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Mesenchymal stem cells (MSCs) represent a promising therapeutic candidate for treating many diseases. However, their proliferation and therapeutic abilities decline during the aging process and disease development. Therefore, fetal MSCs derived from the umbilical cord (UC) attract more attention. Storing and delivering the UC is one critical step for efficient MSC isolation. Although the culture medium-based solution is suitable for UC storage, it is not feasible for large-scale preparation because of its high price. Thus, we demonstrate here that a simple solution containing a pH buffering reagent, calcium, magnesium and glucose could be used as a cost-effective storage solution for UC delivery and efficient MSC isolation.

Objectives Mesenchymal stem cells (MSCs) have been intensively investigated as to their therapeutic potentials. However, the full chemical-defined medium supporting the isolation and expansion of human MSCs has not been developed yet. Materials and methods Here, we developed the full chemical-defined medium, NBVbe medium, via RNA sequencing, bioinformatic analysis, and growth factor screening. Results The NBVbe medium contains N2B27 medium with the BSA (bovine serum albumin) replaced by the recombinant human albumin, bFGF (basic fibroblast growth factor), vitamin C, and EGF (epidermal growth factor). The NBVbe medium could support the isolation and expansion of human MSCs from the umbilical cords. Conclusions The full chemical-defined medium supporting the isolation and expansion of human MSCs has been developed. This would be helpful for further optimization of the MSC medium, their clinical applications, and molecular characterization.

Background/Aims: The target genome editing technology not only plays an important role in basic biology studies but also holds a great promise for potential clinical applications. The new generation of engineered nuclease RGEN (RNA Guided EndoNuclease) is much easier to construct and modify, and attracts more attentions. In the current study, we compared different plasmid construction strategies of Cas9-gRNA (guide RNA). Methods: Different plasmid construction strategies of Cas9-gRNA were compared. And more modifications were introduced into the plasmid construction strategy. Results: The plasmid construction efficiency of expressing the gRNA and Cas9 in one plasmid was lower than expressing them in two separate plasmids. However, they showed the similar genome editing efficiency. We further introduced the Golden-gate assembly and blue-white screening approaches into the Cas9-gRNA construction procedures, without the process of vector digestion and gel purification. Conclusions: Combing with the optimized gRNA structure (gRNA-BL) we identified before, we established one more cost-effective, time-saving and efficient plasmid construction strategy for Cas9-gRNA.

// Jianyong Xu 1,2,3 , Wei Lian 1,2,3 , Lingyun Li 1,2,3 and Zhong Huang 1,2,3 1 Institute of Biological Therapy, Shenzhen University, Shenzhen, China 2 Department of Pathogen Biology and Immunology, Shenzhen University School of Medicine, Shenzhen, China 3 Shenzhen City Shenzhen University Immunodiagnostic Technology Platform, Shenzhen, China Correspondence to: Jianyong Xu, email: // Zhong Huang, email: // Keywords : cardiac progenitor cell, CPC, somatic reprogramming, induced CPC Received : November 30, 2016 Accepted : January 24, 2017 Published : February 10, 2017 Abstract It has been demonstrated that cardiac progenitor cells (CPCs) represent a more effective cell-based therapy for treatment of myocardial infarction. Unfortunately, their therapeutic application is limited by low yield of cell harvesting, declining quality and quantity during the ageing process, and the need for highly invasive heart biopsy. Therefore, there is an emerging interest in generating CPC-like stem cells from somatic cells via somatic reprogramming. This novel approach would provide an unlimited source of stem cells with cardiac differentiation potential. Here we would firstly discuss the different types of CPC and their importance in stem cell therapy for treatment of myocardial infarction; secondly, the necessity of generating induced CPC from somatic cells via somatic reprogramming; and finally the current progress of somatic reprogramming in cardiac cells, especially induced CPC generation.

