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Neurog2 directly converts astrocytes into functional neurons in midbrain and spinal cord.

ABSTRACT: Conversion of astrocytes into neurons in vivo offers an alternative therapeutic approach for neuronal loss after injury or disease. However, not only the efficiency of the conversion of astrocytes into functional neurons by single Neurog2, but also the conundrum that whether Neurog2-induced neuronal cells (Neurog2-iNs) are further functionally integrated into existing matured neural circuits remains unknown. Here, we adopted the AAV(2/8) delivery system to overexpress single factor Neurog2 into astrocytes and found that the majority of astrocytes were successfully converted into neuronal cells in multiple brain regions, including the midbrain and spinal cord. In the midbrain, Neurog2-induced neuronal cells (Neurog2-iNs) exhibit neuronal morphology, mature electrophysiological properties, glutamatergic identity (about 60%), and synapse-like configuration local circuits. In the spinal cord, astrocytes from both the intact and lesioned sources could be converted into functional neurons with ectopic expression of Neurog2 alone. Notably, further evidence from our study also proves that Neurog2-iNs in the intact spinal cord are capable of responding to diverse afferent inputs from dorsal root ganglion (DRG). Together, this study does not merely demonstrate the feasibility of Neurog2 for efficient in vivo reprogramming, it gives an indication for the Neurog2-iNs as a functional and potential factor in cell-replacement therapy.

SUBMITTER: Liu F

PROVIDER: S-EPMC7921562 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Neurog2 directly converts astrocytes into functional neurons in midbrain and spinal cord.

Liu Fei F Zhang Yijie Y Chen Fuliang F Yuan Jiacheng J Li Sanlan S Han Sue S Lu Dengyu D Geng Junlan J Rao Zhiping Z Sun Li L Xu Jianhua J Shi Yuhan Y Wang Xiaojing X Liu Yueguang Y

Cell death & disease 20210301 3

Conversion of astrocytes into neurons in vivo offers an alternative therapeutic approach for neuronal loss after injury or disease. However, not only the efficiency of the conversion of astrocytes into functional neurons by single Neurog2, but also the conundrum that whether Neurog2-induced neuronal cells (Neurog2-iNs) are further functionally integrated into existing matured neural circuits remains unknown. Here, we adopted the AAV(2/8) delivery system to overexpress single factor Neurog2 into ...[more]

PMID: 33649354

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Project description:Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system.

Project description:IntroductionHuman induced pluripotent stem cells (hiPSCs) offer great promise for regenerative therapies or in vitro modelling of neurodegenerative disorders like Parkinson's disease. Currently, widely used cell sources for the generation of hiPSCs are somatic cells obtained from aged individuals. However, a critical issue concerning the potential clinical use of these iPSCs is mutations that accumulate over lifetime and are transferred onto iPSCs during reprogramming which may influence the functionality of cells differentiated from them. The aim of our study was to establish a differentiation strategy to efficiently generate neurons including dopaminergic cells from human cord blood-derived iPSCs (hCBiPSCs) as a juvenescent cell source and prove their functional maturation in vitro.MethodsThe differentiation of hCBiPSCs was initiated by inhibition of transforming growth factor-β and bone morphogenetic protein signaling using the small molecules dorsomorphin and SB 431542 before final maturation was carried out. hCBiPSCs and differentiated neurons were characterized by immunocytochemistry and quantitative real time-polymerase chain reaction. Since functional investigations of hCBiPSC-derived neurons are indispensable prior to clinical applications, we performed detailed analysis of essential ion channel properties using whole-cell patch-clamp recordings and calcium imaging.ResultsA Sox1 and Pax6 positive neuronal progenitor cell population was efficiently induced from hCBiPSCs using a newly established differentiation protocol. Neuronal progenitor cells could be further maturated into dopaminergic neurons expressing tyrosine hydroxylase, the dopamine transporter and engrailed 1. Differentiated hCBiPSCs exhibited voltage-gated ion currents, were able to fire action potentials and displayed synaptic activity indicating synapse formation. Application of the neurotransmitters GABA, glutamate and acetylcholine induced depolarizing calcium signal changes in neuronal cells providing evidence for the excitatory effects of these ligand-gated ion channels during maturation in vitro.ConclusionsThis study demonstrates for the first time that hCBiPSCs can be used as a juvenescent cell source to generate a large number of functional neurons including dopaminergic cells which may serve for the development of novel regenerative treatment strategies.

Project description:BACKGROUND:Spinal cord injury (SCI) is a destructive disease that incurs huge personal and social costs, and there is no effective treatment. Although the pathogenesis and treatment mechanism of SCI has always been a strong scientific focus, the pathogenesis of SCI is still under investigation. AIM:To determine the key genes based on the modularization of in-depth analysis, in order to identify the repair mechanism of astrocytes and non-astrocytes in SCI. METHODS:Firstly, the differences between injured and non-injured spinal cord of astrocyte (HA), injured and non-injured spinal cord of non-astrocyte (FLOW), injured spinal cord of non-injured astrocyte (HA) and non-injured spinal cord of non-astrocyte (FLOW), and non-injured spinal cord of astrocyte (HA) and non-astrocyte (FLOW) were analyzed. The total number of differentially expressed genes was obtained by merging the four groups of differential results. Secondly, the genes were co-expressed and clustered. Then, the enrichment of GO function and KEGG pathway of module genes was analyzed. Finally, non-coding RNA, transcription factors and drugs that regulate module genes were predicted using hypergeometric tests. RESULTS:In summary, we obtained 19 expression modules involving 5216 differentially expressed genes. Among them, miR-494, XIST and other genes were differentially expressed in SCI patients, and played an active regulatory role in dysfunction module, and these genes were recognized as the driving genes of SCI. Enrichment results showed that module genes were significantly involved in the biological processes of inflammation, oxidation and apoptosis. Signal pathways such as NF-kappa B/A20, AMPK and MAPK were significantly regulated. In addition, non-coding RNA pivot (including miR-136-5p and let-7d-5p, etc.) and transcription factor pivot (including NFKB1, MYC, etc.) were identified as significant regulatory dysfunction modules. CONCLUSION:Overall, this study uncovered a co-expression network of key genes involved in astrocyte and non-astrocyte regulation in SCI. These findings helped to reveal the core dysfunction modules, potential regulatory factors and driving genes of the disease, and to improve our understanding of its pathogenesis.

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Neurog2 directly converts astrocytes into functional neurons in midbrain and spinal cord.

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Neurog2 directly converts astrocytes into functional neurons in midbrain and spinal cord.

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