Mapping the temporal and spatial dynamics of the human endometrium in vivo and in vitro

The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated dense single-cell and spatial reference maps of the human uterus and three-dimensional endometrial organoid cultures. We dissect the signaling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids reveals the pathways and cell states regulating differentiation of the secretory and ciliated lineages both in vivo and in vitro. In vitro downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. We utilize our cellular maps to deconvolute bulk data from endometrial cancers and endometriotic lesions, illuminating the cell types dominating in each of these disorders. These mechanistic insights provide a platform for future development of treatments for common conditions including endometriosis and endometrial carcinoma.

Histone Demethylase KDM5B Catalyzed H3k4me3 Demethylation to Promote Differentiation of Bone Marrow Mesenchymal Stem Cells into Cardiomyocytes

BACKGROUND: Studies have shown that histone H3 methylation is involved in regulating the differentiation of Bone Marrow Mesenchymal Stem Cells (BMSCs). KDM5B can specifically reduce the level of histone 3 lysine 4 trimethylation (H3K4me3), thereby activating the expression of related genes and participating in biological processes such as cell differentiation, embryonic development and tumor formation. Whether KDM5B is involved in the regulation of BMSCs differentiation into cardiomyocytes through the above manner has not been reported.OBJECTIVE: To investigate the effect of KDM5B on the induction and differentiation of swine BMSCs into myocardial cells in vitro.METHODS: Swine bone marrow BMSCs were isolated and cultured, and the overexpression, interference expression and blank vector of KMD5B were constructed and transfected by lentivirus. BMSCs was induced to differentiate into cardiomyocytes by 5-azacytidine (5-AZA) in vitro, and the differentiation efficiency was compared by immunofluorescence, RT-PCR, Western Blot and whole-cell patch clamp detection.RESULT: Compared with the control group, the expression levels of histone H3K4me3 and pluripotency gene Nanog in KDM5B overexpression group were significantly decreased, while the expression level of key myocardial gene HCN4 was significantly increased, and the Na+ current density on the surface of differentiated myocardial cell membrane was significantly increased. Meanwhile, the corresponding results of the KDM5B silent expression group were just opposite. CONCLUSIONS: It indicated that enhanced KDM5B expression could promote the differentiation of BMSCs into cardiomyocytes and improve the differentiation efficiency by controlling H3K4 methylation levels..

MyD88 Mediates Colitis- and RANKL-Induced Microfold Cell Differentiation

Intestinal microfold (M) cells are critical for sampling antigen in the gut and initiating the intestinal mucosal immune response. In this study, we found that the differentiation efficiency of M cells was closely related to the colitis severity. The expression levels of M cells differentiation-related genes were synchronized with the kinetics of pro-inflammatory cytokines expression originated from dextran sulfate sodium (DSS) induction and Salmonella infection. Compared with wild-type (WT) mice, MyD88-/- mice exhibited significantly lower expression levels of M cells differentiation-related genes. However, DSS could induce colitis in MyD88-/- mice but failed to promote M cells differentiation. Furthermore, the receptor activator of the Nuclear Factor-κB ligand (RANKL) induced M cells differentiation in murine intestinal organoids prepared from both WT and MyD88-/- mice. However, less M cells differentiation were found in MyD88-/- mice as compared with WT mice. Hence, we concluded that myeloid differentiation factor 88 (MyD88) is an essential molecule for colitis- and RANKL-related M cells differentiation.

Loss of Atg2b and Gskip impairs the maintenance of the hematopoietic stem cell pool size

A germline copy number duplication of chromosome 14q32, which contains ATG2B and GSKIP , was identified in families with myeloproliferative neoplasm (MPN). Herein, we show that mice lacking both Atg2b and Gskip , but not either alone, exhibited decreased hematopoiesis, resulting in death in utero accompanied by anemia. In marked contrast to MPN patients with duplication of ATG2B and GSKIP , the number of hematopoietic stem cells (HSCs), in particular long-term HSCs, in double knockout fetal livers were significantly decreased due to increased cell death. Although the remaining HSCs still had the ability to differentiate into hematopoietic progenitor cells, the differentiation efficiency was quite low. Remarkably, mice with knockout of Atg2b or Gskip alone did not show any hematopoietic abnormality. Mechanistically, while loss of both genes had no effect on autophagy, it increased the expression of genes encoding enzymes involved in oxidative phosphorylation. Taken together, our results indicate that Atg2b and Gskip play a synergistic effect in maintaining the pool size of HSCs.

Enhancer-associated H3K4 methylation safeguards in vitro germline competence

Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.

MyD88 Mediates Colitis- and RANKL-induced Microfold Cell Differentiation

Intestinal microfold (M) cells are critical for sampling antigen in the gut and initiating the intestinal mucosal immune response. In this study, we found that the differentiation efficiency of M cells was closely related to the colitis severity. The expression levels of M cells differentiation-related genes were synchronized with the kinetics of pro-inflammatory cytokines expression originated from dextran sulfate sodium (DSS) induction and Salmonella infection. Compared with wild-type (WT) mice, MyD88-/- mice exhibited significantly lower expression levels of M cells differentiation-related genes. However, DSS could induce colitis in MyD88-/- mice but failed to promote M cells differentiation. Furthermore, the receptor activator of the Nuclear Factor-κB ligand (RANKL) induced M cells differentiation in murine intestinal organoids prepared from both WT and MyD88-/- mice. However, less M cells differentiation were found in MyD88-/- mice as compared with WT mice. Hence, we concluded that myeloid differentiation factor 88 (MyD88) is an essential molecule for colitis- and RANKL-related M cells differentiation.

