Matrix-free 3D culture supports human follicular development from the unilaminar to the antral stage in vitro yielding morphologically normal metaphase II oocytes.

STUDY QUESTION: Can group culture with stage-specific anti-Müllerian hormone (AMH) modulation support human follicular development and oocyte maturation in vitro? SUMMARY ANSWER: In the presence of FSH, AMH supplementation at the secondary-to-early antral stage followed by AMH depletion promotes the coordinated growth and function of human follicles during group culture, thereby yielding mature oocytes. WHAT IS KNOWN ALREADY: Stage-specific AMH modulation promotes in-vitro development of nonhuman primate follicles. The group culture method supports nonhuman primate follicle growth from the primary to antral stage, producing developmentally competent oocytes. STUDY DESIGN, SIZE, DURATION: Ovarian tissue samples were collected from 19 patients of reproductive age (22-47 years old having menstrual cycles) who underwent oophorectomy or hysterectomy for clinical purposes. Tissue pieces were cultured in a matrix-free system for 3 weeks followed by isolation of follicles for the subsequent 6-week individual or group culture. PARTICIPANTS/MATERIALS, SETTING, METHODS: Pieces of ovarian cortical tissue were cultured to support primordial follicle activation and early-stage follicle growth. Secondary follicles isolated from cultured tissue were then randomly assigned to two groups for individual culture: control and AMH modulation, i.e., recombinant human AMH protein supplementation during the secondary-to-early antral stage followed by the addition of neutralizing anti-human AMH antibody. Secondary follicles were also cultured in groups with the same AMH modulation. Follicle survival, growth, steroid hormone and paracrine factor production, steroidogenic protein expression, as well as oocyte maturation and morphology were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Follicles grew to the secondary stage during 3 weeks of ovarian tissue culture. In-vitro-developed follicles expressed AMH and levels of secreted AMH increased (P < 0.05) in the culture media over time. Secondary follicles isolated from cultured ovarian tissue survived and grew to the antral stage during 6 weeks of individual follicle culture. In-vitro-developed antral follicles produced granulosa and theca cell-derived steroid hormones and paracrine factors, which were detectable in the culture media. Germinal vesicle oocytes obtained from cultured follicles exhibited a perinucleolar chromatin rim configuration. AMH modulation did not alter follicle survival or oocyte maturation relative to those of the control follicles. However, follicle diameters, as well as steroid hormone and paracrine factor production, increased (P < 0.05) in the AMH-modulation group compared with the control group. Secondary follicles isolated from cultured ovarian tissue formed aggregates and grew to the antral stage during 6 weeks of group culture. In-vitro-developed antral follicles expressed steroidogenic enzymes and secreted steroid hormones were detectable in the culture media. Oocytes obtained from cultured follicle aggregates with AMH-modulation progressed to the metaphase II stage after IVM, containing a normal-sized first polar body and meiotic spindle. Oocytes exhibited a typical ultrastructure. LIMITATIONS, REASONS FOR CAUTION: Follicles were obtained from fresh ovarian tissue of adult patients. Oocyte maturation rates were relatively low and oocytes were assessed by morphological evaluation. Owing to the lack of a control group, the beneficial effects of AMH modulation remained undetermined for the group culture in this study. WIDER IMPLICATIONS OF THE FINDINGS: Stage-specific AMH modulation supports human follicular development in the matrix-free group culture, which is consistent with previously reported AMH actions on growing follicles in nonhuman primates. Oocytes generated by in-vitro-developed follicles achieve meiotic maturation with a typical morphology and ultrastructure, which supports in-vitro follicle maturation as a potential approach for fertility preservation in women. STUDY FUNDING/COMPETING INTEREST(S): NICHD R01HD082208 and NIH Office of the Director P51OD011092. The authors have no competing interest to declare. TRIAL REGISTRATION NUMBER: N/A.

X-chromosome inactivation in monkey embryos and pluripotent stem cells.

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.

Principles of and strategies for germline gene therapy.

Monogenic disorders occur at a high frequency in human populations and are commonly inherited through the germline. Unfortunately, once the mutation has been transmitted to a child, only limited treatment options are available in most cases. However, means of correcting disease-causing nuclear and mitochondrial DNA mutations in gametes or preimplantation embryos have now been developed and are commonly referred to as germline gene therapy (GGT). We will discuss these novel strategies and provide a path forward for safe, high-efficiency GGT that may provide a promising new paradigm for preventing the passage of deleterious genes from parent to child.

Gene targeting in adult rhesus macaque fibroblasts.

