Optimizing in vitro T cell differentiation by using induced pluripotent stem cells with GFP-RUNX1 and mCherry-TCF7 labelling.

In vitro T-cell differentiation from pluripotent stem cells (PSCs) could potentially provide an unlimited source of T cells for cancer immunotherapy, which, however is still hindered by the inefficient obtaining functionally-matured, terminally-differentiated T cells. Here, we established a fluorescence reporter human induced pluripotent stem cell (iPSC) line termed TCF7mCherryRUNX1GFP, in which the endogenous expression of RUNX1 and TCF7 are illustrated by the GFP and mCherry fluorescence, respectively. Utilizing TCF7mCherryRUNX1GFP, we defined that the feeder cells incorporating CXCL12-expressing OP9 cells with DL4-expressing OP9 cells at a 1:3 ratio (OP9-C1D3) significantly enhanced efficiency of CD8+ T cell differentiation from PSCs. Additionally, we engineered a chimeric antigen receptor (CAR) targeting EGFR into iPSCs. The CAR-T cells differentiated from these iPSCs using OP9-C1D3 feeders demonstrated effective cytotoxicity toward lung cancer cells. We anticipate this platform will help the in vitro HSPC and T cell differentiation optimization, serving the clinical demands of these cells.

Lin- PU.1dim GATA-1- defines haematopoietic stem cells with long-term multilineage reconstitution activity.

Despite extensive characterization of the state and function of haematopoietic stem cells (HSCs), the use of transcription factors to define the HSC population is still limited. We show here that the HSC population in mouse bone marrow can be defined by the distinct expression levels of Spi1 and Gata1. By using a double fluorescence knock-in mouse model, PGdKI, in which the expression levels of PU.1 and GATA-1 are indicated by the expression of GFP and mCherry, respectively, we uncover that the HSCs with lymphoid and myeloid repopulating activity are specifically enriched in a Lin- PU.1dim GATA-1- (LPG) cell subset. In vivo competitive repopulation assays demonstrate that bone marrow cells gated by LPG exhibit haematopoietic reconstitution activity which is comparable to that of classical Lin- Sca1+ c-kit+ (LSK). The integrated analysis of single-cell RNA sequence data from LPG and LSK-gated cells reveals that a transcriptional network governed by core TFs contributes to regulation of HSC multipotency. These discoveries provide new clues for HSC characterization and functional study.

Melatonin promotes the proliferation of primordial germ cell-like cells derived from porcine skin-derived stem cells: A mechanistic analysis.

In vitro differentiation of stem cells into functional gametes remains of great interest in the biomedical field. Skin-derived stem cells (SDSCs) are an adult stem cells that provides a wide range of clinical applications without inherent ethical restrictions. In this paper, porcine SDSCs were successfully differentiated into primordial germ cell-like cells (PGCLCs) in conditioned media. The PGCLCs were characterized in terms of cell morphology, marker gene expression, and epigenetic properties. Furthermore, we also found that 25 µM melatonin (MLT) significantly increased the proliferation of the SDSC-derived PGCLCs while acting through the MLT receptor type 1 (MT1). RNA-seq results found the mitogen-activated protein kinase (MAPK) signaling pathway was more active when PGCLCs were cultured with MLT. Moreover, the effect of MLT was attenuated by the use of S26131 (MT1 antagonist), crenolanib (platelet-derived growth factor receptor inhibitor), U0126 (mitogen-activated protein kinase kinase inhibitor), or CCG-1423 (serum response factor transcription inhibitor), suggesting that MLT promotes the proliferation processes through the MAPK pathway. Taken together, this study highlights the role of MLT in promoting PGCLCs proliferation. Importantly, this study provides a suitable in vitro model for use in translational studies and could help to answer numerous remaining questions related to germ cell physiology.

Protective Mechanism of Luteinizing Hormone and Follicle-Stimulating Hormone Against Nicotine-Induced Damage of Mouse Early Folliculogenesis.

