Handbook of Stem Cells

1013 Pages·2012·39.189 MB·English

Chapter 1
Why Stem Cell Research?
Advances in the Field
Alan Trounson
CaliforniaInstituteforRegenerativeMedicine,SanFrancisco,CA,USA
Despite the escalating costsof newdrugs producedby the medicine. The potential was quickly underlined by Tony
pharmaceutical industry, which is approaching US$4 Atala’s demonstration of engineering bladder for patients
billionforeachnewmarketabledrug(Munos,2009),stem (Atala, 2010).
cellresearchthataimsatdevelopingnewcelltherapieshas Basic scientists gathered around Len Zon to form and
accelerateddramaticallyintermsofthebreadthofinterests launch the International Stem Society of Stem Cell
andthediscoveriesthatcontinuetoevolvewithapacethat ResearchSociety,aformidablegroupofthebestscientists
is astonishing. Research in stem cell biology is opening in stem cell science. Cell and tissue transplant scientists
new platforms to launch even more spectacular develop- have remained largely separate but are another effective
mentsandthesecrowdthepagesofthemajorjournalseach science and therapeutic organization under the Interna-
month. One might wonder why the field took so long to tional Society Cell Therapy. Separately the stem cell
explodeinsuch anincrediblefashion. ThestudiesofJohn biotechnologyindustryhasjoinedtogethertheumbrellaof
Gurdon and colleagues (Gurdon, 1962) on reprograming the Alliance for Regenerative Medicine to become an
amphibian cells using oocytes, stand as a very significant effective advocate for the emerging industry interests in
milestone that was emphatically amplified by Ian Wilmut cellandtissue therapies.
and his colleagues (Campbell et al., 1996), who unex- The Bush administration in the USA raised concerns
pectedly reprogrammed mammalian somatic cell nuclei with the fledgling stem cell science community by
intototipotentembryoswhenintroducedintooocytesofthe restrictingthefundingofembryonicstemcellresearchand
same species. Martin Evans and colleagues (Evans, 2008) limitingthenumberofembryonicstemcelllinesthatcould
showed that early embryonic cells of the blastocyst stage be studied with federal funding. The key scientists of
could be converted to pluripotent embryonic stem cells. California coopted Robert (Bob) Klein, a financier and
Travelingonanindependentplaneofdiscoveryweremany lawyer to their cause and he was able to galvanize the
great scientists of whom Irv Weismann stands out for his Californianvoterstopass(59%support)Proposition71e
discoveries in adult hematopoietic stem cells in mice and a game changing state bond initiative that required
humans (Spangrude et al., 1988; Weissman et al., 2001). Californiatosellgeneralobligationbondsupto$3billion
Bone marrow transplants have been long established as to fund pluripotent stem and progenitor cell research
atherapeutic strategyfor blood cancersand diseases. (Klein and Trounson, 2010). This extremely cleaver
WhatameltingpotofingredientsforJamesThompson approach to funding intellectual capital was supported by
to launch the discovery of human embryonic stem cells the Republican Governor Arnold Schwarzenegger and
(Thompson et al., 1998), cloning for stem cells in the established the Californian Institute for Regenerative
mousebymembersofmyowngroup(Munsieetal.,2000), Medicine (CIRM). Californian institutions have become
and most significantly the demonstration by Shinya a major hub for stem cell research, attracting many of the
Yamanaka (Takahashi et al., 2007) of the ability to repro- world’s best scientists in stem cell research, rivaling the
gram somatic cells to pluripotency (induced pluripotent hubs around Boston and New York. Twelve new research
stem cells) using four critical transcription factors. Again institutes have been built in California under CIRM spon-
independently, Art Caplan (Caplan, 1991) isolated mesen- sorship, concentrating the critical mass of intellectual
chymal stem cells from bone marrow, showing their mul- excellence and driving an incredible productivity of
tipotent capacities to form bone, cartilage, and adipose discovery research. Both Thompson and Yamanaka have
tissue. Now we have the ingredients to explore the possi- appointments inCalifornia.Twoclustersofbiotechnology
bility of applying stem cell discoveries to regenerative companies involved in cell therapies have evolved in the
1
HandbookofStemCells,Two-VolumeSet.DOI:http://dx.doi.org/10.1016/B978-0-12-385942-6.00001-9
Copyright(cid:1)2013ElsevierInc.Allrightsreserved.
2 VOLUME | 1 Pluripotent StemCells
Bay Area and San Diego (Institute for Strategy and arefrequentlyunregulated(Tayloretal.,2010).Thereisan
Competitiveness, 2010), with a third expected to be importantconsiderationthatoftenfailsintheseuninformed
recognized shortly in Los Angeles. Companies are relo- commentaries edono harm.
cating to California and are actively opening offices and Stem cell science will ultimately prevail despite the
labs to contribute to the energized environment there. opposition from some quarters because researchers will
CIRM has also developed a very major network of derivetheevidencefortheunderstandingofdisease,andby
collaborations with 12 international countries and states, rigorousdesignandadequatelycontrolledexperimentation
a number of US states, foundations, and, most recently, the truevaluewill bedemonstrated. Ifnot,the hypotheses
with the US National Institutes of Health. These collabo- will fail and wewill moveon.
rations are driving globally a vast array of basic research Iwish Iwere starting again instem cell research.
andtranslationalmedicine.Thisnetworkisgamechanging
thequalityanddepthofaglobalresearchtofindsolutions
to theworld’s most feared and intractable diseases. At the
REFERENCES
front edge of this endeavor are critical studies to find
themeanstoeradicatethemostdangerouscellofallethe
Atala,A.,2011.Tissueengineeringofhumanbladder.Br.Med.Bull.97,
malignancy seeding cancer stem cell in blood and solid
81e104.
