A gonogenic stimulated transition of mouse embryonic stem cells with enhanced control of diverse differentiation pathways

Abstract

Embryonic stem (ES) cells share markers with undifferentiated primordial germ cells (PGCs). Here, we discovered that a cellular state with some molecular markers of male gonocyte induction, including a G1/S phase arrest and upregulation of specific genes such as Nanos2, Tdrd1, Ddx4, Zbtb16 and Plk1s1, can be chemically induced in male mouse ES cells in vitro, which we termed gonogenic stimulated transition (GoST). After longer culture of the resulting GoST cells without chemical stimulation, several molecular markers typical for early gonocytes were detected including the early gonocyte marker Tex101. Motivated by previous studies that found multipotency in cell lines derived from neonatal male germ cells in vitro, we then compared the differentiation potential of GoST cells to that of ES cells in vitro. Interestingly, GoST cells showed equal neurogenic, but enhanced cardiogenic and hepatogenic differentiation compared to ES cells in vitro. This work shows for the first time that some important molecular markers of the first developmental sexual differentiation program can be induced in male mouse ES cells in vitro and defines a novel concept to generate cells with enhanced multipotency.

Differentiation of mouse embryonic stem cells into cells with spermatogonia-like morphology with chemical intervention-dependent increased gene expression of LIM homeobox 1 (Lhx1)

“…Typical for ES cells [6], on day 0, the culture consisted of compact multicellular colonies, while on day 7 it had grown to confluent multilayered cell sheets of variable thickness with some visible debris from dead cells in both groups (Fig 2). From day 0 to day 7, the continued presence of βME supported cell viability of the control group, while tBHQ-induced nuclear factor erythroid 2-related factor 2 (NRF2) signaling rescued cell viability of the intervention group in the absence of βME as previously shown [13]. Thicker cell sheets with holes in some areas and reduced debris were observable on day 19 in both groups.…”

“…To investigate aspects of spermatogonia development in male mouse ES cells, we decided to use 2iL and serum as the basis for the culture, based on previous reports that serum permits a broader spectrum of pluripotent states and downstream lineages [11], that 2i induces a PGC-like state in ES cells and facilitates downstream PGC-like differentiation from ES cells [11,12], and that 2iL supports the ground state of pluripotency [1]. Using this 2iL and serum culture basis, we designed a combined cell culture protocol without (control) and with chemical intervention (intervention) (Fig 1), based on a previous report that the chemical intervention was associated with the induction of molecular markers of the PGC to gonocyte differentiation process in male mouse ES cells in serum culture [13].…”

“…The copyright holder for this this version posted October 13, 2021. ; https://doi.org/10.1101/2021.10.12.464060 doi: bioRxiv preprint 4 male mouse ES cells cultured in serum that a chemical intervention, including a timed combination of the SIRT1 inhibitor Ex-527, the DNA methyltransferase inhibitor RG-108 and the electrophilic redox cycling compound tert-butylhydroquinone (tBHQ), was associated with molecular markers of the PGC to gonocyte differentiation process [13]. The aim of this study was to investigate whether the combination of a new cell culture protocol based on 2iL and serum with the previously reported chemical intervention [13] can induce aspects of spermatogonia development in male mouse ES cells in vitro. We found that male mouse ES cells cultured in 2iL and serum could be differentiated into cells with spermatogonia-like morphology (CSMs) by removal of 2iL and a specific schedule of partial medium replacements, and that a combination of this new cell culture protocol with the previously reported chemical intervention resulted in the first observation of CSMs in vitro with enriched Lhx1 expression.…”

“…2i also increased the expression of DAZL in ES cells, an RNA-binding protein and marker of late PGCs which is expressed only in a subset of cells of the ICM, while serum increased heterogeneity of pluripotent states [12]. Furthermore, it was shown for 4 male mouse ES cells cultured in serum that a chemical intervention, including a timed combination of the SIRT1 inhibitor Ex-527, the DNA methyltransferase inhibitor RG-108 and the electrophilic redox cycling compound tert-butylhydroquinone (tBHQ), was associated with molecular markers of the PGC to gonocyte differentiation process [13]. The aim of this study was to investigate whether the combination of a new cell culture protocol based on 2iL and serum with the previously reported chemical intervention [13] can induce aspects of spermatogonia development in male mouse ES cells in vitro.…”

Single-cell RNA sequencing of mitotic-arrested prospermatogonia with DAZL::GFP chickens and revealing unique epigenetic reprogramming of chickens

“…Germ cell specific-marker genes ( DAZL , DDX4 , and PIWIL1 ) were strongly expressed at all time points. By contrast, both pluripotency-marker genes ( Pou5f3 , NANOG , and SOX2 ) and mitotic-germ cell-marker genes ( CFC1 , TFAP2C , and CXCR4 ) [ 20 , 55 , 56 ], were significantly less expressed after E12 (Fig. 1 E).…”