Mesenchymal stem cells (MSCs) can be derived from various adult tissues with multipotent and self‐renewal abilities. The characteristics of presenting no major ethical concerns, having low immunogenicity and possessing immune modulation functions make MSCs promising candidates for stem cell therapies. MSCs could promote inflammation when the immune system is underactivated and restrain inflammation when the immune system is overactivated to avoid self‐overattack. These cells express many immune suppressors to switch them from a pro‐inflammatory phenotype to an anti‐inflammatory phenotype, resulting in immune effector cell suppression and immune suppressor cell activation. We would discuss the mechanisms governing the immune modulation function of these cells in this review, especially the immune‐suppressive effects of MSCs.

Mesenchymal stem cells (MSCs) have been intensively investigated and widely applied in regenerative medicine and immune modulation. However, their efficacy declines during the aging or disease process. Thus, genome-edited MSCs with over-expression or inhibition of specific genes hold a great deal of promise in terms of their therapeutic application. Here we optimized the direct PCR approach for rapid identification of genome-edited MSCs with only ten cells required, which reduces the time and labor to expand the MSC colonies. Combined with our previously optimized guide RNA structure and plasmid construction strategy for Cas9, we successfully identified MSC colonies over-expressing IL-10 in the AAVS1 locus.

Mesenchymal stem cells (MSCs) have been intensively studied and applied in regenerative medicine and tissue engineering. Recently, their immune modulation functions make them as attractive potential approaches for autoimmune disease treatment. Systemic lupus erythematosus (SLE) is one type of chronic autoimmune diseases with multi-organ damaged by the immune system. Although current available treatments are effective for some patients, others are refractory for these therapies. The immuno-modulatory and regenerative characteristics of MSCs make them as one promising candidate for treating SLE. Thus, we would discuss their immune modulation effects, pre-clinical and clinical applications, and the potentials for immune tolerance re-establishment in SLE here.

Large-scale genome-wide association studies have reported EPHA1 rs11767557 variant to be associated with Alzheimer's disease (AD) risk in the European population. However, it is still unclear how this variant functionally contributes to the underlying disease pathogenesis. The rs11767557 variant is located approximately 3 kb upstream of EPHA1 gene. We think that rs11767557 may modify the expression of nearby genes such as EPHA1 and further cause AD risk. Until now, the potential association between rs11767557 and the expression of nearby genes has not been reported in previous studies. Here, we evaluate the potential expression association between rs11767557 and EPHA1 using multiple large-scale eQTLs datasets in human brain tissues and the whole blood. The results show that rs11767557 variant could significantly regulate EPHA1 gene expression specifically in human whole blood. These findings may further provide important supplementary information about the regulating mechanisms of rs11767557 variant in AD risk.

A recent study analyzed 2053 multiple sclerosis (MS) cases and 799 healthy controls to investigate whether five genetic variants (rs11039149, rs12221497, rs2279238, rs7120118 and rs7114704) in NR1H3 are associated with MS risk. However this study reported negative results. It is very important that the appropriate samples and approach should be used in replication studies, which may provide the correct interpretation of the results. Here, we evaluated the above findings using large-scale MS genome-wide association studies with a total of 27,148 samples including 9772 MS cases and 17,376 controls, and multiple expression quantitative trait loci datasets. The results suggest that rs7120118 and rs2279238 variants are significantly associated with MS risk, and could significantly regulate NR1H3 expression in kinds of human tissues and cells. In summary, these findings provide important supplementary information about the association between NR1H3 variants and MS risk.

The term laminopathies defines a group of genetic disorders caused by defects in the nuclear envelope, mostly the lamins. Lamins are the main constituents of the nuclear lamina, a filamentous meshwork associated with the inner nuclear membrane that provides mechanical stability and plays important roles in processes such as transcription, DNA replication and chromatin organization. More than 300 mutations in lamin A/C have been associated with diverse clinical phenotypes, understanding the molecular basis of these diseases may provide a rationale for treating them. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient with inherited dilated cardiomiopathy and 2 patients with distinct accelerated forms of aging, atypical Werner syndrome and Hutchinson Gilford progeria, all of which are caused by mutations in lamin A/C. These cell lines were pluripotent and displayed normal nuclear membrane morphology compared to donor fibroblasts. Their differentiated progeny reproduced the disease phenotype, reinforcing the idea that they represent excellent tools for understanding the role of lamin A/C in normal physiology and the clinical diversity associated with these diseases. © Ho et al., published_or_final_version

