In vitro derivation of germ cells from embryonic stem cells in mammals

In mammalian development, primordial germ cells (PGCs) represent the initial population of cells that are committed to the germ cell lineage. PGCs segregate early in development, triggered by signals from the extra-embryonic ectoderm. They are distinguished from surrounding cells by their unique gene expression patterns. Some of the more common genes used to identify them are Blimp1, Oct3/4, Fragilis, Stella, c-Kit, Mvh, Dazl and Gcna1. These genes are involved in regulating their migration and differentiation, and in maintaining the pluripotency of these cells. Recent research has demonstrated the possibility of obtaining PGCs, and subsequently, mature germ cells from a starting population of embryonic stem cells (ESCs) in culture. This phenomenon has been investigated using a variety of methods, and ESC lines of both mouse and human origin. Embryonic stem cells can differentiate into germ cells of both the male and female phenotype and in one case has resulted in the birth of live pups from the fertilization of oocytes with ESC derived sperm. This finding leads to the prospect of using ESC derived germ cells as a treatment for sterility. This review outlines the evolvement of germ cells from ESCs in vitro in relation to in vivo events.

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Testicular somatic cell-like cells derived from embryonic stem cells induce differentiation of epiblasts into germ cells

Communications Biology ◽

10.1038/s42003-021-02322-8 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Holly Rore ◽

Nicholas Owen ◽

Raul Eduardo Piña-Aguilar ◽

Kevin Docherty ◽

Ryohei Sekido

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Somatic Cell ◽

Germ Cells ◽

Pluripotent Stem Cells ◽

Seminiferous Tubule ◽

Embryonic Stem ◽

In Vitro Reconstitution ◽

Real Challenge

AbstractRegeneration of the testis from pluripotent stem cells is a real challenge, reflecting the complexity of the interaction of germ cells and somatic cells. Here we report the generation of testicular somatic cell-like cells (TesLCs) including Sertoli cell-like cells (SCLCs) from mouse embryonic stem cells (ESCs) in xeno-free culture. We find that Nr5a1/SF1 is critical for interaction between SCLCs and PGCLCs. Intriguingly, co-culture of TesLCs with epiblast-like cells (EpiLCs), rather than PGCLCs, results in self-organised aggregates, or testicular organoids. In the organoid, EpiLCs differentiate into PGCLCs or gonocyte-like cells that are enclosed within a seminiferous tubule-like structure composed of SCLCs. Furthermore, conditioned medium prepared from TesLCs has a robust inducible activity to differentiate EpiLCs into PGCLCs. Our results demonstrate conditions for in vitro reconstitution of a testicular environment from ESCs and provide further insights into the generation of sperm entirely in xeno-free culture.

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Germ cells from mouse and human embryonic stem cells

Reproduction ◽

10.1530/rep-06-0022 ◽

2006 ◽

Vol 132 (5) ◽

pp. 699-707 ◽

Cited By ~ 34

Author(s):

Behrouz Aflatoonian ◽

Harry Moore

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Germ Cells ◽

Primordial Germ Cells ◽

Endocrine Disrupting Chemicals ◽

Embryonic Stem ◽

Founder Population ◽

Testicular Dysgenesis Syndrome

Mammalian gametes are derived from a founder population of primordial germ cells (PGCs) that are determined early in embryogenesis and set aside for unique development. Understanding the mechanisms of PGC determination and differentiation is important for elucidating causes of infertility and how endocrine disrupting chemicals may potentially increase susceptibility to congenital reproductive abnormalities and conditions such as testicular cancer in adulthood (testicular dysgenesis syndrome). Primordial germ cells are closely related to embryonic stem cells (ESCs) and embryonic germ (EG) cells and comparisons between these cell types are providing new information about pluripotency and epigenetic processes. Murine ESCs can differentiate to PGCs, gametes and even blastocysts – recently live mouse pups were born from sperm generated from mESCs. Although investigations are still preliminary, human embryonic stem cells (hESCs) apparently display a similar developmental capacity to generate PGCs and immature gametes. Exactly how such gamete-like cells are generated during stem cell culture remains unclear especially as in vitro conditions are ill-defined. The findings are discussed in relation to the mechanisms of human PGC and gamete development and the biotechnology of hESCs and hEG cells.

