Tracking induced pluripotent stem cell differentiation with a fluorescent genetically encoded epigenetic probe.

Epigenetic modifications (methylation, acetylation, etc.) of core histones play a key role in regulation of gene expression. Thus, the epigenome changes strongly during various biological processes such as cell differentiation and dedifferentiation. Classical methods of analysis of epigenetic modifications such as mass-spectrometry and chromatin immuno-precipitation, work with fixed cells only. Here we present a genetically encoded fluorescent probe, MPP8-Green, for detecting H3K9me3, a histone modification associated with inactive chromatin. This probe, based on the chromodomain of MPP8, allows for visualization of H3K9me3 epigenetic landscapes in single living cells. We used this probe to track changes in H3K9me3 landscapes during the differentiation of induced pluripotent stem cells (iPSCs) into induced neurons. Our findings revealed two major waves of global H3K9me3 reorganization during 4-day differentiation, namely on the first and third days, whereas nearly no changes occurred on the second and fourth days. The proposed method LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscapes), which combines genetically encoded epigenetic probes and machine learning approaches, enables classification of multiparametric epigenetic signatures of single cells during stem cell differentiation and potentially in other biological models.

A comparative analysis of genes differentially expressed between rete testis cells and Sertoli cells of the mouse testis.

The rete testis (RT) is a region of the mammalian testis that plays an important role in testicular physiology. The RT epithelium consists of cells sharing some well-known gene markers with supporting Sertoli cells (SCs). However, little is known about the differences in gene expression between these two cell populations. Here, we used fluorescence-activated cell sorting (FACS) to obtain pure cultures of neonatal RT cells and SCs and identified differentially expressed genes (DEGs) between these cell types using RNA sequencing (RNA-seq). We then compared our data with the RNA-seq data of other studies that examined RT cells and SCs of mice of different ages and generated a list of DEGs permanently upregulated in RT cells throughout testis development and in culture, which included 86 genes, and a list of 79 DEGs permanently upregulated in SCs. The analysis of studies on DMRT1 function revealed that nearly half of the permanent DEGs could be regulated by this SC upregulated transcription factor. We suggest that useful cell lineage markers and candidate genes for the specification of both RT cells and SCs may be present among these permanent DEGs.

Establishment of a pure culture of immature Sertoli cells by PDGFRA staining and cell sorting.

Sertoli cells are key somatic cells in the testis that form seminiferous tubules and support spermatogenesis. The isolation of pure Sertoli cells is important for their study. However, it is a difficult effort because of the close association of Sertoli cells with peritubular myoid cells surrounding seminiferous tubules. Here, we propose a novel approach to the establishment of a pure Sertoli cell culture from immature mouse testes. It is based on the staining of testicular cells for platelet-derived growth factor receptor alpha (PDGFRA), followed by fluorescence-activated cell sorting and culturing of a PDGFRA-negative cell population. Cells positive for a Sertoli cell marker WT1 accounted for more than 96% of cells in cultures from 6 to 12 days postpartum (dpp) mice. The numbers of peritubular myoid cells identified by ACTA2 staining did not exceed 4%. Cells in the cultures were also positive for Sertoli cell proteins SOX9 and DMRT1. Amh and Hsd17b3 expression decreased and Ar and Gata1 expression increased in 12 dpp cultures compared to 6 dpp cultures, which suggests that cultured Sertoli cells at least partially retained their differentiation status. This method can be employed in various applications including the analysis of differential gene expression and functional studies.

Neural Stem Cells and Methods for Their Generation From Induced Pluripotent Stem Cells in vitro.

Neural stem cells (NSCs) provide promising approaches for investigating embryonic neurogenesis, modeling of the pathogenesis of diseases of the central nervous system, and for designing drug-screening systems. Such cells also have an application in regenerative medicine. The most convenient and acceptable source of NSCs is pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells). However, there are many different protocols for the induction and differentiation of NSCs, and these result in a wide range of neural cell types. This review is intended to summarize the knowledge accumulated, to date, by workers in this field. It should be particularly useful for researchers who are beginning investigations in this area of cell biology.