Dataset Information

Small molecule-based lineage switch of human adipose-derived stem cells into neural stem cells and functional GABAergic neurons.

ABSTRACT: Cellular reprogramming using small molecules (SMs) without genetic modification provides a promising strategy for generating target cells for cell-based therapy. Human adipose-derived stem cells (hADSCs) are a desirable cell source for clinical application due to their self-renewal capacity, easy obtainability and the lack of safety concerns, such as tumor formation. However, methods to convert hADSCs into neural cells, such as neural stem cells (NSCs), are inefficient, and few if any studies have achieved efficient reprogramming of hADSCs into functional neurons. Here, we developed highly efficient induction protocols to generate NSC-like cells (iNSCs), neuron-like cells (iNs) and GABAergic neuron-like cells (iGNs) from hADSCs via SM-mediated inhibition of SMAD signaling without genetic manipulation. All induced cells adopted morphological, molecular and functional features of their bona fide counterparts. Electrophysiological data demonstrated that iNs and iGNs exhibited electrophysiological properties of neurons and formed neural networks in vitro. Microarray analysis further confirmed that iNSCs and iGNs underwent lineage switch toward a neural fate. Together, these studies provide rapid, reproducible and robust protocols for efficient generation of functional iNSCs, iNs and iGNs from hADSCs, which have utility for modeling disease pathophysiology and providing cell-therapy sources of neurological disorders.

SUBMITTER: Park J

PROVIDER: S-EPMC5579051 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Small molecule-based lineage switch of human adipose-derived stem cells into neural stem cells and functional GABAergic neurons.

Park Jihye J Lee Nayeon N Lee Jaekwang J Choe Eun Kyung EK Kim Min Kyung MK Lee Jeonghoon J Byun Min Soo MS Chon Myong-Wuk MW Kim Seong Who SW Lee C Justin CJ Kim Ju Han JH Kwon Jun Soo JS Chang Mi-Sook MS

Scientific reports 20170831 1

Cellular reprogramming using small molecules (SMs) without genetic modification provides a promising strategy for generating target cells for cell-based therapy. Human adipose-derived stem cells (hADSCs) are a desirable cell source for clinical application due to their self-renewal capacity, easy obtainability and the lack of safety concerns, such as tumor formation. However, methods to convert hADSCs into neural cells, such as neural stem cells (NSCs), are inefficient, and few if any studies ha ...[more]

PMID: 28860504

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Project description:BackgroundWe have previously described fundamental differences in the biology of stem cells as compared to other dividing cell populations. We reasoned therefore that a differential screen using US Food and Drug Administration (FDA)-approved compounds may identify either selective survival factors or specific toxins and may be useful for the therapeutically-driven manufacturing of cells in vitro and possibly in vivo.Methodology/principal findingsIn this study we report on optimized methods for feeder-free culture of hESCs and hESC-derived neural stem cells (NSCs) to facilitate automated screening. We show that we are able to measure ATP as an indicator of metabolic activity in an automated screening assay. With this optimized platform we screened a collection of FDA-approved drugs to identify compounds that have differential toxicity to hESCs and their neural derivatives. Nine compounds were identified to be specifically toxic for NSCs to a greater extent than for hESCs. Six of these initial hits were retested and verified by large-scale cell culture to determine dose-responsive NSC toxicity. One of the compounds retested, amiodarone HCL, was further tested for possible effects on postmitotic neurons, a likely target for transplant therapy. Amiodarone HCL was found to be selectively toxic to NSCs but not to differentiated neurons or glial cells. Treated and untreated NSCs and neurons were then interrogated with global gene expression analysis to explore the mechanisms of action of amiodarone HCl. The gene expression analysis suggests that activation of cell-type specific cationic channels may underlie the toxicity of the drug.Conclusions/significanceIn conclusion, we have developed a screening strategy that allows us to rapidly identify clinically approved drugs for use in a Chemistry, Manufacture and Control protocol that can be safely used to deplete unwanted contaminating precursor cells from a differentiated cell product. Our results also suggest that such a strategy is rich in the potential of identifying lineage specific reagents and provides additional evidence for the utility of stem cells in screening and discovery paradigms.

Project description:Unlabelled: Huntington's disease (HD) results from a CAG repeat expansion in the gene encoding the huntingtin protein. This inherited disorder is characterized by progressive neurodegeneration. In particular, HD progression involves the loss of striatal projection neurons. The limited availability of reliable sources of human striatal projection neurons currently hampers our understanding of HD mechanisms and hinders the development of novel HD treatments. In this paper, we described two- and three-step methods for differentiating human neural progenitor cells toward striatal projection neurons. In the two-step differentiation protocol, 90%, 54%, and 6% of MAP2-positive cells were immunopositive for GABA, calbindin (CALB1), and DARPP-32/PPP1R1B, respectively. In the three-step differentiation protocol, 96%, 84%, and 21% of MAP2-positive cells were immunopositive for GABA, calbindin, and DARPP-32/PPP1R1B, respectively. In line with a striatal projection neuron phenotype, cells differentiated with our protocols displayed significantly increased expression of MAP2, CALB1, DARPP-32/PPP1R1B, ARPP21, and CTIP2. Application of glutamate receptor agonists induced calcium influx; accordingly, the cells also expressed various ionotropic glutamate receptor subunits. Differentiated cells also released GABA on stimulation. We suggest that our three-step differentiation protocol presents a reliable and simplified method for the generation of striatal projection neurons, yielding a critical resource for neuronal physiology and neurodegenerative disorder studies.SignificanceThe earliest changes in the neurodegenerative disorder Huntington's disease affect a specific type of brain neurons, the so-called medium spiny neurons of the striatum. In this study, two protocols were developed for the differentiation of neural progenitor cells into striatal medium spiny neurons, and the differentiated neurons were extensively characterized. The data indicate that the three-step differentiation protocol presents a reliable and simplified method for the generation of striatal medium spiny neurons. The generated striatal medium spiny neurons could represent a critical resource for the study of neurodegenerative disorders, a model system for drug discovery, and a step toward cell-based regeneration therapies.

Dataset Information

Small molecule-based lineage switch of human adipose-derived stem cells into neural stem cells and functional GABAergic neurons.

Publications

Small molecule-based lineage switch of human adipose-derived stem cells into neural stem cells and functional GABAergic neurons.

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