Cyril Ramathal

Divergent RNA-Binding Proteins, DAZL and VASA, Induce Meiotic Progression in Human Germ Cells Derived In Vitro

Our understanding of human germ cell development is limited in large part due to inaccessibility of early human development to molecular genetic analysis. Pluripotent human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been shown to differentiate to cells of all three embryonic germ layers, as well as germ cells in vitro, and thus may provide a model for the study of the genetics and epigenetics of human germline. Here, we examined whether intrinsic germ cell translational, rather than transcriptional, factors might drive germline formation and/or differentiation from human pluripotent stem cells in vitro. We observed that, with overexpression of VASA (DDX4) and/or DAZL (Deleted in Azoospermia Like), both hESCs and iPSCs differentiated to primordial germ cells, and maturation and progression through meiosis was enhanced. These results demonstrate that evolutionarily unrelated and divergent RNA‐binding proteins can promote meiotic progression of human‐derived germ cells in vitro. These studies describe an in vitro model for exploring specifics of human meiosis, a process that is remarkably susceptible to errors that lead to different infertility‐related diseases. STEM CELLS 2012;30:441–451

Fate of iPSCs Derived from Azoospermic and Fertile Men following Xenotransplantation to Murine Seminiferous Tubules

Historically, spontaneous deletions and insertions have provided means to probe germline developmental genetics in Drosophila, mouse and other species. Here, induced pluripotent stem cell (iPSC) lines were derived from infertile men with deletions that encompass three Y chromosome azoospermia factor (AZF) regions and are associated with production of few or no sperm but normal somatic development. AZF-deleted iPSC lines were compromised in germ cell development in vitro. Undifferentiated iPSCs transplanted directly into murine seminiferous tubules differentiated extensively to germ-cell-like cells (GCLCs) that localized near the basement membrane, demonstrated morphology indistinguishable from fetal germ cells, and expressed germ-cell-specific proteins diagnostic of primordial germ cells. Alternatively, all iPSCs that exited tubules formed primitive tumors. iPSCs with AZF deletions produced significantly fewer GCLCs in vivo with distinct defects in gene expression. Findings indicate that xenotransplantation of human iPSCs directs germ cell differentiation in a manner dependent on donor genetic status.

Fate of induced pluripotent stem cells following transplantation to murine seminiferous tubules

Studies of human germ cell development are limited in large part by inaccessibility of germ cells during development. Moreover, although several studies have reported differentiation of mouse and human germ cells from pluripotent stem cells (PSCs) in vitro, differentiation of human germ cells from PSCs in vivo has not been reported. Here, we tested whether mRNA reprogramming in combination with xeno-transplantation may provide a viable system to probe the genetics of human germ cell development via use of induced pluripotent stem cells (iPSCs). For this purpose, we derived integration-free iPSCs via mRNA-based reprogramming with OCT3/4, SOX2, KLF4 and cMYC alone (OSKM) or in combination with the germ cell-specific mRNA, VASA (OSKMV). All iPSC lines met classic criteria of pluripotency. Moreover, global gene expression profiling did not distinguish large differences between undifferentiated OSKM and OSKMV iPSCs; however, some differences were observed in expression of pluripotency factors and germ cell-specific genes, and in epigenetic profiles and in vitro differentiation studies. In contrast, transplantation of undifferentiated iPSCs directly into the seminiferous tubules of germ cell-depleted immunodeficient mice revealed divergent fates of iPSCs produced with different factors. Transplantation resulted in morphologically and immunohistochemically recognizable germ cells in vivo, particularly in the case of OSKMV cells. Significantly, OSKMV cells also did not form tumors while OSKM cells that remained outside the seminiferous tubule proliferated extensively and formed tumors. Results indicate that mRNA reprogramming in combination with transplantation may contribute to tools for genetic analysis of human germ cell development.

Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status

IntroductionThe reprogramming of a patient’s somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known.MethodsIn this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions.ResultsWe found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status.ConclusionFor the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.

Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.

