Protein kinase Cα downregulation via siRNA-PKCα released from foldable capsular vitreous body in cultured human retinal pigment epithelium cells

We previously found that downregulation of protein kinase Cα (PKCα) can inhibit retinal pigment epithelium (RPE) cell proliferation involved in the development of proliferative vitreoretinopathy (PVR). In this study, we tested whether PKCα could be downregulated via small interfering RNA (siRNA)-PKCα released from foldable capsular vitreous body (FCVB) in cultured human RPE cells. SiRNA-PKCα content, determined by ultraviolet (UV) spectrophotometer, was released from FCVB containing 200, 300, 400, 500, and 600 nm siRNA-PKCα in a time-dependent manner from 1 to 96 hours and a dose-dependent manner at five concentrations. The content (y) had a good linear relationship with time (x), especially in the 600 nm siRNA-PKCα group (y = 16.214x, R2 = 0.9809). After treatment with siRNA-PKCα released from FCVBs, the PKCα was significantly decreased by RT-PCR, Western blot, and immunofluorescence analysis in RPE cells. These results indicate that PKCα was significantly downregulated by siRNA-PKCα released from FCVB in human RPE cells and provide us with a new avenue to prevent PVR

Generation of Human Epidermis-Derived Mesenchymal Stem Cell-like Pluripotent Cells and their reprogramming in mouse chimeras

Stem cells can be derived from the embryo (embryonic stem cells, ESCs), from adult tissues (adult stem cells, ASCs), and by induction of fibroblasts (induced pluripotent stem cells, iPSs). Ethical problems, immunological rejection, and difficulties in obtaining human tissues limit the use of ESCs in clinical medicine. Induced pluripotent stem cells are difficult to maintain in vitro and carry a greater risk of tumor formation. Furthermore, the complexity of maintenance and propagation is especially difficult in the clinic. Adult stem cells can be isolated from several adult tissues and present the possibility of self-transplantation for the clinical treatment of a variety of human diseases. Recently, several ASCs have been successfully isolated and cultured in vitro, including hematopoietic stem cells (HSCs) , mesenchymal stem cells (MSCs), epidermis stem cells, neural stem cells (NSCs), adipose-derived stem cells (ADSCs), islet stem cells, and germ line stem cells. Human mesenchymal stem cells originate mainly from bone marrow, cord blood, and placenta, but epidermis-derived MSCs have not yet been isolated. We isolated small spindle-shaped cells with strong proliferative potential during the culture of human epidermis cells and designed a medium to isolate and propagate these cells. They resembled MSCs morphologically and demonstrated pluripotency in vivo; thus, we defined these cells as human epidermis-derived mesenchymal stem cell-like pluripotent cells (hEMSCPCs). These hEMSCPCs present a possible new cell resource for tissue engineering and regenerative medicine

Optic nerve head astrocytes contribute to vascular associated effects

PurposeThis study was conducted in order to test the expression of vasoactive substances within rat lamina cribrosa (LC) and optic nerve head (ONH) astrocytes, so as to investigate the role and potential mechanism of ONH astrocytes in vascular associated effects.MethodsLC tissue sections and primary cultured ONH astrocytes were obtained from adult Sprague-Dawley (SD) rats. Immunofluorescent staining was then used to detect the expression of vasoactive substances. Hyperoxia exposure was carried out both in vivo and in vitro, after which nitric oxide (NO) levels in LC tissue and cell supernatant were detected. The variations of protein and gene expression associated with vasoactive substances were subsequently tested. ONH astrocytes and vascular smooth muscle cells (VSMCs) were then incubated in a direct co-culture manner. Morphological parameters of VSMCs were finally analyzed in order to evaluate cell contraction.ResultsEndothelin-1 (ET-1), nitric oxide synthase (NOS) and renin-angiotensin system (RAS) were detected in both LC tissue and ONH astrocytes. Retinal vessel diameter was found obviously decreased following hyperoxia exposure. Moreover, hyperoxia inhibited NO production both in vivo and in vitro. ET-1 and RAS elements were observed to be upregulated, whereas NOS was downregulated. In ONH astrocytes and VSMCs co-culture system, the length-to-width ratio of VSMCs was shown to significantly increase on days 3 and 7 in hyperoxia compared with normoxia.ConclusionsThere is an abundance of expression of vasoactive substances within LC tissue and ONH astrocytes. The contractile response of VSMCs in the co-culture system provided direct evidence for the involvement of ONH astrocytes in vascular associated effects, which may signify a potentially novel direction for future research

