Defining the Diversity of Phenotypic Respecification Using Multiple Cell Lines and Reprogramming Regimens

Abstract

To better understand the basis of variation in cellular reprogramming, we performed experiments with two primary objectives: first, to determine the degree of difference, if any, in reprogramming efficiency among cells lines of a similar type after accounting for technical variables, and second, to compare the efficiency of conversion of multiple similar cell lines to two separate reprogramming regimens-induced neurons and induced skeletal muscle. Using two reprogramming regimens, it could be determined whether converted cells are likely derived from a distinct subpopulation that is generally susceptible to reprogramming or are derived from cells with an independent capacity for respecification to a given phenotype. Our results indicated that when technical components of the reprogramming regimen were accounted for, reprogramming efficiency was reproducible within a given primary fibroblast line but varied dramatically between lines. The disparity in reprogramming efficiency between lines was of sufficient magnitude to account for some discrepancies in published results. We also found that the efficiency of conversion to one phenotype was not predictive of reprogramming to the alternate phenotype, suggesting that the capacity for reprogramming does not arise from a specific subpopulation with a generally ''weak grip'' on cellular identity. Our findings suggest that parallel testing of multiple cell lines from several sources may be needed to accurately assess the efficiency of direct reprogramming procedures, and that testing a larger number of fibroblast lines-even lines with similar origins-is likely the most direct means of improving reprogramming efficiency.

Reprogramming to the nervous system: a computational and candidate gene approach

“…Parallel analysis of selected mouse lines (Alicea et al 2013; see Supplemental Figure 3, panels C and D) showed a result similar to the human lines, with the exception of was because several independent laboratories had confirmed the production of iN cells using similar methodologies (Vierbuchen et al, 2010;Pfistetrer, et al, 2011;Pang et al, 2011;Caiazzo et al, 2011 (Vierbuchen et al, 2010;Caiazzo et al, 2011) of human origin fused to YFP as shown schematically in Alicea et al 2013 (Supplemental Figure 4). As predicted, all of the YFP-fusion proteins localized primarily to the nucleus, some presenting a mottled appearance and others appearing more diffuse (Figure 2.1, panel A).…”

“…and satellite cells, but are not expressed at significant levels in fibroblasts (Day, Shefer, and Yablonka-Reuveni, 2010;Day et al, 2007;Ellis et al, 2004;D'Amour and Gage, 2003;Vogel et al, 2003;Avilion et al, 2003;Zimmerman et al, 1994). The results revealed that essentially all cells appeared immunopositive for the fibroblast-associated markers vimentin and fibronectin and no cells appeared positive for the stem cell markers nestin and Sox-2 that were readily observed in control cultures of human neural stem cells (Alicea et al 2013; see Supplemental Figure 3, panel A and B).…”

“…and POU3F2 combined with either ZIC1 or MYT1L/ND produced many TUJ1-positive iN cells in both human and mouse cultures using either fetal or adult fibroblasts, whereas cells infected with each neurogenic factor alone produced very few iN cells ( Figure 2.1, panel B). Mock-infected cells or cells infected with a YFP vector only and cultured under identical conditions produced no strongly immunoreactive TUJ1-positive cells with neuron-like morphology (Alicea et al 2013; see Supplemental Figure 3, panel A). As described previously, the primary difference between the generation of mouse and human iN cells was the maturation rate (Vierbuchen et al, 2010;Pfistetrer, et al, 2011;Pang et al, 2011;Caiazzo et al, 2011).…”

“…To examine the impact of genetic homogeneity on reprogramming efficiency, mouse fibroblast lines were generated from a single donor animal Appendix B, Table B.3 (bottom panel). To ensure that the individual mouse lines did not functionally approximate secondary lines or subclones that could occur if all lines were derived from a single explant, we harvested eight different mouse organs for establishment of fibroblast lines (Alicea et al 2013; see Supplemental Figure 1, panel B) and then selected only those lines that displayed no evident non-fibroblastic cells and a consistently uniform fibroblastic morphology (Alicea et al 2013; see Supplemental Figure 1, panel C). Lines also had to display a similar capacity for growth, passage, and survival of freeze/thaw that allowed them to be maintained side-by-side with other lines.…”

“…For differentiation, NPCs were plated on PORN/lam plates and FGF-2 was progressively withdrawn to a final concentration of 2ng/ml by day 20-24, when the cells were processed for immunostaining to confirm neuronal identity and subjected to electrophysiological analysis. Undifferentiated NPCs at 30-50% confluency were fixed and used as controls in the experiments shown in Alicea et al 2013 (see Supplemental Figure 3). For iSM cells, total sarcomeric α-actinin fluorescence was taken as the red value,…”