Oxygen tension modulates the mitochondrial genetic bottleneck and influences the segregation of a heteroplasmic mtDNA variant in vitro

AbstractMost humans carry a mixed population of mitochondrial DNA (mtDNA heteroplasmy) affecting ~1–2% of molecules, but rapid percentage shifts occur over one generation leading to severe mitochondrial diseases. A decrease in the amount of mtDNA within the developing female germ line appears to play a role, but other sub-cellular mechanisms have been implicated. Establishing an in vitro model of early mammalian germ cell development from embryonic stem cells, here we show that the reduction of mtDNA content is modulated by oxygen and reaches a nadir immediately before germ cell specification. The observed genetic bottleneck was accompanied by a decrease in mtDNA replicating foci and the segregation of heteroplasmy, which were both abolished at higher oxygen levels. Thus, differences in oxygen tension occurring during early development likely modulate the amount of mtDNA, facilitating mtDNA segregation and contributing to tissue-specific mutation loads.

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Resf1 supports embryonic stem cell self-renewal and effective germline entry

10.1101/2021.05.25.445589 ◽

2021 ◽

Author(s):

Matus Vojtek ◽

Ian Chambers

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Germ Line ◽

Primordial Germ Cell ◽

Embryonic Stem ◽

Cell Specification ◽

Self Renewal ◽

Downstream Target ◽

Germ Cell Specification

Retroelement silencing factor 1 (Resf1) interacts with the key regulators of mouse embryonic stem cells (ESCs) Oct4 and Nanog, and its absence results in sterility of mice. However, the function of Resf1 in ESCs and germ line specification is poorly understood. In this study, we used Resf1 knockout cell lines to determine the requirements of RESF1 for ESCs self-renewal and for in vitro specification of ESCs into primordial germ cell-like cells (PGCLCs). We found that deletion of Resf1 in ESCs cultured in serum and LIF reduces self-renewal potential whereas episomal expression of RESF1 has a modest positive effect on ESC self-renewal. In addition, RESF1 is not required for the capacity of NANOG and its downstream target ESRRB to drive self-renewal in the absence of LIF. However, Resf1 deletion reduces efficiency of PGCLC differentiation in vitro. These results identify Resf1 as a novel player in the regulation of pluripotent stem cells and germ cell specification.

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Generation of multipotent cell lines from a distinct population of male germ line stem cells

Reproduction ◽

10.1530/rep-07-0479 ◽

2008 ◽

Vol 135 (6) ◽

pp. 771-784 ◽

Cited By ~ 90

Author(s):

Fariborz Izadyar ◽

Francis Pau ◽

Joel Marh ◽

Natalia Slepko ◽

Tracy Wang ◽

...

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Cell Lines ◽

Germ Line ◽

Es Cells ◽

Embryonic Stem ◽

Complex Mixture ◽

Neural Cells ◽

Differentiation Potential

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

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Loss of Resf1 reduces the efficiency of embryonic stem cell self-renewal and germline entry

Life Science Alliance ◽

10.26508/lsa.202101190 ◽

2021 ◽

Vol 4 (12) ◽

pp. e202101190

Author(s):

Matúš Vojtek ◽

Ian Chambers

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Primordial Germ Cell ◽

Embryonic Stem ◽

Cell Specification ◽

Self Renewal ◽

Downstream Target ◽

Germ Cell Specification ◽

Positive Effect

Retroelement silencing factor 1 (RESF1) interacts with the key regulators of mouse embryonic stem cells (ESCs) OCT4 and NANOG, and its absence results in sterility of mice. However, the function of RESF1 in ESCs and germline specification is poorly understood. In this study, we used Resf1 knockout cell lines to determine the requirements of RESF1 for ESC self-renewal and for in vitro specification of ESCs into primordial germ cell-like cells (PGCLCs). We found that deletion of Resf1 in ESCs cultured in serum and LIF reduces self-renewal potential, whereas episomal expression of RESF1 has a modest positive effect on ESC self-renewal. In addition, RESF1 is not required for the capacity of NANOG and its downstream target ESRRB to drive self-renewal in the absence of LIF. However, Resf1 deletion reduces the efficiency of PGCLC differentiation in vitro. These results identify Resf1 as a novel player in the regulation of pluripotent stem cells and germ cell specification.

