N OCIETY' - the Society for Reproductive Biology

Implantation and transplantation: endocrine signals in cell lineage choiceJohn P. HearnResearch School of Biological Sciences, Australian National UniversityPO Box 475, Canberra, ACT 2601, AustraliaTHE DEVELOPMENT AND POTENTIAL USE OF HUMAN EMBRYONIC STEM CELLSIntroduction:The rapid establishment of embryo-maternalatr~;;:'hment, implantation and differentiation at thestan of pregnancy is enabled by an endocrinedialogue. The reliance on chorionicgonadotrophin (CG) as an embryonic signal ofviability, and capacity to support the corpusluteum, is apparently restricted to primates. Wequestioned the possible role of gonadotrophinreleasing hormone (GnRH) in regulating CGrelease during implantation. fu associated studies(with James Thomson, University of Wisconsin)we isolated and characterised the similarities inendocrine secretion from totipotent or pluripotentembryonic stem cells in marmoset and rhesusmonkeys.Materials and Methods:The development of non-surgical embryorecovery, microassays to measure GnRH and CG,and embryo culture to the in vitro equivalent ofday 12 after embryo attachment, were describedearlier (1,2,3). fu current studies we incubatedsingle embryos with exogenous GnRH, agonist orantagonist, measuring the resulting change in CGsecretion and the differentiation stages ofembryos. In addition, embryonic stem cells wereisolated (4,5) and their secretion of CG monitored.Results:Control embryos (n=25) secreted a characteristicprofile of CG, with GnRH measurable in theculture fluid by mid blastocyt stages ofdevelopment. fucubation with antagonist (n=15)reduced CG secretion and slowed differentiation.fucubation with agonist (n=15) enhanced CGsecretion but drastically inhibited attachment anddifferentiation. fucubation with exogenous GnRH(n=10) greatly enhanced both CG secretion andembryo differentiation, suggesting an unexpectedstimulus to embryo survival, with possibletherapeutic implications. Differentiatedembryonic stem cells also secreted CG andmaintained the developmental potential to formtrophoblast and all three embryonic germ layers.Discussion:Our results suggest a direct or indirect functionfor GnRH, secreted by the embryo during periimplantationstages of pregnancy, in theregulation of CG secretion. There may also bedirect effects on cell growth anddifferentiation. The ability to enhance ordiminish embryo attachment, outgrowth anddifferentiation may provide a novel (andcontrolled) approach to study intra-embryoniccell signalling by stimulating or inhibitingperi-implantation events (6,7). In embryonicstem cell preparations, using a similarapproach, it may be possible to dissectgenetically in vitro the mechanisms controllingprimitive streak formation in primates and theendocrine signals that influence choice in celllineage.References:1. Hearn JP et al (1994) In: Marshall'sPhysiology of Reproduction, 4 th Ed. Pp.535-676, Chapman and Hall, London2. Webley, GE and Hearn JP (1994) OxfordReviews of Reproductive Biology 16: 1-323. Seshagiri, PB, Terasawa, E and Hearn, JP(1994Hum.Reprod.91300-1307.4. Thomson JA, Hearn JP et al. (1995)Proceedings of the National Academy ofSciences 92: 7844-7848.5. Thomson, JA, Hearn, JP et al (1996) Biol.Reprod. 55, 254-259.6. Hearn, JP (1997) Endocrine Signals inembryo implantation In Marsupial biology,recent research, new perspectives, pp 22-30UNSW Press, Sydney7. Stouffer, RL and Hearn, JP (1998) In: TheEndocrinology of Pregnancy. Pp 35-57Humana Press, NJ. t-\rfiJ\\ C.,.!t'l{ 'j\ ,'." . /~I'i,~(2)(3)Ben Reubinoff l ,2, Martin Pera l , AriffBo~gs03, Chui Fong 3 and Alan Trounson lICentre for Early Human Development, fustitute of Reproduction and Development, MonashUniversi~, 2Department of Obstetrics and Gynecology, H~dassah l!niv~rsity H?spital, Israel andDepartment of Obstetrics and Gynaecology, NatIonal UnIverSIty of SIngaporeHuman embryos can be grown to the blastocyst s.tage efficie~tly in ~itro (1) and. will ~ttach t~ andoutgrow in plastic culture dishes in vitro (2). It IS also pOSSIble to Immunosurgically Isolate ~nnercell mass cells (ICM) of human blastocysts and to maintain these cells in long-term culture 10 anundifferentiated state. The isolation and passage of undifferentiated human embryonic cells waspublished by Thomson et al. (3). We have independently derived undiffere~tiat~d human embryoniccells that maintain their phenotype through extended culture and passage In VItro. These cells aremultipotential and are able to differentiate in vitro and when ~enotra~s~lanted in~o ,a ~ery. widerange of cell and tissue types. The multipotentiality of the dIfferentIatIng cells IS IndIcatIve ofembryonic stem cells.The potential value and use of embryonic stem cells are for the d~t~rminat~on of. the int~in.sic andextrinsic factors that direct lineage specific differentiation. The abIlIty to dIrect dIfferentIation andto maintain stem cell phenotype during large scale multiplication may enable these cells to be usedfor transplantation, be used for tissue engineering and gene therapy.The research with these putative embryonic stem cells is presently aimed to identify the factorsinvolved in the maintenance of stem cell phenotype and those involved in the check points directingearly differentiation events.References(1)Jones GM, Trounson AO, Lolatgis N, Wood C (1998) Factors affecting the success ofhuman blastocyst development and pregnancy following IVF and embryo transfer. FertH.Steril. 70: 1022-29.Bongso A, Fong C-Y, Ng SC, Ratnam S (1994) Isolation and culture of inner cell mass cellsfrom human blastocysts. Human Reprod. 9: 2110-17.Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, JonesJM (1998) Embryonic stem cell lines -derived from human blastocysts. Science 282 (5391):1061-62.66""""-~G67