Qiong Ke

Connexin 43 is involved in the generation of human induced pluripotent stem cells

Although somatic cells can be successfully programmed to create pluripotent stem cells by ectopically expressing defined transcriptional factors, reprogramming efficiency is low and the reprogramming mechanism remains unclear. Previous reports have shown that almost all human connexin (CX) isoforms are expressed by human embryonic stem (hES) cells and that gap junctional intercellular communication (GJIC) is important for ES cell survival and differentiation. However, the CX expression profiles in human induced pluripotent stem (iPS) cells and the role of CXs in the process of reprogramming back to iPS cells remains unknown. Here, we determined the expression levels of most forms of CX in human embryonic fibroblasts (hEFs) and in the hEF-derived iPS cells. A scrape loading/dye transfer assay showed that human iPS cells contained functional gap junctions (GJs) that could be affected by pharmacological inhibitors of GJ function. We found that CX43 was the most dramatically upregulated CX following reprogramming. Most importantly, the ectopic expression of CX43 significantly enhanced the reprogramming efficiency, whereas shRNA-mediated knockdown of endogenous CX43 expression greatly reduced the efficiency. In addition, we found that CX43 overexpression or knockdown affected the expression of E-CADHERIN, a marker of the mesenchymal-to-epithelial transition (MET), during reprogramming. In conclusion, our data indicate that CX43 expression is important for reprogramming and may mediate the MET that is associated with the acquisition of pluripotency.

TALEN-based generation of a cynomolgus monkey disease model for human microcephaly

Gene editing in non-human primates may lead to valuable models for exploring the etiologies and therapeutic strategies of genetically based neurological disorders in humans. However, a monkey model of neurological disorders that closely mimics pathological and behavioral deficits in humans has not yet been successfully generated. Microcephalin 1 (MCPH1) is implicated in the evolution of the human brain, and MCPH1 mutation causes microcephaly accompanied by mental retardation. Here we generated a cynomolgus monkey (Macaca fascicularis) carrying biallelic MCPH1 mutations using transcription activator-like effector nucleases. The monkey recapitulated most of the important clinical features observed in patients, including marked reductions in head circumference, premature chromosome condensation (PCC), hypoplasia of the corpus callosum and upper limb spasticity. Moreover, overexpression of MCPH1 in mutated dermal fibroblasts rescued the PCC syndrome. This monkey model may help us elucidate the role of MCPH1 in the pathogenesis of human microcephaly and better understand the function of this protein in the evolution of primate brain size.

Cell adhesion-mediated mitochondria transfer contributes to mesenchymal stem cell-induced chemoresistance on T cell acute lymphoblastic leukemia cells

BackgroundDespite the high cure rate of T cell acute lymphoblastic leukemia (T-ALL), drug resistance to chemotherapy remains a significant clinical problem. Bone marrow mesenchymal stem cells (MSCs) protect leukemic cells from chemotherapy, but the underlying mechanisms are poorly understood. In this study, we aimed to uncover the mechanism of MSC-induced chemoresistance in T-ALL cells, thus providing a promising clinical therapy target.MethodsCell viability was determined using the viability assay kit CCK-8. The mitochondrial ROS levels were detected using the fluorescent probe MitoSOX™ Red, and fluorescence intensity was measured by flow cytometry. In vitro, MSCs and Jurkat cells were cocultured. MSCs were labeled with green fluorescent protein (GFP), and Jurkat cells were labeled with the mitochondria-specific dye MitoTracker Red. Bidirectional mitochondrial transfer was detected by flow cytometry and confocal microscopy. The mechanism of mitochondria transfer was analyzed by inhibitor assays. Transcripts related to Jurkat cell/MSC adhesion in the coculture system were assessed by qRT-PCR. After treatment with a neutralizing antibody against a key adhesion molecule, mitochondria transfer from Jurkat cells to MSCs was again detected by flow cytometry and confocal microscopy. Finally, we verified our findings using human primary T-ALL cells cocultured with MSCs.ResultsChemotherapeutic drugs caused intracellular oxidative stress in Jurkat cells. Jurkat cells transfer mitochondria to MSCs but receive few mitochondria from MSCs, resulting in chemoresistance. This process of mitochondria transfer is mediated by tunneling nanotubes, which are protrusions that extend from the cell membrane. Moreover, we found that most Jurkat cells adhered to MSCs in the coculture system, which was mediated by the adhesion molecule ICAM-1. Treatment with a neutralizing antibody against ICAM-1 led to a decreased number of adhering Jurkat cells, decreased mitochondria transfer, and increased chemotherapy-induced cell death.ConclusionsWe show evidence that mitochondria transfer from Jurkat cells to MSCs, which is mediated by cell adhesion, may be a potential therapeutic target for T-ALL treatment.