BACKGROUND: Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT) has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology. RESULTS: A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow long term in vitro manipulations. Successful gene targeting of the HPRT locus in rhesus macaques was achieved by electroporating S-phase synchronized cells with a construct containing a SV40 enhancer. CONCLUSION: The cell lines reported here could be used for the production of null mutant rhesus macaque models of human genetic disease using SCNT technology. In addition, given the close evolutionary relationship and biological similarity between rhesus macaques and humans, the protocols described here may prove useful in the genetic engineering of human somatic cells.

Stage-specific modulation of antimüllerian hormone promotes primate follicular development and oocyte maturation in the matrix-free three-dimensional culture.

OBJECTIVE: To study whether follicular growth and oocyte maturation can be improved by antimüllerian hormone (AMH) modulation at specific stages of follicular development. DESIGN: Primary and secondary follicles were cultured in a matrix-free system and were assigned to the control group and the group with AMH supplementation during the preantral stage and neutralizing AMH antibody addition during the antral stage. SETTING: National primate research center. ANIMAL(S): Adult, female rhesus macaques (Macaca mulatta). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicle survival, growth, steroid and paracrine factor production, and oocyte competence were evaluated. Follicles were assessed for expression of genes that are critical for gonadotropin signaling, cumulus cell glycolysis, and oocyte quality. RESULT(S): Primary follicles formed "organoids" and developed to the antral stage in group culture. AMH exposure during the preantral stage increased organoid diameters. Oocytes from the AMH-treated organoids had greater diameters and matured to the metaphase II (MII) stage. Secondary follicles developed to the antral stage during individual culture. The AMH exposure during the preantral stage and AMH antibody treatment during the antral stage increased follicle diameters, vascular endothelial growth factor and follistatin production, differentiation factor 9 expression, and oocyte diameters. The MII oocytes from the AMH-modulated group developed to the morula stage after IVF, with one to the blastocyst stage. CONCLUSION(S): AMH supplementation at the preantral stage and depletion at the antral stage enhanced primate follicular development and oocyte competence in vitro. The improved embryonic development supports in vitro follicle maturation as a potential approach for fertility preservation.

Nuclear transfer in nonhuman primates.

The nonhuman primate is a highly relevant model for the study of human diseases, and currently there is a significant need for populations of animals with specific genotypes that can not be satisfied by the capture of animals from the wild or by conventional breeding. There is an even greater need for genetically identical animals in vaccine development or tissue transplantation research, where immune system function is under study. Efficient somatic cell nuclear transfer (SCNT) procedures could provide a source for genetically identical nonhuman primates for biomedical research. SCNT offers the possibility of cloning animals using cultured cells and potentially provides an alternative approach for the genetic modification of primates. The opportunity to introduce precise genetic modifications into cultured cells by gene targeting procedures, and then use these cells as nuclear donors in SCNT, has potential application in the production of loss-of-function monkey models of human diseases. We were initially successful in producing monkeys by NT using embryonic blastomeres as the source of donor nuclei and have repeated that success. However, when somatic cells are used as nuclear donor cells, the developmental potential of monkey SCNT embryos is limited, and somatic cell cloning has not yet been accomplished in primates. High rates of in vitro development to blastocysts, comparable with in vitro fertilization controls, and successful production of rhesus monkeys by NT from embryonic blastomeres suggests that basic cloning procedures, including enucleation, fusion, and activation, are consistent with the production of viable embryos. Although modifications or additional steps in SCNT are clearly warranted, the basic procedures will likely be similar to those extant for embryonic cell NT. In this chapter, we describe detailed protocols for rhesus macaque embryonic cell NT, including oocyte and embryo production, micromanipulation, and embryo transfer in nonhuman primates.

Genomic imprinting in primate embryos and embryonic stem cells.

Embryonic stem (ES) cells hold promise for cell and tissue replacement approaches to treating human diseases. However, long-term in vitro culture and manipulations of ES cells may adversely affect their epigenetic integrity including imprinting. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. We demonstrated aberrant biallelic expression of IGF2 and H19 in several rhesus monkey ES cell lines while SNRPN and NDN were normally imprinted and expressed from the paternal allele. In contrast, expanded blastocyst-stage embryos, from which these ES cells were derived, exhibited normal paternal expression of IGF2 and maternal expression of H19. To test the possibility that aberrant methylation at an imprinting centre (IC) upstream of H19 accounts for the relaxed imprinting of IGF2 and H19, we performed comprehensive methylation analysis by investigating methylation profiles of CpG sites within the IGF2/H19 IC. Our results demonstrate abnormal hypermethylation within the IGF2/H19 IC in all analysed ES cell lines consistent with biallelic expression of these genes. Cellular overproliferation and tumour formation resulting from tissue or cell transplantation are potential problems that must be addressed before clinical trials of ES cell-based therapy are initiated.

