Differentiation最新文献

Differentiation of human umbilical cord mesenchymal stem cells (Uc-MSCs) into islet-like clusters which are capable of synthesizing and secreting insulin can potentially serve as donors for islet transplantation in the patient deficiency in islet β cell function both in type 1 or type 2 diabetic patients. Therefore, we developed an easy and higher efficacy approach by trypsinazing the Uc-MSCs and followed culture in differentiation medium to induce of Uc-MSCs differentiation into islet-like clusters, and the potential mechanism that in the early stage of differentiation was also investigated by using RNA-sequencing and bioinformatics. Results show that induction efficacy was reached to 98% and TGF-β signaling pathway may play critical role in the early stage differentiation, it was further confirmed that the retardant effect of differentiation progress either in cell morphology or in islet specific genes expression can be observed upon blocking the activation of TGF-β signaling pathway using specific inhibitor of LY2109761 (TβRI/II kinase inhibitor). Our current study, for the first time, development a protocol for differentiation of Uc-MSCs into islet-like clusters, and revealed the importance of TGF-β signaling pathway in the early stage of differentiation of Uc-MSCs into islet-like clusters. Our study will provide alternative approach for clinical treatment of either type I or type II diabtes mellitus with dysfunctional pancreatic islets.

Hepatic organoids might provide a golden opportunity for realizing precision medicine in various hepatic diseases. Previously described hepatic organoid protocols from pluripotent stem cells rely on complicated multiple differentiation steps consisting of both 2D and 3D differentiation procedures. Therefore, the spontaneous formation of hepatic organoids from 2D monolayer culture is associated with a low-throughput production, which might hinder the standardization of hepatic organoid production and hamper the translation of this technology to the clinical or industrial setting. Here we describe the stepwise and fully 3D production of hepatic organoids from human pluripotent stem cells. We optimized every differentiation step by screening for optimal concentrations and timing of differentiation signals in each differentiation step. Hepatic organoids are stably expandable without losing their hepatic functionality. Moreover, upon treatment of drugs with known hepatotoxicity, we found hepatic organoids are more sensitive to drug-induced hepatotoxicity compared with 2D hepatocytes differentiated from PSCs, making them highly suitable for in vitro toxicity screening of drug candidates. The standardized fully 3D protocol described in the current study for producing functional hepatic organoids might serve as a novel platform for the industrial and clinical translation of hepatic organoid technology.

Epithelial–mesenchymal interactions occur during tooth development. The dental epithelium (DE) is regarded as the signal center that regulates tooth morphology. However, the mechanism by which DE regulates the differentiation of mesenchyme-derived dental papilla (DP) into odontoblasts remains unclear. Using miniature pigs as a model, we analyzed the expression profiles of the DE and DP during odontoblast differentiation using high-throughput RNA sequencing. The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is one of the most enriched pathways in both DE and DP. The PI3K/AKT pathway was first activated in the inner enamel epithelium but not in the DP on embryonic day 50. This pathway was then activated in the odontoblast layer on embryonic day 60. We showed that AKT activation promoted odontoblast differentiation of DP cells. We further demonstrated that activation of PI3K/AKT signaling in the DE effectively increased the expression levels of AKT and dentin sialophosphoprotein in DP cells. Additionally, we found that DE cells secreted collagen type IV alpha 6 chain (COL4A6) downstream of epithelial AKT signaling to positively regulate mesenchymal AKT levels. Therefore, our data suggest that PI3K/AKT signaling from the DE to the DP promotes odontoblast differentiation via COL4A6 secretion.

