scholarly journals Oocyte Arrested at Metaphase II Stage Were Derived From Human Pluripotent Stem Cells in Vitro

2021 ◽  
Author(s):  
Xiaoli Yu ◽  
Ning Wang ◽  
Yingxin Zhang ◽  
Xiang Wang ◽  
Yikai Qiu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Polar Body ◽  
Primordial Follicle ◽  
Preimplantation Embryos ◽  
Differentiation Potential ◽  
Cell Medium

Abstract BackgroundGeneration and maturation of human oocyte in vitro could facilitate studies of folliculogenesis and oogenesis. We have previously shown that human aminotic fluid stem cells giving rise to oocyte-like cells (OLCs), However, it was difficult to observe whether these OLCs enter meiotic stage. MethodsHuman induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) were cultured by follicle fluid, cytokines and small molecule to induced oocyte-like cells (OLCs) formation through a three-step induction procedure. Surface marker expression and differentiation potential of germ cells were analyzed in vitro by flow cytometry, gene expression, immunocytochemistry, western blotting and RNA Sequencing.ResultsTo induce hiPSCs differentiation into OLCs, cells were firstly cultured in a primordial germ cell medium for 10 days. The cells showed the morphology similar to primordial germ cells (PGCs), highly expressing germ cell markers and primordial follicle development associated genes. The induced PGCs were then cultured in the primordial follicle-like cell medium for 5 days to form the induced follicle-like structures (iFLs), which retained both primordial oocytes-like cells and granulosa-like cells. In the third step, the detached iFLs were harvested and transferred to the OLC-medium for additional 10 days. The cumulus-oocyte-complexes (COC) structures and OLCs in different sizes (50-150 μm diameter) with zona pellucida were observed. The in vitro matured OLCs presented the polar body and arrested at metaphase II (MII) stage. Some OLCs were self-activated and spontaneously developed into multiple-cell structures similar to preimplantation embryos, indicating that OLCs were parthenogenetically activated though in vitro fertilization potential of OLCs are yet proved.ConclusionsIn vitro maturation of OLCs derived from hiPSCs provides a new means to study human germ cell formation and oogenesis.

183 In Vitro Generation and Characterization of Putative Primordial Germ Cells Derived from Induced Pluripotent Stem Cells in Cattle

2018 ◽  
Vol 30 (1) ◽  
pp. 231
Author(s):  
F. F. Bressan ◽  
M. A. Lima ◽  
L. S. Machado ◽  
N. C. G. Pieiri ◽  
P. Fantinato-Neto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Growth Factor ◽  
Germ Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cells ◽  
Farm Animals ◽  
Induced Pluripotent

Embryonic pluripotent stem cells (ESC) and induced pluripotent stem cells (iPSC) were reported capable of differentiating into primordial germ cell-like (PGCL) and functional gametes in vitro in the murine model (Hikabe et al. 2016 Nature 539, 299-303). The in vitro generation of primordial germ cells (PGC) and gametes from farm animals would greatly contribute to enhance animal production technologies and to the creation of adequate models for several disorders. The present study aimed at the generation of PGC in vitro (iPGC) from iPSC in cattle and their characterisation through pluripotency and germ cell markers. For that, bovine iPSC previously generated and characterised (Bressan et al. 2015 Reprod. Fertil. Dev. 27, 254) were submitted to in vitro differentiation into epiblast-like cells (EpiLC) and iPGC by the protocol adapted from mice (Hayashi et al. 2011 Cell 146, 519-532). The biPS cells were induced into EpiLC by culture in fibronectin-coated (16.7 µg mL−1) 6-well plates in N2B27 culture medium supplemented with 20 ng mL−1 activin A, 12 ng mL−1 basic fibroblast growth factor (bFGF), and 1% knockout serum replacement (KSR) for 48 h and further differentiated into iPGC by non-adherent culture (Agreewell plates, StemCell Technologies, Vancouver, BC, Canada) with GK15 medium (GMEM supplemented with 15% KSR, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 2 mm l-glutamine, and 1% antibiotics) in the presence of 500 ng mL−1 BMP4, 100 ng mL−1 SCF, 500 ng mL−1 BMP8b, and 50 ng mL−1 epidermal growth factor for 4 days. The cells were then characterised regarding morphology, detection of alkaline phosphatase, immunofluorescence for OCT4, DDX4, VASA, and c-Kit proteins, and transcripts of pluripotency-related genes OCT4 and SOX2, as well as of imprinted genes (H19, SNRPN) and imprinted-related (DNMT1, DNMT3B) genes were analysed through RT-qPCR and compared with constitutive genes GAPDH, NAT1, and ACTB. Alkaline phosphatase and immunofluorescence analysis were positive for all specific markers. Interestingly, although OCT4 and SOX2 expression was present in iPS, EpiLC, and iPGC, this last group presented greater OCT4 and lesser SOX2 transcript amounts compared with other groups, suggesting, as expected, that PGC are still pluripotent but may already be differentiating into germ-cell lineages. The expression of H19 was increased in iPGC, whereas the expression of SNRPN was decreased only in the fibroblast group, potentially indicating epigenetic reprogramming process in these cells. Expression of DNMT1 and DNMT3B was not different between pluripotent groups but subtly increased when compared with that in fibroblasts. The results obtained herein represent an important first step in the in vitro generation of PGC and gametes from domestic farm animals, an unprecedented and desirable tool for enhancing new reproductive technologies and providing new understanding of cellular reprogramming and pluripotent germ cell biology. Financially supported by FAPESP grants 2013/08135-2, 2013/13686-8, 2015/26818-5; CNPq 482163/2013-5.

