thy1+RholoCD49f+(Notta F.Dick JE.Science 333,218-221,2011)(Nestin)(GFAP)(Pax6)(A2B5)(NG2)(MBP)(NeuM)(Mash-1)(PDGFRa a)Sue FischerHow to identify and characterize(adult)stem cells?1.Marker analysis2.Label-retaining cells(LRC):Pulse-chase exper.3.Clonal/clonogenic assays4.Functional analysis:
16、Side population(SP)assay5.Functional analysis:Aldefluor assay6.Cell size-based enrichment7.Genetic marking&lineage tracing Till JE&McCulloch EA.A direct measurement of the radiation sensitivity of normal mouse bone marrow cells.Radiat.Res 14,213-222,1961.The earliest report in which putative stem ce
17、lls 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-cycling libmal epithelial basal cells that can be preferentially stimulated to proliferate:Implications on epithelial stem cells.Cell 57,201-
18、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 cycle,and skin carcinogenesis.Cell 61,1329-1337,1990.LRCs in the Bulge&BM ARE Stem CellsTumbar T et al.,Defining the epithelial stem cell niche in s
19、kin.Science 303,359-363,2004.Blanpain,C.,et al.,Self-renewal,multipotency,and the existence of two cell populations 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.Fu
20、chs E&Horsley V.Ferreting out stem cells from their niches.Nat Cell Biology 13:513-518,2011.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
21、cycling hematopoietic stem cells.Nat.Biotechnol.27,84-90,2009.Not 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
22、 the ones that do,approximately only 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 CellsHow to identify and characterize(adult)stem c
23、ells?1.Marker analysis2.Label-retaining cells(LRC):Pulse-chase exper.3.Clonal/clonogenic assays4.Functional analysis:Side population(SP)assays5.Functional analysis:Aldefluor assay6.Cell size-based enrichment7.Genetic marking&lineage tracing ERheinwald JG&Green H.Serial cultivation of human epidermal
24、 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 50,1131-1137,1987Rice RHCell 11,417-422,1977Cell 18,681-
25、694,1979Watt FMJCB 90,738-742,1981CLONAL 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 dilutionHolocloneMero-or paraclonea.Cloning efficiency(CE;%)b.Clonal siz
26、e(cell number/clone)c.Clonal development(tracking)d.Clone typesA clone:a two-dimensional structurePlating efficiencyProlif.potential ClonogenicIn-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(track
27、ing)d.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 fibroblastsMixing Experiments to Demonstrate the Clonality of Clones/SpheresDU145 RFP:D
28、U145 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 stem cells.Cell Stem Cell 8,486-498,2011How to identify and characterize(adult)stem cells?1.Marker analysis2.Label-
29、retaining cells(LRC):Pulse-chase exper.3.Clonal/clonogenic assays4.Functional analysis:Side population(SP)assays5.Functional analysis:Aldefluor assay6.Cell size-based enrichment7.Genetic marking&lineage tracing Goodell MA et al.Isolation and functional properties of murine hematopoietic stem cells t
30、hat are replicating in vivo.J.Exp.Med.183,1797-1806,1996.Golebiewska A,Brons NH,Bjerkvig R,and Niclou SP.Critical appraisal of the side population assayin stem cell and cancer stem cell research.Cell Stem Cell 8,136-147,2011.Bcrp(ABCG2)is a major mediator of the SP phenotypeZhou et al.,Nature Med 7,
31、1028,2001How to identify and characterize(adult)stem cells?1.Marker analysis2.Label-retaining cells(LRC):Pulse-chase exper.3.Clonal/clonogenic assays4.Functional analysis:Side population(SP)assays5.Functional analysis:Aldefluor assay6.Cell size-based enrichment7.Genetic marking&lineage tracing Aliso
32、n MR,Guppy NJ,Lim SML,&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
33、isozymes 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
34、of elevated 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 ac
35、tivity.PNAS 96,9118-9123,1999.How to identify and characterize(adult)stem cells?1.Marker analysis2.Label-retaining cells(LRC):Pulse-chase exper.3.Clonal/clonogenic assays4.Functional analysis:Side population(SP)assays5.Functional analysis:Aldefluor assay6.Cell size-based enrichment7.Genetic marking&
36、lineage tracing Fuchs E&Horsley V.Ferreting out stem cells from their niches.Nature Cell Biology 13:513-518,2011.Fuchs E&Horsley V.Ferreting out stem cells from their niches.Nature Cell Biology 13:513-518,2011.Liver J et al.,Transgenic strategies for combinatorial expression of fluorescent proteins
37、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 143,134-144,2010.1.Characteristics&Definition2.SC Identification3.SC Niche&Plasticity4.SCs&Cancer5.Cancer Stem Cells(CSCs)Ste
38、m Cells&CancerStem Cell Niche in Hair Follicles:The BulgeMoore KA&Lemischka IR.Science 311,1880-1885,2006Bulge Stem CellsTumbar et al.,Science 303,359-363,2004;Fuchs et al.,Cell 116,769,2004Stem Cell Niche in Small Intestine:The CryptMoore KA&Lemischka IR.Science 311,1880-1885,2006Barker N et al.,Ce
39、ll Stem Cell 11:452-460,2012.Stem 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 unique b
40、one marrow niche.Nature 466,829-834,2010.Stem cell lineageDifferentiatedcellsDeath(PCD)SenescenceStem cellsProgenitors/Precursor cellsOther differ.cell(s)*First report:Long-term cultured adult brain(stem)cells can reconstitute the whole blood in lethally irradiated mice(Bjornson et al.,Science 283,5
41、34-537,1999).*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 e
42、t 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;Lagass
43、e 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 o
44、f 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.,N
45、ature 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 bil
46、e 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 CellsDe-differentiation:Cell-cycle re-entry*Many post-mitotic cells such as hepatocytes,endothelial
47、 cells,and Schwann cells have longbeen 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 dedi
48、fferentiation 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 cell
49、s 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 o
50、ver 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 di