tumors. There are also strategies rapidly evolving in
Baraban, S.C., Southwell, D.G., Estrada, R.C., Jones, D.L., Sebe, J.Y.,
translation for the cure of HIV/AIDS, recovery of blind-
Alfaro-Cervello, C., et al., 2009. Reduction of seizures by trans-
ness,potentialcuresforTypeIdiabetes,genetherapyusing
plantationofcorticalGABAergicinterneuronprecursorsintoKv1.1
stem cell vehicles, spinal cord injury, and other motor mutantmice.Proc.Natl.Acad.Sci.U.S.A.106,15472e15477.
neuron and demyelinating diseases. The list of potential Brennand, K.J., Simone, A., Jou, J., Gelboin-Burkhart, C., Tran, N.,
therapiesisexhaustiveandneedstobeaddressedasscience Sangar, S., et al., 2011. Modeling schizophrenia using human
opens an understanding of these diseases. Surprisingly, induced pluripotent stem cells. Nature Oct 19. doi: 10.1038/
iPS cell studies are exposing new insights into mental nature10603.FH.
retardation, autism (Marchetto et al., 2010), epilepsy Campbell, K.H., McWhir, J., Ritchie, W.A., Wilmut, I., 1996. Sheep
cloned by nuclear transfer from a cultured cell line. Nature 380,
(Baraban et al., 2009), and schizophrenia (Brennand
64e66.
et al., 2011). Hope remains strong that cell therapies can
Caplan,A.I.,1991.Mesenchymalstemcells.J.Orthop.Res.9,641e650.
offer substantial benefits to neurodegeneration such as
Evans,M.,2008.Embryonicstemcells:themousesourceeveichlefor
Parkinson’s, Alzheimer’s, and Huntington’sdiseases.
mammaliangeneticsandbeyond(Nobellecture).Chembiochem.9,
Meanwhile the biotechnology industry has began to 1690e1696.
delivertheclinicaltrialsusingadultcells(Trounsonetal., Gurdon,J.B.,1962.Adultfrogsderivedfromthenucleiofsinglesomatic
2011), particularly mesenchymal stem cells, adipose- cells.Dev.Biol.4,256e273.
derivedstromalcells,adultandfetalneuralstemcells,and Ieda, M., Fu, J.D., Delgado-Olguin, P., Vedantham, V., Hayashi, Y.,
other types of cells for an array of applications from soft Bruneau, B.G., et al., 2010. Direct reprogramming of fibroblasts
tissue, bone, heart disease, diabetes, stroke, and gene into functional cardiomyocytes by defined factors. Cell 142,
therapies. 375e386.
Institute for Strategy and Competitiveness, 2010. Cluster mapping
Why wouldn’t there be a strong move of scientists
project.HarvardBusinessSchool.http:data.isc.hbs.edu/isc/.
towards stem cell research with the tools and critical
Klein, R.N., Trounson, A., 2010. A new political-financial paradigm
technologiesthathaveevolved?Itappearsthatendogenous
for medical research: the California model. In: Prescott, C.,
celllineagemaybemanipulatedbyjudicioususeoftissue
Polak, I. (Eds.), The Delivery of Regenerative Medicines and
targeting of key transcription factors. The conversion of
Their Impact on Healthcare. Taylor & Francis Group, LLC,
stromal phenotypes to endocrine (Zhou et al., 2008), London, pp. 11e33.
muscle(Iedaetal.,2010)orneuralcelltypes(Vierbuchen Marchetto,M.C.,Carromeu,C.,Acab,A.,Yu,D.,Yeo,G.W.,Mu,Y.,
etal.,2010)thathavebeenlostindiseaseandinjurycould et al., 2010. A model for neural development and treatment of
be the next major platform of stem and progenitor cell Rett syndrome using human induced pluripotent stem cells. Cell
research. Could these developments sidestep the need to 143, 527e539.
develop transplantation tolerance strategies for enabling Munos, B., 2009. Lessons from 60 years of pharmaceutical innovation.
Nat.Rev.DrugDiscov.8,959e968.
effectivegrafting ofallogeneic cellular therapies?
Munsie, M.J., Michalska, A.E., O’Brien, C.M., Trounson, A.O.,
There remains very vocal and manipulative conserva-
Pera,M.F.,Mountford,P.S.,2000.Isolationofpluripotentembryonic
tive and religious interest groups that decry the potential
stemcellsfromreprogrammedadultmousesomaticcellnuclei.Curr.
benefitsofembryonicstemcellscience,despiteverystrong Biol.10,989e992.
overallcommunitysupportintheUSandelsewhere.They
Singer,T.,McConnell,M.J.,Marchetto,M.C.,Coufal,N.G.,Gage,F.H.,
exclusively support adult stem cell therapies, including 2010. LINE-1 retrotransposons: mediators of somatic variation in
thosewherethereislittlescientificevidenceofbenefitand neuronalgenomes?TrendsNeurosci.33,345e354.
Chapter | 1 Why StemCellResearch?Advances inthe Field 3
Spangrude,G.J.,Helmfeld,S.,Weissman,I.L.,1998.Purificationandchar- Trounson,A.,Thaker,R.,Lomax,G.,Gibbons,D.,2011.Clinicaltrials
acterizationofmousehematopoieticstemcells.Science241,58e62. forstemcelltherapies.BMCMed.109,52.
Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Vierbuchen, T., Ostermeier, A., Pang, Z.P., Kokubu, Y., Su¨dhof, T.C.,
Tomoda, K., et al., 2007. Induction of pluripotent stem cells from Wernig, M., 2010. Direct conversion of fibroblasts to functional
adulthumanfibroblastsbydefinedfactors.Cell131,861e872. neuronsbydefinedfactors.Nature463,1035e1041.
Taylor, P.L., Barker, R.A., Blume, K.G., Cattaneo, E., Colman, A., Weissman, I.L., Anderson, D.J., Gage, F., 2001. Stem and progenitor
Deng, H., et al., 2010. Patients beware: commercialized stem cell cells: origins, phenotypes, lineage commitments, and trans-
treatmentsontheweb.CellStemCell27,43e49. differentiations.Annu.Rev.CellDev.Biol.17,387e403.
Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J., Melton, D.A., 2008.
Swiergiel,J.J.,Marshall,V.S.,etal.,1998.Embryonicstemcelllines In vivo reprogramming of adult pancreatic exocrine cells to beta-
derivedfromhumanblastocysts.Science282,1145e1147. cells.Nature455,627e632.
Chapter 2
“Stemness”: Definitions, Criteria,
and Standards
Douglas Melton
DepartmentofMolecularandCellularBiologyandHowardHughesMedicalInstitute,HarvardUniversity,Cambridge,MA,USA
Chapter Outline
Introduction 5 HowareStemCellsIdentified,Isolated,
WhatIsaStemCell? 5 andCharacterized? 9
Self-Renewal 5 EmbryonicStemCells 9
Clonality 6 AdultStemCells 10
Potency 6 Stemness:ProgressTowardaMolecularDefinition
Definition 6 ofStemCells 10
WhereDoStemCellsComeFrom? 6 Acknowledgments 11
StemCellsoftheEarlyEmbryo 6 References 11
OntogenyofAdultStemCells 7
INTRODUCTION differentiated cells (Weissman et al., 2001; Smith, 2001).
More explicitly, stem cells can generate daughter cells
Stemcellshaverecentlygeneratedmorepublicandprofes-
identical to their mother (self-renewal) as well as produce
sional interest than almost any other topic in biology. One
progeny with more restricted potential (differentiated
reasonstemcellscapturetheimaginationofsomanyisthe
cells).Thissimpleandbroaddefinitionmaybesatisfactory
promise that understanding their unique properties may
forembryonicorfetalstemcellsthatdonotperdureforthe
provide deep insights into the biology of cells as well as
lifetimeofanorganism.Butthisdefinitionbreaksdownin
a path toward treatments for a variety of degenerative
trying to discriminate between transient adult progenitor
illnesses. And although the field of stem cell biology has
cellsthathaveareducedcapacityforself-renewalandadult
grown rapidly, there exists considerable confusion and
stem cells. It is therefore important when describing adult
disagreementastothenatureofstemcells.Thisconfusion
stemcellstofurtherrestrictthisdefinitiontocellsthatself-
canbepartlyattributedtothesometimesidiosyncraticterms
renewthroughoutthelifespanoftheanimal(vanderKooy
and definitions used to describe stem cells. Although defi-
and Weiss, 2000). Another parameter that should be
nitionscanberestrictive,theyareusefulwhentheyprovide
considered is potency: Does the stem cell generate to
a basis for mutual understanding and experimental stan-
multipledifferentiatedcelltypes(multipotent),orisitonly
dardization. With this intention, I present explanations of
capable of producing one type of differentiated cell (uni-
definitions,criteria,andstandardsforstemcells.Moreover,I
potent)? Thus, a more complete description of a stem cell
highlightacentralquestioninstemcellbiology,namelythe
includes a consideration of replication capacity, clonality,
originofthesecells.Ialsosuggestcriteriaorstandardsfor
and potency. Some theoretical as well as practical consid-
identifying,isolating,andcharacterizingstemcells.Finally,I
erations surrounding these concepts are considered in this
summarizethenotionof“stemness”anddescribeitspossible
chapter.
applicationinunderstandingstemcellsandtheirbiology.
Self-Renewal
WHAT IS A STEM CELL?
Stem cell literature is replete with terms such as
Stem cells are defined functionally as cells that have the “immortal,” “unlimited,” “continuous,” and “capable of
capacity to self-renew as well as the ability to generate extensive proliferation,” all used to describe the cell’s
5
HandbookofStemCells,Two-VolumeSet.DOI:http://dx.doi.org/10.1016/B978-0-12-385942-6.00002-0
Copyright(cid:1)2013ElsevierInc.Allrightsreserved.
6 VOLUME | 1 Pluripotent StemCells
replicativecapacity.Theseratherextremeandvagueterms from a tissue if they are cellular preparations containing
are not very helpful, as it can be noted that experiments a mixed population, possibly contaminated by stem cells
designedtotestthe“immortality”ofastemcellwouldby from another tissue.
necessity outlast authors and readers alike. Most somatic
cells cultured invitro display a finite number of (less than
Potency
80) population doublings prior to replicative arrest or
senescence, and this can be contrasted with the seemingly The issue of potency may be the most contentious part of
unlimited proliferative capacity of stem cells in culture a widely accepted definition for stem cells. A multipotent
(Houck et al., 1971; Hayflick, 1973, 1974; Sherr and stem cell sits atop a lineage hierarchy and can generate
DePinho, 2000; Shay and Wright, 2000). Therefore, it is multiple types of differentiated cells, the latter being cells
reasonable to say that a cell that can undergo more than with distinct morphologies and gene expression patterns.
