Abstract
ABSTRACTIn mice, leukemia inhibitory factor (LIF)-dependent primitive neural stem cells (NSCs) have a higher neurogenic potential than bFGF-dependent definitive NSCs. Therefore, expandable primitive NSCs are required for research and for the development of therapeutic strategies for neurological diseases. There is a dearth of suitable techniques for the generation of human long-term expandable primitive NSCs. Here, we have described a method for the conversion of human fibroblasts to LIF-dependent primitive NSCs using a strategy based on techniques for the generation of induced pluripotent stem cells (iPSCs). These LIF-dependent induced NSCs (LD-iNSCs) can be expanded for >100 passages. Long-term cultured LD-iNSCs demonstrated multipotent neural differentiation potential and could generate motor neurons and dopaminergic neurons, as well as astrocytes and oligodendrocytes, indicating a high level of plasticity. Furthermore, LD-iNSCs easily reverted to human iPSCs, indicating that LD-iNSCs are in an intermediate iPSC state. This method may facilitate the generation of patient-specific human neurons for studies and treatment of neurodegenerative diseases.
Highlights
Yamanaka’s discovery of induced pluripotent stem cells was a breakthrough innovation in the field of cellular reprogramming, which is the conversion of differentiated cells into an undifferentiated state (Takahashi et al, 2007; Takahashi and Yamanaka, 2006)
LD-induced neural stem/progenitor cells (iNSCs) are directly induced from human fibroblasts by five factors with two inhibitors (2i) A recent report indicated that leukemia inhibitory factor (LIF)-dependent human primitive neural stem cells differentiate from hiPSCs in a chemically defined medium containing LIF and two inhibitors (MEK1/MEK2 inhibitor PD0325901 and GSK-3β inhibitor CHIR99021), collectively termed 2i (Hirano et al, 2012)
The generation efficiency of iNSCs was assessed using an assay for alkaline phosphatasepositive colonies, based on a previous report that LIF-dependent primitive NSCs derived from hESCs are positive for alkaline phosphatase (Li et al, 2011)
Summary
Yamanaka’s discovery of induced pluripotent stem cells (iPSCs) was a breakthrough innovation in the field of cellular reprogramming, which is the conversion of differentiated cells into an undifferentiated state (Takahashi et al, 2007; Takahashi and Yamanaka, 2006). Because most of the converted cells are terminally differentiated, their proliferation potential is much lower compared Received 25 June 2015; Accepted 21 September 2015 to that of multipotent stem cells, suggesting that direct lineage conversion technology has limitations in drug screening or autologous cell therapy. To address this issue, direct conversion of somatic cells to lineage-committed multipotent stem cells might be an attractive approach for indirect generation of desired cell types. This indirect approach would allow for the production of sufficient amounts of cells for basic research, drug screening, disease modeling, or cell therapy
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