Melatonin improves reprogramming efficiency and proliferation of bovine‐induced pluripotent stem cells

Melatonin can modulate neural stem cell (NSC) functions such as proliferation and differentiation into NSC‐derived pluripotent stem cells (N‐iPS) in brain tissue, but the effect and mechanism underlying this are unclear. Thus, we studied how primary cultured bovine NSCs isolated from the retinal neural layer could transform into N‐iPS cell. NSCs were exposed to 0.01, 0.1, 1, 10, or 100 μm melatonin, and cell viability studies indicated that 10 μm melatonin can significantly increase cell viability and promote cell proliferation in NSCs in vitro. Thus, 10 μm melatonin was used to study miR‐302/367‐mediated cell reprogramming of NSCs. We noted that this concentration of melatonin increased reprogramming efficiency of N‐iPS cell generation from primary cultured bovine NSCs and that this was mediated by downregulation of apoptosis‐related genes p53 and p21. Then, N‐iPS cells were treated with 1, 10, 100, or 500 μm melatonin, and N‐iPS (M‐N‐iPS) cell proliferation was measured. We noted that 100 μm melatonin increased proliferation of N‐iPS cells via increased phosphorylation of intracellular ERK1/2 via activation of its pathway in M‐N‐iPS via melatonin receptors 1 (MT1). Finally, we verified that N‐iPS cells and M‐N‐iPS cells are similar to typical embryonic stem cells including the expression of pluripotency markers (Oct4 and Nanog), the ability to form teratomas in vivo, and the capacity to differentiate into all three embryonic germ layers.     

Study of mitochondrial respiratory defects on reprogramming to human induced pluripotent stem cells

Reprogramming of somatic cells into a pluripotent state is known to be accompanied by extensive restructuring of mitochondria and switch in metabolic requirements. Here we utilized Leber''s hereditary optic neuropathy (LHON) as a mitochondrial disease model to study the effects of homoplasmic mtDNA mutations and subsequent oxidative phosphorylation (OXPHOS) defects in reprogramming. We obtained fibroblasts from a total of 6 LHON patients and control subjects, and showed a significant defect in complex I respiration in LHON fibroblasts by high-resolution respiratory analysis. Using episomal vector reprogramming, our results indicated that human induced pluripotent stem cell (hiPSC) generation is feasible in LHON fibroblasts. In particular, LHON-specific OXPHOS defects in fibroblasts only caused a mild reduction and did not significantly affect reprogramming efficiency, suggesting that hiPSC reprogramming can tolerate a certain degree of OXPHOS defects. Our results highlighted the induction of genes involved in mitochondrial biogenesis (TFAM, NRF1), mitochondrial fusion (MFN1, MFN2) and glycine production (GCAT) during reprogramming. However, LHON-associated OXPHOS defects did not alter the kinetics or expression levels of these genes during reprogramming. Together, our study provides new insights into the effects of mtDNA mutation and OXPHOS defects in reprogramming and genes associated with various aspects of mitochondrial biology.     

iPSCs在心律失常疾病体外模型方面的研究进展

心律失常严重威胁人类健康,许多遗传性心律失常由基因突变引起。以患者的诱导型多能干细胞（iPSCs）源性的心肌细胞作为体外模型,可为研究心律失常的发病机制提供新的思路。本文主要对iPSCs源性诱导型心肌细胞的产生过程以及心律失常患者诱导iPSCs 心肌细胞在长QT综合征、儿茶酚胺依赖性室性心动过速、致心律失常性右室心肌病等疾病模型的研究进展进行了综述。     

Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells

Neural crest stem cells can be isolated from differentiated cultures of human pluripotent stem cells, but the process is inefficient and requires cell sorting to obtain a highly enriched population. No specific method for directed differentiation of human pluripotent cells toward neural crest stem cells has yet been reported. This severely restricts the utility of these cells as a model for disease and development and for more applied purposes such as cell therapy and tissue engineering. In this report, we use small-molecule compounds in a single-step method for the efficient generation of self-renewing neural crest-like stem cells in chemically defined media. This approach is accomplished directly from human pluripotent cells without the need for coculture on feeder layers or cell sorting to obtain a highly enriched population. Critical to this approach is the activation of canonical Wnt signaling and concurrent suppression of the Activin A/Nodal pathway. Over 12-14 d, pluripotent cells are efficiently specified along the neuroectoderm lineage toward p75(+) Hnk1(+) Ap2(+) neural crest-like cells with little or no contamination by Pax6(+) neural progenitors. This cell population can be clonally amplified and maintained for >25 passages (>100 d) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells, and mesenchymal precursor cells. Neural crest-like stem cell-derived mesenchymal precursors have the capacity for differentiation into osteocytes, chondrocytes, and adipocytes. In sum, we have developed methods for the efficient generation of self-renewing neural crest stem cells that greatly enhance their potential utility in disease modeling and regenerative medicine.     

