Motor Neuron Transdifferentiation of Neural Stem Cell from Adipose-Derived Stem Cell Characterized by Differential Gene Expression

Abstract

Adipose-derived stem cells (ADSC) are adult stem cells which can be induced into motor neuron-like cells (MNLC) with a preinduction-induction protocol. The purpose of this study is to generate MNLC from neural stem cells (NSC) derived from ADSC. The latter were isolated from the perinephric regions of Sprague-Dawley rats, transdifferentiated into neurospheres (NS) using B27, EGF, and bFGF. After generating NSC from the NS, they induced into MNLC by treating them with Shh and RA, then with GDNF, CNTF, BDNF, and NT-3. The ADSC lineage was evaluated by its mesodermal differentiation and was characterized by immunostaining with CD90, CD105, CD49d, CD106, CD31, CD45, and stemness genes (Oct4, Nanog, and Sox2). The NS and the NSC were evaluated by immunostaining with nestin, NF68, and Neurod1, while the MNLC were evaluated by ISLET1, Olig2, and HB9 genes. The efficiency of MNLC generation was more than 95 ± 1.4 % (mean ± SEM). The in vitro generated myotubes were innervated by the MNLC. The induced ADSC adopted multipolar motor neuron morphology, and they expressed ISLET1, Olig2, and HB9. We conclude that ADSC can be induced into motor neuron phenotype with high efficiency, associated with differential expression of the motor neuron gene. The release of MNLC synaptic vesicles was demonstrated by FM1-43, and they were immunostained with synaptophysin. This activity was correlated with the intracellular calcium ion shift and membrane depolarization upon stimulation as was demonstrated by the calcium indicator and the voltage-sensitive dye, respectively.

Materials for Neural Differentiation, Trans‐Differentiation, and Modeling of Neurological Disease

“…After a series of in‐depth research studies, the direct trans‐differentiation from somatic cells is a feasible method for achieving induced neurons without those mentioned problems. Since Vierbuchen et al directly reprogrammed mouse fibroblasts into induced neurons, several somatic cells, such as fibroblasts, glial cells, mesenchymal stem cells, hepatocytes, and adipose‐derived stem cells, have been reported to be cell sources for inducing neuron‐like cells …”

“…They are the ideal source for providing unlimited numbers of neural cells for drug screening, disease modeling, and regenerative medicine . In addition to neural differentiation from PSCs, converting other somatic cells into neural cells is also an effective way to provide neural cells for biomedical research . Regardless of direct neural differentiation from PSCs or trans‐differentiation from other cell types, obtaining a relatively enriched neuronal population is the first step toward in vitro disease modeling, drug screening, and cell therapy.…”

Comunicação exossomal na transdiferenciação de células-tronco em cocultivo com células neuronais

“…No processo de diferenciação neuronal muitos protocolos preconizam a utilização de fatores de crescimento (BRONZI et al, 2010), indutores (ERCOLIN et al, 2016;MARESCHI et al, 2006) e meio neurogênico (BAGLIONI et al, 2009;DHANASEKARAN et al, 2013;GOLDMAN, 2016;PRABHAKARAN et al, 2009). No entanto, pesquisas têm utilizado o cocultivo com insertos (transwell) de célulastronco com outros tipos celulares, e demostraram aumento na taxa de diferenciação, devido a fatores secretados pelas células (BAHMANI et al, 2014;DARVISHI et al, 2017;ROBALLO et al, 2013;ZHANG et al, 2017), e aumento da proliferação celular (ZHANG et al, 2017), tornando esse sistema de cocultivo in vitro atrativo para estudos que visam interações celulares e a indução da diferenciação. Desta maneira, nesse trabalho propusemos utilizar como método de indução ao processo de diferenciação neuronal o cocultivo com insertos (transwell) de células neuronais como ADSC, e também utilizar este método de cocultivo para o estudo da comunicação mediada por vesículas extracelulares, ressaltando que não há outros estudos semelhantes ao proposto nesse trabalho.…”

“…As ADSC-CC após três, sete e 14 dias (D3, D7 e D14) de indução em cocultivo com insertos apresentaram morfologia e fenótipo muito semelhantes às células neuronais, com células positivas para as proteínas neuronais TUBIII e SNAP25. De acordo com outras pesquisas (DARVISHI et al, 2017;XIE et al, 2017) as ADSC de ratos também alteram sua morfologia, fenótipo e função se assemelhando a células de Schwann após um período de indução química por três semanas. Além disso, também possuem capacidade de se transdiferenciar em células neuronais-like (DARVISHI et al, 2017).…”

“…De acordo com outras pesquisas (DARVISHI et al, 2017;XIE et al, 2017) as ADSC de ratos também alteram sua morfologia, fenótipo e função se assemelhando a células de Schwann após um período de indução química por três semanas. Além disso, também possuem capacidade de se transdiferenciar em células neuronais-like (DARVISHI et al, 2017). Nesses experimentos foram analisados a morfologia e o fenótipo celular por meio da imunocitoquímica com os marcadores GFAP, S100, MAP2 e nestina (DARVISHI et al, 2017;XIE et al, 2017).…”