Previous studies have shown that nicotine could impair the germ cell cyst breakdown and the primordial follicle assembly by autophagy. In this paper, we discovered that luteinizing hormone (LH) and follicle-stimulating hormone (FSH) could counteract the damage caused by nicotine of mouse germ cell cyst breakdown. The neonatal mice were separately intraperitoneally injected with nicotine, nicotine plus LH, nicotine plus FSH, and saline (control) for 4 days. Compared with the nicotine group, the quality of oocytes and the number of follicles were remarkably increased in the nicotine plus LH group or nicotine plus FSH group. LH and FSH could alleviate nicotine-induced oocyte autophagy by different pathways. LH reduced the nicotine-induced autophagy by restoring the phosphorylation level of adenosine 5'-monophosphate-activated protein kinase α-1, while FSH by downregulating the phosphorylation level of Forkhead box class O 1. In addition, in a subsequent study of 6-week mice in different treated groups, we found that LH and FSH supplementation significantly improved normal maturation rates, fertilization rates, and embryo's developmental potential of oocytes in oocytes exposed to nicotine. Taken together, these results suggested that LH and FSH could counteract the damage caused by nicotine and finally ensure normal germ cell cyst breakdown and early embryo development.

A Single-cell Transcriptome Atlas of Cashmere Goat Hair Follicle Morphogenesis.

Cashmere, also known as soft gold, is produced from the secondary hair follicles (SHFs) of cashmere goats. The number of SHFs determines the yield and quality of cashmere; therefore, it is of interest to investigate the transcriptional profiles present during cashmere goat hair follicle development. However, mechanisms underlying this development process remain largely unexplored, and studies regarding hair follicle development mostly use a murine research model. In this study, to provide a comprehensive understanding of cellular heterogeneity and cell fate decisions, single-cell RNA sequencing was performed on 19,705 single cells of the dorsal skin from cashmere goat fetuses at induction (embryonic day 60; E60), organogenesis (E90), and cytodifferentiation (E120) stages. For the first time, unsupervised clustering analysis identified 16 cell clusters, and their corresponding cell types were also characterized. Based on lineage inference, a detailed molecular landscape was revealed along the dermal and epidermal cell lineage developmental pathways. Notably, our current data also confirmed the heterogeneity of dermal papillae from different hair follicle types, which was further validated by immunofluorescence analysis. The current study identifies different biomarkers during cashmere goat hair follicle development and has implications for cashmere goat breeding in the future.

A hypothetical role for autophagy during the day/night rhythm-regulated melatonin synthesis in the rat pineal gland.

Melatonin is a highly conserved molecule that regulates day/night rhythms; it is associated with sleep improvement, reactive oxygen species (ROS) scavenging, anti-aging effects, and seasonal and circadian rhythms and has been a hot topic of research for decades. Using single-cell RNA sequencing, a recent study describes a single-cell transcriptome atlas for the rat pineal gland. Based on a more comprehensive analysis of the retrieved data (Mays et al., PLoS One, 2018, 13, e0205883), results from the current study unveiled the underappreciated gene regulatory network behind different cell populations in the pineal gland. More importantly, our study here characterized, for the first time, the day/night activation of autophagy flux in the rat pineal gland, indicating a potential role of autophagy in regulating melatonin synthesis in the rat pineal gland. These findings emphasized a hypothetical role of day/night autophagy in linking the biological clock with melatonin synthesis. Furthermore, ultrastructure analysis of pinealocytes provided fascinating insights into differences in their intracellular structure between daytime and nighttime. In addition, we also provide a preliminary description of cell-cell communication in the rat pineal gland. In summary, the current study unveils the day/night regulation of autophagy in the rat pineal gland, raising a potential role of autophagy in day/night-regulated melatonin synthesis.

Detrimental effect of Bisphenol S in mouse germ cell cyst breakdown and primordial follicle assembly.