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247. BMP signalling in the induction of germline precursors from mouse embryonic stem cells in vitro

Reproduction Fertility and Development ◽

10.1071/srb08abs247 ◽

2008 ◽

Vol 20 (9) ◽

pp. 47

Author(s):

V. L. Dias ◽

J. C. Young ◽

K. L. Loveland

Keyword(s):

Germ Cell ◽

Embryoid Body ◽

Primordial Germ Cell ◽

Embryonic Stem ◽

Critical Window ◽

Cell Markers ◽

Germ Cell Specification ◽

Bmp Signalling ◽

Transient Elevation

BMP signalling is critical for germline lineage establishment during mouse embryogenesis. To assess its importance in the induction of germline precursors in vitro, a mouse embryonic stem (ES) cell line harbouring an Oct4 promoter-EGFP reporter construct was used to derive embryoid body (EB) aggregates cultured in the absence or presence of combinations of BMP2, BMP4 and BMP8b for 3 to 10 days. At both day 5 and 10 of culture, clearly defined clusters of Oct4-EGFP were observed in EBs cultured with BMPs, while these clusters were minimal to absent in untreated EBs. Quantitative mRNA analysis of day 3 to day 10 EBs revealed a significant elevation in the expression of genes associated with primordial germ cell specification in EBs grown in the presence of BMPs. Moreover, a transient elevation of early germ cell markers Blimp1, Fragilis and Stella was detected in day 3–4 EBs cultured with BMPs, followed their decline by day 5. In contrast, levels of the pluripotency markers, Oct3/4 and Nanog, and the later germ cell markers, Dazl and Vasa, progressively increased from day 3 to day 5. Levels of TGFβ superfamily signalling components ALK2, Smad1 and Smad5 remained relatively constant during this period. Wholemount immunofluorescence of day 5 Oct4-EGFP EBs exposed to BMP4 demonstrated co-localisation of primordial germ cell markers Oct3/4, Stella, and the cell surface antigen SSEA-1 with EGFP+ clusters. These results demonstrate that signalling by BMP2 and 4 enhances germ cell marker expression within EBs and identifies the day 3 to 5 timeframe in EB differentiation as a critical window when putative germ cells are first specified in vitro.

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Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells

Stem Cell Research & Therapy ◽

10.1186/s13287-019-1427-2 ◽

2019 ◽

Vol 10 (1) ◽

Author(s):

Zili Li ◽

Fang Fang ◽

Qian Zhao ◽

Honggang Li ◽

Chengliang Xiong

Keyword(s):

Flow Cytometry ◽

Vitamin C ◽

Germ Cell ◽

Embryonic Stem ◽

Qrt Pcr ◽

Differentiation Protocol ◽

Germ Cell Specification ◽

Tet Enzymes ◽

In Vitro Induction

Abstract Background As the precursors of sperm and eggs, human primordial germ cells (hPGCs) emerge as early as weeks 2 to 3 of post-implantation development. Recently, robust hPGC induction models have been established in vitro with different protocols, but global 5mC/5hmC epigenetic reprogramming is not initiated in vitro. Previous studies found that vitamin C can enhance Tet (ten-eleven translocation) enzyme expression and improve 5hmC level in cells. But the effect of vitamin C supplementation on hPGC in vitro induction is still unknown. Methods We generated a gene-edited human embryonic stem cell (hESC) line carrying a BLIMP1-mkate2 reporter by CRISPR/Cas9 technology and used flow cytometry to optimize the PGC differentiation protocol; meanwhile, the expression of PGC genes (BLIMP1, TFAP2C, SOX17, OCT4) was evaluated by qRT-PCR. When different concentrations of vitamin C were added to the induction medium, the percentage of hPGCLCs (hPGC-like cells) was analyzed by flow cytometry; dot blot and ELISA were used to detect the levels of 5hmC and 5mC. The expression of TET enzymes was also evaluated by qRT-PCR. Results We optimized the PGC differentiation protocol with the BLIMP1-mkate reporter hESCs, and the efficiency of PGC induction in vitro can be improved to 30~40%. When 50 μg/mL vitamin C was added, the derived hPGCLCs not only upregulated the expression of key genes involved in human early germ cell development such as NANOS3, TFAP2C, BLIMP1, and SOX17, but also increased the levels of 5hmC and TET enzymes. Conclusions Taken together, supplementation of vitamin C can promote the in vitro induction of hPGCLCs from hESCs, which might be related to vitamin C-mediated epigenetic regulations during the differentiation process.

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In vitro germ cell differentiation from embryonic stem cells of mice: induction control by BMP4 signalling

Bioscience Reports ◽

10.1042/bsr20160348 ◽

2016 ◽

Vol 36 (6) ◽

Cited By ~ 3

Author(s):

