Development of membrane ion channels during neural differentiation from human embryonic stem cells.

OBJECTIVE: For human embryonic stem cells (hESCs) to differentiate into neurons, enormous changes has to occur leading to trigger action potential and neurotransmitter release. We attempt to determine the changes in expression of voltage gated channels (VGCs) and their electrophysiological properties during neural differentiation. MATERIALS AND METHODS: The relative expressions of α-subunit of voltage gated potassium, sodium and calcium channels were characterized by qRT-PCR technique. Patch clamp recording was performed to characterize the electrophysiological properties of hESCs during their differentiation into neuron-like cells. RESULTS: Relative expression of α-subunit of channels changed significantly. 4-AP and TEA sensitive outward currents were observed in all stages, although TEA sensitive currents were recorded once in rosette structure. Nifedipine and QX314 sensitive inward currents were recorded only in neuron-like cells. CONCLUSION: K+ currents were recorded in hESCs and rosette structure cells. Inward currents, sensitive to Nifedipine and QX314, were recorded in neuron-like cells.

miR-135a Inhibits Cancer Stem Cell-Driven Medulloblastoma Development by Directly Repressing Arhgef6 Expression.

microRNAs (miRNAs) are short noncoding RNAs, which regulate gene expression post-transcriptionally and play crucial roles in relevant biological and pathological processes. Here, we investigated the putative role of miRNAs in modulating the tumor-initiating potential of mouse medulloblastoma (MB)-derived cancer stem cells (CSCs). We first subjected bona fide highly tumorigenic (HT) CSCs as well as lowly tumorigenic MB CSCs and normal neural stem cells to miRNA profiling, which identified a HT CSC-specific miRNA signature. Next, by cross-checking CSC mRNA/miRNA profiles, we pinpointed miR-135a as a potential tumor suppressor gene, which was strongly downregulated in HT CSCs as well as in the highly malignant experimental tumors derived from them. Remarkably, enforced expression of miR-135a in HT CSCs strongly inhibited tumorigenesis by repressing the miR-135a direct target gene Arhgef6. Considering the upregulation of Arhgef6 in human MBs and its involvement in mediating experimental medulloblastomagenesis, its efficient suppression by miR-135a might make available an effective therapeutic strategy to selectively impair the tumorigenic potential of MB CSCs. Stem Cells 2015;33:1377-1389.

Transplantation of adult monkey neural stem cells into a contusion spinal cord injury model in rhesus macaque monkeys.

OBJECTIVE: Currently, cellular transplantation for spinal cord injuries (SCI) is the subject of numerous preclinical studies. Among the many cell types in the adult brain, there is a unique subpopulation of neural stem cells (NSC) that can self-renew and differentiate into neurons. The study aims, therefore, to explore the efficacy of adult monkey NSC (mNSC) in a primate SCI model. MATERIALS AND METHODS: In this experimental study, isolated mNSCs were analyzed by flow cytometry, immunocytochemistry, and RT-PCR. Next, BrdU-labeled cells were transplanted into a SCI model. The SCI animal model was confirmed by magnetic resonance imaging (MRI) and histological analysis. Animals were clinically observed for 6 months. RESULTS: Analysis confirmed homing of mNSCs into the injury site. Transplanted cells expressed neuronal markers (TubIII). Hind limb performance improved in trans- planted animals based on Tarlov's scale and our established behavioral tests for monkeys. CONCLUSION: Our findings have indicated that mNSCs can facilitate recovery in contusion SCI models in rhesus macaque monkeys. Additional studies are necessary to determine the im- provement mechanisms after cell transplantation.

Differentiation of embryonic stem cells into neural cells on 3D poly (D, L-lactic acid) scaffolds versus 2D cultures.

In this study, a highly porous poly (D, L-lactic acid) (PDLLA) scaffold was designed and fabricated using dioxane and thermal-induced phase separation (TIPS) methods (liquid-liquid and solid-liquid). Additionally, we characterized the ability of mouse embryonic stem cells (ESCs) to differentiate into neural cells in PDLLA scaffold with uniform porosity, interconnectivity, and high porosity, and then compared them with cells seeded under conventional two-dimensional (2D) culture conditions. Histochemistry staining showed the migration of differentiated cells through the scaffold. Immunofluorescence analysis of the differentiated cells by counting positive cells revealed that the PDLLA scaffold resulted in a significantly greater number of neural markers, microtubule associated protein-2, ß-tubulin III, neurofilament protein, and glial fibrillary acidic protein (the astrocyte marker) when compared to those in 2D culture condition. Moreover, the expression of Nestin, Mash1, Pax6, and HB9 increased significantly in 3D scaffolds when compared with 2D cultures as detected by semi-quantitative RT-PCR. Scanning electron microscopy of differentiated neurons on scaffolds also demonstrated favorable results for neurite outgrowth. The results of this study demonstrated a promising effect of 3D scaffold culture for neural cell differentiation from ESCs in prospective tissue engineering applications.