DDX3Y gene rescue of a Y chromosome AZFa deletion restores germ cell formation and transcriptional programs

Deletions of the AZFa region (AZoospermia Factor-a) region of the human Y chromosome cause irreversible spermatogenic failure that presents clinically in men as Sertoli-cell only (SCO) pathology of the testis. Deletions of the AZFa region typically encompass two genes: DDX3Y and USP9Y. However, human genetic evidence indicates that SCO is most tightly linked to deletion of DDX3Y and that deletions/mutations of USP9Y can be transmitted from one generation to the next. Here, we generated stable iPSC lines with AZFa deletions, tested complementation via introduction of DDX3Y, and assessed ability to form germ cells in vivo in a xenotransplantation model. We observed a quantifiable improvement in formation of germ cell like cells (GCLCs) from complemented donor iPSCs. Moreover, expression of UTF1, a prospermatogonial protein, was restored in cells complemented by introduction of DDX3Y on the AZFa background. Whole-genome RNA sequencing of purified GCLCs revealed an enrichment of genes involved in translational suppression and transcriptional control in DDX3Y-rescued GCLCs over mutant GCLCs, which maintained a molecular phenotype more similar to undifferentiated iPSCs. This study demonstrates the ability to probe fundamental genetics of human germ cell formation by complementation and indicates that DDX3Y functions in the earliest stages of human germ cell development.

Embryonic Stem Cells and the Germ Cell Lineage

Stem cells possess the unique ability to either propagate by self-renewal or to differentiate to mature tissues under the influence of appropriate molecular cues. This remarkable feature, also termed “stem cell potency” has been the focus of both a historical and current research spotlight on stem cells, and particularly the potential for human stem cells in regenerative medicine and organ transplantation. There are several classes of stem cells, all varying in degrees of pluripotency. Depending on the developmental stage from where they originate, stem cell potencies range from totipotency (ability to transform into all cell types), pluripotency (most cell types), multipotency (many cell types), oligopotency (few cell types) to unipotency (one cell type) (Smith, 2001). Among these, embryonic stem cells (ESCs) represent the prototypical pluripotent stem cells. Embryonic stem cells are localized to the inner cell mass of the developing mammalian blastocyst and can give rise to all future cell types and tissues of the organism. Additionally, a small population of embryonic stem cells is allocated to the germline (primordial germ cells). The evolution of these germline stem cells is different from the remaining cells which undergo gastrulation and give rise to the three germ layers (endoderm, mesoderm and ectoderm) during development (Ewen & Koopman, 2010). The survival, gonadal migration and proper epigenetic programming of primordial germ cells (PGCs) are major, distinct and early events that have an impact on future fertility and the successful transmission of genetic information from parent to offspring. Finally, stem cells found in neonatal tissues, amniotic fluid, cord blood, and adult tissues are categorized as adult stem cells and are usually multipotent, oligopotent or unipotent. Adult stem cells are clinically important due to the absence of ethical and federal restrictions on their use. Furthermore, they are valued for their relative abundance and accessibility in somatic tissues. In contrast, human embryonic stem cells have been the focus of substantial research and discussion because of their unique potential to differentiate into almost all body cell types and tissues and the relative ease with which they are propagated in cell culture. The search for an alternative source of pluripotent stem cells other than from human embryos is a much sought after goal in the stem cell research community for several reasons. First, diversity in stem cells sources should be explored to better understand and evaluate their various clinical utilities. Second, immunological matching of stem cells to recipients is necessary to avoid rejection in stem cell-based regenerative therapies. Hence, the advent of induced pluripotency and nuclear reprogramming strategies have been well

Over Expression of NANOS3 and DAZL in Human Embryonic Stem Cells

The mechanisms underlying human germ cell development are largely unknown, partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here, we studied NANOS3 and DAZL, which have critical roles in germ cell development in several species, via their over expression in human embryonic stem cells using global transcriptional analysis, in vitro germ cell differentiation, and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition, we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL, our results suggest a post-transcriptional regulation mechanism in hES cells. In addition, we found that DAZL suppressed the translation of OCT4, and affected the transcription of several genes associated with germ cells, cell cycle arrest, and cell migration. Furthermore, DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo.

Preimplantation Embryo Development and Primordial Germ Cell Lineage Specification

Embryo development in mammals begins at fertilization with migration and fusion of the gametic pronuclei and extensive genome-wide epigenetic remodeling. The hallmark of preimplantation development is the transition from gametic to embryonic differentiation programs. Information that functionally specifies germ cells must be degraded while those required by the nascent embryo are expressed for the first time. Unique gene expression and epigenetic patterning also provides the foundation for differentiation of somatic and germ cell lineages. This chapter reviews fundamentals of epigenetic reprogramming, genetic (chromosomal) instability, timing, and mechanisms of embryonic gene activation, compaction, and cavitation. We discuss lineage specification beginning with trophectoderm, then focus on allocation of the germ line, and move toward development of germ cells from pluripotent stem cells. Finally, we conclude by discussing how current knowledge of embryogenesis and pluripotent stem cell biology may be applied to assisted reproduction and regenerative medicine now and in the future.