James Byrne

Isolation and Characterization of Novel Rhesus Monkey Embryonic Stem Cell Lines

ESCs are important as research subjects since the mechanisms underlying cellular differentiation, expansion, and self‐renewal can be studied along with differentiated tissue development and regeneration in vitro. Furthermore, human ESCs hold promise for cell and tissue replacement approaches to treating human diseases. The rhesus monkey is a clinically relevant primate model that will likely be required to bring these clinical applications to fruition. Monkey ESCs share a number of properties with human ESCs, and their derivation and use are not affected by bioethical concerns. Here, we summarize our experience in the establishment of 18 ESC lines from rhesus monkey preimplantation embryos generated by the application of the assisted reproductive technologies. The newly derived monkey ESC lines were maintained in vitro without losing their chromosomal integrity, and they expressed markers previously reported present in human and monkey ESCs. We also describe initial efforts to compare the pluripotency of ESC lines by expression profiling, chimeric embryo formation, and in vitro‐directed differentiation into endodermal, mesodermal, and ectodermal lineages.

Heterozygous Embryonic Stem Cell Lines Derived from Nonhuman Primate Parthenotes

Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC‐derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line‐dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.

Trophoblast cell activation by trophinin ligation is implicated in human embryo implantation

During human embryo implantation, trophectoderm mediates adhesion of the blastocyst to the uterine epithelium. The rapid growth of the embryo and invasion of the maternal tissue suggest adhesion-induced activation of the embryonal cells. We show here that ligation of trophinin, a homophilic cell adhesion molecule expressed on trophoblastic cells, induces tyrosine phosphorylation in trophinin-expressing trophoblastic HT-H cells. The phosphorylation could be induced in HT-H cells with the binding of trophinin-expressing cells or anti trophinin antibodies. Trophinin-dependent tyrosine phosphorylation was associated with actin reorganization. We also isolated trophinin-binding peptides from phage libraries. These peptides exhibited the consensus sequence GWRQ and seemed to reproduce the effects of trophinin-mediated cell adhesion. Upon binding of a GWRQ peptide, HT-H cells became highly proliferative and motile. HT-H cells expressed ErbB family receptors and bound EGF and heparin-binding EGF-like growth factor (HB-EGF), but ErbB family receptor phosphorylation in these cells required GWRQ. In the absence of GWRQ, trophinin interacted with the cytoplasmic protein bystin, which binds to ErbB4 and blocks its autophosphorylation. In HT-H cells, GWRQ peptide dissociated trophinin from bystin, and ErbB4 was activated. Culturing monkey blastocysts in the presence of the peptide increased total number and motility of the trophectoderm cells. These results suggest that trophinin-mediated cell adhesion functions as a molecular switch for trophectoderm activation in human embryo implantation.

Induction of trophinin in human endometrial surface epithelia by CGβ and IL-1β

During embryo implantation, trophinin mediates cell adhesion by homophilic binding at the apical surfaces of trophectoderm and endometrium. Trophinin is expressed on the human endometrial epithelia in rare occasions. We developed hCG‐coated agarose beads that mimic the physical and physiological features of an implantation‐stage human blastocyst. When hCG‐coated beads were applied to human endometrial epithelial cells in the presence of IL‐1β, endometrial cells acquired strong trophinin expression and the ability for apical cell adhesion with trophinin‐expressing human trophoblastic cells. These results provide a mechanism for trophinin‐mediated adhesion of human blastocyst to endometrium by a spatially and temporally restricted paracrine effect of hCG derived from the blastocyst.

Transcriptional profiling of rhesus monkey embryonic stem cells.

Abstract Embryonic stem cells (ESCs) may be able to cure or alleviate the symptoms of various degenerative diseases. However, unresolved issues regarding survival, functionality, and tumor formation mean a prudent approach should be adopted towards advancing ESCs into human clinical trials. The rhesus monkey provides an ideal model organism for developing strategies to prevent immune rejection and test the feasibility, safety, and efficacy of ESC-based medical treatments. Transcriptional profiling of rhesus monkey ESCs provides a foundation for pre-clinical ESC research in this species. In the present study, we used microarray technology, immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) to characterize and transcriptionally profile rhesus monkey ESCs. We identified 367 stemness gene candidates that were highly (>85%) conserved across five different ESC lines. Rhesus monkey ESC lines maintained a pluripotent undifferentiated state over a wide range of POU5F1 (also known as OCT4) expression levels, and comparisons between rhesus monkey, mouse, and human stemness genes revealed five mammalian stemness genes: CCNB1, GDF3, LEFTB, POU5F1, and NANOG. These five mammalian genes are strongly expressed in rhesus monkey, mouse, and human ESCs, albeit only in the undifferentiated state, and represent the core key mammalian stemness factors.

Current progress with primate embryonic stem cells.

Embryonic stem cells (ESCs) can proliferate indefinitely, maintain an undifferentiated pluripotent state and differentiate into any cell type. Differentiation of ESCs into various specific cell-types may be able to cure or alleviate the symptoms of various degenerative diseases. Unresolved issues regarding maintaining function, possible apoptosis and tumor formation in vivo mean a prudent approach should be taken towards advancing ESCs into human clinical trials. Rhesus macaques provide the ideal model organism for testing the feasibility, efficacy and safety of ESC based therapies and significant numbers of primate ESC lines are now available. In this review, we will summarize progress in evaluating the genetic and epigenetic integrity of primate ESCs, examine their current use in pre-clinical trials and discuss the potential of producing ESC-derived cell populations that are genetically identical (isogenic) to the host by somatic cell nuclear transfer.

Primate Models for the Assisted Reproductive Technologies and Embryonic Stem Cell Biology

Nonhuman primates (NHPs) represent clinically relevant animal models used in studies on the etiology and treatment of human diseases. In the context of reproduction, NHP models are relevant to research interests as diverse as the etiology and treatment of infertility and contraceptive development to an evaluation of cell or tissue-based therapies of disease employing embryonic stem cell-derived phenotypes. The assisted reproductive technologies (ARTs) are used in the production of animals carrying desired MHC alleles for HIV vaccine development and could be used in the production of genetically identical animals by somatic cell nuclear transfer (SCNT). SCNT in turn is relevant to rescuing the unique genetics of aging or even deceased animals, to testing the efficacy of therapeutic cloning, and in combination with gene targeting, to creating monkey models of genetically based, neurodegenerative diseases. Since most NHP experience in applying the ARTs and in deriving embryonic stem cells (ESCs) involves Old World monkeys, principally rhesus and cynomolgus macaques and baboons, studies with these species will be featured in this chapter.