J Biol Chem. 2014 Feb 21;289(8):5250-60.
Direct Conversion of Human Fibroblasts into Neuronal Restricted Progenitors.
Qingjian Zou1,2, Quanmei Yan1, Juan Zhong1, Kepin Wang1, Haitao Sun3, Xiaoling Yi1, and Liangxue Lai1
1Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China;
2School of Life Sciences, University of Science and Technology of China, Hefei 230027, China;
3Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China
Neuronal restricted progenitors (NRPs) represent a type of transitional intermediate cells that lie between multipotent neural progenitors (NPs) and terminal differentiated neurons during neurogenesis. These NRPs have the ability to self-renew and differentiate into neurons, but not into glial cells, which is considered as an advantage for cellular therapy of human neurodegenerative diseases. However, difficulty in the extraction of highly purified NPRs from normal nervous tissue prevents further studies and applications. In this study, we reported conversion of human fetal fibroblasts into human induced neuronal restricted progenitors (hiNRPs) in eleven days by using just three defined factors: Sox2, c-Myc, and either Brn2 or Brn4. The hiNRPs exhibited distinct neuronal characteristics, including cell morphology, multiple neuronal markers expression, self-renewal capacity, and genome-wide transcriptional profile. Moreover, hiNRPs were able to differentiate into various terminal neurons with functional membrane properties, but not glial cells. Direct generation of hiNRPs from somatic cells will provide a new source of cells for cellular replacement therapy of human neurodegenerative diseases.
KEYWORDS: Brn2; Brn4; Induced Neuronal Restricted Progenitors; Induced Pluripotent Stem (iPS) Cell; Neural Stem Cell; Neurons; Reprogramming; Sox2; Trans-differentiation; Transcription Factors
Neurodegenerative disease is caused by the function loss of neuronal cells. Generation of a large amount of patient specific- neurons will light on the cellular replacement therapy of human neurodegenerative diseases. Mouse and human fibroblasts have been directly induced into pluripotent stem cells, neural stem cells (NSCs) and terminal-differentiated neurons with different combinations of transcription factors (Figure 1). However, several disadvantage need to be solved before application of these cells, e.g. tumorigenic potential of PSCs , glial potential differentiation of NSCs [2, 3] and proliferative limitation of mature neurons.
Figure 1. Neural-committed differentiation of pluriportent stem cells and the reprogramming of fibroblasts in vitro. Model describing neurogenesis from pluripotent stem cells to neurons, astrocytes and oligodendrocytes. Mesodermal fibroblasts could be induced to not only ESCs, NSCs and neurons, but also NRPs in our experiment. ESCs: embryonic stem cells; NSCs: neural stem cells; NPs: neural progenitors; GRPs: Glial restricted progenitors; NRPs: neuronal restricted progenitors
In this study, we established an approach to directly convert human fetal fibroblasts (HFFs) into human induced neuronal restricted progenitors (hiNRPs) by using just three defined factors: Sox2, c-Myc, and either Brn2 or Brn4 within 7 days (Figure 1 and Figure 2A). The cells in 3F-hiNRP colonies were round, small, and could be easily distinguished from the original fibroblasts. The hiNRPs expressed neuronal specific markers such as nestin, Sox2, DCX, Tuj1, MAP2 and Msi1 (Figure 2B), while silenced with Oct4, Pax6 and NeuN. After allowed to differentiate in the neuron-generating medium for two weeks, these cells become Tuj1 positive with neuronal identity. However, when cultured in astrocyte differentiating condition for two weeks, they were not able to become GFAP positive glial cells. While as a control test, ES-derived NPs could differentiate into both neurons in neuron-differentiating medium and glial cells in glial cell-differentiating medium (Figure 2C).
To test their survival ability and differentiation potential in vivo, hiNRPs were labeled with GFP and then transplanted into the left lateral ventricle of two-month-old mice. After 4 weeks of transplantation, the grafts survived and integrated in the host brain around lateral ventricle and formed many long neuritis (Figure 2D). Terminal differentiated neuronal markers (NeuN) were observed in most grafts. Nevertheless, GFP-positive cells showed negative results for GFAP expression (Figure 2E), indicating that the grafts had been induced to the neuronal lineage, but could not form glial cells in vivo.
The successful generation of hiNRPs from somatic cells may provide a new source of neurons for replacement therapy of human neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease and Huntington’s chorea.
Figure 2. Induction, characteristics and differentiation of hiNRPs. (A) After induction, cells proliferated to form colonies and decreased in size compared with the original fibroblasts. (B) The 3F-hiNRPs were immunopositive for neuronal markers such as Sox2, Nestin, Msi1, Tuj1, MAP2, and DCX and proliferation marker Ki-67. (C) In vitro differentiation: Neural progenitors could differentiate into neuronal and glial cells (Tuj1+, GFAP+ and O4+), while hiNRPs only differentiated into neuronal cells (Tuj1+, GFAP− and O4−). (D and E) In vivo differentiation: hiNRP grafts integrated and migrated inside the brain from the lateral wall of the lateral ventricle. Grafts differentiated into neurons and form many neuritis which extend inside of the brain (D). The transplanted grafts differentiated into NeuN positive neurons but not GFAP positive glia in thalamus and cerebral cortex (E).
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