Direct Cell Fate Conversion of Human Somatic Cells into Neural Stem Cells

Abstract

Neurodegenerative diseases are caused by the progressive loss of neurons and function including a neuronal death. Most of neurodegenerative diseases are incurable and the pathophysiology is not well understood. For these reasons, cell replacement therapy and disease modeling are expected to be key objectives to cure and understand the neurodegenerative disorder, and neural stem cells (NSCs) and neuronal cells (NCs) are great promising cell sources for the regenerative medicine. Due to inaccessibility of NSCs and NCs in human, many researchers recently study on generating induced NSCs (iNSCs) and induced NCs (iNs) by introducing retrovirus, lentivirus, episomal vector, mRNA, protein, small molecules and so on. iNs are terminally differentiated and unable to proliferate, and it is not appropriate to apply for cell replacement therapy which requires massive amount cell, whereas iNSCs are self-renewal and have a tri-lineage differential potential. Using the direct conversion method, iNSCs were able to generate from not only normal people but also from patients, and generated iNSCs are personalized cells which can be used for drug screening and understanding the diseases mechanisms for patient specific. Therefore, iNSCs have more amendable and a great potential for clinical application and disease modeling. Here I show the two different methods to generate the NSCs. In the first part of this study, I generated the iNSCs by introducing the retrovirus into human somatic cells. A recent study has suggested that fibroblasts can be converted into mouse induced neural stem cells (miNSCs) through the expression of defined factors. However, successful generation of human iNSCs (hiNSCs) has proved challenging to achieve. Here, using miRNA expression profile analyses, I showed that let-7 microRNA has critical roles for the formation of PAX6/NESTIN-positive colonies from human adult fibroblasts and the proliferation and self-renewal of hiNSCs. HMGA2, a let-7 targeting gene, enables induction of hiNSCs that displayed morphological/molecular features and in vitro/in vivo differentiation potential similar to H9-derived NSCs (H9-NSCs). Interestingly, HMGA2 facilitated the efficient conversion of senescent somatic cells or blood CD34+ cells into hiNSCs through an interaction with SOX2, while other combinations or SOX2 alone showed a limited conversion ability. These findings suggest that HMGA2/let-7 facilitates direct reprogramming toward hiNSCs in minimal conditions and maintains hiNSC self-renewal, providing a strategy for the clinical treatment of neurological diseases. In the second part of this study, without introducing exogenous transcription factors, using only chemical combination directly converse the human somatic cells into NSCs. Overexpression of exogenous transcription factors could result in the random insertion and mutation into host genome which causes the tumorigenesis and mutagenesis

therefore, recent researches focused on non-integration methods to generate the target cells. Here, I report a direct conversion of human fibroblasts into expandable induced neural stem cells by four chemicals (ciNSCs), without passing through the pluripotent stage. These ciNSCs resemble to H9-NSCs and induced NSCs from human fibroblast by virus transfection, in neurosphere formation and NSC-specific gene and protein expression. After further maturation, ciNSCs can be differentiate into three main neural lineage, neurons, astrocytes and oligodendrocyte in vitro and in vivo. Additionally, they can be terminally differentiated into specific neuronal subtypes, such as dopaminergic-, glutamatergic- and GABAergic- neurons, under defined culture conditions. Thus, my data suggests that a specific chemical cocktail can drive the neural lineage-specific direct conversion of human somatic cells into progenitors with self-renewal and multipotency. Taken together, these findings reveal that additional transcription factor can induced the human somatic cells into neural stem cell more efficiently and rapidly and also the limitation of clinical application by introducing exogenous transcription factor can be replaced by application of small molecules. iNSCs have a potential to replace the damaged and non-functional cells in neurodegenerative diseases and patient derived NSC can be the disease model and used for drug screening. Even though application of iNSC in clinical approach need to be more studied for safety, it would provide the better understanding of neurodegenerative disease. Therefore, my studies suggest novel insights to treatment the neurodegenerative diseases modeling and treatment.