In order to clarify such method dependent
In order to clarify such method-dependent reprogramming hallmarks, we applied standard assays to compare polycistronic cassettes (constructed in-house or obtained from public resources) in a drug-inducible piggyBac (PB) transposon reprogramming system. The induced expression of various polycistronic cassettes in mouse fibroblasts evoked phenotypic and gene expression changes that we divided into two basic behavioral classes. An examination of individual factor sequences across the panel of polycistronic constructs revealed a re-occurring discrepancy in the Klf4 cDNA defining two coding region length variants. Increased length at the KLF4 N terminus was associated with higher protein levels and thus altered relative factor stoichiometry, ultimately impacting both the initiation and stabilization phases of iPSC derivation. Here, we report the consequences of KLF4 N-terminal variation in mono- and polycistronic reprogramming experiments, and apply these findings to recognize and reconcile differences in reprogramming characteristics implied hitherto.
Discussion Principally, we have uncovered a fundamental characteristic of cloned Klf4 that governs relative factor stoichiometry and reprogramming responses during mouse iPSC derivation with polycistronic constructs. At the root of our discovery lies the disparate coding prediction of full-length Klf4 cDNA (Garrett-Sinha et al., 1996; Shields et al., 1996), resulting in two proteins with distinct N termini defined herein as KLF4S and KLF4L. These two variants have seen unprejudiced use among vectors constructed for reprogramming (Table 1 and associated references). OKMS (Klf4) presented low KLF4 levels, which were restored along with Klf4-related phenotypes by dihydroergotamine of the KLF4S N terminus by 9aa. Thus, OK+9MS excluded the influence of 2A peptide choice and factor order variation. Moreover, high KLF4-associated phenotypes were reproduced by supplementation of OKMS with either monocistronic Klf4 or Klf4, limiting the effect to a polycistronic context. Relative stoichiometry was explored in a recent study, and it was found that chemically controlled degradation of KLF4 resulted in premature termination of reprogramming (Nishimura et al., 2014). Although precise factor titration and more direct biochemical assays may be required, our monocistronic reprogramming data argue against modification of an N-terminal functional domain, and rather support the idea that threshold levels of KLF4 are important for minimizing the occurrence of partial reprogramming. Polycistronic cassettes encoding KLF4S tend to permit rapid expansion of transgenic cells with robust SSEA-1 activation. Yet, this majority population is ultimately impeded in the acquisition and stabilization of pluripotency, as indicated by transgene dependence and high-level expression of markers associated with pre-iPSCs (Mikkelsen et al., 2008; Theunissen et al., 2011). Polycistronic reprogramming with Klf4 or OKMS supplementation with improved reporter activation and dox independence. Nanog-GFP reporter activation is consistent with a central role for KLF4 in the core ESC pluripotency network, as ectopic expression of Klf4 promotes epiblast stem cell conversion to ground-state pluripotency (Guo et al., 2009) and prevents loss of pluripotency in the absence of LIF (Niwa et al., 2009). Transgene silencing is considered a hallmark of complete reprogramming (Golipour et al., 2012). How the silencing phenomenon is related to the acquisition of pluripotency, and whether it is coordinated directly or indirectly through KLF4 are issues of considerable interest. Individual expression of either Klf4 or Klf4 in MEFs led to a potent epidermal gene response (Figure S2B). Moreover, a similar response was noted as a common feature of d6 reprogramming populations induced with Klf4 cassettes (Figure 3B). A retrospective examination of early reprogramming experiments recalls reports of epidermis-related gene activation (Mikkelsen et al., 2008). Klf4 knockout mice die postnatally from failed epidermal stratification (Segre et al., 1999). Reciprocally, ectopic Klf4 expression during embryogenesis results in premature barrier formation (Jaubert et al., 2003). Therefore, it is pertinent that high KLF4 stoichiometry induces a cascade of epithelialization and expression of epidermis-associated genes such as Krt6a, Krt17, Sprr1a, Cnfn, and Tgm1 during early reprogramming (Figures 3A and 3B; Table S1). Interestingly, many of these genes are not expressed in ESCs, nor are they maintained in GFP+ iPSCs (data not shown), which brings into question their relevance in iPSC derivation. To address potential cellular heterogeneity, it will be important to link gene expression responses and reprogramming outcomes to specific subpopulations of cells. Thus, it remains to be determined whether the epidermal response elicited by excess KLF4 in the early phase directly contributes to high-fidelity reprogramming or to an alternate cell fate.