Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients.
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ABSTRACT:
SUBMITTER: de Souza AF
PROVIDER: S-EPMC8624672 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Despite increasing insight into the genetics of infertility, the developmental disease processes remain unclear due to the lack of adequate experimental models. The advent of induced pluripotent stem cell (iPSC) technology has provided a unique tool for in vitro disease modeling enabling major advances in our understanding of developmental disease processes. We report the full characterization of complex genetic abnormalities in two infertile patients with either azoospermia or XX male syndrome and we identify genes of potential interest implicated in their infertility. Using the erythroblasts of both patients, we generated primed iPSCs and converted them into a naive-like pluripotent state. Naive-iPSCs were then differentiated into primordial germ-like cells (PGC-LCs). The expression of early PGC marker genes SOX17, CD-38, NANOS3, c-KIT, TFAP2C, and D2-40, confirmed progression towards the early germline stage. Our results demonstrate that iPSCs from two infertile patients with significant genetic abnormalities are capable of efficient production of PGCs. Such in vitro model of infertility will certainly help identifying causative factors leading to early germ cells development failure and provide a valuable tool to explore novel therapeutic strategies.
| S-EPMC9395518 | biostudies-literature
Project description:Primordial germ cells (PGCs) are the founder cells of the germline. The ability to generate PGC-like cells (PGCLCs) from pluripotent stem cells has advanced our knowledge of gametogenesis and holds promise for developing infertility treatments. However, generating an ample supply of PGCLCs for demanding applications such as high-throughput genetic screens has been a limitation. Here, we demonstrated that simultaneous overexpressing 4 transcriptional factors - Nanog and three PGC master regulators Prdm1, Prdm14 and Tfap2c - in suspended mouse epiblast like cells (EpiLCs) and formative embryonic stem cells (ESCs) results in efficient and cost-effective production of PGCLCs. The overexpression of Nanog enhances the PGC regulatory network and suppresses differentiation of somatic lineages, enabling a significant improvement in the efficiency of PGCLC production. Transcriptomic analysis reveals that differentiated PGCLCs exhibit similarities to in vivo PGCs and are more advanced compared to cytokine-induced PGCLCs. These differentiated PGCLCs could be sustained over prolonged periods of culture and could differentiate into spermatogonia-like cells in vitro. Importantly, the ability to produce PGCLCs at scale, without using costly cytokines, enables biochemical and functional genomic screens to dissect mechanisms of germ cell development and infertility.
| S-EPMC10888945 | biostudies-literature
Project description:Stem cells are unique cell types capable to proliferate, some of them indefinitely, while maintaining the ability to differentiate into a few or any cell lineages. In 2003, a group headed by Hans R. Schöler reported that oocyte-like cells could be produced from mouse embryonic stem (ES) cells in vitro. After more than 10 years, where have these researches reached? Which are the major successes achieved and the problems still remaining to be solved? Although during the last years, many reviews have been published about these topics, in the present work, we will focus on an aspect that has been little considered so far, namely a strict comparison between the in vitro and in vivo developmental capabilities of the primordial germ cells (PGCs) isolated from the embryo and the PGC-like cells (PGC-LCs) produced in vitro from different types of stem cells in the mouse, the species in which most investigation has been carried out. Actually, the formation and differentiation of PGCs are crucial for both male and female gametogenesis, and the faithful production of PGCs in vitro represents the basis for obtaining functional germ cells.
| S-EPMC4632295 | biostudies-other
Project description:ObjectivesPrevious studies have shown that adipose mesenchymal stem cells (AMSCs) share the potency of typical bone marrow mesenchymal stem cells (MSCs); however, there is little information concerning characteristics of canine AMSCs (CAMSCs); it has not previously been made clear whether CAMSCs would be able to differentiate into other cell types.Materials and methodsIn this study, typical AMSC lines were established, and their characteristics including morphology, typical markers and differentiation potentiality were tested.ResultsThe cells exhibited typical MSC morphology and were positive for CD90, CD44 and CD166, considered to be MSCs surface markers. They were negative for CD34 and CD45. The CAMSCs also exhibited embryonic stem cell (ESC) markers, including Oct4 and Sox2, at passage 2. In an appropriate microenvironment, CAMSCs differentiated into EBs and were able to produce cells of the three germ layers. These results indicate that established cells were putative adipocyte-derived MSCs, which also displayed properties of ESCs. Moreover, when the CAMSCs were induced by bone morphogenetic protein 4 (BMP4), they differentiated into PGC-like cells (PGCLCs) and male germ-like cells, which were positive for PR domain-containing 1 (Prdm1), PR domain-containing 14 (Prdm14), doublesex and mab-3 related transcription factor (Dmrt1), as well as for promyelocytic leukaemia zinc finger (Plzf). Quantitative real-time PCR (qRT-PCR) and western blotting analysis verified higher expression levels of these markers.ConclusionThis study provides an efficient approach to study germ cell development using CAMSCs.
