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Expandable and Rapidly Differentiating Human Induced Neural Stem Cell Lines for Multiple Tissue Engineering Applications.

ABSTRACT: Limited availability of human neurons poses a significant barrier to progress in biological and preclinical studies of the human nervous system. Current stem cell-based approaches of neuron generation are still hindered by prolonged culture requirements, protocol complexity, and variability in neuronal differentiation. Here we establish stable human induced neural stem cell (hiNSC) lines through the direct reprogramming of neonatal fibroblasts and adult adipose-derived stem cells. These hiNSCs can be passaged indefinitely and cryopreserved as colonies. Independently of media composition, hiNSCs robustly differentiate into TUJ1-positive neurons within 4 days, making them ideal for innervated co-cultures. In vivo, hiNSCs migrate, engraft, and contribute to both central and peripheral nervous systems. Lastly, we demonstrate utility of hiNSCs in a 3D human brain model. This method provides a valuable interdisciplinary tool that could be used to develop drug screening applications as well as patient-specific disease models related to disorders of innervation and the brain.

SUBMITTER: Cairns DM

PROVIDER: S-EPMC5032182 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Expandable and Rapidly Differentiating Human Induced Neural Stem Cell Lines for Multiple Tissue Engineering Applications.

Cairns Dana M DM Chwalek Karolina K Moore Yvonne E YE Kelley Matt R MR Abbott Rosalyn D RD Moss Stephen S Kaplan David L DL

Stem cell reports 20160825 3

Limited availability of human neurons poses a significant barrier to progress in biological and preclinical studies of the human nervous system. Current stem cell-based approaches of neuron generation are still hindered by prolonged culture requirements, protocol complexity, and variability in neuronal differentiation. Here we establish stable human induced neural stem cell (hiNSC) lines through the direct reprogramming of neonatal fibroblasts and adult adipose-derived stem cells. These hiNSCs c ...[more]

PMID: 27569063

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Project description:Treatment strategies of critical nerve injuries involving nerve gaps more than 3 cm are still not optimal. The need for an alternative to the standard autologous nerve grafts has led to the exploration of adipose derived stem cells (ASC) seeded in bioartificial nerve conduits for enhancement of peripheral nerve regeneration. ASC can be differentiated into Schwann cell like cells, which is believed to improve their neurotrophic potency. Nevertheless, the differentiation process requires several weeks and there is no evidence that the cells maintain their differentiated phenotype in vivo. Thus, the present study evaluated the synergistic effect of undifferentiated ASC with exogenously added growth factors in vitro with the aim to improve the neurotrophic potency of undifferentiated ASC through short ex-vivo stimulation with growth factors. The study furthermore provides a comparative in vitro assay of the six well-known neurotrophic factors NGF, GDNF, BDNF, CNTF, NT3 and NT4. ASC were cultured and thus stimulated in the presence of the respective exogenous growth factor for three days and resulting conditioned medium was evaluated in an in vitro axonal outgrowth assay. In addition, also unstimulated ASC’s conditioned medium was tested in combination with the respective exogenous growth factor for the same assay, which was perfomed on dorsal root ganglion (DRG) explants from 10 days old embryonic chicken. DRG explants from most promising conditions were further analyzed for upregulated STAT-3 and GAP-43 by qRT-PCR. Furthermore, also proteomics and phosphoproteomics were performed for ASC and DRG explants using mass spectrometry. The quantity of selected proteins contained in the ASC’s conditioned medium was investigated by ELISA. Combination of ASC’s conditioned medium with growth factors generally resulted in significantly enhanced axonal outgrowth when compared with their control of DRG explants cultured with the respective growth factor only. Specifically, conditioned medium derived from NT3-stimulated as well as ASC’s conditioned medium supplemented with NT3 or BDNF elicited significant axonal outgrowth from DRG explants in contrast to all other experimental conditions. Significant upregulation of STAT-3 and GAP-43 was evidenced for DRG explants grown in NT3-stimulated ASC’s conditioned medium and to a certain extent also for DRG cultured in ASC’s conditioned medium supplemented with NT3.

Dataset Information

Expandable and Rapidly Differentiating Human Induced Neural Stem Cell Lines for Multiple Tissue Engineering Applications.

Publications

Expandable and Rapidly Differentiating Human Induced Neural Stem Cell Lines for Multiple Tissue Engineering Applications.

Similar Datasets

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