Cardiovascular disease is the leading cause of death worldwide. Despite significant progress in understanding of the disease mechanisms, most therapies remain at best palliative. Few therapeutic approaches offer direct tissue repair and regeneration. Cell-based therapy offers a promising approach that involves transplantation of healthy and functional cells to replenish damaged cells and repair injured tissue. Endothelial dysfunction is one of the most important mechanisms of cardiovascular disease, thus endothelial progenitor cells (EPC) and their derivatives have been investigated as a potential source for cell therapy. In pre-clinical and pilot clinical studies, treatment with EPCs or their derivatives as well as their co-transplantation with other cell types has shown some initial promising results. In this review, we will first describe the importance of endothelial cells and EPC homeostasis in the pathophysiology of cardiovascular disease. The potential sources of EPCs, including their isolation and purification, differentiation from pluripotent stem cells and adult stem cells, and trans-differentiation from somatic cells will then be summarized. Lastly, the application of target genome editing tools, such as Zinc Finger Nuclease (ZFN), Transcription Activator Like Effector Nucleases (TALEN) and RNA Guided Endo Nuclease (RGEN) to modify EPCs and their derivatives will be described. These technologies promise to further improve the therapeutic potential of EPCs and their derivatives to treat cardiovascular disease.

C-C chemokine receptor type 5 (CCR5) is a major co-receptor for the entry of human immunodeficiency virus type-1 (HIV-1) into target cells. Human hematopoietic stem cells (hHSCs) with naturally occurring CCR5 deletions (Δ32) or artificially disrupted CCR5 have shown potential for curing acquired immunodeficiency syndrome (AIDS). However, Δ32 donors are scarce, heterologous bone marrow transplantation is not exempt of risks, and genetic engineering of autologous hHSCs is not trivial. Here, we have disrupted the CCR5 locus of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) using specific zinc finger nucleases (ZFNs) combined with homologous recombination. The modified hESCs and hiPSCs retained pluripotent characteristics and could be differentiated in vitro into CD34+ cells that formed all types of hematopoietic colonies. Our results suggest the potential of using patient-specific hHSCs derived from ZFN-modified hiPSCs for treating AIDS.

Directed hepatocyte differentiation from human induced pluripotent stem cells (iPSCs) potentially provides a unique platform for modeling liver genetic diseases and performing drug-toxicity screening in vitro. Wilson's disease is a genetic disease caused by mutations in the ATP7B gene, whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. Interestingly, the spectrum of ATP7B mutations is vast and can influence clinical presentation (a variable spectrum of hepatic and neural manifestations), though the reason is not well understood. We describe the generation of iPSCs from a Chinese patient with Wilson's disease that bears the R778L Chinese hotspot mutation in the ATP7B gene. These iPSCs were pluripotent and could be readily differentiated into hepatocyte-like cells that displayed abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Moreover, gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B or treatment with the chaperone drug curcumin could reverse the functional defect in vitro. Hence, our work describes an attractive model for studying the pathogenesis of Wilson's disease that is valuable for screening compounds or gene therapy approaches aimed to correct the abnormality. In the future, once relevant safety concerns (including the stability of the mature liver-like phenotype) and technical issues for the transplantation procedure are solved, hepatocyte-like cells from similarly genetically corrected iPSCs could be an option for autologous transplantation in Wilson's disease.

The ciliary hypothesis for cystic renal diseases postulates that most of these conditions result from abnormalities in the primary cilium, a microtubule-based structure that acts as a sensor for extracellular cues. Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes to renal cysts and clear cell renal cell carcinoma. VHL plays a critical role in the formation of primary cilia in kidney epithelium, but the underlying mechanisms are poorly understood. Here, we demonstrate that VHL inactivation induces HEF1/Cas-L/NEDD9 and Aurora kinase A via the stabilization of hypoxia-inducible factors 1 and 2. Aurora kinase A is a mitotic kinase commonly upregulated in cancer that causes regression of the primary cilium by promoting histone deacetylase-dependent tubulin depolymerization of the ciliary axoneme. HEF1/Cas-L/NEDD9 is a component of focal adhesions that has a prominent role in inducing metastasis and that colocalizes with Aurora kinase A at the centrosome, thereby enhancing the harmful effect of Aurora kinase A on the cilium. Suppression of this pathway improved the formation of primary cilia and reduced cell motility in VHL-defective renal cancer cells. Our results highlight the gatekeeper role of VHL in the kidney epithelium.