The female reproductive lifespan is largely determined by the size of primordial follicle pool, which is established in early life. Bisphenol S (BPS), frequently present in plastic products used in daily life, has been demonstrated as an exogenous estrogen-like endocrine disrupting chemical interfering with the endocrine and reproductive systems. However, the molecular mechanisms of its reproductive toxicity remain to be determined. In the present study, we focused on the effect of BPS on the early ovarian folliculogenesis of mice. Our in vivo experiments showed that the treatment with BPS at 2 and 10 µg/kg body weight/day for 3 days induced abnormal germ cell cyst breakdown and primordial follicle assembly in the mouse ovary, further affecting later ovarian differentiation and reducing oocyte quality. In addition, our in vitro study demonstrated that BPS could interact with estrogen receptors (ERs) to induce phosphorylation of JNKs, which is responsible for reducing oocyte adhesion in cysts. Meanwhile, BPS exposure up-regulated Notch signaling pathway to increase the proliferation of granulosa cells precursors. Our study provided new evidence for the adverse effects of BPS on female reproduction, especially after perinatal exposure, and elucidated how it works.

Dissecting the initiation of female meiosis in the mouse at single-cell resolution.

Meiosis is one of the most finely orchestrated events during gametogenesis with distinct developmental patterns in males and females. However, the molecular mechanisms involved in this process remain not well known. Here, we report detailed transcriptome analyses of cell populations present in the mouse female gonadal ridges (E11.5) and the embryonic ovaries from E12.5 to E14.5 using single-cell RNA sequencing (scRNA seq). These periods correspond with the initiation and progression of meiosis throughout the first stage of prophase I. We identified 13 transcriptionally distinct cell populations and 7 transcriptionally distinct germ cell subclusters that correspond to mitotic (3 clusters) and meiotic (4 clusters) germ cells. By analysing cluster-specific gene expression profiles, we found four cell clusters correspond to different cell stages en route to meiosis and characterized their detailed transcriptome dynamics. Our scRNA seq analysis here represents a new important resource for deciphering the molecular pathways driving female meiosis initiation.

Single-cell Transcriptome Profiling reveals Dermal and Epithelial cell fate decisions during Embryonic Hair Follicle Development.

It is estimated that 50% of men and 25% of women worldwide suffer from hair loss, and therefore it is of great significance to investigate the molecular pathways driving hair follicle de novo morphogenesis. However, due to high cellular heterogeneity and the asynchronous development of hair follicles, our current understanding of the molecular mechanisms involved in follicle development remains limited. Methods: Single-cell suspensions from the dorsal skin of E13.5 (induction stage), E16.5 (organogenesis) fetal mice, and newborn mice (cytodifferentiation stage, postnatal day 0, P0) were prepared for unbiased single-cell RNA sequencing. To delineate the single-cell transcriptional landscape during hair follicle de novo morphogenesis, we performed t-distributed Stochastic Neighbor Embedding (tSNE), pseudotime cell trajectory inference, and regulon enrichment analysis to dissect cellular heterogeneity and reveal the molecular pathways underlying major cell type cell fate decisions. To validate our analysis, we further performed immunohistochemistry analysis of the key molecules involved during hair follicle morphogenesis. Meanwhile, intercellular communication between different cell populations was inferred based on a priori knowledge of ligand-receptor pairs. Results: Based on tSNE analysis, we identified 14 cell clusters from skin tissue and delineated their cellular identity from specific gene expression profiles. By using pseudotime ordering analysis, we successfully constructed the epithelium/dermal cell lineage differentiation trajectory. For dermal cell lineage, our analysis here recapitulated the dynamic gene expression profiles during dermal condensate (DC) cell fate commitment and delineated the heterogeneity of the different dermal papilla (DP) cell populations during in utero hair follicle development. For the epithelium cell lineage, our analysis revealed the dynamic gene expression profiles of the underappreciated matrix, interfollicular epidermis (IFE), hair shaft and inner root sheath (IRS) cell populations. Furthermore, single-cell regulatory network inference and clustering analysis revealed key regulons during cell fate decisions. Finally, intercellular communication analysis demonstrated that strong intercellular communication was involved during early hair follicle development. Conclusions: Our findings here provide a molecular landscape during hair follicle epithelium/dermal cell lineage fate decisions, and recapitulate the sequential activation of core regulatory transcriptional factors (TFs) in different cell populations during hair follicle morphogenesis. More importantly, our study here represents a valuable resource for understanding the molecular pathways involved during hair follicle de novo morphogenesis, which will have implications for future hair loss treatments.

Zearalenone exposure triggered porcine granulosa cells apoptosis via microRNAs-mediated focal adhesion pathway.

