Generation of induced vascular progenitor cells and induced neural stem cells for clinical application

Abstract

Cell therapy is being developed as a means of treating degenerative diseases that have been difficult to treat with surgical or chemical drugs developed so far. In particular, stem cells, with their ability to differentiate into multiple cells as well as high proliferation ability, are in the spotlight as cell therapy products. In addition, because neural stem cells have the property of moving to the lesion site, they can be used as mediators to deliver genes for gene therapy. Pluripotent stem cells which can be differentiated into every cell type consisting of our whole body have been believed as a great candidate for cell therapy. However, the remaining undifferentiated cells could form tumors after implantation into patient. Direct lineage conversion techniques are emerging as a next-generation technique for cell therapy products. Direct lineage conversion can rule out tumorigenesis because there is no intermediate step through pluripotency. By applying this direct lineage conversion technique, I generated induced vascular progenitor cells (iVPCs) to cure vascular diseases. Fibroblasts were infected with lentiviral vectors encoding Etv2 and Fli1, which are transcription factors related to vascular development in early stage. Etv2 and Fli1-infected cells expressed vasculature genes. The pure CD144 positive iVPCs were sorted by FACS. iVPCs was self-renewal and maintained high proliferation rate in multiple passages. iVPCs differentiated into endothelial cells (iVPC-ECs) and smooth muscle cells (iVPC-SMCs). The number of CD31 positive and LDL uptake cells, typical makers of endothelial cells, were increased after endothelial differentiation. Moreover, iVPC-ECs formed tube-like structure on Matrigel. iVPC-SMC expressed Calponin and SM-MHC which are SMC markers. The contractability of iVPC-SMCs was confirmed by carbachol treatment. Based on the in vitro results, I further analyzed the effect of iVPC transplantation in ischemic disease model. The blood flow was recovered in ischemic hind limb after iVPC transplantation. I also found that iVPCs participated into vasculogenesis in the transplanted limb. Therefore, iVPCs, which have a potential to be developed for cell therapy to cure vascular disease, can be generated by direct lineage conversion technique. In addition, I generated induced neural stem cells (iNSCs) to evaluate the function of Spon1 on amyloid beta in vitro. iNSCs was derived from fibroblasts by overexpression of Oct4. iNSCs expressed NSC markers. Spon1 was transduced into iNSCs in order to generate Spon1-secreting iNSCs. I confirmed that Spon1 was secreted from Spon1-secreting iNSCs by ELISA. Spon1-secreting iNSCs maintained the expression of NSC markers after Spon1 transduction. To evaluate the effect of Spon1 to inhibit amlyoidogeneis, Spon1-secreting iNSCs co-cultured with Neuro2a cells which express Amyloid beta (Aβ). The amount of Aβ was significantly reduced by co-culturing with Spon1-secreting iNSCs compared to co-culturing with control iNSCs. I also confirmed that the effect of human SPON1 on inhibition of amyloidogeneis by direct infection as a gene therapy concept. I infected human SPON1 into 293T-APP cells which express the amyloid precursor protein having family Alzheimer’s mutation. The amount of Aβ was reduced in 293T-APP cells after human SPON1 infection. Based on the in vitro results, I further analyzed the effect of SPON1 in 5xFAD, AD model mouse. I injected lentiviral vector encoding human SPON1 into hippocampus and entorhinal cortex in 3-month-old 5xFAD in which AD is early pathology. . I confirmed that the cognitive impairment and memory deficiency of mice injected by SPON1 ameliorated relative to MOCK-injected mice. Aβ was reduced in SPON1 injected hippocampus and entorhinal cortex. Overall, SPON1 inhibits amyloidogenesis and recovers cognitive impairment and memory deficient in AD mice.