twice this number of population doublings (160) without At the same time, many would argue that a self-renewing
oncogenic transformation can be termed “capable of cell that can only produce one type of differentiated
extensive proliferation.” In a few cases, this criterion has descendantisnonethelessastemcell(Slack,2000).Acase
been met, most notably with embryonic stem (ES) cells can be made, for clarity, that a unipotent cell is probably
derived from either humans or mice as well as with adult bestdescribedasaprogenitor.Progenitorsaretypicallythe
neural stem cells (NSCs) (Smith, 2001; Morrison et al., descendants of stem cells, only they are more constrained
1997).Anincompleteunderstandingofthefactorsrequired intheirdifferentiationpotentialorcapacityforself-renewal
forself-renewalexvivoformanyadultstemcellsprecludes andare oftenmorelimited inboth senses.
establishing similar proliferative limits in vitro. In some
cases, a rigorous assessment of the capacity for self-
Definition
renewal of certain adult stem cells can be obtained by
single-cell or serial transfer into acceptable hosts, an In conclusion, a working definition of a stem cell is
excellent example of which is adult hematopoietic stem a clonal, self-renewing entity that is multipotent and thus
cells (HSCs) (Allsopp and Weissman, 2002; Iscove and can generate several differentiated cell types. Admittedly,
Nawa, 1997). Adult stem cells are probably still best this definition is not applicable in all instances and is best
defined in vivo, where they must display sufficient prolif- used as aguide tohelp describecellular attributes.
erative capacity to last the lifetime of the animal. Terms
suchas“immortal”and“unlimited”areprobablybestused
WHERE DO STEM CELLS COME FROM?
sparinglyif at all.
TheoriginorlineageofstemcellsiswellunderstoodforES
cells; their origin in adults is less clear and in some cases
Clonality
controversial. It may be significant that ES cells originate
A second parameter, perhaps the most important, is the before germ layer commitment, raising the intriguing
idea that stem cells are clonogenic entities: single cells possibility that this may be a mechanism for the develop-
withthecapacitytocreatemorestem cells.Thisissuehas mentofmultipotentstemcells,includingsome adultstem
beenexhaustivelydealtwithelsewhereandisessentialfor cells. The paucity of information on the developmental
any definitive characterization of self-renewal, potential, originsofadultstemscellsleavesopenthepossibilitythat
and lineage. Methods for tracing the lineage of stem cells theytooescapelineagerestrictionintheearlyembryoand
are described in subsequent chapters. Although the clonal subsequentlycolonizespecializedniches,whichfunctionto
“gold standard” is well understood, there remain several bothmaintaintheirpotencyaswellasrestricttheirlineage
confusing practical issues. For instance, what constitutes potential. Alternatively, the more widely believed, though
a cell line? The lowest standard would include any pop- stillunsubstantiated,modelfortheoriginofadultstemcells
ulation of cells that can be grown in culture, frozen, assumesthattheyarederivedaftersomaticlineagespecifi-
thawed, and subsequently repassaged in vitro. A higher cation, whereupon multipotent stem cellseprogenitors
standard would be a clonal or apparently homogeneous arise and colonize their respective cellular niches. In this
population of cells with these characteristics, but it must section,Ibrieflysummarizetheoriginofstemcellsfromthe
be recognized that cellular preparations that do not derive earlyembryoandexplainwhatisknownabouttheontogeny
from a single cell may be a mixed population containing ofadultstemcellsfocusingattentiononHSCsandNSCs.
stem cells and a separate population of “supportive” cells
required for the propagation of the purported stem cells.
Stem Cells of the Early Embryo
Hence, any reference to a stem cell line should be made
withanexplanationoftheirderivation.Forexample,itcan Mouse and human ES cells are derived directly from the
be misleading to report on stem cells or “stem cell lines” inner cell mass of preimplantation embryos after the
Chapter | 2 “Stemness”:Definitions, Criteria,and Standards 7
formationofacysticblastocyst(Papaioannou,2001).This be the case that adult stem cells are derived from PGCs.
population of cells would normally produce the epiblast Although intriguing, it is important to stress that this idea
andeventuallyalladulttissues,whichmayhelptoexplain lacksexperimental evidence.
thedevelopmentalplasticityexhibitedbyEScells.Infact,
EScellsappeartobetheinvitroequivalentoftheepiblast,
Ontogeny of Adult Stem Cells
as they have the capacity to contributeto all somatic line-
ages and in mice to produce germ-line chimeras. By the The origin of most adult stem cells is poorly understood.
time the zygote has reached the blastocyst stage, the Withtheissueofadultstemcellplasticityattheforefront,
developmentalpotentialofcertaincellshasbeenrestricted. as described in this section, studies designed to elucidate
Theoutercellsoftheembryohavebeguntodifferentiateto the ontogeny of adult stem cells may help to reveal their
form trophectoderm, from which a population of embry- specific lineage relationships and shed light on their plas-
onic trophoblast stem cells has also been derived in mice ticityandpotential.Informationontheoriginsofadultstem
(Tanaka et al.,1998).These specializedcellscan generate cells would also help to define the molecular programs
all cell types of the trophectoderm lineage, including involved in lineage determination, which may in turn
differentiated giant trophoblast cells. At the egg cylinder provide insights into methods for manipulating their
stageofembryonicdevelopment(embryonicday(E)6.5in differentiation. To this end, I summarize what is known
mice), a population of cells near the epiblast (Figure 2.1) about the development of adult stem cells within the
can be identified as primordial germ cells (PGCs), which contextof the hematopoietic andneuralsystems.
