• •• • 1Stem cells: units of development and regenerationFernando D. Camargo Ph.D.Whitehead FellowWhitehead Institute for Biomedical ResearchConcepts 1. Embryonic vs. adult stem cells2. Hematopoietic stem cells3. The stem cell niche4. Cancer stem cells• •• • 2What are stem cells? Units of development and regenerationWhy are stem cells special? 1. They are primitive2. They can self-renew3. They can produce differentiated cells• •• • 3LiverGutPancreasBloodMuscleBoneNervous systemSkinDifferentiationHuman development: a stem cell hierarchyEndodermMesodermEctodermEmbryonic stem cellsDevelopmental potentialHSCThymusT cellsB cellsNK cellsNeutrophilEosinophilErythrocyteMegakaryocyteMast cellMacrophageDevelopmental hierarchies:the hematopoietic systemDifferentiationDevelopmental potential• •• • 4Embryonic stem (ES) cells arepluripotentialHuman ES cells (Thompson et al, 1998)Gut Neural Bone Muscle• •• • 5Embryonic versus adult• Isolated from earlyembryos• Can expandindefinitely in culture• Can give rise to allcell types• Isolated from adulthuman tissue• Can not beexpanded in culture• Can only give rise tosame tissueThe hematopoietic stemcell (HSC)• First stem cell to be isolated and studied• Most of what we know about stem cellscomes from studies with HSCs• First and only stem cell used in the clinic• Discovered by Till and McCulloch in 1961(winners of the 2005 Lasker Award)• •• • 6The 1960s: stem cells, ahypothetical concept• Several lineages in the blood: lymphoid, myeloid,erythroid. How were these derived? Multipotentprogenitor or single lineage progenitor?• Jacobson: shielding the spleen from total bodyirradiation or infusion of BM cells providedradioprotection. Cellular or humoral effects?Till and McCulloch:Those famous bumps• •• • 7Red cells, platelet progenitors and granulocytes Spherical shape suggested that each nodule was from a single founder cell - maybe the long sought stem cell?? HOW TO PROVE????First demonstration of clonality of a stem cellIrradiate BM cellsInduce randomchromosomalaberrationsClonality Analyze karyotypeof all cells within acolonyAll cell types within one colony (red cells, platelets,granulocytes) had the same chromosomal aberrations!!!!• •• • 8First demonstration of stem cell self-renewalA single colony, upon secondary transplantation, gives riseto functionally equivalent coloniesFirst demonstration of stem cell self-renewalCriteria established by Till and McCulloch are still used todefine a stem cell nowadays:Clonality, self-renewal, differentiation• •• • 9Is there a universal blood stem cell that gave riseto both myeloid and lymphoid cell lineages?Keller and coworkers (1985)Infected bone marrow cells with a virus containing neoR gene - virus insets itself randomly into the bone marrow cell genome - recombinant bone marrow cells are resistant to neomycin- can select the infected cells - virus is replicated in the same genomic position at each celldivision - provides each cloned bone marrow cells with a uniquemarkerThey injected the engineered bone marrow cells into an irradiatedmouse and analyzed the resulting recolonization of the hematopoieticsystemRetroviral markingTransduce BMTransplantIsolate bloodlineagesSouthern analysesT B MT B M T B M1 month3 months 1 yearTBM• •• • 10Retroviral marking studies: lessons learnedExistance of two types of stem cells : long-term and short-termHSCsOnly a few LT-HScs are enough to sustain hematopoiesis forlife (in primary and secondary recipients)All LT-HSCs are multipotent, no unilineage HSCs• •• • 111990’s: Focus on the prospective isolation of HSCsUse of monoclonal antibodies and fluorescence-activated cell sorting(FACS)-Isolation of homogeneous populations of LT, ST-HSCs, and all committedprogenitors-Single-physically isolated HSCs could reconstitute the entire blood systemof a mouseGeneral characteristics ofadult stem cells• Stem cells are very rare• Only stem cells have the ability to self renew• Stem cells divide very infrequently• •• • 12Not all adult tissues areregenerated by stem cellsLiver also: are thesedifferentiated cells stem cells?Embryonic versus adult• Isolated from earlyembryos• Can expandindefinitely in culture• Can give rise to allcell types• Isolated from adulthuman tissue• Can not beexpanded in culture• Can only give rise tosame tissue• •• • 13Stem CellPlasticityFrom Blau et al.(C e ll, 2000)Can adult stem cells learn new tricks?• •• • 14Adult neural stem cells can contribute to allthree germ layers Isolate NSCsInject into blastocystTransplanted HSCs can generate liver cellsTransplant BMTO SWAP OUT!Analyze liver forDonor-derived cells• •• • 15Potential therapeutic implicationsof “plasticity”• Transplantation of bone marrow formuscular dystrophies• Direct injection of bone marrow stemcells for cardiac regeneration• Bone marrow injections forneurodegenerative diseasesWhat is the mechanism for plasticity? HSC• •• • 16Reevaluating the role ofHSCs in plasticity• Muscle and liver engraftment require previous BM and bloodengraftment by donor HSCs.• HSCs directly injected into the liver or muscle do not engraft.• Non-hematopoietic engraftment by a single or hundreds ofHSCs is identical.• Experimental models of plasticity require severe injuries, whichresult in extensive recruitment of inflammatory cells.Marking mature hematopoieticcells in vivoLineage-CrelacZrxCD19-Cre = B cellsLck-Cre = T cellsLysM-Cre = MyeloidCre reporter• •• • 17Lineage tracing in miceHSCThymusT cellsB cellsNK cellsNeutrophilEosinophilErythrocyteMegakaryocyteMast cellMacrophageDo differentiated myeloid cellscontribute to muscle?Injure TA muscleLysCre/R26RPos ctrl.Neg CtrlLysCre/lacZr• •• • 18Purified macrophages fusewith myofibersIsolate macrophagesMLacZMDXMyeloid cells regenerate liver Pos. R26!FAH Wild typeLysCre/R26R!"#$• •• • 19What is the mechanism for plasticity? Cell fusionTrans-differentiationBM-derived hepatocytes arise through fusion ofmyeloid cellsCD45.1CD45.1LysCreR26R/FAHX-galDonorRecipient#of lacZposnodulesRosa26(n=5)FAH279 ± 38LysM-Cre(n=2)FAH/R26R239 ± 43• •• • 20Cell fusion underlies plasticityTotal engraftmentBM-derivedengraftmentCell fusion underlies plasticity80 XXXY 120XXXXYYXY into XX transplants• •• • 21Lessons