Magazine R503

Primer

Introducing embryonicstem cellsJennifer Nichols

The regeneration of damaged ordiseased human tissues using specificlineage-directed precursors couldsoon be possible. Pluripotent stemcell lines from human embryos haverecently been established in vitro.Applying specific differentiationtechniques developed for murineembryonic stem (ES) cells result inlineage-restricted cells that canintegrate into damaged tissues andrestore function. In addition to theseembryo-derived stem cells, severalreports have recently described thegeneration of cells of one tissue typefrom cells normally restricted toanother lineage. However, themechanisms directing thistransdifferentiation, and the meansby which these cells may beexpanded are not clear. Furthermore,the control of such events in culturehas yet to be developed. For thisreason stem cells derived fromembryos are likely to prove to be themost efficient route for tissuereplacement therapy. This reviewoutlines the embryological origins ofthe pluripotent stem cell linesderived from murine embryos andcompares some of theircharacteristics with those of theirhuman counterparts.

Stem cells of the early mouse embryoThe embryos of many vertebratesdevelop in a pattern that is dictatedby cytoplasmic determinants in theegg. The mammalian embryo,however, stands alone in itsimpressive capacity to developnormally after cell ablation or re-location of cells during cleavage. Forexample, at the 2-cell stage themouse embryo can be separated intoblastomeres that can each develop

into a perfectly normal animal. Inrabbits and sheep, blastomeresisolated at the 8-cell stage can giverise to normal individuals. Removalof blastomeres from the 16-cell stagemammalian embryo does not resultin loss of any specific structure, asoccurs for example after cell ablationin frog embryos. This facility forregulation has been exploited inagricultural applications with cattleand sheep to generate multipleindividuals with a desirable genotypefrom a single embryo, simply byseparation of the blastomeres duringpreimplantation stages. It has alsobeen invaluable in allowing biopsy inhumans for pre-gestational screeningof inherited diseases in in vitrofertilisation programmes.

This flexibility, or totipotency ofmammalian development is reducedas the morula becomes a blastocystwhen the first tissue, thetrophectoderm, differentiates on theoutside of the embryo. Thetrophectoderm will give rise solely tostructures involved in invasion of theuterus and establishment of theplacenta. The remaining insidepopulation of cells — the inner cellmass, ICM, or pluriblast — is initiallytotipotent, and has the capacity toregenerate the trophectoderm if theoutside differentiated layer isremoved. Gradually, this regenerativeproperty is lost, but the cells of theICM remain equivalent to oneanother and have the capacity toform all tissues of the foetus as wellas additional extraembryonicstructures. The ICM cells are nowdefined as pluripotent.

Just before implantation a seconddifferentiative event occurs in theICM, resulting in formation of theprimitive endoderm or hypoblast onthe surface lining the blastocystcavity. This tissue is involved information of the parietal and visceralyolk sacs that will surround thefoetus in utero. The remainder of theICM is now known as the primitiveectoderm or epiblast. In the mousethe epiblast does not retain the

ability to produce further hypoblastcells, but this is not a universalproperty of mammalian embryos, andis not shared by the rat for example,whose development is otherwise verysimilar.

At around the time ofimplantation the epiblast is merely aball of cells, but within a few hoursthe cells begin to organise into a cup-shaped epithelium. This event isthought to be controlled by signalsfrom the visceral endoderm, aderivative of the primitive endodermthat surrounds the epiblast.Following implantation the epiblastenters a period of rapid cell divisionthat persists during gastrulation. Thisis a time of progressivedifferentiation as cells of the epiblastbecome allocated to specific fates,depending upon whether they havepassed through the primitive streakand the subsequent position theycome to occupy. Interestingly, duringthe early stages of gastrulationablation of up to 80% of thedeveloping embryo does not preventrelatively normal subsequentdevelopment. By the end ofgastrulation all of the epiblast cellshave differentiated; only theprimordial germ cells, located in theposterior region of the embryo retainthe potential for development into alltissues under appropriate conditions.