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Gamete derivation from embryonic stem cells, induced pluripotent stem cells or somatic cell nuclear transfer-derived embryonic stem cells: state of the art

Reproduction Fertility and Development ◽

10.1071/rd14317 ◽

2015 ◽

Vol 27 (1) ◽

pp. 89 ◽

Cited By ~ 11

Author(s):

Charles A. Easley ◽

Calvin R. Simerly ◽

Gerald Schatten

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Somatic Cell ◽

Germ Cells ◽

Pluripotent Stem Cells ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

Induced Pluripotent ◽

Normal Offspring

Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and several biomedical rationales. Here, we consider several strategies for deriving gametes from PSCs from mice and primates (human and non-human) and their anticipated strengths, challenges and limitations. Although the ‘Weismann barrier’, which separates the mortal somatic cell lineages from the potentially immortal germline, has long existed, breakthroughs first in mice and now in humans are artificially creating germ cells from somatic cells. Spermatozoa with full reproductive viability establishing multiple generations of seemingly normal offspring have been reported in mice and, in humans, haploid spermatids with correct parent-of-origin imprints have been obtained. Similar progress with making oocytes has been published using mouse PSCs differentiated in vitro into primordial germ cells, which are then cultured after xenografting reconstructed artificial ovaries. Progress in making human oocytes artificially is proving challenging. The usefulness of these artificial gametes, from assessing environmental exposure toxicity to optimising medical treatments to prevent negative off-target effects on fertility, may prove invaluable, as may basic discoveries on the fundamental mechanisms of gametogenesis.

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Regulation of in vitro and in vivo differentiation of mouse embryonic stem cells, embryonic germ cells and teratocarcinoma cells by TGFβ family signaling factors

Russian Journal of Developmental Biology ◽

10.1134/s1062360409060010 ◽

2009 ◽

Vol 40 (6) ◽

pp. 325-338 ◽

Cited By ~ 6

Author(s):

O. F. Gordeeva ◽

T. M. Nikonova ◽

N. V. Lifantseva

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Germ Cells ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Embryonic Germ Cells ◽

Teratocarcinoma Cells ◽

In Vivo Differentiation

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Dynamic expression and regulatory mechanism of TGF-β signaling in chicken embryonic stem cells differentiating into spermatogonial stem cells

Bioscience Reports ◽

10.1042/bsr20170179 ◽

2017 ◽

Vol 37 (4) ◽

Cited By ~ 5

Author(s):

Qisheng Zuo ◽

Kai Jin ◽

Yani Zhang ◽

Jiuzhou Song ◽

Bichun Li

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Signaling Pathway ◽

Germ Cells ◽

Regulatory Mechanism ◽

Embryonic Stem ◽

Marker Genes ◽

Qrt Pcr ◽

Dynamic Expression

The present study investigated the dynamic expression and regulatory mechanism of transforming growth factor β (TGF-β) signaling involved in embryonic stem cells (ESCs) differentiation into male germ cells. Candidate genes involved in TGF-β signaling pathway were screened from RNA-sequencing (RNA-seq), which were further validated by quantitative real-time PCR (qRT-PCR). Bone morphogenetic protein 4 (BMP4) was used to induce differentiation of ESCs in vitro. Inhibition of TGF-β signaling pathway was reflected by Western blot of SMAD2 and SMAD5 expression. Differentiating efficiency of germ cells was evaluated by immunofluorescence and fluorescence-activated cell sorting (FACS). Germ cell marker genes were assessed by qRT-PCR in the differentiation process, with activation or inhibition of TGF-β signaling pathway. In the process of in vitro induction, SMAD2 and SMAD5 were found to significantly up-regulated in BMP4 group versus the control and inhibition groups after 4 and 14 days. Expression of CKIT, CVH, DAZL, STRA8, and INTEGRIN α6 were significantly increased in the BMP4 group compared with the control group, while down-regulated in the inhibition groups. The proportion of germ cell-like cells was decreased from 17.9% to 2.2% after 4 days induction, and further decreased from 14.1% to 2.1% after 14 days induction. Correspondingly, expression of marker genes in germ cells was significantly lower. In vivo inhibition of TGF-β signaling pathway reduced germ cells formation from 5.5% to 1.6%, and down-regulated the expression of CKIT, CVH, DAZL, STRA8, and INTEGRIN α6. In conclusion, our study reveals the mechanism regulating spermatogonial stem cells (SSCs) and lays the basis for further understanding of the regulatory network.

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