are subsequently excluded from somatic specification or Thedevelopmentofhematopoieticcellsinmiceoccurs
restriction (Saitou et al., 2002). PGCs migrate to and soon after gastrulation (E7.5), although HSCs with the
colonize the genital ridges, where they produce mature same activities as those in the adult have only been
germ cells and generate functional adult gametes. PGCs observed and isolated at midgestational stages (E10.5)
canbeisolatedeitherpriororsubsequenttotheirarrivalin (Orkin, 1996; Dzierzak, 2002; Weissman, 2000). These
the genital ridges and, when cultured with appropriate observations suggest that the embryo has a unique hema-
factors in vitro, can generate embryonic germ (EG) cells topoietic lineage hierarchy, which may not be founded by
(Matsui et al., 1992; Resnick et al., 1992). EG cells have anadulttypeHSC.Thus,hematopoiesisappearstooccurat
manyofthecharacteristicsofEScellswithrespecttotheir multiple times or in successive waves within the embryo,
differentiation potential and their contribution to the germ and the emergence of an HSC may not precede or be
lineofchimericmice(Laboskyetal.,1994;Stewartetal., concomitant with the appearance of differentiated hema-
1994). The most notable difference between ES and EG topoietic cells.
cells is that the latter may display (depending upon the Thefirstsiteofhematopoiesisinthemouseistheextra-
developmental stage of their derivation) considerable embryonic yolk sac, soon followed by the intraembryonic
imprinting of specific genes (Surani, 1998, 2001; Howell aortaegonademesonephros(AGM)region.Whichofthese
et al., 2001). Consequently, certain EG cell lines are inca- sitesleadstothegenerationoftheadulthematopoieticsystem
pable ofproducingnormal chimeric mice. and, importantly, HSCs is still unclear. Results from non-
Importantly, no totipotent stem cell has been isolated mammalian embryo-grafting experiments, with various
from the early embryo. ES and EG cells generate all findingsinthemouse,suggestthatthemammalianembryo,
somatic lineages as well as germ cells but rarely if ever specifically the AGM, generates the adult hematopoietic
contribute to the trophectoderm, extraembryonic endo- systemandHSCs(KauandTurpen,1983;Medvinskyetal.,
derm, or extraembryonic mesoderm. Trophectoderm stem 1993; Medvinsky and Dzierzak, 1996). Interestingly, the
(TS)cellshavebeenisolated,andtheseonlygeneratecells midgestationalAGMisalsotheregionthatharborsmigrating
ofthetrophectodermlineage.Itremainstobeseenwhether PGCsandisthoughttoproducepopulationsofmesenchymal
cells can be derived and maintained from totipotent stemcells,vascularprogenitors,andperhapshemangioblasts
embryonicstages.Althoughourunderstandingofcellfates (Molyneauxetal.,2001;Minasietal.,2002;Alessandrietal.,
in the early embryo is incomplete, it appears that the only 2001;Haraetal.,1999;Munoz-Chapulietal.,1999).Inthe
pluripotent stem cells found after gastrulation are PGCs absenceofstudiesdesignedtoclonallyevaluatethelineage
(with the possible exceptions of multipotential adult potential of cells from the AGM, and without similarly
progenitorcells(Jiangetal.,2002)andteratocarcinomas). accuratefatemappingofthisregion,itremainspossiblethat
ItmaybethatPGCsescapegermlayercommitmentduring alloftheadultstemcelltypesthoughttoemergewithinthe
gastrulation by developing near the epiblast and subse- AGM arise from a common unrestricted precursor. This
quentlymigratetopositionsinsidetheembryoproper.This hypotheticalprecursorcouldhelptoexplainreportsofnon-
developmentalstrategymaynotbeuniquetoPGCs,andit fusion-basedadultstemcellplasticity.Theobservedlineage
raisestheinterestingpossibilitythatotherstemcellsmight specificityofmostadultstemcellscouldlikewisebeattrib-
have similar developmental origins. Alternatively, it may utedtothehigh-fidelitylineagerestrictionimposedonthem
8 VOLUME | 1 Pluripotent StemCells
(A) E7.5 Embryo
Visceral Visceral
Chorion Altantois
Endoderm Endoderm
Amnion
PGCs
Mesendoderm Niche/
Support Cells
Head Primitive
Process Streak
V
Primitive
A P
Streak
Node
D
(B) nervous tissue/NSCs
ectoderm
skin/skin SCs
bone marrow and blood/HSCs and MSCs
gastrulation
mesoderm
muscle and bone/tissue SCs
lung, liver, and pancreas/organ specific SCs
1 endoderm
esophagus, stomach, intestine/intestinal SCs
primordial germ cells
nervous tissue
ectoderm
skin
gastrulation bone marrow, blood
mesoderm
muscle, bone
lung, liver, pancreas
endoderm
esophagus, stomach, intestine
2
primordial germ cells
NSCs/skin SCs
multipotent stem cells HSCs/MSCs/tissue SCs
organ SCs/intestinal SCs
Chapter | 2 “Stemness”:Definitions, Criteria,and Standards 9
by the specific niche they colonize or are derived from. There are several caveats that must be considered when
Simple ideas such as these have not been ruled out by describing the developmental origins of NSCs. First, dis-
experimental evidence, underscoring both the opportunity rupting the neuroepithelia to purify NSCs may have the
and the necessity for further study of the developmental undesirable effect of dysregulating spatial patterning
originsofadultstemcells. acquiredbythesecells.Second,growthofpurifiedNSCsin
A key lesson from studies of the developing hemato- culture may reprogram the stem cells through exposure to
poieticsystemisthattheappearanceofdifferentiatedcells nonphysiologicalinvitrocultureconditions. Bothofthese
doesnottelluswhereorwhenthecorrespondingadultstem problemscanbeaddressedeitherbyinvivolineagetracing
cells originate. Definitive lineage tracing, with assays of orbyprospectivelyisolatingNSCsandtransplantingthem
clonogenic potential, remains the method of choice for intoacceptablehostswithoutinterveningculture.Carefully
identifying the origin of stem cells. Another potential designed experiments promise to answer questions impor-
pitfallrevealedbythesestudiesisthatthedefinitionofthe tant not only for stem cell biology but also for neuro-
stem cell can make all the difference inits identification. embryology and development. These include which
ThedevelopmentofNSCsbeginswiththeformationof features of the developmental program are intrinsic to
nervous tissue from embryonic ectoderm following individualcells,whichdifferentiationorpatterningsignals
gastrulation. Induction of the neural plate is thought to act exclusively to instruct specific cell fates, and how
coincidewiththeappearanceofNSCsaswellasrestricted developmental changes in cell-intrinsic programs restrict
progenitor types(Temple, 2001).Theexactfrequencyand the responses of progenitorstocell-extrinsic signals.
