The ability to reprogram somatic cells into self-renewable iNSCs has major implications for regenerative medicine. iNSCs can serve as a model system for unveiling disease pathogenesis, for drug screening and toxicity tests, and ultimately for cell transplantation therapies. Many studies have focused on generating NSCs from pluripotent sources such as ES cells or iPS cells (Hochedlinger and Plath, 2009
; Yamanaka, 2009
). However, these methods are plagued by ethical and practical issues, such as the origin of ES cells and the tendency for teratoma formation of cells derived from iPS cells (Fong et al., 2010
; Miura et al., 2009
; Yamanaka, 2009
). Interestingly, transplantation of Sox2-reprogrammed iNSCs into mouse brains does not generate tumors, making iNSCs more attractive than NSCs derived from iPS cells. iN cells can be generated from fibroblasts and other somatic cell sources (Ambasudhan et al., 2011
; Caiazzo et al., 2011
; Kim et al., 2011b; Marro et al., 2011
; Pang et al., 2011
; Qiang et al., 2011
; Son et al., 2011
; Vierbuchen et al., 2010
; Yoo et al., 2011
), but iN cells are terminally differentiated and restricted to the subtypes of neurons they can generate. Having a patient-derived population of multipotent iNSCs would bypass some of the disadvantages of pluripotent and terminally differentiated cell populations. Thus, direct reprogramming of somatic cells into self-renewable and multipotent iNSCs should not only complement the iPS cell and iN technologies but also sidestep their shortcomings.
It has recently been reported that the four Yamanaka reprogramming factors in combination with NSC-permissive culture conditions can reprogram fibroblasts to induced neural progenitors (iNPCs) that can generate multiple neuronal cell types as well as astrocytes (Kim et al., 2011a
). However, these iNPCs can only self-renew for 3–5 passages in culture and have not been shown to differentiate into oligodendrocytes. In a separate study, a combination of nine factors reprogramed Sertoli cells into iNSCs (Sheng et al., 2011). However, exogenous expression of eight out of the nine factors was not silenced even after multiple passages, raising the question of whether these iNSCs would revert back to their original state without constant overexpression of those factors. It also has been reported that three factors, Brn2, Sox2, and FoxG1, can reprogram mouse fibroblasts to tripotent, self-renewing iNPCs (Lujan et al., 2012
). However, when the authors attempted to generate iNPCs with only two factors, they found that Sox2 and FoxG1 generated only bipotent iNPCs and that the combination of FoxG1 and Brn2 generated tripotent iNPCs that were unable to form mature and functional neurons in vitro
. Interestingly, the three-factor-reprogrammed iNPCs could generate oligodendrocytes in vivo
although it was not tested for generation of neurons or astrocytes. Most recently, two studies have shown that the combinations of Sox2, Klf4 and c-Myc or Brn4, Sox2, Klf4, c-Myc, and E47/Tcf3 can reprogram mouse fibroblasts into iNSCs (Thier et al., 2012
; Han et al., 2012
). While these studies do show that iNSCs can self-renew, generate functional neurons in vitro
, and integrate in vivo
, both reprogramming methods require overexpression of the potent c-Myc oncogene, which has been reported to be a cause of brain tumorigenesis from transplanted iPS cell-derived NSCs (Okita et al., 2007).
The studies mentioned above are in line with our findings that mouse fibroblasts can be directly reprogrammed into iNSCs that exhibit typical NSC properties and differentiation abilities in vitro and in vivo. However, our iNSC reprogramming protocol is advantageous because it requires a single factor to generate tripotent iNSCs from both mouse and human fibroblasts. The miNSCs described here can be passaged more than 40 times, can generate functional neurons with synaptic connections in vitro, and can survive, integrate, and are multipotent in vivo without tumor formation. Furthermore, our results uniquely show that our miNSCs are a homogeneous tripotent population, rather than a heterogenous population of different neural progenitor cells. Additionally, retroviral expression of Sox2 in miNSCs is silenced at later passage, suggesting that Sox2-reprogrammed miNSCs have turned on endogenous expression of NSC genes and can maintain a stable cell fate. Finally, our SOX2 reprogramming protocol can reprogram human fibroblasts into hiNSCs that express the typical NSC markers, can self-renew over 20 passages, generate neurospheres comparable to NSCs derived from human iPS cells, and are tripotent in vitro.
Sox2 functions as a master regulator gene for NSC identity and maintenance, as knocking down Sox2 expression leads to immediate cell cycle exit and terminal differentiation of NSCs (Bylund et al., 2003
; Graham et al., 2003
). Thus, it is conceivable that under conditions conducive to NSC expansion, including the presence of growth factors and proper surface and substrates, overexpression of Sox2 can reprogram fibroblasts to multipotent NSCs. If one factor can generate a multipotent population of NSCs from somatic cells, then certain combinations of more lineage-defined factors may generate subtype-specific NSCs, such as motor neuron, dopaminergic neuron, oligodendrocyte, or astrocyte progenitors. Overexpression of specific transcription factors such as Lmx1a in combination with extrinsic factors can bias NSCs toward differentiation into dopaminergic neurons that constitute 75–90% of the total neuronal cell population (Panman et al., 2011
). Thus, Sox2 might be used in combination with such factors to create neural progenitors that can develop into subtype-specific neurons, which would be invaluable for mechanistic studies, drug screening, and potential cell therapies for different neurodegenerative diseases.