Adult male Sertoli cell-specific Connexin43 knockout mice (SCCx43KO) exhibit higher Sertoli cell (SC) numbers per seminiferous tubule compared to their wild type (WT) littermates. Thus, deletion of this testicular gap junction protein seems to affect the proliferative potential and differentiation of “younger” SC. Although SC have so far mostly been characterised as postmitotic cells that cease to divide and become an adult, terminally differentiated cell population at around puberty, there is rising evidence that there exist exceptions from this for a very long time accepted paradigm. Aim of this study was to investigate postnatal SC development and to figure out underlying causes for observed higher SC numbers in adult KO mice. Therefore, the amount of SC mitotic figures was compared, resulting in slightly more and prolonged detection of SC mitotic figures in KO mice compared to WT. SC counting per tubular cross section revealed significantly different time curves, and comparing proliferation rates using Bromodesoxyuridine and Sox9 showed higher proliferation rates in 8-day old KO mice. SC proliferation was further investigated by Ki67 immunohistochemistry. SC in KO mice displayed a delayed initiation of cell-cycle-inhibitor p27^Kip1 synthesis and prolonged synthesis of the phosphorylated tumour suppressor pRb and proliferation marker Ki67. Thus, the higher SC numbers in adult male SCCx43KO mice may arise due to two different reasons: Firstly, in prepubertal KO mice, the proliferation rate of SC was higher. Secondly, there were differences in their ability to cease proliferation as shown by the delayed initiation of p27^Kip1 synthesis and the prolonged production of phosphorylated pRb and Ki67. Immunohistochemical results indicating a prolonged period of SC proliferation in SCCx43KO were confirmed by detection of proliferating SC in 17-days-old KO mice. In conclusion, deletion of the testicular gap junction protein Cx43 might prevent normal SC maturation and might even alter also the proliferation potential of adult SC.

The vascular system plays a crucial role in bone tissue. Angiogenic and osteogenic processes are coupled through a spatial-temporal connection. Recent studies have identified three types of capillaries in the skeletal system. Compared with type L and E vessels, type H vessels express high levels of CD31 and endomucin, and function to couple angiogenesis and osteogenesis. Endothelial cells in type H vessels interact with osteolineage cells (e.g., osteoblasts, osteoclasts, and osteocytes) through cytokines or signaling pathways to maintain bone growth and homeostasis. In imbalanced bone homeostases, such as osteoporosis and osteoarthritis, it may be a new therapeutic strategy to regulate the endothelial cell activity in type H vessels to repair the imbalance. Here, we reviewed the latest progress in relevant factors or signaling pathways in coupling angiogenesis and osteogenesis. This review would contribute to further understanding the role and mechanisms of type H vessels in coupling angiogenic and osteogenic processes. Furthermore, it will facilitate the development of therapeutic approaches for bone disorders by targeting type H vessels.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial disease that is characterized by increased cellular proliferation and differentiation together with excessive extracellular matrix (ECM) deposition leading to buildup of scar tissue (fibrosis) and remodeling in the lungs. The activated and differentiated (myo)fibroblasts are one of the main sources of tissue remodeling in IPF and a crucial mechanism known to contribute to this feature is an aberrant crosstalk between pulmonary fibroblasts and the abnormal or injured pulmonary epithelium. This epithelial-fibroblast interaction mimics the temporal, spatial and cell-type specific crosstalk between the endoderm and mesoderm in the so-called epithelial-mesenchymal trophic unit (EMTU) during lung development that is proposed to be activated in healthy lung repair and dysregulated in various lung diseases including IPF. To study the dysregulated lung EMTU in IPF, various complex in vitro models have been established. Hence, in this review, we will provide a summary of studies that have used complex (3-dimensional) in vitro co-culture, and organoid models to assess how abnormal epithelial-fibroblast interactions in lung EMTU contribute to crucial features of the IPF including defective cellular differentiation, proliferation and migration as well as increased ECM deposition.

