Cellular Environment Directs Differentiation of Human Umbilical Cord Blood�CDerived Neural Stem Cells In Vitro

Cord blood–derived neural stem cells (NSCs) are proposed as an alternative cell source to repair brain damage upon transplantation. However, there is a lack of data showing how these cells are driven to generate desired phenotypes by recipient nervous tissue. Previous research indicates that local environment provides signals driving the fate of stem cells. To investigate the impact of these local cues interaction, the authors used a model of cord blood–derived NSCs co-cultured with different rat brain–specific primary cultures, creating the neural-like microenvironment conditions in vitro. Neuronal and astro-, oligo-, and microglia cell cultures were obtained by the previously described methods. The CMFDA-labeled neural stem cells originated from, non-transformed human umbilical cord blood cell line (HUCB-NSCs) established in a laboratory. The authors show that the close vicinity of astrocytes and oligodendrocytes promotes neuronal differentiation of HUCB-NSCs, whereas postmitotic neurons induce oligodendrogliogenesis of these cells. In turn, microglia or endothelial cells do not favor any phenotypes of their neural commitment. Studies have confirmed that HUCB-NSCs can read cues from the neurogenic microenvironment, attaining features of neurons, astrocytes, or oligodendrocytes. The specific responses of neurally committed cord blood–derived cells, reported in this work, are very much similar to those described previously for NSCs derived from other “more typical” sources. This further proves their genuine neural nature. Apart from having a better insight into the neurogenesis in the adult brain, these findings might be important when predicting cord blood cell derivative behavior after their transplantation for neurological disorders.     

Effects of Oxygen Concentration on the Proliferation and Differentiation of Mouse Neural Stem Cells In Vitro

Background and purpose Cerebral ischemia is known to elicit the activation of neural stem cells (NSCs); however its mechanism is not fully determined. Although oxygen concentration is known to mediate many ischemic actions, there has been little attention given to the role of pathological oxygen changes under cerebral ischemia on the activation of NSCs. We investigated the effects of various oxygen concentrations on mouse neural stem cells in vitro. Methods NSCs were cultured from the ganglionic eminence of fetal ICR mice on embryonic day 15.5 using a neurosphere method. The effects of oxygen concentrations on proliferation, differentiation, and cell death of NSCs were evaluated by bromodeoxyuridine (BrdU) incorporation, immunocytochemistry, and TUNEL assay, respectively. Results The highest proliferation and the neuronal differentiation of the NSCs were observed in 2% oxygen, which yielded significantly higher proportions of both BrdU-labeled cells and Tuj1-positive cells when compared with 20% and 4% oxygen. On the other hand, the differentiation to the astrocytes was not affected by oxygen concentrations, except in the case of anoxia (0% oxygen). The cell death of the NSCs increased in lower oxygen conditions and peaked at anoxia. Furthermore, the switching of the neuronal subtype differentiation from GABA-positive to glutamate-positive neurons was observed in lower oxygen conditions. Conclusions These findings raise the possibility that reduced oxygen levels occurring with cerebral ischemia enhance NSC proliferation and neural differentiation, and that mild hypoxia (2% oxygen), which is known to occur in the ischemic penumbra, is suitable for abundant neuronal differentiation.     

Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody Targets Adult Human Pancreatic β Cells for In Vitro and In Vivo Analysis

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Genomic Instability Associated with p53 Knockdown in the Generation of Huntington’s Disease Human Induced Pluripotent Stem Cells

Alterations in DNA damage response and repair have been observed in Huntington’s disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown.     

Inhibition of Methyltransferase Setd7 Allows the In Vitro Expansion of Myogenic Stem Cells with Improved Therapeutic Potential

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Generation of Human Induced Pluripotent Stem (iPS) Cells in Serum- and Feeder-Free Defined Culture and TGF-β1 Regulation of Pluripotency