scholarly journals Germ Cell Derivation from Pluripotent Stem Cells for Understanding In Vitro Gametogenesis

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1889
Author(s):  
Tae-Kyung Hong ◽  
Jae-Hoon Song ◽  
So-Been Lee ◽  
Jeong-Tae Do
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Assisted Reproductive Technologies ◽  
Primordial Germ Cells ◽  
Reproductive Technologies ◽  
Obstructive Azoospermia ◽  
Female Germ Cell

Assisted reproductive technologies (ARTs) have developed considerably in recent years; however, they cannot rectify germ cell aplasia, such as non-obstructive azoospermia (NOA) and oocyte maturation failure syndrome. In vitro gametogenesis is a promising technology to overcome infertility, particularly germ cell aplasia. Early germ cells, such as primordial germ cells, can be relatively easily derived from pluripotent stem cells (PSCs); however, further progression to post-meiotic germ cells usually requires a gonadal niche and signals from gonadal somatic cells. Here, we review the recent advances in in vitro male and female germ cell derivation from PSCs and discuss how this technique is used to understand the biological mechanism of gamete development and gain insight into its application in infertility.

scholarly journals Human Pluripotent Stem Cells in Reproductive Science—A Comparison of Protocols Used to Generate and Define Male Germ Cells from Pluripotent Stem Cells

2020 ◽  
Vol 21 (3) ◽  
pp. 1028
Author(s):  
Magdalena Kurek ◽  
Halima Albalushi ◽  
Outi Hovatta ◽  
Jan-Bernd Stukenborg
Keyword(s):  
Stem Cells ◽  
Growth Factors ◽  
Germ Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Male Germ Cells ◽  
Germ Cell Differentiation

Globally, fertility-related issues affect around 15% of couples. In 20%–30% of cases men are solely responsible, and they contribute in around 50% of all cases. Hence, understanding of in vivo germ-cell specification and exploring different angles of fertility preservation and infertility intervention are considered hot topics nowadays, with special focus on the use of human pluripotent stem cells (hPSCs) as a source of in vitro germ-cell generation. However, the generation of male germ cells from hPSCs can currently be considered challenging, making a judgment on the real perspective of these innovative approaches difficult. Ever since the first spontaneous germ-cell differentiation studies, using human embryonic stem cells, various strategies, including specific co-cultures, gene over-expression, and addition of growth factors, have been applied for human germ-cell derivation. In line with the variety of differentiation methods, the outcomes have ranged from early and migratory primordial germ cells up to post-meiotic spermatids. This variety of culture approaches and cell lines makes comparisons between protocols difficult. Considering the diverse strategies and outcomes, we aim in this mini-review to summarize the literature regarding in vitro derivation of human male germ cells from hPSCs, while keeping a particular focus on the culture methods, growth factors, and cell lines used.

scholarly journals Advances in Female Germ Cell Induction from Pluripotent Stem Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Maisumu Gulimiheranmu ◽  
Xinjie Wang ◽  
Junmei Zhou
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Ethical Issues ◽  
Developmental Process ◽  
Human Embryos ◽  
Human Female ◽  
Germ Cell Differentiation

Germ cells are capable of maintaining species continuity through passing genetic and epigenetic information across generations. Female germ cells mainly develop during the embryonic stage and pass through subsequent developmental stages including primordial germ cells, oogonia, and oocyte. However, due to the limitation of using early human embryos as in vivo research model, in vitro research models are needed to reveal the early developmental process and related mechanisms of female germ cells. After birth, the number of follicles gradually decreases with age. Various conditions which damage ovarian functions would cause premature ovarian failure. Alternative treatments to solve these problems need to be investigated. Germ cell differentiation from pluripotent stem cells in vitro can simulate early embryonic development of female germ cells and clarify unresolved issues during the development process. In addition, pluripotent stem cells could potentially provide promising applications for female fertility preservation after proper in vitro differentiation. Mouse female germ cells have been successfully reconstructed in vitro and delivered to live offspring. However, the derivation of functional human female germ cells has not been fully achieved due to technical limitations and ethical issues. To provide an updated and comprehensive information, this review centers on the major studies on the differentiation of mouse and human female germ cells from pluripotent stem cells and provides references to further studies of developmental mechanisms and potential therapeutic applications of female germ cells.