Skewed megakaryopoiesis in human induced pluripotent stem cell‐derived haematopoietic progenitor cells harbouring calreticulin mutations

Somatic mutations in the calreticulin (CALR) gene have been found in most patients with JAK2‐ and MPL‐unmutated Philadelphia chromosome‐negative myeloproliferative neoplasms (MPNs). It has recently been shown that mutant CALR constitutively activates the thrombopoietin receptor MPL and, thus, plays a causal role in the development of MPNs. However, the roles of mutant CALR in human haematopoietic cell differentiation remain predominantly elusive. To examine the impact of the 5‐base insertion mutant CALR gene (Ins5) on haematopoietic cell differentiation, we generated induced pluripotent stem cells from an essential thrombocythaemia (ET) patient harbouring a CALR‐Ins5 mutation and from a healthy individual (WT). Megakaryopoiesis was more prominent in Ins5‐haematopoietic progenitor cells (Ins5‐HPCs) than in WT‐HPCs, implying that the system recapitulates megakaryocytosis observed in the bone marrow of CALR‐mutant ET patients. Ins5‐HPCs exhibited elevated expression levels of GATA1 and GATA2, suggesting a premature commitment to megakaryocytic differentiation in progenitor cells. We also demonstrated that 3‐hydroxy anagrelide markedly perturbed megakaryopoiesis, but not erythropoiesis. Collectively, we established an in vitro model system that recapitulates megakaryopoiesis caused by mutant CALR. This system can be used to validate therapeutic compounds for MPN patients harbouring CALR mutations and in detailed studies on mutant CALR in human haematological cell differentiation.     

机械牵张对诱导多能干细胞向心肌细胞分化的研究

目的研究机械牵张对诱导多能干细胞向心肌细胞分化效率的影响。方法诱导多能干细胞形成拟胚体后将拟胚体分为四组:对照组(未行牵张处理),牵张组1(5-6天行24小时牵张),牵张组2(5-6天行24小时牵张,7-8天再行24小时牵张),牵张组3(5-6天行24小时牵张,7-10天再行72小时牵张),通过对小鼠诱导多能干细胞施加20%形变率的机械牵张力后,于第15天,分别计数每组跳动克隆数目从而初步在上述四组中选出诱导效率最高组,此后用荧光免疫染色、Westernblot、RT-PCR和激光共聚焦法,进一步鉴定对照组和初步筛选出的牵张组最终分化效率和细胞成熟度差异。结果机械牵张刺激下,分化15天时,牵张组2的跳动克隆数上升(P<0.05),初步筛选出牵张组2可以提高分化效率;统计α-MHC免疫荧光染色面积发现牵张组2是对照组的2.1倍(P<0.05);牵张组2TroponinI的蛋白表达量为对照组的1.7倍(P<0.05);半定量PCR结果发现,心肌细胞标志基因β-MHC,MLC-2v及心肌细胞早期转录因子Nkx2.5的表达量分别提高了6.7倍、4.4倍和11.4倍(P值均<0.05);激光扫描共聚焦显微镜对分化来的单个心肌细胞进行α-actinin观察发现,牵张组2有利于心肌细胞的伸展和成熟。结论初步验证机械牵张力作为一种刺激诱导因素,20%拉伸形变率,牵张组2(贴壁的拟胚体5-6天行24小时牵张,7-8天再行24小时牵张)的处理方法可以显著促进诱导多能干细胞向心肌细胞的分化效率,为以后的深入研究和临床应用提供了实验基础。     

Establishment and identification of induced pluripotent stem cells in liver cancer patients