Cloning of non-human primates: the road "less traveled by".

Early studies on cloning of non-human primates by nuclear transfer utilized embryonic blastomeres from preimplantation embryos which resulted in the reproducible birth of live offspring. Soon after, the focus shifted to employing somatic cells as a source of donor nuclei (somatic cell nuclear transfer, SCNT). However, initial efforts were plagued with inefficient nuclear reprogramming and poor embryonic development when standard SCNT methods were utilized. Implementation of several key SCNT modifications was critical to overcome these problems. In particular, a non-invasive method of visualizing the metaphase chromosomes during enucleation was developed to preserve the reprogramming capacity of monkey oocytes. These modifications dramatically improved the efficiency of SCNT, yielding high blastocyst development in vitro. To date, SCNT has been successfully used to derive pluripotent embryonic stem cells (ESCs) from adult monkey skin fibroblasts. These remarkable advances have the potential for development of human autologous ESCs and cures for many human diseases. Reproductive cloning of nonhuman primates by SCNT has not been achieved yet. We have been able to establish several pregnancies with SCNT embryos which, so far, did not progress to term. In this review, we summarize the approaches, obstacles and accomplishments of SCNT in a non-human primate model.

Differential requirements for hematopoietic commitment between human and rhesus embryonic stem cells.

Progress toward clinical application of ESC-derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model. However, in contrast to human ESCs (hESCs), efforts to induce conclusive hematopoietic differentiation from rhesus macaque ESCs (rESCs) have been unsuccessful. Characterizing these poorly understood functional differences will facilitate progress in this area and likely clarify the critical steps involved in the hematopoietic differentiation of ESCs. To accomplish this goal, we compared the hematopoietic differentiation of hESCs with that of rESCs in both EB culture and stroma coculture. Initially, undifferentiated rESCs and hESCs were adapted to growth on Matrigel without a change in their phenotype or karyotype. Subsequent differentiation of rESCs in OP9 stroma led to the development of CD34(+)CD45(-) cells that gave rise to endothelial cell networks in methylcellulose culture. In the same conditions, hESCs exhibited convincing hematopoietic differentiation. In cytokine-supplemented EB culture, rESCs demonstrated improved hematopoietic differentiation with higher levels of CD34(+) and detectable levels of CD45(+) cells. However, these levels remained dramatically lower than those for hESCs in identical culture conditions. Subsequent plating of cytokine-supplemented rhesus EBs in methylcellulose culture led to the formation of mixed colonies of erythroid, myeloid, and endothelial cells, confirming the existence of bipotential hematoendothelial progenitors in the cytokine-supplemented EB cultures. Evaluation of four different rESC lines confirmed the validity of these disparities. Although rESCs have the potential for hematopoietic differentiation, they exhibit a pause at the hemangioblast stage of hematopoietic development in culture conditions developed for hESCs.

Evaluation of the vervet (Clorocebus aethiops) as a model for the assisted reproductive technologies.

The vervet monkey was evaluated as a primate model for use in assisted reproductive technologies (ARTs). Eight adult female vervets were hormonally monitored for their potential use as egg donors and those six females displaying regular menstrual cycles were subjected to controlled ovarian stimulation with recombinant human gonadotropins. Three animals failed to respond while laparoscopic follicular aspiration was performed on the other three females at 27-30 h post-human chorionic gonadotropin administration. A total of 62, 40, and 18 oocytes was recovered from these three animals of which 30, 20, and 4, respectively, matured to the metaphase II stage and were subsequently inseminated using intracytoplasmic sperm injection. An average of 40+/-15% (SEM) of the inseminated oocytes were fertilized based on pronucleus formation and timely cleavage. One embryo from each of the two stimulated females developed into expanded blastocysts. Two adult male vervets were assessed as sperm donors. Neither adjusted well to the restraint and collection procedure required for penile electroejaculation. Samples collected via rectal electroejaculation were very low in sperm motility and concentration; however, cauda epididymal aspirations from one male yielded an adequate concentration of motile sperm. These results emphasize the need to establish species-specific ovarian stimulation protocols and semen collection techniques if vervets are to be considered for basic and applied (ARTs) research on primate gametes or embryos.

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.

Aberrant genomic imprinting in rhesus monkey embryonic stem cells.