Mutations in SHH and several other genes encoding components of the Hedgehog signaling pathway have been associated with holoprosencephaly syndromes, with craniofacial anomalies ranging in severity from cyclopia to facial cleft to midfacial and mandibular hypoplasia. Studies in animal models have revealed that SHH signaling plays crucial roles at multiple stages of craniofacial morphogenesis, from cranial neural crest cell survival to growth and patterning of the facial primordia to organogenesis of the palate, mandible, tongue, tooth, and taste bud formation and homeostasis. This article provides a summary of the major findings in studies of the roles of SHH signaling in craniofacial development, with emphasis on recent advances in the understanding of the molecular and cellular mechanisms regulating the SHH signaling pathway activity and those involving SHH signaling in the formation and patterning of craniofacial structures.

The neural crest is a stem cell population that forms in the neurectoderm of all vertebrates and gives rise to a diverse set of cells such as sensory neurons, Schwann cells and melanocytes. Neural crest development in snakes is still poorly understood. From the point of view of evolutionary and comparative anatomy is an interesting topic given the unique anatomy of snakes. The aim of the study was to characterize how trunk neural crest cells (TNCC) migrate in the developing elapid snake Naja haje haje and consequently, look at the beginnings of development of neural crest derived sensory ganglia (DRG) and spinal nerves. We found that trunk neural crest and DRG development in Naja haje haje is like what has been described in other vertebrates and the colubrid snake strengthening our knowledge on the conserved mechanisms of neural crest development across species. Here we use the marker HNK1 to follow the migratory behavior of TNCC in the elapid snake Naja haje haje through stages 1–6 (1–9 days postoviposition). We observed that the TNCC of both snake species migrate through the rostral portion of the somite, a pattern also conserved in birds and mammals. The development of cobra peripheral nervous system, using neuronal and glial markers, showed the presence of spectrin in Schwann cell precursors and of axonal plexus along the length of the cobra embryos. In conclusion, cobra embryos show strong conserved patterns in TNCC and PNS development among vertebrates.

Precise spatiotemporal control of gene expression patterns is critical for normal development. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with the ability of unlimited self-renewal and differentiation into any cell type, provide a unique tool for understanding the underlying mechanism of development and disease in a dish. N6-methyl-adenosine (m⁶A) modification is the most extensive internal mRNA modification, which regulates almost all aspects of mRNA metabolism and thus extensively participates in gene expression regulation. However, the role of m⁶A during cardiogenesis still needs to be fully elucidated. Here, we found that core components of m⁶A methyltransferase decreased during cardiomyocyte differentiation. Impeding m⁶A deposition, by either deleting the m⁶A methyltransferase Mettl3 or overexpressing m⁶A demethylase alkB homolog 5 (Alkbh5), at early stages of cardiac differentiation of mouse pluripotent stem cells, led to inhibition of cardiac gene activation and retardation of the outgrowth of embryoid bodies, whereas interfering m⁶A modification at later stages of differentiation had minimal effects. Consistently, stage-specific inhibition of METTL3 with METTL3 inhibitor STM2457 during human ESCs (hESCs) cardiac differentiation demonstrated a similarly pivotal role of METTL3 for the induction of mesodermal cells while dispensable function for later stages. In summary, our study reveals a stage-specific requirement of m⁶A on the cardiac differentiation of pluripotent stem cells and demonstrates that precise tuning of m⁶A level is critical for cardiac differentiation.

The Drosophila melanogaster eye has been instrumental for determining both how cells communicate with one another to determine cell fate, as well as cell morphogenesis and patterning. Here, we describe the effects of the small GTPase Rap1 on the development of multiple cell types in the D. melanogaster eye. Although Rap1 has previously been linked to RTK-Ras-MAPK signaling in eye development, we demonstrate that manipulation of Rap1 activity is modified by increase or decrease of Delta/Notch signaling during several events of cell fate specification in eye development. In addition, we demonstrate that manipulating Rap1 function either in primary pigment cells or in interommatidial cells affects cone cell contact switching, primary pigment cell enwrapment of the ommatidial cluster, and sorting of secondary and tertiary pigment cells. These data suggest that Rap1 has roles in both ommatidial cell recruitment/survival and in ommatidial morphogenesis in the pupal stage. They lay groundwork for future experiments on the role of Rap1 in these events.