Objective:To induce pluripotent stem(IPS)cells from fibrocytes that are separated from liver cancer patients.Methods:The fibrocytes were reprogrammed to IPS cells by lentiviral vector,stained and identified by immunohistochemistry.Results:The IPS cells were successfully established from fibrocytes after infection,and IPS cell clones formed in round shape under a microscopy.The induction rate was 0.013%±0.007%.No tumor formed at the back of nude mice within 8 weeks after the inoculation of cell clone.However,tetatoma appeared in nude mice within 1 week after IPS inoculation.A few tumors formed in nude mice within 4 weeks after the inoculation of cell clones.However,subcutaneous tumors formed within 1 week after IPS inoculation.The induced IPS cells showed three germ layers in tetatoma.Nanog and OCT4 in the induced IPS cells showed hypomethylation.SSEA-A,TRA-1-6-,TRA-1-81 and Nanog were highly expressed in the induced IPS cells,indicating the IPS cells possessed the similar ability as the stem cells.Conclusion:The IPS cells of liver cancer patients can be established effectively from fibrocytes and can be cultured stably in vitro,which provides an approach for the treatment of intermediate or advanced stage liver cancer.     

Melatonin protects bone marrow mesenchymal stem cells against iron overload‐induced aberrant differentiation and senescence

Bone marrow mesenchymal stem cells (BMSCs) are an expandable population of stem cells which can differentiate into osteoblasts, chondrocytes and adipocytes. Dysfunction of BMSCs in response to pathological stimuli contributes to bone diseases. Melatonin, a hormone secreted from pineal gland, has been proved to be an important mediator in bone formation and mineralization. The aim of this study was to investigate whether melatonin protected against iron overload‐induced dysfunction of BMSCs and its underlying mechanisms. Here, we found that iron overload induced by ferric ammonium citrate (FAC) caused irregularly morphological changes and markedly reduced the viability in BMSCs. Consistently, osteogenic differentiation of BMSCs was significantly inhibited by iron overload, but melatonin treatment rescued osteogenic differentiation of BMSCs. Furthermore, exposure to FAC led to the senescence in BMSCs, which was attenuated by melatonin as well. Meanwhile, melatonin was able to counter the reduction in cell proliferation by iron overload in BMSCs. In addition, protective effects of melatonin on iron overload‐induced dysfunction of BMSCs were abolished by its inhibitor luzindole. Also, melatonin protected BMSCs against iron overload‐induced ROS accumulation and membrane potential depolarization. Further study uncovered that melatonin inhibited the upregulation of p53, ERK and p38 protein expressions in BMSCs with iron overload. Collectively, melatonin plays a protective role in iron overload‐induced osteogenic differentiation dysfunction and senescence through blocking ROS accumulation and p53/ERK/p38 activation.     

叶酸缺乏对鼠胚成纤维细胞MAPK通路及多潜能干细胞诱导效率的影响

目的:观察短暂性的叶酸缺乏对鼠胚成纤维细胞(MEF)MAPK通路及其诱导多潜能干(iPS)细胞诱导效率的影响。方法:将鼠胚成纤维细胞分为正常叶酸组、叶酸缺乏3 d组和叶酸缺乏6 d组,分别于叶酸缺乏3、6 d western blot法检测提取的上述3组鼠胚成纤维细胞中的磷酸化细胞外信号调节激酶(p-ERK)蛋白和总细胞外信号调节激酶(ERK)蛋白的变化,用经典四因子的逆转录病毒载体进行感染,并对获得iPS细胞进行鉴定。结果:叶酸缺乏3 d组p-ERK明显降低,感染后第12天出现具有胚胎干细胞形态的克隆,叶酸缺乏3 d组、6 d组产生的iPS细胞和正常叶酸组的诱导效率分别为(0.0477±0.0005)%、(0.0155±0.0019)%和(0.01±0.0005)%,所获的iPS细胞多能性基因和免疫荧光多能性标志鉴定为阳性。结论:叶酸缺乏3 d能明显压低p-ERK的表达,提高小鼠iPS细胞的诱导效率,且诱导的细胞多能性基因和免疫荧光多能性标志鉴定为阳性。     

Stem cell-based treatments for Type 1 diabetes mellitus: bone marrow, embryonic, hepatic, pancreatic and induced pluripotent stem cells

