Transcriptional control of human gametogenesis.

The pathways of gametogenesis encompass elaborate cellular specialization accompanied by precise partitioning of the genome content in order to produce fully matured spermatozoa and oocytes. Transcription factors are an important class of molecules that function in gametogenesis to regulate intrinsic gene expression programs, play essential roles in specifying (or determining) germ cell fate and assist in guiding full maturation of germ cells and maintenance of their populations. Moreover, in order to reinforce or redirect cell fate in vitro, it is transcription factors that are most frequently induced, over-expressed or activated. Many reviews have focused on the molecular development and genetics of gametogenesis, in vivo and in vitro, in model organisms and in humans, including several recent comprehensive reviews: here, we focus specifically on the role of transcription factors. Recent advances in stem cell biology and multi-omic studies have enabled deeper investigation into the unique transcriptional mechanisms of human reproductive development. Moreover, as methods continually improve, in vitro differentiation of germ cells can provide the platform for robust gain- and loss-of-function genetic analyses. These analyses are delineating unique and shared human germ cell transcriptional network components that, together with somatic lineage specifiers and pluripotency transcription factors, function in transitions from pluripotent stem cells to gametes. This grand theme review offers additional insight into human infertility and reproductive disorders that are linked predominantly to defects in the transcription factor networks and thus may potentially contribute to the development of novel treatments for infertility.

Treatment of Parkinson's Disease through Personalized Medicine and Induced Pluripotent Stem Cells.

Parkinson's Disease (PD) is an intractable disease resulting in localized neurodegeneration of dopaminergic neurons of the substantia nigra pars compacta. Many current therapies of PD can only address the symptoms and not the underlying neurodegeneration of PD. To better understand the pathophysiological condition, researchers continue to seek models that mirror PD's phenotypic manifestations as closely as possible. Recent advances in the field of cellular reprogramming and personalized medicine now allow for previously unattainable cell therapies and patient-specific modeling of PD using induced pluripotent stem cells (iPSCs). iPSCs can be selectively differentiated into a dopaminergic neuron fate naturally susceptible to neurodegeneration. In iPSC models, unlike other artificially-induced models, endogenous cellular machinery and transcriptional feedback are preserved, a fundamental step in accurately modeling this genetically complex disease. In addition to accurately modeling PD, iPSC lines can also be established with specific genetic risk factors to assess genetic sub-populations' differing response to treatment. iPS cell lines can then be genetically corrected and subsequently transplanted back into the patient in hopes of re-establishing function. Current techniques focus on iPSCs because they are patient-specific, thereby reducing the risk of immune rejection. The year 2018 marked history as the year that the first human trial for PD iPSC transplantation began in Japan. This form of cell therapy has shown promising results in other model organisms and is currently one of our best options in slowing or even halting the progression of PD. Here, we examine the genetic contributions that have reshaped our understanding of PD, as well as the advantages and applications of iPSCs for modeling disease and personalized therapies.

Derivation of human induced pluripotent stem cell line (HUSTi001-A) from a 40-year-old patient with idiopathic infertility.

Human fibroblasts were isolated from foreskin of a clinically diagnosed 40-year old patient with idiopathic infertility. The fibroblasts were reprogrammed with the Yamanaka KOSM transcriptional factors using the retroviral vectors. The obtained induced pluripotent stem cell (iPSC) line showed pluripotency verified by the expression of pluripotency markers, NANOG, SOX2, OCT4, TRA-1-60, and SSEA-4. And the iPSC line was demonstrated to have the three germ layers differentiation capacity in vivo by teratoma assay. The iPSC line also showed normal karyotype. This patient-specific iPSC line can be used to explore the mechanism for idiopathic male infertility.

Efficacy and Safety of Immuno-Magnetically Sorted Smooth Muscle Progenitor Cells Derived from Human-Induced Pluripotent Stem Cells for Restoring Urethral Sphincter Function.

Human-induced pluripotent stem cells (hiPSCs)-based cell therapy holds promise for treating stress urinary incontinence (SUI). However, safety concerns, especially tumorgenic potential of residual undifferentiated cells in hiPSC derivatives, are major barriers for its clinical translation. An efficient, fast and clinical-scale strategy for purifying committed cells is also required. Our previous studies demonstrated the regenerative effects of hiPSC-derived smooth muscle progenitor cells (pSMCs) on the injured urethral sphincter in SUI, but the differentiation protocol required fluorescence-activated cell sorting (FACS) which is not practical for autologous clinical applications. In this study, we examined the efficacy and safety of hiPSC-derived pSMC populations sorted by FDA-approved magnetic-activated cell sorting (MACS) using cell-surface marker CD34 for restoring urethral sphincter function. Although the heterogeneity of MACS-sorted pSMCs was higher than that of FACS-sorted pSMCs, the percentage of undifferentiated cells dramatically decreased after directed differentiation in vitro. In vivo studies demonstrated long-term cell integration and no tumor formation of MACS-sorted pSMCs after transplantation. Furthermore, transplantation of MACS-sorted pSMCs into immunodeficient SUI rats was comparable to transplantation with FACS-sorted pSMCs for restoration of the extracellular matrix metabolism and function of the urethral sphincter. In summary, purification of hiPSC derivatives using MACS sorting for CD34 expression represent an efficient approach for production of clinical-scale pSMCs for autologous stem cell therapy for regeneration of smooth muscle tissues. Stem Cells Translational Medicine 2017;6:1158-1167.