Long-term self-renewable feeder-free human induced pluripotent stem cell-derived neural progenitors.

Human induced pluripotent stem cells (hiPSCs) have led to an important revolution in stem cell research and regenerative medicine. To create patient-specific neural progenitors (NPs), we have established a homogenous, expandable, and self-renewable population of multipotent NPs from hiPSCs, using an adherent system and defined medium supplemented with a combination of factors. The established hiPSC-NPs highly expressed Nestin and Sox1. These NPs were continuously propagated for ~1 year without losing their potential to generate astrocytes, oligodendrocytes, and functional neurons and maintained a stable chromosome number. Voltage clamp analysis revealed outward potassium currents in hiPSC-NPs. The self-renewal characteristic of the NPs was demonstrated by a symmetrical mode of Nestin-positive cell division. Additionally, these hiPSC-NPs can be easily frozen and thawed in the presence of Rho-associated kinase (ROCK) inhibitor without losing their proliferation, karyotype stability, and developmental potential. The characteristics of our generated hiPSC-NPs provide the opportunity to use patient-specific or ready-to-use hiPSC-NPs in future biomedical applications.

Dehydroepiandrosterone stimulates neurogenesis in mouse embryonal carcinoma cell- and human embryonic stem cell-derived neural progenitors and induces dopaminergic neurons.

To evaluate the effect of dehydroepiandrosterone (DHEA) as a neurosteroid on the rate of neurogenesis, neural survival, and proliferation of pluripotent stem cell-derived neurons, we have added DHEA to mouse P19 embryonal carcinoma cell- and human embryonic stem cell-derived neural progenitors (ECC- and ESC-NPs). In ECC-derived NPs, flow cytometric analysis of nestin and Tuj1-positive cells revealed that the percentages of these cells increased significantly for the markers following DHEA treatment of the cells. Moreover, the percentages of tyrosine hydroxylase (TH)-positive cells, the marker of dopaminergic neurons, significantly increased in the presence of DHEA. The expression of neural-specific genes such as Mash1, Pax6, Tuj1, and TH was also detected by RT-PCR analysis. BrdU incorporation and estrogen receptor (EsR) were found to be increased after DHEA induction. Moreover, apoptosis was significantly decreased after DHEA treatment. DHEA effect was also confirmed on human ESC-NPs by the enhancement of Tuj1- and TH-immunofluorescent-positive cells and TH and Nurr1 transcripts, as detected by quantitative RT-PCR. In conclusion, these results have presented evidence that DHEA was able to induce neurogenesis in mouse ECC and human ESC-NPs. This observation was related to the division of NPs and the reduction of apoptosis. Moreover, DHEA has dopaminergic potential in the cells of both orders. This provides a better insight into the differentiation and maintenance of neural cells and treatment of a wide variety of neurological diseases such as Alzheimer's and Parkinson's by stem cells.

Human embryonic stem cell-derived neural precursor transplants in collagen scaffolds promote recovery in injured rat spinal cord.

BACKGROUND AIMS: Several studies have reported functional improvement after transplantation of in vivo-derived neural progenitor cells (NPC) into injured spinal cord. However, the potential of human embryonic stem cell-derived NPC (hESC-NPC) as a tool for cell replacement of spinal cord injury (SCI) should be considered. METHODS: We report on the generation of NPC as neural-like tubes in adherent and feeder-free hESC using a defined media supplemented with growth factors, and their transplantation in collagen scaffolds in adult rats subjected to midline lateral hemisection SCI. RESULTS: hESC-NPC were highly expressed molecular features of NPC such as Nestin, Sox1 and Pax6. Furthermore, these cells exhibited the multipotential characteristic of differentiating into neurons and glials in vitro. Implantation of xenografted hESC-NPC into the spinal cord with collagen scaffold improved the recovery of hindlimb locomotor function and sensory responses in an adult rat model of SCI. Analysis of transplanted cells showed migration toward the spinal cord and both neural and glial differentiation in vivo. CONCLUSIONS: These findings show that transplantation of hESC-NPC in collagen scaffolds into an injured spinal cord may provide a new approach to SCI.