Type 1 diabetes mellitus--characterized by the permanent destruction of insulin-secreting β-cells--is responsive to cell-based treatments that replace lost β-cell populations. The current gold standard of pancreas transplantation provides only temporary independence from exogenous insulin and is fraught with complications, including increased mortality. Stem cells offer a number of theoretical advantages over current therapies. Our review will focus on the development of treatments involving tissue stem cells from bone marrow, liver and pancreatic cells, as well as the potential use of embryonic and induced pluripotent stem cells for Type 1 diabetes therapy. While the body of research involving stem cells is at once promising and inconsistent, bone marrow-derived mesenchymal stem cell transplantation seems to offer the most compelling evidence of efficacy. These cells have been demonstrated to increase endogenous insulin production, while partially mitigating the autoimmune destruction of newly formed β-cells. However, recently successful experiments involving induced pluripotent stem cells could quickly move them into the foreground of therapeutic research. We address the limitations encountered by present research and look toward the future of stem cell treatments for Type 1 diabetes.     

Melatonin reverses H2O2‐induced premature senescence in mesenchymal stem cells via the SIRT1‐dependent pathway

Mesenchymal stem cells (MSCs) represent an attractive source for stem cell‐based regenerative therapy, but they are vulnerable to oxidative stress‐induced premature senescence in pathological conditions. We previously reported antioxidant and antiarthritic effects of melatonin on MSCs against proinflammatory cytokines. In this study, we hypothesized that melatonin could protect MSCs from premature senescence induced by hydrogen peroxide (H₂O₂) via the silent information regulator type 1 (SIRT1)‐dependent pathway. In response to H₂O₂ at a sublethal concentration of 200 μm , human bone marrow‐derived MSCs (BM‐MSCs) underwent growth arrest and cellular senescence. Treatment with melatonin before H₂O₂ exposure cannot significantly prevent premature senescence; however, treatment with melatonin subsequent to H₂O₂ exposure successfully reversed the senescent phenotypes of BM‐MSCs in a dose‐dependent manner. This result was made evident by improved cell proliferation, decreased senescence‐associated β‐galactosidase activity, and the improved entry of proliferating cells into the S phase. In addition, treatment with 100 μm melatonin restored the osteogenic differentiation potential of BM‐MSCs that was inhibited by H₂O₂‐induced premature senescence. We also found that melatonin attenuated the H₂O₂‐stimulated phosphorylation of p38 mitogen‐activated protein kinase, decreased expression of the senescence‐associated protein p16^INK4α, and increased SIRT1. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked melatonin‐mediated antisenescence effects. Inhibition of SIRT1 by sirtinol counteracted the protective effects of melatonin, suggesting that melatonin reversed the senescence in cells through the SIRT1‐dependent pathway. Together, these findings lay new ground for understanding oxidative stress‐induced premature senescence and open perspectives for therapeutic applications of melatonin in stem cell‐based regenerative medicine.     

Cardiotrophin-1 promotes cardiomyocyte differentiation from mouse induced pluripotent stem cells via JAK2/STAT3/Pim-1 signaling pathway

Background The induced pluripotent stem cell (iPSC) has shown great potential in cellular therapy of myocardial infarction (MI), while its application is hampered by the low efficiency of cardiomyocyte differentiation. The present study was designed to investigate the effects of cardiotrophin-1 (CT-1) on cardiomyocyte differentiation from mouse induced pluripotent stem cells (miPSCs) and the underlying mechanisms involved. Methods The optimal treatment condition for cardiomyocyte differentiation from miPSCs was established with ideal concentration (10 ng/mL) and duration (from day 3 to day 14) of CT-1 administration. Up-regulated expression of cardiac specific genes that accounted for embryonic cardiogenesis was observed by quantitative RT-PCR. Elevated amount of α-myosin heavy chain (α-MHC) and cardiac troponin I (cTn I) positive cells were detected by immunofluorescence staining and flow cytometry analysis in CT-1 group. Results Transmission electron microscopic analysis revealed that cells treated with CT-1 showed better organized sacromeric structure and more mitochondria, which are morphological characteristic of matured cardiomyocytes. Western blot demonstrated that CT-1 promotes cardiomyocyte differentiation from miPSCs partly via JAK2/STAT3/Pim-1 pathway as compared with control group. Conclusions These findings suggested that CT-1 could enhance the cardiomyocyte differentiation as well as the maturation of mouse induced pluripotent stem cell derived cardiomyocytes by regulating JAK2/STAT3/Pim-1signaling pathway.