Reprogramming of pig somatic cells to induced pluripotent stem cells provides a tremendous advance in the field of regenerative medicine since the pig represents an ideal large animal model for the preclinical testing of emerging cell therapies. However, the current generation of pig-induced pluripotent stem cells (piPSCs) require the use of time-consuming and laborious retroviral or lentiviral transduction approaches, in order to ectopically express the pluripotency-associated transcription factors Oct4, Sox2, Klf4 and c-Myc, in the presence of feeder cells. Here, we describe a simple method to produce piPSC with a single transfection of a CAG-driven polycistronic plasmid expressing Oct4, Sox2, Klf4, c-Myc and a green fluorescent protein (GFP) reporter gene, in gelatine-coated plates, with or without feeder cells. In our system, the derivation of piPSCs from adult pig ear fibroblasts on a gelatine coating showed a higher efficiency and rate of reprogramming when compared with three consecutive retroviral transductions of a similar polycistronic construct. Our piPSCs expressed the classical embryonic stem cell markers, exhibit a stable karyotype and formed teratomas. Moreover, we also developed a simple method to generate in vitro spontaneous beating cardiomiocyte-like cells from piPSCs. Overall, our preliminary results set the bases for the massive production of xeno-free and integration-free piPSCs and provide a powerful tool for the preclinical application of iPSC technology in a large animal setting.

IκBs exert principal functions as cytoplasmic inhibitors of NF‐kB transcription factors. Additional roles for IκB homologues have been described, including chromatin association and transcriptional regulation. Phosphorylated and SUMOylated IκBα (pS‐IκBα) binds to histones H2A and H4 in the stem cell and progenitor cell compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unknown. Here, we show that serine 32–36 phosphorylation of IκBα favors its binding to nucleosomes and demonstrate that p‐IκBα association with H4 depends on the acetylation of specific H4 lysine residues. The N‐terminal tail of H4 is removed during intestinal cell differentiation by proteolytic cleavage by trypsin or chymotrypsin at residues 17–19, which reduces p‐IκBα binding. Inhibition of trypsin and chymotrypsin activity in HT29 cells increases p‐IκBα chromatin binding but, paradoxically, impaired goblet cell differentiation, comparable to IκBα deletion. Taken together, our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular basis for the understanding of the restricted nuclear distribution of p‐IκBα in specific stem cell compartments.

IκBs exert a principal function as cytoplasmic inhibitors of the NF-kB transcription factors. Additional functions for IκB homologues have been described including association to chromatin and transcriptional regulatioin. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds histones H2A and H4 in the stem and progenitor compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unstudied. We here show that serine 32-36 phosphorylation of IκBα favors its binding with nucleosomes and demonstrated that p-IκBα association to H4 is favored by acetylation at specific H4 lysine residues. N-terminal tail of H4 is lost during intestinal cell differentiation by proteolytic cleavage at residues 17-19 imposed ny trypsin or chymotrypsin, which interferes p-IκBα binding. Paradoxically, inhibition of trypsin and chymotrypsin activity in HT29 cells increased p-IκBα chromatin binding and impaired goblet cell differentiation, comparable to IκBα deletion. Together our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular base for the restricted nuclear distribution of p-IκBα at specific stem cell compartments.

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Genome editing requires precision to broadly move on to industrial and clinical applications. For this reason, homologous directed repair (HDR) is one of the preferred methods for small edits, other than knock-outs. However, HDR has low efficiency. Current investigations to enhance HDR have mainly gone in the direction of finding non-homologous end joining (NHEJ) inhibitors. NHEJ is crucial for cellular integrity, then the inhibition of this pathway is detrimental for the correct survival of living entities. In other studies, a second opportunity is given to HDR by targeting the byproducts of NHEJ, using an extra gRNA. In this study, we propose the use of a meiotic factor, PRDM9, to directly enhance homology recombination. Through the exploration of combinatorial factors and donor design, we have established an optimized protocol for HDR. PRDM9-Cas9 fusion combined with CtIP improves HDR/NHEJ ratio. In addition, we have validated this combinatorial approach for small edits through a traffic light reporter system, as well as for longer edits with a split-GFP reporter system.