location of stem cells within the developing neuro-
epithelium remains unknown; specific markers must be
HOW ARE STEM CELLS IDENTIFIED,
discoveredtofullyunravelthisquestion.Anemergingview
ISOLATED, AND CHARACTERIZED?
inthefieldisthatembryonicneuroepitheliagenerateradial
glial that subsequently develop into periventricular astro- Howstemcellsareidentified,isolated,andcharacterizedare
cytesandthatthesecellsaretheembryonicandadultNSCs the key methodological questions in stem cell biology, so
within the central nervous system (Alvarez-Buylla et al., muchsothatsubsequentchaptersaredevotedtoaddressing
2001; Tramontin, 2003; Doetsch et al., 1999; Gaiano and theseproblemsindetail.Here,Ibrieflyoutlinestandardsand
Fishell, 2002). Developing and adult NSCs also appear to criteria that may be employed when approaching the chal-
acquirepositionalandtemporalinformation.Forexample, lengeofidentifying,isolating,andcharacterizingastemcell.
stem cells isolated from different neural regions generate
region-appropriateprogeny(Kalyanietal.,1998;Heetal.,
Embryonic Stem Cells
2001; Anderson et al., 1997). In addition, several studies
suggestthattemporalinformationisencodedwithinNSCs, ThebasiccharacteristicsofanEScellincludeself-renewal,
thatearlierstemcellsgiverisemorefrequentlytoneurons, multilineage differentiation in vitro and in vivo, clonoge-
andthatmorematurestemcellspreferentiallydifferentiate nicity, a normal karyotype, extensive proliferation invitro
into glia (Temple, 2001; Qian et al., 2000; White et al., underwell-definedcultureconditions,andtheabilitytobe
2001). Moreover, more mature NSCs appear incapable frozenandthawed.Inanimalspecies,invivodifferentiation
of making cells appropriate for younger stages when can be assessed rigorously by the ability of ES cells to
transplanted into the early cerebral cortex (Desai and contribute to all somatic lineages and produce germ-line
McConnell, 2000). Thus, the nervous system appears to chimerism.ThesecriteriaarenotappropriateforhumanES
follow a classical lineage hierarchy, with a common cells; consequently, these cells must generate embryoid
progenitorcellgeneratingmostifnotalldifferentiatedcell bodies and teratomas containing differentiated cells of all
types in a regional- and temporal-specific manner. There threegerm layers. Moreover, as a stringent invivo assess-
mayalsoberarestemcellsinthenervoussystem,perhaps mentofpluripotencyisimpossible,humanEScellsmustbe
notofneuralorigin,thathavegreaterplasticityintermsof showntobepositiveforwell-knownmolecularmarkersof
producingdiversesomaticcelltypesandlackingtemporal pluripotent cells. These markers are defined as factors
and spatial constraints (Weissman, 2000; Temple, 2001). expressed consistently, and enriched, in human ES cells
:
FIGURE2.1 (A)Developmentofprimordialgermcells.Aschematicofanembryonicday7.5mouseembryohighlightsthepositionofthedeveloping
primordialgermcells(PGCs)proximaltotheepiblast.TheexpandedviewontherightservestoillustratethepointthatPGCsescapelineagecommitment/
restrictionbyavoidingthemorphogeneticeffectsofmigratingthroughtheprimitivestreakduringgastrulation.(B)Putativedevelopmentalontogenyof
stemcells.Inlineagetree1,thedevelopmentofstemcellsoccursaftertheformationofgermlayers.Thesestemcellsarethusrestrictedbygermlayer
commitmentto theirrespectivelineage(e.g.,mesodermisformed,givingriseto hematopoietic progenitorsthatbecomehematopoietic stemcells).
Lineagetree2illustratestheideathatstemcellsmightdevelopsimilarlytoPGCs,inthattheyavoidthelineagecommitmentsduringgastrulationand
subsequentlymigratetospecifictissueandorganniches.
10 VOLUME | 1 Pluripotent StemCells
(Brivanlou et al., 2003). As a substitute for whole-animal Experimentsdesignedtocarefullyevaluatethesepossibili-
chimerism, human ES cells could be tested for their tieswillyieldinsightintothenatureofstemcells.
contributions to specific tissues when transplanted in
discrete regions of nonhuman adults or embryos. A
complementary analysis might include transplanting STEMNESS: PROGRESS TOWARD
human ES cells into nonhuman blastocysts and evaluating
A MOLECULAR DEFINITION
theircontributiontovariousorgansandtissues,thoughthis
OF STEM CELLS
experiment has raised ethical concerns in some quarters.