Spatiotemporal Reconstruction of the Human Blastocyst by Single-Cell Gene-Expression Analysis Informs Induction of Naive Pluripotency.

Human preimplantation embryo development involves complex cellular and molecular events that lead to the establishment of three cell lineages in the blastocyst: trophectoderm, primitive endoderm, and epiblast. Owing to limited resources of biological specimens, our understanding of how the earliest lineage commitments are regulated remains narrow. Here, we examined gene expression in 241 individual cells from early and late human blastocysts to delineate dynamic gene-expression changes. We distinguished all three lineages and further developed a 3D model of the inner cell mass and trophectoderm in which individual cells were mapped into distinct expression domains. We identified in silico precursors of the epiblast and primitive endoderm lineages and revealed a role for MCRS1, TET1, and THAP11 in epiblast formation and their ability to induce naive pluripotency in vitro. Our results highlight the potential of single-cell gene-expression analysis in human preimplantation development to instruct human stem cell biology.

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.

X chromosome inactivation: recent advances and a look forward.

X chromosome inactivation, the transcriptional inactivation of one X chromosome in somatic cells of female mammals, has revealed important advances in our understanding of development, epigenetic control, and RNA biology. Most of this knowledge comes from extensive studies in the mouse; however, there are some significant differences when compared to human biology. This is especially true in pluripotent cell types and, over the past few years, a significant amount of work has been dedicated to understanding these differences. This review focuses specifically on recent advances in the mechanism of Xist spreading, the role of Xist in cancer, the effects of reprogramming on X chromosome inactivation in human induced pluripotent stem cells, and new tools for studying X chromosome inactivation.

Directed dopaminergic neuron differentiation from human pluripotent stem cells.

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson's disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

Human germ cell formation in xenotransplants of induced pluripotent stem cells carrying X chromosome aneuploidies.

Turner syndrome is caused by complete or partial loss of the second sex chromosome and is characterized by spontaneous fetal loss in >90% of conceptions. Survivors possess an array of somatic and germline clinical characteristics. Induced pluripotent stem cells (iPSCs) offer an opportunity for insight into genetic requirements of the X chromosome linked to Turner syndrome. We derived iPSCs from Turner syndrome and control individuals and examined germ cell development as a function of X chromosome composition. We demonstrate that two X chromosomes are not necessary for reprogramming or maintenance of pluripotency and that there are minimal differences in gene expression, at the single cell level, linked to X chromosome aneuploidies. Formation of germ cells, as assessed in vivo through a murine xenotransplantation model, indicated that undifferentiated iPSCs, independent of X chromosome composition, are capable of forming germ-cell-like cells (GCLCs) in vivo. In combination with clinical data regarding infertility in women with X chromosome aneuploidies, results suggest that two intact X chromosomes are not required for human germ cell formation, qualitatively or quantitatively, but rather are likely to be required for maintenance of human germ cells to adulthood.

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.

A modified method for implantation of pluripotent stem cells under the rodent kidney capsule.

Teratoma formation, the standard in vivo pluripotency assay, is also frequently used as a tumorigenicity assay. A common concern in therapeutic stem cell applications is the tumorigenicity potential of a small number of cell impurities in the final product. Estimation of this small number is hampered by the inaccurate methodology of the tumorigenicity assay. Hence, a protocol for tumorigenicity assay that can deliver a defined number of cells, without error introduced by leakage or migration of cells is needed. In this study, we tested our modified transplantation method that allows for transplant of small numbers of pluripotent stem cells (PSCs) under the kidney capsule with minimal cell leakage. A glass capillary with a finely shaped tip and an attached mouth pipette was used to inject PSCs into the rodent kidney capsule. H9 embryonic and induced PSCs were tagged with Fluc and green fluorescence protein reporter genes and divided in different cell doses for transplantation. Bioluminescence imaging (BLI) on the day of surgery showed that the cell signal was confined to the kidney and signal intensity correlated with increasing transplant cell numbers. The overall cell leakage rate was 17% and the rodent survival rate was 96%. Teratoma formation was observed in rodents transplanted with cell numbers between 1 × 10(5)-2 × 10(6). We conclude that this modified procedure for transplanting PSCs under the kidney capsule allows for transplantation of a defined number of PSCs with significant reduction of error associated with cell leakage from the transplant site.

