Two-step generation of induced pluripotent stem cells from mouse fibroblasts using Id3 and Oct4.

Dear Editor, Somatic cells can be reprogrammed into pluripotent stem cells, called induced pluripotent stem cells (iPSCs), by defined transcription factors (Takahashi et al., 2006). The reprogramming of somatic cells may be a continuous stochastic process in which nearly all somatic donor cells have the ability to give rise to iPSCs with continuous passaging and the expression of defined factors (Hanna et al., 2009). However, the reprogramming process can be divided into discrete stages, including the generation of dedifferentiated pre-pluripotent cells followed by authentic pluripotent cells (Silva et al., 2009). The sequential expression of marker genes, such as AP, SSEA1, and Oct4 or Nanog, has been demonstrated during the reprogramming process (Brambrink et al., 2008). Neural stem cells (NSCs) endogenously express SSEA1 and exhibit AP activity (Kim et al., 2009), suggesting that NSCs are a quasi-intermediate state and might represent a more advanced stage in the reprogramming process compared with terminally differentiated cells. Subsequently, iPSCs have been generated from NSCs using Oct4 alone (Kim et al., 2009). Members of the HLH protein family of the inhibitor of differentiation (Id) dimerize with basic HLH transcription factors and function as negative regulators of differentiation during development. Four subtypes of the Id family are known in vertebrates (Id1, Id2, Id3, and Id4), but their functions are not significantly different (Perk et al., 2005). Id proteins are required for neural progenitor proliferation and accurate timing of neural differentiation (Jung et al., 2010), suggesting that Id proteins play critical roles in cell cycle regulation in neural precursor cells. Moreover, over-expression of Id proteins inhibits the expression of the tumor suppressor INK4a (also known as p16 and CDKN2A; Perk et al., 2005). Given the biological significance of Id proteins in the regulation of normal self-renewal and differentiation, we hypothesized that Id overexpression results in the transdifferentiation of fibroblasts into NSC-like cells, a more advanced stage, and endows stem cell-like renewal activity, generating iPSCs in combination with Oct4. We focused on the expression and functional activity of Id3 in the transdifferentiation of fibroblasts into NSC-like cells due to its prominent role in governing NSC fate (Jung et al., 2010) and promotion of cell cycle progression by inhibiting cyclindependent kinase inhibitors, such as p21 and p16. Id3 protein and mRNA and several NSC markers were detected in NSCs but not MEFs (Supplementary Figure S1A). These results with previous findings (Jung et al., 2010) tempt us to determine whether enforced Id3 expression drives MEF transdifferentiation. The transduction of Id3 in MEFs accelerated cell proliferation compared with vector alone (Supplementary Figure S1B). When Id3transduced MEFs were cultured under NSC culture conditions, cell morphology changed and the small spheres became hyperplastic, forming a large, floating spherical mass similar to the neurosphere morphology of mouse NSCs; conversely, neurosphere formation was not observed in cultures of mock-transduced MEFs, which remained attached to the culture dishes and underwent cell death (Supplementary Figure S1C). Neurosphere formation by Id3-transduced MEFs suggested the possibility of MEF transdifferentiation to NSC-like cells. The reprogramming efficiency was assessed by counting the number of both Nestinand Sox2-positive cells, and we determined that 19% of cells were immunostaining positive (Supplementary Figure S1D). To isolate, expand, and verify the self-renewal potential of the Id3-transduced neurosphere-forming cells, a neurosphere assay was performed in which the dissociation of primary Id3-transduced neurospheres led to secondary neurospheres. Each small cluster could form a colony within several days after isolation; however, despite mild enzymatic passaging and the avoidance of single cell dissociation, Id3-transduced neurospheres appeared to lose their ability to form colonies within two to three passages (Supplementary Figure S1E). Notably, these cells expanded in the presence of basic fibroblast growth factor (bFGF) or bFGF + EGF, but not EGF alone or in the absence of mitogens, though the number of neurospheres formed in the presence of bFGF alone was slightly less than the number formed in the presence of both mitogens (56 vs. 91, diameter .200 mm) (Supplementary Figure S1F). Moreover, expression of exogenous Id3 was silenced in Id3-transduced neurospheres (Supplementary Figure S1G), suggesting that the potentials of Id3-transduced neurospheres were independent of exogenous expression of Id3. RT-PCR, immunocytochemistry, and FACS analyses revealed that Id3-transduced neurospheres expressed marker genes at levels typical of NSCs, including nestin, CD133, Sox2, and SSEA1, as well as AP activity (Figure 1A and Supplementary Figure S1H and I). To further compare Id3-transduced neurospheres with NSCs, the methylation state of CpG dinucleotides in the Nestin promoter regions was analyzed. Bisulfite genomic sequencing analyses showed that the Nestin promoter doi:10.1093/jmcb/mjr038 Journal of Molecular Cell Biology (2012), 4, 59–62 | 59 Published online November 30, 2011