Current Issue : October - December Volume : 2017 Issue Number : 4 Articles : 5 Articles
A large number of myocytes are necessary to treat intractable muscular disorders such as Duchenne muscular dystrophy with cellbased\ntherapies. However, starting materials for cellular therapy products such as myoblasts, marrow stromal cells, menstrual\nblood-derived cells, and placenta-derived cells have a limited lifespan and cease to proliferate in vitro. From the viewpoints of\nmanufacturing and quality control, cells with a long lifespan are more suitable as a starting material. In this study, we generated\nstromal cells for future myoblast therapy from a working cell bank of human embryonic stem cells (ESCs). The ESC-derived\nCD105+ cells with extensive in vitro proliferation capability exhibited myogenesis and genetic stability in vitro. These results\nimply that ESC-derived CD105+ cells are another cell source for myoblasts in cell-based therapy for patients with genetic\nmuscular disorders. Since ESCs are immortal, mesenchymal stromal cells generated from ESCs can be manufactured at a large\nscale in one lot for pharmaceutical purposes....
Background: Bone marrow-derived mesenchymal stromal cells (MSCs) are multipotent cells with a high\nconstitutive level of autophagy and low expression of CD99. Under certain conditions, MSCs may develop\ntumorigenic properties. However, these transformation-induced conditions are largely unknown. Recently, we\nhave identified an association between Hsp70, a main participant in cellular stress response and tumorigenesis,\nand CD99. Preliminary observations had revealed upregulation of both proteins in stressed long-term cultured\nMSCs. And so we hypothesized that CD99 is implicated in stress-induced mechanisms of cellular transformation\nin MSCs. Hence, we investigated the effects of prolonged stress on MSCs and the role of CD99 and autophagy in\ntheir survival.\nMethods: Human telomerase reverse transcriptase (hTERT) overexpressing immortalized MSCs and primary bone\nmarrow stromal cells were used to investigate the influence of long-term serum deprivation and hypoxia on\ngrowth and differentiation of MSCs. Cell proliferation and apoptosis were evaluated using flow cytometry,\ndifferentiation capabilities of MSCs were assessed by immunohistochemical staining followed by microscopic\nexamination. CD99, Hsp70 expression were analyzed using flow cytometry, western blotting, and reverse\ntranscriptase polymerase chain reaction. Autophagy was explored with specific inhibitors using cell morphology\nexamination and western blotting.\nResults: Chronic stress factors are able to change the morphology of MSCs and to inhibit spontaneous\ndifferentiation into adipocyte lineage. Furthermore, CD99 elevation and downregulation of p53 and p21\naccompanied defective autophagy, which is usually associated with tumor formation. We found that inhibition\nof autophagy by chloroquine promoted cell detachment and modulated CD99 expression level whereas\nincorporation of CD99 recombinant protein into the cells suppressed autophagy.\nConclusions: Obtained results provide a model for chronic stress-induced transformation of MSCs via CD99 and\nmay therefore be highly relevant to mesenchymal tumorigenesis....
Background: Pluripotent stem cells (PSCs) such as embryonic stem cells and induced pluripotent stem cells are\npromising target cells for cell regenerative medicine together with recently advanced technology of in-vitro\ndifferentiation. However, residual undifferentiated stem cells (USCs) during in-vitro differentiation are considered a\npotential risk for development of cancer cells and nonspecific lineage cell types. In this study we observed that\nUSCs still exist during hepatic differentiation, consequently resulting in poor quality of the hepatic population and\nforming teratoma in vivo. Therefore, we hypothesized that effectively removing USCs from in-vitro differentiation\ncould improve the quality of the hepatic population and guarantee safety from risk of teratoma formation.\nMethods: Human PSCs were differentiated to hepatocytes via four steps. YM155, a known BIRC5 inhibitor, was\napplied for removing the residual USCs on the hepatic differentiation. After YM155 treatment, hepatocyte\ndevelopment was evaluated by measuring gene expression, immunostaining and hepatic functions at each stage of\ndifferentiation, and forming teratomas were confirmed by cell transplantation with or without YM155.\nResults: The selected concentrations of YM155 removed USCs (NANOG+ and OCT4+) in a dose-dependent manner.\nAs a result, expression of endodermal markers (SOX17, FOXA2 and CXCR4) at stage II of differentiation and hepatic\nmarkers (ALB, AFP and HNF4A) at stage III was up-regulated by YM155 treatment as well as the hepatic population\n(ALB+), and functions (ALB/urea secretion and CYP450 enzyme activity) were enhanced at the final stage of\ndifferentiation (stage IV). Furthermore, we demonstrated that NANOG and OCT4 expression remaining until stage III\n(day 15 of differentiation) completely disappeared when treated with YM155 and teratoma formation was\neffectively prevented by YM155 pretreatment in the in-vitro study....
Three to eight percent of female carriers of Duchenne muscular dystrophy (DMD) develop dystrophic symptoms ranging from\nmild muscle weakness to a rapidly progressive DMD-like muscular dystrophy due to skewed inactivation of X chromosomes\nduring early development. Here, we generated human induced pluripotent stem cells (hiPSCs) from a manifesting female carrier\nusing retroviral or Sendai viral (SeV) vectors and determined their X-inactivation status. Although manifesting carrier-derived\niPS cells showed normal expression of human embryonic stem cell markers and formed well-differentiated teratomas in vivo,\nmany hiPS clones showed bi-allelic expression of the androgen receptor (AR) gene and loss of X-inactivation-specific transcript\nand trimethyl-histone H3 (Lys27) signals on X chromosomes, suggesting that both X chromosomes of the hiPS cells are in an\nactive state. Importantly, normal dystrophin was expressed in multinucleated myotubes differentiated from a manifesting carrier\nof DMD-hiPS cells with XaXa pattern. AR transcripts were also equally transcribed from both alleles in induced myotubes. Our\nresults indicated that the inactivated X chromosome in the patient�s fibroblasts was activated during reprogramming, and XCI\noccurred randomly during differentiation....
Mutations in WD40-repeat protein 62 (WDR62) are commonly associated with primary microcephaly and other developmental\ncortical malformations. We used human pluripotent stem cells (hPSC) to examine WDR62 function during human neural\ndifferentiation and model early stages of human corticogenesis. Neurospheres lacking WDR62 expression showed decreased\nexpression of intermediate progenitor marker, TBR2, and also glial marker, S100�². In contrast, inhibition of c-Jun N-terminal\nkinase (JNK) signalling during hPSC neural differentiation induced upregulation of WDR62 with a corresponding increase in\nneural and glial progenitor markers, PAX6 and EAAT1, respectively. These findings may signify a role of WDR62 in specifying\nintermediate neural and glial progenitors during human pluripotent stem cell differentiation....
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