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.

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.

Concurrent generation of functional smooth muscle and endothelial cells via a vascular progenitor.

Smooth muscle cells (SMCs) and endothelial cells (ECs) are typically derived separately, with low efficiencies, from human pluripotent stem cells (hPSCs). The concurrent generation of these cell types might lead to potential applications in regenerative medicine to model, elucidate, and eventually treat vascular diseases. Here we report a robust two-step protocol that can be used to simultaneously generate large numbers of functional SMCs and ECs from a common proliferative vascular progenitor population via a two-dimensional culture system. We show here that coculturing hPSCs with OP9 cells in media supplemented with vascular endothelial growth factor, basic fibroblast growth factor, and bone morphogenetic protein 4 yields a higher percentage of CD31(+)CD34(+) cells on day 8 of differentiation. Upon exposure to endothelial differentiation media and SM differentiation media, these vascular progenitors were able to differentiate and mature into functional endothelial cells and smooth muscle cells, respectively. Furthermore, we were able to expand the intermediate population more than a billion fold to generate sufficient numbers of ECs and SMCs in parallel for potential therapeutic transplantations.

Vascular progenitors from cord blood-derived induced pluripotent stem cells possess augmented capacity for regenerating ischemic retinal vasculature.

BACKGROUND: The generation of vascular progenitors (VPs) from human induced pluripotent stem cells (hiPSCs) has great potential for treating vascular disorders such as ischemic retinopathies. However, long-term in vivo engraftment of hiPSC-derived VPs into the retina has not yet been reported. This goal may be limited by the low differentiation yield, greater senescence, and poor proliferation of hiPSC-derived vascular cells. To evaluate the potential of hiPSCs for treating ischemic retinopathies, we generated VPs from a repertoire of viral-integrated and nonintegrated fibroblast and cord blood (CB)-derived hiPSC lines and tested their capacity for homing and engrafting into murine retina in an ischemia-reperfusion model. METHODS AND RESULTS: VPs from human embryonic stem cells and hiPSCs were generated with an optimized vascular differentiation system. Fluorescence-activated cell sorting purification of human embryoid body cells differentially expressing endothelial/pericytic markers identified a CD31(+)CD146(+) VP population with high vascular potency. Episomal CB-induced pluripotent stem cells (iPSCs) generated these VPs with higher efficiencies than fibroblast-iPSC. Moreover, in contrast to fibroblast-iPSC-VPs, CB-iPSC-VPs maintained expression signatures more comparable to human embryonic stem cell VPs, expressed higher levels of immature vascular markers, demonstrated less culture senescence and sensitivity to DNA damage, and possessed fewer transmitted reprogramming errors. Luciferase transgene-marked VPs from human embryonic stem cells, CB-iPSCs, and fibroblast-iPSCs were injected systemically or directly into the vitreous of retinal ischemia-reperfusion-injured adult nonobese diabetic-severe combined immunodeficient mice. Only human embryonic stem cell- and CB-iPSC-derived VPs reliably homed and engrafted into injured retinal capillaries, with incorporation into damaged vessels for up to 45 days. CONCLUSIONS: VPs generated from CB-iPSCs possessed augmented capacity to home, integrate into, and repair damaged retinal vasculature.

Reprogramming of fibroblasts from older women with pelvic floor disorders alters cellular behavior associated with donor age.

We aimed to derive induced pluripotent stem cell (iPSC) lines from vaginal fibroblasts from older women with pelvic organ prolapse. We examined the effect of donor age on iPSCs and on the cells redifferentiated from these iPSCs. Vaginal fibroblasts were isolated from younger and older subjects for reprogramming. iPSCs were generated simultaneously using an excisable polycistronic lentiviral vector expressing Oct4, Klf4, Sox2, and cMyc. The pluripotent markers of iPSCs were confirmed by immunocytochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Spectral karyotyping was performed. The ability of the iPSCs to differentiate into three germ layers was confirmed by embryoid body and teratoma formation. Senescence marker (p21, p53, and Bax) expressions were determined by qRT-PCR and Western blot. The iPSCs were redifferentiated to fibroblasts and were evaluated with senescence-associated ß-galactosidase (SA) activity and mitotic index using time-lapse dark-field microscopy. iPSCs derived from both the younger and older subjects expressed pluripotency markers and showed normal karyotype and positive teratoma assays. There was no significant difference in expression of senescence and apoptosis markers (p21, p53, and Bax) in iPSCs derived from the younger subject compared with the older subject. Furthermore, fibroblasts redifferentiated from these iPSCs did not differ in SA activity or mitotic index. We report successful derivation of iPSCs from women with pelvic organ prolapse. Older age did not interfere with successful reprogramming. Donor age differences were not observed in these iPSCs using standard senescence markers, and donor age did not appear to affect cell mitotic activity in fibroblasts redifferentiated from iPSCs.

