Patient-derived induced pluripotent stem cells with a MERTK mutation exhibit cell junction abnormalities and aberrant cellular differentiation potential

BACKGROUND Human induced pluripotent stem cell(hiPSC)technology is a valuable tool for generating patient-specific stem cells,facilitating disease modeling,and invest-igating disease mechanisms.However,iPSCs carrying specific mutations may limit their clinical applications due to certain inherent characteristics.AIM To investigate the impact of MERTK mutations on hiPSCs and determine whether hiPSC-derived extracellular vesicles(EVs)influence anomalous cell junction and differentiation potential.METHODS We employed a non-integrating reprogramming technique to generate peripheral blood-derived hiPSCs with and hiPSCs without a MERTK mutation.Chromo-somal karyotype analysis,flow cytometry,and immunofluorescent staining were utilized for hiPSC identification.Transcriptomics and proteomics were employed to elucidate the expression patterns associated with cell junction abnormalities and cellular differentiation potential.Additionally,EVs were isolated from the supernatant,and their RNA and protein cargos were examined to investigate the involvement of hiPSC-derived EVs in stem cell junction and differentiation.RESULTS The generated hiPSCs,both with and without a MERTK mutation,exhibited normal karyotype and expressed pluripotency markers;however,hiPSCs with a MERTK mutation demonstrated anomalous adhesion capability and differentiation potential,as confirmed by transcriptomic and proteomic profiling.Furthermore,hiPSC-derived EVs were involved in various biological processes,including cell junction and differentiation.CONCLUSION HiPSCs with a MERTK mutation displayed altered junction characteristics and aberrant differentiation potential.Furthermore,hiPSC-derived EVs played a regulatory role in various biological processes,including cell junction and differentiation.

Locus- and cell type-specific epigenetic switching during cellular differentiation in mammals

BACKGROUND： Epigenomic reconfiguration, including changes in DNA methylation and histone modifications, is crucial for the differentiation of embryonic stem cells （ESCs） into somatic cells. However, the extent to which the epigenome is reconfigured and the interplay between components of the epigenome during cellular differentiation remain poorly defined. METHODS： We systematically analyzed and compared DNA methylation, various histone modification, and transcriptome profiles in ESCs with those of two distinct types of somatic cells from human and mouse. RESULTS： We found that global DNA methylation levels are lower in somatic cells compared to ESCs in both species. We also found that 80% of regions with histone modification occupancy differ between human ESCs and the two human somatic cell types. Approximately 70% of the reconfigurations in DNA methylation and histone modifications are locus- and cell type- specific. Intriguingly, the loss ofDNA methylation is accompanied by the gain of different histone modifications in a locus- and cell type-specific manner. Further examination of transcriptional changes associated with epigenetic reconfiguration at promoter regions revealed an epigenetic switching for gene regulation--a transition from stable gene silencing mediated by DNA methylation in ESCs to flexible gene repression facilitated by repressive histone modifications in somatic cells. CONCLUSIONS： Our findings demonstrate that the epigenome is reconfigured in a locus- and cell type-specific manner and epigenetic switching is common during cellular differentiation in both human and mouse.

Effects of normobaric cyclic hypoxia exposure on mesenchymal stem-cell differentiation–pilot study on bone parameters in elderly