Harnessing pluripotency in cultureTo understand the biology ofmammalian pluripotent cells and toexploit their potential uses it isdesirable to be able to grow them inculture and manipulate themgenetically. In the 1970s embryonalcarcinoma (EC) cells were derivedfrom teratocarcinomas generated bytransferring ICM cells or epiblasts toa permissive site, such as the testis orkidney of a host animal.Teratocarcinomas consist of manydifferentiated cells and a populationof stem cells that can induce a secondtumour if transferred to another host;and can also proliferate in vitro. ECcells were the first pluripotent

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mammalian stem cells that could bepropagated in culture. Lines havebeen successfully obtained usingmurine embryos undergoinggastrulation. When integrated into ahost morula or blastocyst, derivativesfrom EC cells can occasionally befound in many tissues of theresulting adult mouse. However, theyrarely, if ever, contribute to thegermline. The establishment of murineembryonic stem (ES) cell lines was amajor breakthrough in the early 80s.ES cells were derived directly inculture from ICMs, either isolatedfrom the trophectoderm or as intactblastocysts. They have becomeinvaluable becasue of their ability tocolonise all tissues including thegermline when aggregated with ahost preimplantation embryo.Consequently, it is possible, usingtransgenic and gene targetingtechnology, to create mice bearingspecific mutations that may be usedfor studies in gene function or asmodels for disease. In spite ofpersistent effort, however, in contrastto EC cells, ES cells have not yetbeen derived from postimplantationembryos. It is possible thatepithelialisation of the epiblast maysomehow inhibit the pluripotentpopulation from overcoming itsprogrammed differentiation whensubjected to the culture conditionsestablished for derivation of ES cellsfrom blastocysts.

An important ingredient for EScell derivation and propagation isleukaemia inhibitory factor (LIF).LIF is produced by the fibroblastsupon which ES cells are frequentlycultured, but it may also be added tothe culture medium in purified form.LIF and related cytokines operatevia a receptor incorporating the cellsurface glycoprotein gp130 thatrecruits the signal transducer andactivator of transcription STAT3.Enforced activation of STAT3 in EScells allows stem cell self-renewaleven in the absence of cytokines,showing that LIF family members

maintain pluripotency primarilythrough this signal transductionpathway.

In addition to STAT3 activation,expression of the transcription factorOct-4 is absolutely required for EScell self-renewal. Oct-4 expression isrestricted to pluripotent stem cellsin vivo and in vitro; pluripotent cellsnot expressing Oct-4 are never seen.Differentiation of ES cells isassociated with down regulation ofOct-4. Targeted deletion of Oct-4results in embryos composed entirelyof trophectoderm that can implant inthe uterus, but fail to develop anyfurther. In normal embryoscompletion of gastrulation iscoincident with restriction of Oct-4expression to the primordial germcells. Cells resembling ES cells havebeen derived from the primordial

germ cells of mice. These embryonicgerm (EG) cells can contribute to alltissues, including the germline ofchimaeras.

Pluripotent stem cell lines fromhuman embryosCells resembling mouse ES cellshave now been derived from surplushuman embryos donated byinformed, consenting couplesfollowing in vitro fertilisationtreatment. It is obviously notpossible to test the developmentalpotential of human ES cells by theircontribution to chimaeras, butderivatives of all three germ layers —ectoderm, mesoderm and endoderm— have been generated in teratomasformed by injecting these cells intosevere combined immunodeficient(SCID) mice. More specifically,

Figure 1

Representation of the progression throughmouse development indicating the means ofgenerating various pluripotent stem cell linesfrom specific stages. Totipotent cells

(orange), pluripotent cells and germ cells(red), trophoblast lineage (blue) and extra-embryonic endoderm (yellow) are indicated.

Gastrulation

Epithelialisation

Graft tokidney or testisEC cells

EG cells EG cells

ImplantationES cells?

Blastocyst

Cleavage

ES cells

Eggcylinder

Morula

2-cell

Zygote

Genitalridges

ES cells

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ES cells

Disaggregate

Dissect epiblast

Disaggregate

Outgrow

Dissect germcell region

neural epithelium, embryonicganglia, stratified squamousepithelium, hair, cartilage, bone,smooth and striated muscle and gutepithelium have been identified inthese tumours. Human ES cells havebeen defined on the strength of thefollowing criteria: they are derivedfrom preimplantation embryos; theyundergo prolonged undifferentiatedproliferation; and they have a stabledevelopmental potential to formderivatives of all three germ layers.The human ES cells derived byThomson and colleagues exhibit ahigh levels of telomerase activity,which plays an important role inreplicative life span of such cells:these cells have been maintained inculture for over a year. Such cellsmay prove to be invaluable for thein vitro study of early humandevelopment, which differssignificantly from that of the mouse,as well as for pharmacologicalpurposes, such as identifying genetargets for new drugs and testingteratogenic or toxic agents.