Genomic imprinting involves modification of a gene or a chromosomal region that results in the differential expression of parental alleles. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. Additionally, abnormal imprinting occurs in mouse embryonic stem cells (ESCs) and in clonally derived animals. Imprinted gene expression patterns in primate ESCs are largely unknown, despite the clinical potential of the latter in the cell-based treatment of human disease. Because of the possible implications of abnormal gene expression to cell or tissue replacement therapies involving ESCs, we examined allele specific expression of four imprinted genes in the rhesus macaque. Genomic and complementary DNA from embryos and ESC lines containing useful single nucleotide polymorphisms were subjected to polymerase chain reaction-based amplification and sequence analysis. In blastocysts, NDN expression was variable indicating abnormal or incomplete imprinting whereas IGF2 and SNRPN were expressed exclusively from the paternal allele and H19 from the maternal allele as expected. In ESCs, both NDN and SNRPN were expressed from the paternal allele while IGF2 and H19 showed loss of imprinting and biallelic expression. In differentiated ESC progeny, these expression patterns were maintained. The implications of aberrant imprinted gene expression to ESC differentiation in vitro and on ESC-derived cell function in vivo after transplantation are unknown.

Transcriptional profiling of rhesus monkey embryonic stem cells.

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.

Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells.

Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.

Monozygotic twinning in rhesus monkeys by manipulation of in vitro-derived embryos.

The nonhuman primate is a relevant model for human disease that can be used for diverse biomedical investigations. The ability to propagate a founder animal by application of assisted reproductive technologies is pressing, but an even greater need in many studies is access to genetically identical animals. In an effort to create genetically identical monkeys, we evaluated two approaches to monozygotic twinning; blastomere separation, and blastocyst bisection. Embryos were produced by intracytoplasmic sperm injection of oocytes recovered following controlled ovarian stimulation. The quality of demiembryos produced in these efforts was evaluated by quantitating the efficiency of creating identical pairs for embryo transfer, by morphological assessment, by the allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) in the blastocyst, and by the outcome of embryo transfer to synchronized host animals. Pairs were produced in high yield (85%-95%) by both twinning methods. Demiembryos resulting from blastomere separations at the 2- or 4-cell stage grew to blastocysts at the control frequency. Demiblastocysts contained, on average, half the number of cells of the intact controls while maintaining the same ICM:TE or ICM:total cell ratio. The equivalency of demiblastocysts within a set was also evaluated by differential cell counting. Embryo transfers of identical sets led to a 33% clinical pregnancy rate, with two twin pregnancies initiated. Neither pregnancy resulted in term birth of monozygotic twins, but our results are sufficiently encouraging to justify a large-scale twinning trial in the rhesus macaque.

Oct-4 expression in pluripotent cells of the rhesus monkey.

The POU (Pit-Oct-Unc)-domain transcription factor, Oct-4, has become a useful marker of pluripotency in the mouse. It is found exclusively in mouse preimplantation-stage embryos after embryonic genome activation and is a characteristic of mouse embryonic stem (ES) cells, and its absence in knockout mice precludes inner cell mass (ICM) formation in blastocysts. Expression of Oct-4 has also been associated with pluripotency in primate cells. Here, we undertook a systematic study of Oct-4 expression in rhesus macaque preimplantation embryos produced by intracytoplasmic sperm injection and in ES cells before and after exposure to differentiating conditions in vitro. We also evaluated Oct-4 expression as a means of monitoring the extent of reprogramming following somatic cell nuclear transfer. Oct-4 was detected by reverse transcription-polymerase chain reaction and immunocytochemistry with a monoclonal antibody. Monkey pronuclear-stage zygotes and cleaving embryos up to the 8-cell stage showed no detectable Oct-4. Nuclear staining for Oct-4 first became obvious at the 16-cell stage, and a strong signal was observed in morula and compact morula stages. Both ICM and trophectodermal cell nuclei of monkey early blastocysts were positive for Oct-4. However, the signal was diminished in trophectodermal cells of expanded blastocysts, whereas expression remained high in ICM nuclei. Similar to the mouse, hatched monkey blastocysts showed strong Oct-4 expression in the ICM, with no detectable signal in the trophectoderm. Undifferentiated monkey ES cells derived from the ICM of in vitro-produced blastocysts expressed Oct-4, consistent with their pluripotent nature, whereas ES cell differentiation was associated with signal loss. Therefore, Oct-4 expression in the monkey, as in the mouse, provides a useful marker for pluripotency after activation of the embryonic genome. Finally, the observed lack or abnormal expression of Oct-4 in monkey nuclear transfer embryos suggests inadequate nuclear reprogramming.

Differentiation of monkey embryonic stem cells into neural lineages.

Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.