Project description:Direct conversion from fibroblasts to neurons is a potential cell replacement therapy for neurological disorders, and a variety of combinations of transcription factors have been tried. We notice that the efficiency of conversion from aging fibroblasts was much lower than in early stage cells, which is consistent with the notion that cellular senescence impairs conversion of fibroblasts to neurons. Here, we found that the transient knockdown of the p16Ink4a/p19Arf locus was sufficient to convert human fibroblasts to neurons. Futhermore, expression of hTERT alone, another mechanism behind immortalization, also induced neuron conversion. Our results show that the acquisition of immortality is a crucial step for the conversion of human fibroblasts into induced neurons. Transient knockdown of p16/p19 or p53 expression or exogenous overexpression of hTERT can induce primary fibroblasts to immortality. In the following, treated cells were cultured in neuron-induction medium. We can observe the morphology change and detect the neuronal markers. Also, some of the induced neurons could generate action potentials and neurotransmitter-induced currents in optimal conditions.
Project description:Direct conversion from fibroblast to neuron has recently been successfully induced bypassing the pluripotent state. However, the conversion takes a few months with low percentages of success. Here we found that depletion of p53, which can converted fibroblasts into three major neural lineages: neurons, astrocytes and oligodendrocytes. Furthermore, our method provided a high efficiency of conversion in aging fibroblasts, where published methods failed. This finding may help developing a prototype for neuron replacement therapy, including foraging people vulnerable to neurological disorders. p53 has been shown to inhibit reprogramming of fibroblasts to iPS cells, by depletion of p53 in human fibroblasts, we study the function of p53 in induced neuron process. By induction of p53 knockdown fibroblasts with special neuron medium, we can get mature neurons directly. In the induction process, many neurogenic transcription factors were up-regulated, and we prove that p21 is not involved in this process.
Project description:The direct conversion of human skin fibroblasts to neurons has a low efficiency and unclear mechanism. Here, we show that the knockdown of PTBP2 (nPTB) significantly enhanced the transdifferentiation induced by ASCL1, MiR124-9/9* and p53 shRNA to generate mostly GABAergic neurons. Longitudinal RNAseq analyses identified the continuous induction of many RNA Splicing Regulators (RSRs). Among these, the knockdown of RBFOX3, which encodes the mature neuronal marker NeuN, significantly abrogated the transdifferentiation. Overexpression of RBFOX3 significantly enhanced the conversion induced by AMp; the enhancement was occluded by PTBP2 knockdown. We found that PTBP2 attenuation significantly favored neuron-specific alternative splicing (AS) of many genes involved in synaptic transmission, signal transduction, and axon formation. RBFOX3 knockdown significantly reversed the effect, while RBFOX3 overexpression enhanced it. The study reveals the critical role of neuron-specific AS in the direct conversion of human skin fibroblasts to neurons by showing that PTBP2 attenuation enhances this mechanism in concert with RBFOX3.
Project description:Direct conversion from fibroblasts to neurons is a potential cell replacement therapy for neurological disorders, and a variety of combinations of transcription factors have been tried. We notice that the efficiency of conversion from aging fibroblasts was much lower than in early stage cells, which is consistent with the notion that cellular senescence impairs conversion of fibroblasts to neurons. Here, we found that the transient knockdown of the p16Ink4a/p19Arf locus was sufficient to convert human fibroblasts to neurons. Futhermore, expression of hTERT alone, another mechanism behind immortalization, also induced neuron conversion. Our results show that the acquisition of immortality is a crucial step for the conversion of human fibroblasts into induced neurons.
Project description:Direct conversion from fibroblast to neuron has recently been successfully induced bypassing the pluripotent state. However, the conversion takes a few months with low percentages of success. Here we found that depletion of p53, which can converted fibroblasts into three major neural lineages: neurons, astrocytes and oligodendrocytes. Furthermore, our method provided a high efficiency of conversion in aging fibroblasts, where published methods failed. This finding may help developing a prototype for neuron replacement therapy, including foraging people vulnerable to neurological disorders.