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Reprogramming somatic cells to cells with neuronal characteristics by defined medium both in vitro and in vivo.

ABSTRACT: BACKGROUND:Currently, direct conversion from somatic cells to neurons requires virus-mediated delivery of at least one transcriptional factor or a combination of several small-molecule compounds. Delivery of transcriptional factors may affect genome stability, while small-molecule compounds may require more evaluations when applied in vivo. Thus, a defined medium with only conventional growth factors or additives for cell culture is desirable for inducing neuronal trans-differentiation. RESULTS:Here, we report that a defined medium (5C) consisting of basic fibroblast growth factor (bFGF), N2 supplement, leukemia inhibitory factor, vitamin C (Vc), and ?-mercaptoethanol (?Me) induces the direct conversion of somatic cells to cells with neuronal characteristics. Application of 5C medium converted mouse embryonic fibroblasts (MEFs) into TuJ+ neuronal-like cells, which were capable of survival after being transplanted into the mouse brain. The same 5C medium could convert primary rat astrocytes into neuronal-like cells with mature electrophysiology characteristics in vitro and facilitated the recovery of brain injury, possibly by inducing similar conversions, when infused into the mouse brain in vivo. Crucially, 5C medium could also induce neuronal characteristics in several human cell types. CONCLUSIONS:In summary, this 5C medium not only provides a means to derive cells with neuronal characteristics without viral transfection in vitro but might also be useful to produce neurons in vivo for neurodegenerative disease treatment.

SUBMITTER: He S

PROVIDER: S-EPMC4696146 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Reprogramming somatic cells to cells with neuronal characteristics by defined medium both in vitro and in vivo.

He Songwei S Guo Yiping Y Zhang Yixin Y Li Yuan Y Feng Chengqian C Li Xiang X Lin Lilong L Guo Lin L Wang Haitao H Liu Chunhua C Zheng Yi Y Luo Chuanming C Liu Qiang Q Wang Fuhui F Sun Hao H Liang Lining L Li Lingyu L Su Huanxing H Chen Jiekai J Pei Duanqing D Zheng Hui H

Cell regeneration (London, England) 20151230

<h4>Background</h4>Currently, direct conversion from somatic cells to neurons requires virus-mediated delivery of at least one transcriptional factor or a combination of several small-molecule compounds. Delivery of transcriptional factors may affect genome stability, while small-molecule compounds may require more evaluations when applied in vivo. Thus, a defined medium with only conventional growth factors or additives for cell culture is desirable for inducing neuronal trans-differentiation.< ...[more]

PMID: 26719791

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Project description:BACKGROUND: The common marmoset (Callithrix jacchus) is a New World primate sharing many similarities with humans. Recently developed technology for generating transgenic marmosets has opened new avenues for faithful recapitulation of human diseases, which could not be achieved in rodent models. However, the longer lifespan of common marmosets compared with rodents may result in an extended period for in vivo analysis of common marmoset disease models. Therefore, establishing rapid and efficient techniques for obtaining neuronal cells from transgenic individuals that enable in vitro analysis of molecular mechanisms underlying diseases are required. Recently, several groups have reported on methods, termed direct reprogramming, to generate neuronal cells by defined factors from somatic cells of various kinds of species, including mouse and human. The aim of the present study was to determine whether direct reprogramming technology was applicable to common marmosets. RESULTS: Common marmoset induced neuronal (cjiN) cells with neuronal morphology were generated from common marmoset embryonic skin fibroblasts (cjF) by overexpressing the neuronal transcription factors: ASCL1, BRN2, MYT1L and NEUROD1. Reverse transcription-polymerase chain reaction of cjiN cells showed upregulation of neuronal genes highly related to neuronal differentiation and function. The presence of neuronal marker proteins was also confirmed by immunocytochemistry. Electrical field stimulation to cjiN cells increased the intracellular calcium level, which was reversibly blocked by the voltage-gated sodium channel blocker, tetrodotoxin, indicating that these cells were functional. The neuronal function of these cells was further confirmed by electrophysiological analyses showing that action potentials could be elicited by membrane depolarization in current-clamp mode while both fast-activating and inactivating sodium currents and outward currents were observed in voltage-clamp mode. The 5-bromodeoxyuridine (BrdU) incorporation assay showed that cjiN cells were directly converted from cjFs without passing a proliferative state. CONCLUSIONS: Functional common marmoset neuronal cells can be obtained directly from embryonic fibroblasts by overexpressing four neuronal transcription factors under in vitro conditions. Overall, direct conversion technology on marmoset somatic cells provides the opportunity to analyze and screen phenotypes of genetically-modified common marmosets.

Project description:BackgroundDifferentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells.Methodology/principal findingsESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34(+) cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34(+) cells. ESC-derived CD34(+) cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34(-) cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult ?-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors.Conclusions/significanceShort-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature blood cell types. However, additional factors have yet to be identified to allow their differentiation into definitive erythroid cultures.

Dataset Information

Reprogramming somatic cells to cells with neuronal characteristics by defined medium both in vitro and in vivo.

Publications

Reprogramming somatic cells to cells with neuronal characteristics by defined medium both in vitro and in vivo.

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