N-Terminal Amino Acids Determine KLF4 Protein Stability in 2A Peptide-Linked Polycistronic Reprogramming Constructs.

A common strategy for multi-protein expression is to link genes by self-cleaving 2A peptide sequences. Yet, little is known how the 2A peptide-derived N-terminal proline or adjacent non-native residues introduced during cDNA cloning affects protein stoichiometry. Polycistronic reprogramming constructs with altered KLF4 protein stoichiometry can influence induced pluripotent stem cell (iPSC) generation. We studied the impact of N-terminal 2A peptide-adjacent residues on the protein stability of two KLF4 isoforms, and assayed their capacity to generate iPSCs. Here, we show that the N-terminal proline remnant of the 2A peptide, alone or in combination with leucine, introduced during polycistronic cloning, destabilizes KLF4 resulting in increased protein degradation, which hinders reprogramming. Interestingly, the addition of charged and hydrophilic amino acids, such as glutamate or lysine stabilizes KLF4, enhancing reprogramming phenotypes. These findings raise awareness that N-terminal modification with 2A peptide-derived proline or additional cloning conventions may affect protein stability within polycistronic constructs.

OVOL1 Influences the Determination and Expansion of iPSC Reprogramming Intermediates.

During somatic cell reprogramming to induced pluripotent stem cells (iPSCs), fibroblasts undergo dynamic molecular changes, including a mesenchymal-to-epithelial transition (MET) and gain of pluripotency; processes that are influenced by Yamanaka factor stoichiometry. For example, in early reprogramming, high KLF4 levels are correlated with the induction of functionally undefined, transiently expressed MET genes. Here, we identified the cell-surface protein TROP2 as a marker for cells with transient MET induction in the high-KLF4 condition. We observed the emergence of cells expressing the pluripotency marker SSEA-1+ mainly from within the TROP2+ fraction. Using TROP2 as a marker in CRISPR/Cas9-mediated candidate screening of MET genes, we identified the transcription factor OVOL1 as a potential regulator of an alternative epithelial cell fate characterized by the expression of non-iPSC MET genes and low cell proliferation. Our study sheds light on how reprogramming factor stoichiometry alters the spectrum of intermediate cell fates, ultimately influencing reprogramming outcomes.

Platforms of in vivo genome editing with inducible Cas9 for advanced cancer modeling.

The emergence of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has dramatically advanced how we manipulate the genome. Regarding in vivo experiments, Cas9-transgenic animals could provide efficient and complex genome editing. However, this potential has not been fully realized partly due to a lack of convenient platforms and limited examples of successful disease modeling. Here, we devised two doxycycline (Dox)-inducible Cas9 platforms that efficiently enable conditional genome editing at multiple loci in vitro and in vivo. In these platforms, we took advantage of a site-specific multi-segment cloning strategy for rapid and easy integration of multiple single guide (sg)RNAs. We found that a platform containing rtTA at the Rosa26 locus and TRE-Cas9 together with multiple sgRNAs at the Col1a1 locus showed higher efficiency of inducible insertions and deletions (indels) with minimal leaky editing. Using this platform, we succeeded to model Wilms' tumor and the progression of intestinal adenomas with multiple mutations including an activating mutation with a large genomic deletion. Collectively, the established platform should make complicated disease modeling in the mouse easily attainable, extending the range of in vivo experiments in various biological fields including cancer research.

Reprogramming suppresses premature senescence phenotypes of Werner syndrome cells and maintains chromosomal stability over long-term culture.

Werner syndrome (WS) is a premature aging disorder characterized by chromosomal instability and cancer predisposition. Mutations in WRN are responsible for the disease and cause telomere dysfunction, resulting in accelerated aging. Recent studies have revealed that cells from WS patients can be successfully reprogrammed into induced pluripotent stem cells (iPSCs). In the present study, we describe the effects of long-term culture on WS iPSCs, which acquired and maintained infinite proliferative potential for self-renewal over 2 years. After long-term cultures, WS iPSCs exhibited stable undifferentiated states and differentiation capacity, and premature upregulation of senescence-associated genes in WS cells was completely suppressed in WS iPSCs despite WRN deficiency. WS iPSCs also showed recapitulation of the phenotypes during differentiation. Furthermore, karyotype analysis indicated that WS iPSCs were stable, and half of the descendant clones had chromosomal profiles that were similar to those of parental cells. These unexpected properties might be achieved by induced expression of endogenous telomerase gene during reprogramming, which trigger telomerase reactivation leading to suppression of both replicative senescence and telomere dysfunction in WS cells. These findings demonstrated that reprogramming suppressed premature senescence phenotypes in WS cells and WS iPSCs could lead to chromosomal stability over the long term. WS iPSCs will provide opportunities to identify affected lineages in WS and to develop a new strategy for the treatment of WS.