Identification of renal cyst cells of type I Nephronophthisis by single-nucleus RNA sequencing

Background: Nephronophthisis (NPH) is the most common genetic cause of end-stage renal disease (ESRD) in childhood, and NPHP1 is the major pathogenic gene. Cyst formation at the corticomedullary junction is a pathological feature of NPH, but the mechanism underlying cystogenesis is not well understood. The isolation and identification of cystic cell subpopulation could help to identify their origins and provide vital clues to the mechanisms underlying cystogenesis in NPH.Methods: Single-nucleus RNA sequencing (snRNA-seq) was performed to produce an atlas of NPHP1 renal cells. Kidney samples were collected from WT (Nphp1+/+) mice and NPHP1 (Nphp1del2-20/del2-20) model mice.Results: A comprehensive atlas of the renal cellular landscape in NPHP1 was generated, consisting of 14 basic renal cell types as well as a subpopulation of DCT cells that was overrepresented in NPHP1 kidneys compared to WT kidneys. GO analysis revealed significant downregulation of genes associated with tubular development and kidney morphogenesis in this subpopulation. Furthermore, the reconstruction of differentiation trajectories of individual cells within this subpopulation confirmed that a specific group of cells in NPHP1 mice become arrested at an early stage of differentiation and proliferate to form cysts. We demonstrate that Niban1 is a specific molecular marker of cystic cells in both mice and human NPHP1.Conclusion: In summary, we report a novel subpopulation of DCT cells, marked by Niban1, that are classified as cystic cells in the NPHP1 mice kidney. These results offer fresh insights into the cellular and molecular basis of cystogenesis in NPH

Serum Starvation Induced Cell Cycle Synchronization Facilitates Human Somatic Cells Reprogramming

Human induced pluripotent stem cells (iPSCs) provide a valuable model for regenerative medicine and human disease research. To date, however, the reprogramming efficiency of human adult cells is still low. Recent studies have revealed that cell cycle is a key parameter driving epigenetic reprogramming to pluripotency. As is well known, retroviruses such as the Moloney murine leukemia virus (MoMLV) require cell division to integrate into the host genome and replicate, whereas the target primary cells for reprogramming are a mixture of several cell types with different cell cycle rhythms. Whether cell cycle synchronization has potential effect on retrovirus induced reprogramming has not been detailed. In this study, utilizing transient serum starvation induced synchronization, we demonstrated that starvation generated a reversible cell cycle arrest and synchronously progressed through G2/M phase after release, substantially improving retroviral infection efficiency. Interestingly, synchronized human dermal fibroblasts (HDF) and adipose stem cells (ASC) exhibited more homogenous epithelial morphology than normal FBS control after infection, and the expression of epithelial markers such as E-cadherin and Epcam were strongly activated. Futhermore, synchronization treatment ultimately improved Nanog positive clones, achieved a 15–20 fold increase. These results suggested that cell cycle synchronization promotes the mesenchymal to epithelial transition (MET) and facilitates retrovirus mediated reprogramming. Our study, utilization of serum starvation rather than additional chemicals, provide a new insight into cell cycle regulation and induced reprogramming of human cells

MUSiC : a model-unspecific search for new physics in proton-proton collisions at root s=13TeV

Results of the Model Unspecific Search in CMS (MUSiC), using proton-proton collision data recorded at the LHC at a centre-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 35.9 fb(-1), are presented. The MUSiC analysis searches for anomalies that could be signatures of physics beyond the standard model. The analysis is based on the comparison of observed data with the standard model prediction, as determined from simulation, in several hundred final states and multiple kinematic distributions. Events containing at least one electron or muon are classified based on their final state topology, and an automated search algorithm surveys the observed data for deviations from the prediction. The sensitivity of the search is validated using multiple methods. No significant deviations from the predictions have been observed. For a wide range of final state topologies, agreement is found between the data and the standard model simulation. This analysis complements dedicated search analyses by significantly expanding the range of final states covered using a model independent approach with the largest data set to date to probe phase space regions beyond the reach of previous general searches.Peer reviewe