Enhanced generation of human induced pluripotent stem cells by ectopic expression of Connexin 45

Somatic cells can be successfully reprogrammed into pluripotent stem cells by the ectopic expression of defined transcriptional factors. However, improved efficiency and better understanding the molecular mechanism underlying reprogramming are still required. In the present study, a scrape loading/dye transfer assay showed that human induced pluripotent stem cells (hiPSCs) contained functional gap junctions partially contributed by Connexin 45 (CX45). We then found CX45 was expressed in human embryonic stem cells (hESCs) and human dermal fibroblasts (hDFs) derived hiPSCs. Then we showed that CX45 was dramatically upregulated during the reprogramming process. Most importantly, the ectopic expression of CX45 significantly enhanced the reprogramming efficiency together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC - OSKM), whereas knockdown of endogenous CX45 expression significantly blocked cellular reprogramming and reduced the efficiency. Our further study demonstrated that CX45 overexpression or knockdown modulated the cell proliferation rate which was associated with the reprogramming efficiency. In conclusion, our data highlighted the critical role of CX45 in reprogramming and may increase the cell division rate and result in an accelerated kinetics of iPSCs production.

A Nestin–Cyclin‐Dependent Kinase 5–Dynamin‐Related Protein 1 Axis Regulates Neural Stem/Progenitor Cell Stemness via a Metabolic Shift

Neural stem/progenitor cells (NSPCs) transplantation provides an alternative approach for various central nervous system (CNS) diseases treatment, while the difficulties in NSPC acquisition and expansion limit their further application. Unveiling the mechanism of NSPC stemness regulation may contribute to its further application. Nestin, generally recognized as a marker of NSPCs, plays a crucial role in the CNS development and NSPC stemness maintenance. Here, we report that Nestin loss triggers mitochondrial network remodeling and enhances oxidative phosphorylation (OXPHOS) in NSPCs treated with Nestin RNA interference (RNAi). Mitochondrial morphology is dynamically controlled by the balance between fission and fusion mediators; one of these mediators, the pro‐fission factor, dynamin‐related protein 1 (Drp1), shows decreased activation in Nestin‐knockdown cells. Upstream, Drp1 phosphorylation is under control of the cytosolic cyclin‐dependent kinase 5 (Cdk5). Inhibition of Cdk5 using RNAi or a chemical inhibitor (roscovitine) induces mitochondrial elongation and promotes mitochondrial respiration, indicating that Cdk5‐dependent Drp1 phosphorylation participates in mitochondrial metabolism and NSPC stemness regulation. Strikingly, Nestin knockdown results in Cdk5 redistribution, with less remaining in the cytosol, leading to mitochondrial remodeling. We identify Nestin1‐640 sequesters Cdk5 in the cytosol and phosphorylates Drp1 subsequently. Together, our results show that a Nestin‐Cdk5‐Drp1 axis negatively regulates mitochondrial OXPHOS, which is indispensable for the maintenance of NSPC stemness. Stem Cells 2018;36:589–601

Modeling the Pathogenesis of Charcot-Marie-Tooth Disease Type 1A Using Patient-Specific iPSCs

Summary Charcot-Marie-Tooth disease type 1A (CMT1A), one of the most frequent inherited peripheral neuropathies, is associated with PMP22 gene duplication. Previous studies of CMT1A mainly relied on rodent models, and it is not yet clear how PMP22 overexpression leads to the phenotype in patients. Here, we generated the human induced pluripotent stem cell (hiPSC) lines from two CMT1A patients as an in vitro cell model. We found that, unlike the normal control cells, CMT1A hiPSCs rarely generated Schwann cells through neural crest stem cells (NCSCs). Instead, CMT1A NCSCs produced numerous endoneurial fibroblast-like cells in the Schwann cell differentiation system, and similar results were obtained in a PMP22-overexpressing iPSC model. Therefore, despite the demyelination-remyelination and/or dysmyelination theory for CMT1A pathogenesis, developmental disabilities of Schwann cells may be considered as an underlying cause of CMT1A. Our results may have important implications for the uncovering of the underlying mechanism and the development of a promising therapeutic strategy for CMT1A neuropathy.

