Direct Transdifferentiation Of Human Fibroblasts Into Neuronal Restricted Progenitors

Neurodegeneration is the umbrella term for the progressive loss of structure or function of neurons, including death of neurons. Many neurodegenerative diseases including Parkinson’s, Alzheimer’s, and Huntington’s occur as a result of neuronal damage. So far, neurodegenerative disease has become the main cause of threat to human health, especially the elderly, due to the lack of effective medication. Therefore, stem-cells therapy may provide an alternative way for neurological disorders. Allogeneic embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells and special neurons may replace damaged neurons after transplantation.Pluripotent stem cells have the abilities of self-renew and can differentiate into various types of cells. They are considered to be the potential source of cells for clinical application in the future. Direction of the differentiation of these cells, however, is difficult to control. It is not suitable to be used for the clinical treatment due to their risk of tumorigenic tendency. Thus, adult stem cells are more useful because of low risk of tumor formation. Hematopoietic stem cells, mesenchymal stem cells and neural stem cells are the most known of three types of adult stem cells. Hematopoietic stem cells and mesenchymal stem cells are mesoderm-derived cells, and can transdifferentiate into neural cells under certain conditions. This two types of stem cells are often use of stem cells for the treatment of neurodegenerative diseases on preclinical studies. However, the inefficiency of the transdifferentiation blocks the therapeutic effects. Neural stem cells or neural precursors (NPs) in the brain are mutipotent cells lay, and can differentiate into two main cellular types of nervous system:neurons and glial cells. However, transplantation experiments have demontrated that neural stem cells differentiate more likely into glial cells, rather than functional neurons. So, it is not ideal using NSCs for clinical therapy. Alternatively, functional neurons can be used for the treatment of disease cause by specific neuronal damage. However, the terminal differentiated neurons are lack of proliferation capacity, resulting in insufficient cells for fixing the damages after transplanted into brain.During neurogenesis, there is another major types of cells, neuronal restricted progenitors (NRPs, also called neuroblasts), which are derived from NPs and have the ability of proliferation and renewal, and can migrate toward pathological sites to form functional neurons in nervous system. NRPs express both stem cell markers and neuronal markers, i.e. Nestin, musashil (MSI1), neural cell adhesion molecule (N-CAM), neuronal class Ⅲ β-tubulin (Tujl), microtubule-associated protein2(MAP2) and doublecortin (DCX), and could only develop into neurons rather than glial cells and other cell types in vivo and in vitro. When injected into the subventricular zone (SVZ), NRPs can migrate extensively and incorporate into different brain regions to form subtypes of neuron as needed. They are thought to functionally contribute to brain plasticity and repair. However, it is very difficult and cumbersome to isolate highly purified NRPs from normal nervous tissue, which blocks its clinical and commercial application.Here, we try to generate hiNRPs directly from human fibroblasts through transdifferentiation, which is a technology of transformation of adult (stem) cells into other types of somatic cells in experiments. Comparing with somatic cellular reprogramming, transdifferentiation is a simpler approach to achieve a functional type of cells with shorter time and lower cost.We initially selected eight key transcription factors, including Sox2, c-Myc, Klf4, TLX, Bmi1, Brn2, Brn4, and FoxG1for pilot studies. After11to12days upon the transfection of these factors into HFFs, the colonies with compacted cells occurred. After several rounds of trial with different combinations of transcription factors, we found three factors Sox2, c-Myc, and either Brn2or Brn4were sufficient to induce large number of NRP-colonies from fibroblasts within only7days. These clonogenic cells have the ability of self-renewal for more than30passages in the presence of bFGF. Immunostaining showed that these cells homogeneously expressed neural markers Nestin. However, the Nestin-positive cells specifically differentiated into Tujl positive neurons, and not into GFAP positive glial cells, indicating that the Nestin-positive cells are not mutiportent neural progenitors, but neuronal restricted progenitors.To further prove our hypothesis, we performed microarray assay, and found that global gene expression pattern of hiNRPs is quite different from that of fibroblasts. As detected by RT-PCR, real-time qPCR and immunofluorescence, most hiNRPs expressed neuronal lineage markers (Sox2, Nestin, Msi1, CD133, Sox1, N-CAM, DCX, Tuj1and MAP2), proliferous marker Ki-67and Chemokine receptor CXCR4, but negative for ESC marker Oct4, neural stem cell specific marker Pax6, mature neuronal marker NeuN and glial markers GFAP. Moreover, hiNRPs can specific differentiate into terminal neurons rather than glial cells in vitro and in vivo. After transplanted into brains of AD mice, hiNRPs show positive treatment effectiveness in short term memory capacity of AD mice by Morris water maze.In conclusion, we have successfully generated hiNRPs from human fibroblasts at the first time. These hiNRPs have the ability of self-renewal for long time and differentiation into only subtypes of neuron, but do not glial cells. This method may help us get more deep insight into critical processes for neurogenesis and provide us a new source of cells for cell replacement therapy of neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease and Huntington’s chorea.