The potential of human embryonic stem cells (ESCs) for regenerative medicine is unquestionable, but practical and ethical considerations have hampered clinical application and research. In an attempt to overcome these issues, the conversion of somatic cells into pluripotent stem cells similar to ESCs, commonly termed nuclear reprogramming, has been a top objective of contemporary biology. More than 40 years ago, King, Briggs, and Gurdon pioneered somatic cell nuclear reprogramming in frogs, and in 1981 Evans successfully isolated mouse ESCs. In 1997 Wilmut and collaborators produced the first cloned mammal using nuclear transfer, and then Thomson obtained human ESCs from in vitro fertilized blastocysts in 1998. Over the last 2 decades we have also seen remarkable findings regarding how ESC behavior is controlled, the importance of which should not be underestimated. This knowledge allowed the laboratory of Shinya Yamanaka to overcome brilliantly conceptual and technical barriers in 2006 and generate induced pluripotent stem cells (iPSCs) from mouse fibroblasts by overexpressing defined combinations of ESC-enriched transcription factors. Here, we discuss some important implications of human iPSCs for biology and medicine and also point to possible future directions.

The umbilical cord and placenta are extra-embryonic tissues of particular interest for regenerative medicine. They share an early developmental origin and are a source of vast amounts of cells with multilineage differentiation potential that are poorly immunogenic and without controversy. Moreover, these cells are likely exempt from incorporated mutations when compared with juvenile or adult donor cells such as skin fibroblasts or keratinocytes. Here we report the efficient generation of induced pluripotent stem cells (iPSCs) from mesenchymal cells of the umbilical cord matrix (up to 0.4% of the cells became reprogrammed) and the placental amniotic membrane (up to 0.1%) using exogenous factors and a chemical mixture. iPSCs from these 2 tissues homogeneously showed human embryonic stem cell (hESC)-like characteristics including morphology, positive staining for alkaline phosphatase, normal karyotype, and expression of hESC-like markers including Nanog, Rex1, Oct4, TRA-1-60, TRA-1-80, SSEA-3, and SSEA-4. Selected clones also formed embryonic bodies and teratomas containing derivatives of the 3 germ layers, and could as well be readily differentiated into functional motor neurons. Among other things, our cell lines may prove useful for comparisons between iPSCs derived from multiple tissues regarding the extent of the epigenetic reprogramming, differentiation ability, stability of the resulting lineages, and the risk of associated abnormalities. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc., link_to_OA_fulltext

Recently, three independent laboratories reported the generation of induced pluripotent stem cells (iPSCs) from pig (Sus scrofa). This finding sums to the growing list of species (mouse, human, monkey, and rat, in this order) for which successful reprogramming using exogenous factors has been achieved, and multiple others are possibly forthcoming. But apart from demonstrating the universality of the network identified by Shinya Yamanaka, what makes the porcine model so special? On one side, pigs are an agricultural commodity and have an easy and affordable maintenance compared with nonhuman primates that normally need to be imported. On the other side, resemblance (for example, size of organs) of porcine and human physiology is striking and because pigs are a regular source of food the ethical concerns that still remain in monkeys are not applicable. Besides, the prolonged lifespan of pigs compared with other domestic species can allow exhaustive follow up of side effects after transplantation. Porcine iPSCs may thus fill the gap between the mouse model, which due to its ease is preferred for mechanistic studies, and the first clinical trials using iPSCs in humans. However, although these studies are relevant and have created significant interest they face analogous problems that we discuss herein together with potential new directions.

SummarySomatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However, the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound, vitamin C (Vc), enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence, a recently identified roadblock for reprogramming. In addition, Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process.

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model.