In order for them to be of use fortissue replacement therapy it mustbe possible to direct theirdifferentiation along specificpathways to produce a purepopulation of the desired lineage thatcan be incorporated into thedamaged tissue and function asnormal. No contamination withundifferentiated ES cells can betolerated because of the risk ofsubsequent tumour formation.Murine ES cells can be directedalong various pathways ofdifferentiation in culture by applyingspecific protocols. For example,neurons, glial cells, cardiac tissue,cells of osteogenic andhaematopoietic lineages have beengenerated in vitro by various researchgroups. Primitive and mature neuralcells have been derived from humanES cells, but so far there have beenno reports of any functional orreplacement studies with these cells.

Differentiation of human EScells into other specific lineages has

yet to be demonstrated. Human EScells are reported to be more difficultto grow in culture than mouse EScells. They apparently do notrespond well when they aredisaggregated into single cells orgrown at low density and theybecome necrotic or differentiate ifremoved from the fibroblast feederlayer, even in the presence of LIF orrelated cytokines. The factorsproduced by the feeder cellsrequired for stem cell self-renewal inhuman ES cells are still unknown.Human ES cells also differ frommurine ES cells in the specificmarkers that they express. However,Oct-4, which appears to be a keyrequirement for pluripotency, isexpressed in ES cells from bothspecies.

Pluripotent cell lines have alsobeen established from primordialgerm cells dissected from abortedhuman embryos. Interestingly, thesecells resemble mouse ES cells moreclosely than do the humanblastocyst-derived cells, at least intheir responsiveness to LIF and theease with which they can bepassaged. So far nothing is knownabout the methylation status of theimprinted genes in human EG cells.This may be a matter for concern,since relaxation of imprinting ofspecific genes has been associatedwith several human malignancies.Human EG cells have not yet beentested for their ability todifferentiate along specific lineagesby injection into SCID mice, but bygrowing them at high density inculture differentiation can beobserved and markers for derivativesof all three germ layers have beenidentified.

The ability to derive human EScells is a very important step towardscell replacement therapy.Demonstration of specific lineage-directed differentiation will certainlybe achieved soon. By adapting thetechnology developed for murine EScells, such as gene targeting, toremove incompatible genes of the

histocompatibility complex, it maybe possible to overcome theanticipated problems of immunerejection when clinical trials begin.ES cell lines have been derived frommurine blastocysts generated bynuclear transfer into enucleatedoocytes. Applying this techniqueusing genetically matched nucleimay provide an alternative way toovercome immune rejection.However, the implications ofdeveloping such a technique usinghuman embryos will invoke fear ofthe possibility of generating wholebeings It may be a long time beforethe obvious advantages of usingnuclear transfer for cell replacementtherapy are allowed to emerge inclinical practice.

Key referencesPera MF, Reubinoff B, Trounson A: Human

embryonic stem cells. J Cell Sci 2000,113:5-10.

Robertson EJ: Teratocarcinoma and Embryo-Derived Stem Cells: A Practical Approach.Oxford: IRL Press; 1987.

Shamblott MJ, Axelman J, Littlefield JW,Blumenthal PD, Huggins GR, Cui Y,Cheng L, Gearhart JD: Derivation ofpluripotent stem cells from culturedhuman primordial germ cells. Proc NatlAcad Sci USA 1988, 95:13726-13731.

Smith, AG: Embryonic stem cells. In Stem CellBiology. Edited by Marshak DR, GardnerRL, Gottlieb D. New York: Cold SpringHarbor Laboratory Press; 2001:205-230.

Thomson JA, Itskovitz-Eldor J, Shapiro SS,Waknitz MA, Swiergiel JJ, Marshall VS,Jones JM: Embryonic stem cell linesderived from human blastocysts. Science1998, 282:1145-1147.

Address: Centre for Genome Research,University of Edinburgh, The King’s Buildings,West Mains Road, Edinburgh EH9 3JQ, UK.

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