Human amniotic mesenchymal stem cell-derived induced pluripotent stem cells may generate a universal source of cardiac cells.

Human amniotic mesenchymal stem cells (hAMSCs) demonstrated partially pluripotent characteristics with a strong expression of Oct4 and Nanog genes and immunomodulatory properties characterized by the absence of HLA-DR and the presence of HLA-G and CD59. The hAMSCs were reprogrammed into induced pluripotent stem cells (iPSCs) that generate a promising source of universal cardiac cells. The hAMSC-derived iPSCs (MiPSCs) successfully underwent robust cardiac differentiation to generate cardiomyocytes. This study investigated 3 key properties of the hAMSCs and MiPSCs: (1) the reprogramming efficiency of the partially pluripotent hAMSCs to generate MiPSCs; (2) immunomodulatory properties of the hAMSCs and MiPSCs; and (3) the cardiac differentiation potential of the MiPSCs. The characteristic iPSC colony formation was observed within 10 days after the transduction of the hAMSCs with a single integration polycistronic vector containing 4 Yamanaka factors. Immunohistology and reverse transcription-polymerase chain reaction assays revealed that the MiPSCs expressed stem cell surface markers and pluripotency-specific genes. Furthermore, the hAMSCs and MiPSCs demonstrated immunomodulatory properties enabling successful engraftment in the SVJ mice. Finally, the cardiac differentiation of MiPSCs exhibited robust spontaneous contractility, characteristic calcium transience across the membrane, a high expression of cardiac genes and mature cardiac phenotypes, and a contractile force comparable to cardiomyocytes. Our results demonstrated that the hAMSCs are reprogrammed with a high efficiency into MiPSCs, which possess pluripotent, immunomodulatory, and precardiac properties. The MiPSC-derived cardiac cells express a c-kit cell surface marker, which may be employed to purify the cardiac cell population and enable allogeneic cardiac stem cell therapy.

Theranostic effect of serial manganese-enhanced magnetic resonance imaging of human embryonic stem cell derived teratoma.

Although human embryonic stem cell (hESC) hold therapeutic potential, teratoma formation has deterred clinical translation. Manganese (Mn(2+)) enters metabolically active cells through voltage-gated calcium channels and subsequently, induces T(1) shortening. We hypothesized that serial manganese-enhanced MRI would have theranostic effect to assess hESC survival, teratoma formation, and hESC-derived teratoma reduction through intracellular accumulation of Mn(2+). Firefly luciferase transduced hESCs (hESC-Lucs) were transplanted into severe combined immunodeficient mouse hindlimbs to form teratoma. The chemotherapy group was injected with MnCl(2) intraperitoneally three times a week. The control group was given MnCl(2) only prior to manganese-enhanced MRI. Longitudinal evaluation by manganese-enhanced MRI and bioluminescence imaging was performed. The chemotherapy group showed significant reduction in the teratoma volume and luciferase activity at weeks 6 and 8. Histology revealed increased proportion of dead cells and caspase 3 positive cells in the chemotherapy group. Systemic administration of MnCl(2) enabled simultaneous monitoring and elimination of hESC-derived teratoma cells by higher intracellular accumulation of Mn(2+).

A characterization of the relationship of ovarian reserve markers with age.

OBJECTIVE: To identify markers of ovarian age that best match the pattern of oocyte loss seen in histology specimens. DESIGN: Cross-sectional study. SETTING: University. PATIENT(S): Caucasian women (n = 252) aged 25-45 years. INTERVENTION(S): none. MAIN OUTCOME MEASURE(S): The relationship between antral follicle count (AFC), antimüllerian hormone (AMH), inhibin B, FSH, and E(2) with age was estimated using the power model, which previously has been shown to most accurately describe oocyte loss in histologic specimens. The power model was fit to each marker and used to compare the rates of change at ages 30 and 40 with the histologic pattern. Among those markers following the pattern, R(2) was used to compare the degree of relationship with age. RESULT(S): Both AMH levels and AFC exhibited significant progressive declines with age. The average rates of loss per year for AFC and AMH were, respectively, -0.57 and -1.09 at age 30, and -1.33 and -3.06 at age 40. FSH, inhibin B, and E(2) did not exhibit progressive rates of change. The R(2) for AFC was 27.3% and for AMH was 22.7%. CONCLUSION(S): Only AFC and AMH follow the pattern of oocyte loss observed histologically. Although AMH may be more cost-effective, AFC is a slightly more accurate noninvasive measure for ovarian aging.

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.