Zohreh Makoolati ◽

Mansoureh Movahedin ◽

Mehdi Forouzandeh-Moghadam

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Germ Cell ◽

Germ Line ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Culture Systems ◽

Germ Cell Differentiation ◽

Cell Commitment

The present study aims to confirm and analyse germ cell-related patterns and specific gene expressions at a very early stage of cell commitment. Following the XY cytogenetic confirmation of the CCE mouse embryonic stem cells (mESCs) line, cells were cultured to form embryoid bodies (EBs). Expression pattern assessment of the mouse vasa homologue (Mvh), Stra8, α6 and β1 integrin genes in ESC and 1–3-day-old EB showed that all genes except α6 integrin were expressed in the ESC. The mean calibration of Mvh, Stra8 and α6 integrin expression significantly increased upon EB formation compared with the ESCs. During mouse embryogenesis, the signalling of bone morphogenetic protein (BMP) is essential for germ-line formation. To investigate its role in germ-line induction in vitro, mESCs were cultured as 1-day-old EB aggregates with BMP4 for 4 days in STO co-culture systems, in the presence and absence of 5 ng/ml BMP4. At the end of the culture period, colony assay (number and diameter) was performed and the viability percentage and proliferation rate was determined. There were no significant statistical differences in the abovementioned criteria between these two groups. Moreover, the expression of Mvh, α6 and β1 integrins, Stra8 and Piwil2 genes was evaluated in co-culture groups. The molecular results of co-culture groups showed higher–but insignificant–Piwil2 and significant α6 integrin expressions in BMP4 treated co-culture systems. These results confirmed that the EB system and the presence of BMP4 in a STO co-culture system improve the differentiation of ESCs to germ cell.

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GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program

Life Science Alliance ◽

10.26508/lsa.202000974 ◽

2021 ◽

Vol 4 (5) ◽

pp. e202000974

Author(s):

Yoji Kojima ◽

Chika Yamashiro ◽

Yusuke Murase ◽

Yukihiro Yabuta ◽

Ikuhiro Okamoto ◽

...

Keyword(s):

Transcription Factors ◽

Germ Cell ◽

Cell Fate ◽

Germ Line ◽

Primordial Germ Cell ◽

Bmp Signaling ◽

Germ Cell Specification ◽

Bona Fide ◽

Underlying Mechanisms

The in vitro reconstitution of human germ-cell development provides a robust framework for clarifying key underlying mechanisms. Here, we explored transcription factors (TFs) that engender the germ-cell fate in their pluripotent precursors. Unexpectedly, SOX17, TFAP2C, and BLIMP1, which act under the BMP signaling and are indispensable for human primordial germ-cell-like cell (hPGCLC) specification, failed to induce hPGCLCs. In contrast, GATA3 or GATA2, immediate BMP effectors, combined with SOX17 and TFAP2C, generated hPGCLCs. GATA3/GATA2 knockouts dose-dependently impaired BMP-induced hPGCLC specification, whereas GATA3/GATA2 expression remained unaffected in SOX17, TFAP2C, or BLIMP1 knockouts. In cynomolgus monkeys, a key model for human development, GATA3, SOX17, and TFAP2C were co-expressed exclusively in early PGCs. Crucially, the TF-induced hPGCLCs acquired a hallmark of bona fide hPGCs to undergo epigenetic reprogramming and mature into oogonia/gonocytes in xenogeneic reconstituted ovaries. By uncovering a TF circuitry driving the germ line program, our study provides a paradigm for TF-based human gametogenesis.

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Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre

Nature Communications ◽

10.1038/s41467-021-23653-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Peng-Fei Xu ◽

Ricardo Moraes Borges ◽

Jonathan Fillatre ◽

Maraysa de Oliveira-Melo ◽

Tao Cheng ◽

...

Keyword(s):

Stem Cells ◽

Cell Tracking ◽

Cardiac Tissue ◽

Embryonic Stem ◽

Mammalian Embryo ◽

Wide Range ◽

Vitro Model ◽

Neurula Stage

AbstractGenerating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.

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Platelets and Endothelial Cells Act in Concert to Delay Thrombolysis – Evidence from an In Vitro Model of the Human Occlusive Thrombus

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614953 ◽

1998 ◽

Vol 79 (03) ◽

pp. 602-608 ◽

Cited By ~ 3

Author(s):

W. G. Jerome ◽

S. Handt ◽

R. R. Hantgan

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

In Vitro Model ◽

Umbilical Vein ◽

Plasminogen Activator Inhibitor ◽

Genetically Engineered ◽

Cellular Mechanisms ◽

Vitro Model ◽

Activator Inhibitor

SummaryThe molecular and cellular mechanisms that over a period of hours render a human thrombus progressively resistant to fibrinolysis have been probed with a novel in vitro model. The kinetics of clot formation and fibrinolysis were monitored by laser light scattering with platelet-rich model thrombi contained in cylindrical flow chambers. In selected experiments, human umbilical vein endothelial cells were also cultured to confluence on the inner walls of these “glass blood vessels”. Following an “aging” period (0.5, 2 or 4 h), each thrombus was gently perfused with a bolus of plasminogen/recombinant tissue plasminogen activator to induce fibrinolysis. Platelets delayed lysis of 2 h-aged thrombi by ~70% and (non-stimulated) endothelial cells by ~30%, compared to cell-free control clots. However, even greater lytic delays (~260%) resulted when both vascular cells were present in the same 2 h-aged thrombus. In contrast, rapid lysis was consistently achieved with R298E,R299E t-PA, a genetically engineered plasminogen activator that is insensitive to inhibition by plasminogen activator inhibitor type 1. These observations suggest platelets and endothelial cells act in concert to enrich the fibrin scaffold of an aging human thrombus in plasminogen activator inhibitor. We propose that the presence of both platelets and endothelial cells may contribute to progressive thrombolytic resistance.

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