Zearalenone (ZEA), a metabolite of Fusarium, which is commonly found in moldy feed crops, is a well-known exogenous endocrine disruptor and has serious negative effects on animal reproduction. In order to understand the toxic effects of ZEA exposure on porcine granulosa cells (pGCs), which were exposed to 10 µM and 30 µM ZEA for 48 h in vitro, several methods were used for analysis. Flow cytometry and TUNEL analysis showed that the apoptosis of pGCs significantly increased in a dose-dependent manner after ZEA exposure compared with that of the control group. Whole transcriptome RNA-seq analysis was performed to reveal the mRNAs and miRNAs expression changes of pGCs after ZEA exposure and it was found that the expression of apoptosis-related genes was altered after ZEA exposure, and miRNAs were also significantly different among the experimental groups. In particular, ZEA exposure affected the expression of miRNAs associated with apoptosis-related pathways, such as miR-744, miR-1343 and miR-331-3p, as well as focal adhesion pathways related genes, Pak4 and Elk1, which were also involved in the apoptosis-related pathways. Moreover, the regulation networks between apoptosis-related mRNA and miRNAs were confirmed with the results of RT-qPCR and immunofluorescence. In conclusion, our results here demonstrated that ZEA exposure impaired pGCs growth and apoptosis via miRNAs-mediated focal adhesion pathway.

Melatonin ameliorates murine fetal oocyte meiotic dysfunction in F1 and F2 offspring caused by nicotine exposure during pregnancy.

Although there is abundant evidence to demonstrate that maternal smoking during pregnancy will harm the health of future generations, the impact of nicotine use by pregnant woman upon the oogenesis and folliculogenesis of female offspring has not been as widely scrutinized. Here we focus on the effects of nicotine on the meiotic progression of fetal oocytes. The data indicated that in pregnant mice treated with nicotine, intracellular ROS increased in follicles within the fetal ovary. Excessive intracellular hydrogen peroxide (H2O2) and superoxide anion (O2-) decreased mitochondrial membrane potential, inducing mitochondrial dysfunction, triggering an autophagic cascade and inhibiting anti-autophagic proteins. Fetal oocytes in F1 offspring of pregnant mice treated with nicotine exhibited a delay in meiotic prophase I, especially from the stage of pachytene to diplotene. In pubertal F1 offspring we observed a reduced number of follicles; the same reduction was also observed in F2 offspring. Of note, we found that melatonin ameliorated nicotine-induced oocyte damage and increased the expression of MnSOD, which decreased the production of nicotine-induced intracellular ROS. In addition, melatonin also maintained normal H3K4 and H3K9 di- and tri-methylation in F1 and F2 ovaries. Taken together, the current evidence suggests that, in the mouse, melatonin could prevent nicotine-impaired fetal oogenesis and folliculogenesis in offspring.

Zearalenone-induced aberration in the composition of the gut microbiome and function impacts the ovary reserve.

Zearalenone (ZEA), as a contaminant commonly found in our daily diet, has been widely studied for its toxicity. However, the exact mechanism underlying ZEA induced reproduction disorders remains unclear. Our study aimed to elucidate the underlying relationship between aberrations in the gut microbiota and the degeneration of the ovarian reserve following exposure to ZEA. Four-week-old mice were treated with different doses (0, 20, 40 µg/kg bw/day) of ZEA for 2 weeks and it was found that the primordial follicles were dramatically decreased when compared to untreated controls. Moreover, we applied metagenomic shotgun sequencing to investigate the effects of ZEA exposure on the population composition and function of gut microbiota. The results showed that the abundance of three susceptible bacterial strains, parabacteroides, bacteroides and lachnospiraceae were increased in a dose-dependent manner after ZEA exposure, whereas the bacterial glycerophospholipid metabolism pathway was greatly suppressed. Of note, utilizing LC/MS we found lysophosphatidylcholines (LPCs), important metabolites in the process of glycerophospholipid metabolism, were markedly decreased in the plasma of the ZEA treated mice. In conclusion, our findings here provide evidences that the dysfunction in gut microbiome after ZEA exposure may affect the ovarian reserve.