Finally,a practical consideration isthe passagenumberof Stemness refers to the common molecular processes
EScells.Althoughitisimportanttoestablishthecapacity underlyingthecorestemcellpropertiesofself-renewaland
ofEScellstoproliferateextensively,itisequallyimportant the generation of differentiated progeny. Although stems
that low-passage cells are evaluated experimentally to cellsindifferentcellularmicroenvironmentsornicheswill
guard against any artifacts introduced through in vitro by necessity have different physiological demands and
manipulation. therefore distinct molecular programs, there are likely
certain geneticcharacteristicsspecific toandsharedbyall
stem cells. Through transcriptional profiling, many of the
Adult Stem Cells
genes enriched in ES cell, TS cell, HSC, and NSC pop-
The basic characteristics of an adult stem cell are a single ulations have been identified (Ivanova et al., 2002; Ram-
cell (clonal) that self-renews and generates differentiated alho-Santos et al., 2002; Tanaka et al., 2002; Anisimov
cells.Themostrigorousassessmentofthesecharacteristics et al., 2002; Luo et al., 2002; Park et al., 2002). By
is to prospectively purify a population of cells (usually by extending this approach to other stem cells and more
cell surface markers), transplant a single cell into an organisms, it may be possible to develop a molecular
acceptable host without any intervening in vitro culture, fingerprintforstemcells.Thisfingerprintcouldbeusedas
and observe self-renewal and tissue, organ, or lineage thebasisforamoleculardefinitionofstemcellsthat,when
reconstitution. Admittedly, this type of in vivo reconstitu- combined with their functional definition, would provide
tionassayisnotwelldefinedformanytypesofadultstem amorecomprehensivesetofcriteriaforunderstandingtheir
cells. Thus, it is important to arrive at an accurate func- unique biology. Perhaps more importantly, these types of
tionaldefinitionforcellswhosedevelopmentalpotentialis studiescouldbeusedtohelpidentifyandisolatenewstem
assessed in vitro only. Above all, clonal assays should be cells. This goal is far from being accomplished, but the
the standard by which fetal and adult stem cells are eval- preliminary findings for specific stem cells have been
uated because this assay removes doubts about contami- described. The transcriptional profiling of stem cells has
nation with other cell types. suggested that they share several distinct molecular char-
Two concepts about the fate or potential of stem cells acteristics.Stemcellsappeartohavethecapacitytosense
havemovedtotheforefrontofadultstemcellresearch.The a broad range of growth factors and signaling molecules
firstisplasticity,theideathatrestrictionsincellfatesarenot and to express many of the downstream signaling compo-
permanentbutareflexibleandreversible.Themostobvious nents involved in the transduction of these signals. Signal
and extreme example of reversing a committed cell fate transduction pathways present and perhaps active in stem
(cid:3)
comes from experiments in which a terminally differenti- cells include TGF , Notch, Wnt, and Jak/Stat family
ated somatic cell generates to another animal following members. Stem cells also express many components
nuclear transfer or cloning (Solter, 2000; Rideout et al., involvedinestablishingtheirspecializedcellcycles,either
2001).Nucleartransferexperimentsshowthatdifferentiated related to maintaining cell cycle arrest in G1 (for most
cells, given the appropriate conditions, can be returned to quiescent adult stem cells) or connected to progression
their most primal state. Thus, it may not be surprising if throughcellcyclecheckpointspromotingrapidcycling(as
conditionsarefoundformorecommittedorspecifiedcellsto is the case for ES cells and mobilized adult stem cells)
dedifferentiate and gain a broader potential. A related (Burdonetal.,1999;Savatieretal.,2002).Moststemcells
conceptisthatoftransdifferentiation.Transdifferentiationis also express molecules involved in telomere maintenance
the generation of functional cells of a tissue, organ, or anddisplayelevatedlevelsoftelomeraseactivity.Thereis
lineage that is distinct from that of the founding stem cell also considerable evidence that stem cells have signifi-
(LiuandRao,2003;Blauetal.,2001).Importantissueshere cantly remodeled chromatin acted upon by DNA methyl-
are whether the cells proposed to transdifferentiate are asesortranscriptionalrepressorsofhistonedeacetylaseand
clonalandwhetherthemechanismbywhichtheyformthe Groucho family members. Another common molecular
functionalcellrequiresfusion(MedvinskyandSmith,2003; feature is the expression of specialized posttranscriptional
Terada et al., 2002; Wang et al., 2003; Ying et al., 2002). regulatory machinery regulated by RNA helicases of the
Chapter | 2 “Stemness”:Definitions, Criteria,and Standards 11
Vasa type. Finally, a shared molecular and functional Hara, T., et al., 1999. Identification of podocalyxin-like protein 1 as
characteristicofstemcellsappearstobetheirresistanceto a novel cell surface marker for hemangioblasts in the murine
stress, mediated by multidrug resistance transporters, aortaegonademesonephrosregion.Immunity11,567e578.
protein-folding machinery, ubiquitin, and detoxifier Hayflick,L.,1973.Thebiologyofhumanaging.Am.J.Med.Sci.265,
432e445.
systems.
Hayflick,L.,1974.Thelongevityofculturedhumancells.J.Am.Geriatr.
Although in its infancy, the search for a molecular
Soc.22,1e12.
signaturetodefinestemcellscontinues.Wehavebegunto
He, W., et al., 2001. Multipotent stem cells from the mouse basal
understand in general terms what molecular components
forebrain contribute GABAergic neurons and oligodendrocytes to
are most often associated with stem cells. In the future, it
the cerebral cortex during embryogenesis. J. Neurosci. 21,
may be possible to precisely define stem cells as a whole 8854e8862.
andindividuallybytheirtell-talemolecularidentities.Until Houck, J.C., Sharma, V.K., Hayflick, L., 1971. Functional failures of
thattime,stemnessremainsaconceptoflimitedutilitywith cultured human diploid fibroblasts after continued population
tremendous potential. doublings.Proc.Soc.Exp.Biol.Med.137,331e333.