Nuclear Nestin deficiency drives tumor senescence via lamin A/C-dependent nuclear deformation

Emerging evidence has revealed that Nestin not only serves as a biomarker for multipotent stem cells, but also regulates cell proliferation and invasion in various tumors. However, the mechanistic contributions of Nestin to cancer pathogenesis are still unknown. In the present study, previously thought to reside exclusively in the cytoplasm, Nestin can also be found in the nucleus and participate in protecting tumor cells against cellular senescence. Specifically, we reveal that Nestin has a nuclear localization signal (aa318–aa347) at the downstream of rod domain. We then find nuclear Nestin could interact with lamin A/C. Mechanistic investigations demonstrate that Nestin depletion results in the activation of cyclin-dependent kinase 5 (Cdk5), which causes the phosphorylation of lamin A/C (mainly at S392 site) and its subsequent translocation to the cytoplasm for degradation. The findings establish a role for nuclear Nestin in tumor senescence, which involves its nucleus-localized form and interaction with lamin A/C.Nestin can be localised in the nucleus of cancer cells, but its nuclear role in tumorigenesis is unclear. Here, the authors show that nuclear Nestin prevents senescence in tumor cells by stabilising lamin A/C from proteasomal degradation to maintain nuclear integrity.

The effect of semimature dendritic cell and the levels of Treg on transplantation tolerance of hepatocytes differentiated from mouse embryonic stem cell

Objective: To investigate the inducing effect and mechanism of semimature dendritic cell (smDCs) on transplantation tolerance of hepatocytes differentiated from mouse embryonic stem cells (ESCs), and to study the connections between smDCs and regulatory dendritic cells (regDCs). Methods: ESCs of 129 mouse labelled green fluorescent protein (GFP) were induced to hepatocytes by using previous methods. Meanwhile, bone marrow mononuclear cells of 129 mouse were induced to smDCs and regDCs. Moreover, the hepatocytes differentiated from 129 mouse ESCs were transplanted into liver of BALB/c mouse 3 days after infusing smDCs and regDCs suspension of 129 mouse into BALB/c mouse by tail vein respectively. After that, the growth status and survival time of transplanted cells in the recipient and infiltration of lymphocytes in transplant sites were observed. Furthermore, Foxp3 expression of peripheral blood CD4+ T cells was also tested. Results: In the control group, the transplanted cells in liver of BALB/c mouse survived only about 1 week. In contrast, the transplanted cells of smDC groups and regDCs groups survived about 4 weeks and the transplant sites of smDC groups also had less CD3(+) T cells. The morphology of smDCs were similar with regDCs. The expression of MHC-Ⅱ, CD40, CD80 and CD86 on smDCs and regDCs were moderate. Moreover, the Foxp3 expression of peripheral blood CD4+ T cells in smDC groups was higher than that in the control groups, from 1.11% up to 5.38%. The Foxp3 expression in regDC groups rose to 3.87%. Conclusion: The smDCs could induce transplantation tolerance of hepatocytes differentiated from 129 mouse ESCs in the recipient. The mechanism was associated with high level of Foxp3(+) Tregs, which could be increased by means of smDCs appropriate expression of MHC-Ⅱ, CD40, CD80 and CD86. The smDCs and regDCs were the same type of tolerance dendritic cells.

Efficient production of cynomolgus monkeys with a toolbox of enhanced assisted reproductive technologies.

The efficiency of assisted reproductive technologies (ARTs) in nonhuman primates is low due to no screening criterions for selecting sperm, oocyte, and embryo as well as its surrogate mothers. Here we analyzed 15 pairs of pregnant and non-pregnant cynomolgus monkeys, each pair of which received embryos from one batch of fertilized oocytes, and found ratio of endometrial to myometrial thicknesses in abdominal ultrasonic transverse section of uterus is a reliable indicator for selection of recipients for embryo transfer. We performed 305 ovarian stimulations in 128 female cynomolgus monkeys and found that ovarian stimulation can be performed in a whole year and repeated up to six times in the same monkey without deteriorating fertilization potential of eggs until a poor response to stimulation happened. Fertilization can be efficiently achieved with both conventional and piezo-driven intracytoplasmic sperm injection procedures. In semen collection, semen quality is higher with the penile robe electrical stimulus method compared with the rectal probe method. Moreover, caesarean section is an effective strategy for increasing baby survival rates of multiple pregnancies. These findings provide a practical guidance for the efficient use of ARTs, facilitating their use in genetic engineering of macaque monkeys for basic and translational neuroscience research.