Tissue of Origin, but Not XCI State, Influences Germ Cell Differentiation from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) are not only a promising tool to investigate differentiation to many cell types, including the germline, but are also a potential source of cells to use for regenerative medicine purposes in the future. However, current in vitro models to generate human primordial germ cell-like cells (hPGCLCs) have revealed high variability regarding differentiation efficiency depending on the hPSC lines used. Here, we investigated whether differences in X chromosome inactivation (XCI) in female hPSCs could contribute to the variability of hPGCLC differentiation efficiency during embryoid body (EB) formation. For this, we first characterized the XCI state in different hPSC lines by investigating the expression of XIST and H3K27me3, followed by differentiation and quantification of hPGCLCs. We observed that the XCI state did not influence the efficiency to differentiate to hPGCLCs; rather, hPSCs derived from cells isolated from urine showed an increased trend towards hPGCLCs differentiation compared to skin-derived hPSCs. In addition, we also characterized the XCI state in the generated hPGCLCs. Interestingly, we observed that independent of the XCI state of the hPSCs used, both hPGCLCs and soma cells in the EBs acquired XIST expression, indicative of an inactive X chromosome. In fact, culture conditions for EB formation seemed to promote XIST expression. Together, our results contribute to understanding how epigenetic properties of hPSCs influence differentiation and to optimize differentiation methods to obtain higher numbers of hPGCLCs, the first step to achieve human in vitro gametogenesis.

3D Spheroid Formation Using BMP-Loaded Microparticles Enhances Odontoblastic Differentiation of Human Dental Pulp Stem Cells

Human dental pulp stem cells (hDPSCs) are the primary cells responsible for dentin regeneration. Typically, in order to allow for odontoblastic differentiation, hDPSCs are cultured over weeks with differentiation-inducing factors in a typical monolayered culture. However, monolayered cultures have significant drawbacks including inconsistent differentiation efficiency, require a higher BMP concentration than should be necessary, and require periodic treatment with BMPs for weeks to see results. To solve these problems, we developed a 3D-cell spheroid culture system for odontoblastic differentiation using microparticles with leaf-stacked structure (LSS), which allow for the sustained release of BMPs and adequate supply of oxygen in cell spheroids. BMPs were continuously released and maintained an effective concentration over 37 days. hDPSCs in the spheroid maintained their viability for 5 weeks, and the odontoblastic differentiation efficiency was increased significantly compared to monolayered cells. Finally, dentin-related features were detected in the spheroids containing BMPs-loaded microparticles after 5 weeks, suggesting that these hDPSC-LSS spheroids might be useful for dentin tissue regeneration.

Aging-related upregulation of the homeobox gene caudal represses intestinal stem cell differentiation in Drosophila

The differentiation efficiency of adult stem cells undergoes a significant decline in aged animals, which is closely related to the decline in organ function and age-associated diseases. However, the underlying mechanisms that ultimately lead to this observed decline of the differentiation efficiency of stem cells remain largely unclear. This study investigated Drosophila midguts and identified an obvious upregulation of caudal (cad), which encodes a homeobox transcription factor. This factor is traditionally known as a central regulator of embryonic anterior-posterior body axis patterning. This study reports that depletion of cad in intestinal stem/progenitor cells promotes quiescent intestinal stem cells (ISCs) to become activate and produce enterocytes in the midgut under normal gut homeostasis conditions. However, overexpression of cad results in the failure of ISC differentiation and intestinal epithelial regeneration after injury. Moreover, this study suggests that cad prevents intestinal stem/progenitor cell differentiation by modulating the Janus kinase/signal transducers and activators of the transcription pathway and Sox21a-GATAe signaling cascade. Importantly, the reduction of cad expression in intestinal stem/progenitor cells restrained age-associated gut hyperplasia in Drosophila. This study identified a function of the homeobox gene cad in the modulation of adult stem cell differentiation and suggested a potential gene target for the treatment of age-related diseases induced by age-related stem cell dysfunction.

MicroRNAs as Important Regulators Mediate the Multiple Differentiation of Mesenchymal Stromal Cells

MicroRNAs (miRNAs) are endogenous short non-encoding RNAs which play a critical role on the output of the proteins, and influence multiple biological characteristics of the cells and physiological processes in the body. Mesenchymal stem/stromal cells (MSCs) are adult multipotent stem cells and characterized by self-renewal and multidifferentiation and have been widely used for disease treatment and regenerative medicine. Meanwhile, MSCs play a critical role in maintaining homeostasis in the body, and dysfunction of MSC differentiation leads to many diseases. The differentiation of MSCs is a complex physiological process and is the result of programmed expression of a series of genes. It has been extensively proven that the differentiation process or programmed gene expression is also regulated accurately by miRNAs. The differentiation of MSCs regulated by miRNAs is also a complex, interdependent, and dynamic process, and a full understanding of the role of miRNAs will provide clues on the appropriate upregulation or downregulation of corresponding miRNAs to mediate the differentiation efficiency. This review summarizes the roles and associated signaling pathways of miRNAs in adipogenesis, chondrogenesis, and osteogenesis of MSCs, which may provide new hints on MSCs or miRNAs as therapeutic strategies for regenerative medicine and biotherapy for related diseases.