Howell,C.Y.,etal.,2001.Genomicimprintingdisruptedbyamaternal
effectmutationintheDnmt1gene.Cell104,829e838.
Iscove, N.N., Nawa, K., 1997. Hematopoietic stem cells expand during
ACKNOWLEDGMENTS
serialtransplantationinvivowithoutapparentexhaustion.Curr.Biol.
I would like to thank Jayaraj Rajagopal and Kevin Eggan for 7,805e808.
helpful discussion and suggestions. I apologize to those Ivanova,N.B.,etal.,2002.Astemcellmolecularsignature.Science298,
authors whosework was inadvertently overlooked or omitted 601e604.
because of space limitations. Jiang, Y., et al., 2002.Pluripotency of mesenchymalstem cells derived
fromadultmarrow.Nature418,41e49.
Kalyani, A.J., et al., 1998. Spinal cord neuronal precursors generate
REFERENCES multipleneuronalphenotypesinculture.J.Neurosci.18,7856e7868.
Kau, C.L., Turpen, J.B., 1983. Dual contribution of embryonic ventral
Alessandri, G., et al., 2001. Humanvasculogenesis ex vivo: embryonal blood island and dorsal lateral plate mesoderm during ontogeny of
aorta as a tool for isolation of endothelial cell progenitors. Lab. hemopoieticcellsinXenopuslaevis.J.Immunol.131,2262e2266.
Invest.81,875e885. Labosky,P.A.,Barlow,D.P.,Hogan,B.L.,1994.Mouseembryonicgerm
Allsopp,R.C.,Weissman,I.L.,2002.Replicativesenescenceofhemato- (EG)celllines:transmissionthroughthegermline,anddifferencesin
poieticstemcellsduringserialtransplantation:doestelomereshort- the methylation imprint of insulin-like growth factor 2 receptor
eningplayarole?Oncogene21,3270e3273. (Igf2r)genecomparedwithembryonicstem(ES)celllines.Devel-
Alvarez-Buylla, A., Garcia-Verdugo, J.M., Tramontin, A.D., 2001. A opment120,3197e3204.
unified hypothesis on the lineage of neural stem cells. Nat. Rev. Liu, Y., Rao, M.S., 2003. Transdifferentiation: fact or artifact. J. Cell
Neurosci.2,287e293. Biochem.88,29e40.
Anderson,D.J.,etal.,1997.Celllineagedeterminationandthecontrolof Luo,Y.,etal.,2002.Microarrayanalysisofselectedgenesinneuralstem
neuronalidentityintheneuralcrest.ColdSpringHarb.Symp.Quant. andprogenitorcells.J.Neurochem.83,1481e1497.
Biol.62,493e504. Matsui, Y., Zsebo, K., Hogan, B.L., 1992. Derivation of pluripotential
Anisimov,S.V.,etal.,2002.SAGEidentificationofgenetranscriptswith embryonicstemcellsfrommurineprimordialgermcellsinculture.
profiles unique to pluripotent mouse R1 embryonic stem cells. Cell70,841e847.
Genomics79,169e176. Medvinsky,A.L.,etal.,1993.Anearlypreliverintraembryonicsourceof
Blau,H.M.,Brazelton,T.R.,Weimann,J.M.,2001.Theevolvingconcept CFU-Sinthedevelopingmouse.Nature364,64e67.
ofastemcell:entityorfunction?Cell105,829e841. Medvinsky, A., Dzierzak, E., 1996. Definitivehematopoiesis is autono-
Brivanlou, A.H., et al., 2003. Stem cells: setting standards for human mouslyinitiatedbytheAGMregion.Cell86,897e906.
embryonicstemcells.Science300,913e916. Medvinsky,A.,Smith,A.,2003.Stemcells:fusionbringsdownbarriers.
Burdon, T., et al., 1999. Signaling mechanisms regulating self-renewal Nature422,823e835.
and differentiation of pluripotent embryonic stem cells. Cells Tiss. Minasi, M.G., et al., 2002. The mesoangioblast: a multipotent, self-
Organs165,131e143. renewingcellthatoriginatesfromthedorsalaortaanddifferentiates
Desai, A.R., McConnell, S.K., 2000. Progressive restriction in fate intomostmesodermaltissues.Development129,2773e2783.
potentialbyneuralprogenitorsduringcerebralcorticaldevelopment. Molyneaux,K.A.,etal.,2001.Time-lapseanalysisoflivingmousegerm
Development127,2863e2872. cellmigration.Dev.Biol.240,488e498.
Doetsch,F.,etal.,1999.Subventricularzoneastrocytesareneuralstem Morrison, S.J., Shah, N.M., Anderson, D.J., 1997. Regulatory mecha-
cellsintheadultmammalianbrain.Cell97,703e716. nismsinstemcellbiology.Cell88,287e298.
Dzierzak, E., 2002. Hematopoietic stem cells and their precursors: Munoz-Chapuli,R.,etal.,1999.Differentiationofhemangioblastsfrom
developmental diversity and lineage relationships. Immunol. Rev. embryonicmesothelialcells?Amodelontheoriginofthevertebrate
187,126e138. cardiovascularsystem.Differentiation64,133e141.
Gaiano,N.,Fishell,G.,2002.Theroleofnotchinpromotingglialand Orkin,S.H.,1996.Developmentofthehematopoieticsystem.Curr.Opin.
neuralstemcellfates.Annu.Rev.Neurosci.25,471e490. Genet.Dev.6,597e602.