| S-EPMC6496567 | biostudies-literature
Project description:Primordial germ cells (PGCs) are the embryonic precursors of sperm and oocytes, which transmit genetic/epigenetic information across generations. Mouse PGC and subsequent gamete development can be fully reconstituted in vitro, opening up new avenues for germ cell studies in biomedical research. However, PGCs show molecular differences between rodents and humans. Therefore, to establish an in vitro system that is closely related to humans, we studied PGC development in vivo and in vitro in the common marmoset monkey Callithrix jacchus (cj). Gonadal cjPGCs at embryonic day 74 express SOX17, AP2Ɣ, BLIMP1, NANOG, and OCT4A, which is reminiscent of human PGCs. We established transgene-free induced pluripotent stem cell (cjiPSC) lines from foetal and postnatal fibroblasts. These cjiPSCs, cultured in defined and feeder-free conditions, can be differentiated into precursors of mesendoderm and subsequently into cjPGC-like cells (cjPGCLCs) with a transcriptome similar to human PGCs/PGCLCs. Our results not only pave the way for studying PGC development in a non-human primate in vitro under experimentally controlled conditions, but also provide the opportunity to derive functional marmoset gametes in future studies.
| S-EPMC10948935 | biostudies-literature
Project description:Reconstitution of germ cell fate from pluripotent stem cells provides an opportunity to understand the molecular underpinnings of germ cell development. Here, we established robust methods for induced pluripotent stem cell (iPSC) culture in the common marmoset (Callithrix jacchus [cj]), allowing stable propagation in an undifferentiated state. Notably, iPSCs cultured on a feeder layer in the presence of a WNT signaling inhibitor upregulated genes related to ubiquitin-dependent protein catabolic processes and enter a permissive state that enables differentiation into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs in vivo. Induction of cjPGCLCs is accompanied by transient upregulation of mesodermal genes, culminating in the establishment of a primate-specific germline transcriptional network. Moreover, cjPGCLCs can be expanded in monolayer while retaining the germline state. Upon co-culture with mouse testicular somatic cells, these cells acquire an early prospermatogonia-like phenotype. Our findings provide a framework for understanding and reconstituting marmoset germ cell development in vitro, thus providing a comparative tool and foundation for a preclinical modeling of human in vitro gametogenesis.
| S-EPMC9937652 | biostudies-literature
Project description:BackgroundWe have previously demonstrated that stem cells isolated from fetal porcine skin have the potential to form oocyte-like cells (OLCs) in vitro. However, primordial germ cells (PGCs), which must also be specified during the stem cell differentiation to give rise to these putative oocytes at more advanced stages of culture, were not systematically characterized. The current study tested the hypothesis that a morphologically distinct population of cells derived from skin stem cells prior to OLC formation corresponds to putative PGCs, which differentiate further into more mature gametes.Methodology/principal findingsWhen induced to differentiate in an appropriate microenvironment, a subpopulation of morphologically distinct cells, some of which are alkaline phosphatase (AP)-positive, also express Oct4, Fragilis, Stella, Dazl, and Vasa, which are markers indicative of germ cell formation. A known differentially methylated region (DMR) within the H19 gene locus, which is demethylated in oocytes after establishment of the maternal imprint, is hypomethylated in PGC-like cells compared to undifferentiated skin-derived stem cells, suggesting that the putative germ cell population undergoes imprint erasure. Additional evidence supporting the germ cell identity of in vitro-generated PGC-like cells is that, when labeled with a Dazl-GFP reporter, these cells further differentiate into GFP-positive OLCs.SignificanceThe ability to generate germ cell precursors from somatic stem cells may provide an in vitro model to study some of the unanswered questions surrounding early germ cell formation.
| S-EPMC2788220 | biostudies-literature
Project description:BackgroundPrimordial germ cell (PGC) specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES) cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo.Methodology and principal findingsHere, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stella-negative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB) method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation.Conclusions and significanceThe in vitro model we have established can recapitulate the developmental processes in vivo and provides new insights into the mechanism of PGC specification.
| S-EPMC2602984 | biostudies-literature
Project description:At present, the etiology of most non-obstructive azoospermia (NOA) remains unclear. In vitro generation of patient-specific induced pluripotent stem cells (iPSCs) is an effective approach for exploring the mechanisms of human disease. Here, we established iPSCs from two patients with idiopathic NOA and differentiated them into primordial germ cell-like cells (PGCLCs) in vitro. Compared with iPSCs derived from normal fertile men, the NOA patient-specific iPSCs show decreased efficiency of PGCLC formation in vitro. Particularly, the embryoids derived from NOA patient-specific iPSCs show defects in the expression of early primordial germ cell (PGC) genes. The transcriptome analysis reveals the expression patterns of key human PGC genes are generally similar in PGCLCs differentiated from all iPSC lines, and the differentially expressed genes were enriched with gene ontology (GO) of cell cycle and apoptosis regulation. Moreover, the PGCLCs derived from NOA patient-specific iPSCs might have initiated epigenetic reprogramming at a very early stage. Thus, the NOA patient-specific iPSCs exhibit poor response to germ cell induction in vitro, which may be related to the regulation of apoptotic process. These findings provide a foundation for future research on mechanism of male infertility.
| S-EPMC7331483 | biostudies-literature
Project description:Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogen-receptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells.
| S-EPMC4987791 | biostudies-literature