Mini Review; Differentiation of Human Pluripotent Stem Cells into Oocytes

2020 ◽  
Vol 15 (4) ◽  
pp. 301-307 ◽  
Author(s):  
Gaifang Wang ◽  
Maryam Farzaneh
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Human Pluripotent Stem Cells ◽  
Human Oocyte ◽  
Differentiation Potential ◽  
Cell Lineages ◽  
Cell Based Therapy ◽  
Induced Pluripotent

Primary Ovarian Insufficiency (POI) is one of the main diseases causing female infertility that occurs in about 1% of women between 30-40 years of age. There are few effective methods for the treatment of women with POI. In the past few years, stem cell-based therapy as one of the most highly investigated new therapies has emerged as a promising strategy for the treatment of POI. Human pluripotent stem cells (hPSCs) can self-renew indefinitely and differentiate into any type of cell. Human Embryonic Stem Cells (hESCs) as a type of pluripotent stem cells are the most powerful candidate for the treatment of POI. Human-induced Pluripotent Stem Cells (hiPSCs) are derived from adult somatic cells by the treatment with exogenous defined factors to create an embryonic-like pluripotent state. Both hiPSCs and hESCs can proliferate and give rise to ectodermal, mesodermal, endodermal, and germ cell lineages. After ovarian stimulation, the number of available oocytes is limited and the yield of total oocytes with high quality is low. Therefore, a robust and reproducible in-vitro culture system that supports the differentiation of human oocytes from PSCs is necessary. Very few studies have focused on the derivation of oocyte-like cells from hiPSCs and the details of hPSCs differentiation into oocytes have not been fully investigated. Therefore, in this review, we focus on the differentiation potential of hPSCs into human oocyte-like cells.

scholarly journals Formative pluripotent stem cells show features of epiblast cells poised for gastrulation

Cell Research ◽  
2021 ◽  
Author(s):  
Xiaoxiao Wang ◽  
Yunlong Xiang ◽  
Yang Yu ◽  
Ran Wang ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Large Set ◽  
Mouse Escs ◽  
Epiblast Stem Cells ◽  
Early Gastrula

AbstractThe pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

154 MOLECULAR CHARACTERIZATION OF CAT PRIMORDIAL GERM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 207
Author(s):  
J. Galiguis ◽  
C. E. Pope ◽  
C. Dumas ◽  
G. Wang ◽  
R. A. MacLean ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Transcript Abundance ◽  
Domestic Cat ◽  
Sample Type

As precursors to germline stem cells and gametes, there are many potential applications for primordial germ cells (PGC). Primordial germ cell-like cells have been generated from mouse embryonic stem cells and induced pluripotent stem cells, which subsequently were used to produce functional spermatozoa, oocytes, and healthy offspring (Hayashi et al. 2012 Science 338(6109), 971–975). Applying this approach to generate sperm and oocytes of endangered species is an appealing prospect. Detection of molecular markers associated with PGC is essential to optimizing the process of PGC induction. In the current study, in vitro-derived domestic cat embryos were assessed at various developmental stages to characterise the expression of markers related to the specification process of cat PGC. In vivo-matured, IVF oocytes were cultured until Days 7, 9, and 12 post-insemination. Then, embryos were assessed by RT-qPCR to determine relative transcript abundance of the pluripotency markers NANOG, POU5F1, and SOX2; the epiblast marker DNMT3B; the primitive endoderm marker GATA4; the PGC marker PRDM14; and the germ cell marker VASA; RPS19 was used as the internal reference gene. To validate the qPCR results, fibroblasts served as the negative control cells, whereas spermatogonial stem cells (SSC) served as the positive control cells for GATA4, PRDM14, and VASA. Total mRNA were isolated using the Cells-to-cDNA™ II Kit (Ambion/Thermo Fisher Scientific, Waltham, MA, USA) from either pools of 2 to 6 embryos or ~25 000 fibroblasts/SSC. A minimum of 2 biological replicates for each sample type was analysed, with transcript abundance detected in 2 technical replicates by SYBR Green chemistry. Student’s t-tests were performed on the ΔCts for statistical analysis. PRDM14, specific to the germ cell lineage, was detected as early as Day 7, suggesting the presence of PGC precursor cells. Compared with their levels at Day 7, PRDM14 expression was 0.34-fold lower in SSC (P < 0.05), whereas expression of VASA and GATA4 were 1964-fold and 144-fold higher, respectively (P < 0.05). This seems to emphasise the relative importance of PRDM14 in pre-germ cell stages. In general, all genes analysed were up-regulated from Day 7 to Day 9. This up-regulation was statistically significant for SOX2 and GATA4 (P < 0.05). Relative to that at Day 9, all transcripts were relatively less abundant at Day 12 (P < 0.05 for NANOG, POU5F1, SOX2, DNMT3B, and PRDM14). The data suggest that PGC specification takes place near Day 9, with peak specification activity concluding by Day 12. Although much needs be explored about PGC specification in the cat before applying induction and in vitro germ cell production techniques, these findings represent the first step towards a new potential strategy for preserving endangered and threatened felids.

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