BACKGROUND Mesenchymal stem cells(MSC)of bone marrow are the progenitor of osteoblasts and adipocytes.MSC tend to differentiate into adipocytes,instead of osteoblasts,with aging.This favors the loss of bone mass and development of osteoporosis.Hypoxia induces hypoxia inducible factor 1αgene encoding transcription factor,which regulates the expression of genes related to energy metabolism and angiogenesis.That allows a better adaptation to low O2 conditions.Sustained hypoxia has negative effects on bone metabolism,favoring bone resorption.Yet,surprisingly,cyclic hypoxia(CH),short times of hypoxia followed by long times in normoxia,can modulate MSC differentiation and improve bone health in aging.AIM To evaluate the CH effect on MSC differentiation,and whether it improves bone mineral density in elderly.METHODS MSC cultures were induced to differentiate into osteoblasts or adipocytes,in CH(3%O2 for 1,2 or 4 h,4 d a week).Extracellular-matrix mineralization and lipid-droplet formation were studied in MSC induced to differentiate into osteoblast or adipocytes,respectively.In addition,gene expression of marker genes,for osteogenesis or adipogenesis,have been quantified by quantitative real time polymerase chain reaction.The in vivo studies with elderly(>75 years old;n=10)were carried out in a hypoxia chamber,simulating an altitude of 2500 m above sea level,or in normoxia,for 18 wk(36 CH sessions of 16 min each).Percentages of fat mass and bone mineral density from whole body,trunk and right proximal femur(femoral,femoral neck and trochanter)were assessed,using dual-energy X-ray absorptiometry.RESULTS CH(4 h of hypoxic exposure)inhibited extracellular matrix mineralization and lipid-droplet formation in MSC induced to differentiate into osteoblasts or adipocytes,respectively.However,both parameters were not significantly affected by the other shorter hypoxia times assessed.The longest periods of hypoxia downregulated the expression of genes related to extracellular matrix formation,in MSC induced to differentiate into osteoblasts.Interestingly,osteocalcin(associated to energy metabolism)was upregulated.Vascular endothelial growth factor an expression and low-density lipoprotein receptor related protein 5/6/dickkopf Wnt signaling pathway inhibitor 1(associated to Wnt/β-catenin pathway activation)increased in osteoblasts.Yet,they decreased in adipocytes after CH treatments,mainly with the longest hypoxia times.However,the same CH treatments increased the osteoprotegerin/receptor activator for nuclear factor kappa B ligand ratio in both cell types.An increase in total bone mineral density was observed in elderly people exposed to CH,but not in specific regions.The percentage of fat did not vary between groups.CONCLUSION CH may have positive effects on bone health in the elderly,due to its possible inhibitory effect on bone resorption,by increasing the osteoprotegerin/receptor activator for nuclear factor kappa B ligand ratio.

THE EXPRESSION OF CYTOKERATINS IN HUMAN HEPATOCELLULAR AND CHOLANGIOCELLULAR CARCINOMAS

In order to determine the usefulness value of the antibodies to cytokeratins(CK) of'bile duct type'in the differential diagnosis between hepatocellular carcinoma(HCC) and cholangiocellular carcinoma(CC),we have made an immunocytochemical investigation,using the antibodies specifically recognizing CK19 and CK18,seperately,in liver,and laminin(LN) antibody.All the CC examined(10 cases) were found CK19-positive;interestingly,CK19-positive cancer cells were also observed in 38% of HCCs(14/37).Therefore,CK19 was not a reliable marker in differentiating HCC from CC,in our consideration.The CK19 expression in HCC was showed to be irrelevant to their differentiation degres,but related to the histologic subtypes which indicated the directions of their differentiation.CK19 expression was observed in all the HCC cell nests with glandular differentiation,and an untontinuous LN-Positive basement membrane-like structure was immunolocalized around these cells.Which indicated that the glandular differentiation and CK19 expression in HCC were also related to the LN deposition,as in fetal liver and some chronic liver disorders.

A Hybrid Mathematical Model of Tumor-Induced Angiogenesis with Blood Perfusion

Angiogenesis, the growth of new blood vessel from existing ones, is a pivotal stage in cancer development,and is an important target for cancer therapy. We develop a hybrid mathematical model to understand the mechanisms behind tumor-induced angiogenesis. This model describes uptake of Tumor Angiogenic Factor（TAF）at extracellular level, uses partial differential equation to describe the evolution of endothelial cell density including TAF induced proliferation, chemotaxis to TAF, and haptotaxis to extracellular matrix. In addition we also consider the phenomenon of blood perfusion in the micro-vessels. The model produces sprout formation with realistic morphological and dynamical features, including the so-called brush border effect, the dendritic branching and fusing of the capillary sprouts forming a vessel network. The model also demonstrates the effects of individual mechanisms in tumor angiogenesis： Chemotaxis to TAF is the key driving mechanisms for the extension of sprout cell; endothelial proliferation is not absolutely necessary for sprout extension; haptotaxis to Extra Cellular Matrix（ECM） gradient provides additional guidance to sprout extension, suggesting potential targets for anti-angiogenic therapies.