Human Embryonic Stem cells (hESCs) and human induced Pluripotent Stem cells (hiPSCs) are commonly maintained on inactivated mouse embryonic fibroblast as feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture medium containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. In addition, there is no consensus as to the optimal formulation, or the nature of the cytokine requirements of the cells to promote their self-renewal and inhibit their differentiation. In this study, we successfully generated hiPSCs from human dental pulp cells (DPCs) using Yamanaka''s factors (Oct3/4, Sox2, Klf4, and c-Myc) with retroviral vectors in serum- and feeder-free defined culture conditions. These hiPSCs retained the property of self-renewal as evaluated by the expression of self-renewal marker genes and proteins, morphology, cell growth rates, and pluripotency evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo. In this study, we found that TGF-β1 increased the expression levels of pluripotency markers in a dose-dependent manner. However, increasing doses of TGF-β1 suppressed the growth rate of hiPSCs cultured under the defined conditions. Furthermore, over short time periods the hiPSCs cultured in hESF9 or hESF9T exhibited similar morphology, but hiPSCs maintained in hESF9 could not survive beyond 30 passages. This result clearly confirmed that hiPSCs cultured in hESF9 medium absolutely required TGF-β1 to maintain pluripotency. This simple serum-free adherent monoculture system will allow us to elucidate the cell responses to growth factors under defined conditions and can eliminate the risk might be brought by undefined pathogens.     

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.     

Microvesicles Derived from Human Umbilical Cord Wharton’s Jelly Mesenchymal Stem Cells Attenuate Bladder Tumor Cell Growth In Vitro and In Vivo

Several studies suggest that mesenchymal stem cells (MSCs) possess antitumor properties; however, the exact mechanisms remain unclear. Recently, microvesicles (MVs) are considered as a novel avenue intercellular communication, which may be a mediator in MSCs-related antitumor effect. In the present study, we evaluated whether MVs derived from human umbilical cord Wharton’s jelly mesenchymal stem cells (hWJMSCs) may inhibit bladder tumor T24 cells growth using cell culture and the BALB/c nu/nu mice xenograft model. CCK-8 assay and Ki-67 immunostaining were performed to estimate cell proliferation in vitro and in vivo. Flow cytometry and TUNEL assay were used to assess cell cycle and apoptosis. To study the conceivable mechanism by which hWJMSC-MVs attenuate bladder tumor T24 cells, we estimated the expression of Akt/p-Akt, p-p53, p21 and cleaved Caspase 3 by Western blot technique after exposing T24 cells to hWJMSC-MVs for 24, 48 and 72h. Our data indicated that hWJMSC-MVs can inhibit T24 cells proliferative viability via cell cycle arrest and induce apoptosis in T24 cells in vitro and in vivo. This study showed that hWJMSC-MVs down-regulated phosphorylation of Akt protein kinase and up-regulated cleaved Caspase 3 during the process of anti-proliferation and pro-apoptosis in T24 cells. These results demonstrate that hWJMSC-MVs play a vital role in hWJMSC-induced antitumor effect and may be a novel tool for cancer therapy as a new mechanism of cell-to-cell communication.     

Redifferentiation of Adult Human β Cells Expanded In Vitro by Inhibition of the WNT Pathway

In vitro expansion of adult human islet β cells is an attractive solution for the shortage of tissue for cell replacement therapy of type 1 diabetes. Using a lineage tracing approach we have demonstrated that β-cell-derived (BCD) cells rapidly dedifferentiate in culture and can proliferate for up to 16 population doublings. Dedifferentiation is associated with changes resembling epithelial-mesenchymal transition (EMT). The WNT pathway has been shown to induce EMT and plays key roles in regulating replication and differentiation in many cell types. Here we show that BCD cell dedifferentiation is associated with β-catenin translocation into the nucleus and activation of the WNT pathway. Inhibition of β-catenin expression in expanded BCD cells using short hairpin RNA resulted in growth arrest, mesenchymal-epithelial transition, and redifferentiation, as judged by activation of β-cell gene expression. Furthermore, inhibition of β-catenin expression synergized with redifferentiation induced by a combination of soluble factors, as judged by an increase in the number of C-peptide-positive cells. Simultaneous inhibition of the WNT and NOTCH pathways also resulted in a synergistic effect on redifferentiation. These findings, which were reproducible in cells derived from multiple human donors, suggest that inhibition of the WNT pathway may contribute to a therapeutically applicable way for generation of functional insulin-producing cells following ex-vivo expansion.     

In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors

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