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Beyond its well-established role in cancer, KRAS is also crucial for embryogenesis, as its absence leads to embryonic lethality. However, the precise mechanisms underlying the developmental functions of KRAS, as well as the respective roles of its two splicing isoforms, KRAS4A and KRAS4B, remain incompletely characterized. To address these issues, we generated Kras4A knock-out ( Kras4A −/− ) and Kras4B −/− mouse models using CRISPR/Cas9 technology, and compared their phenotypes to those of a Kras −/− model, in which both isoforms are simultaneously inactivated. We observed that Kras −/− and Kras4B −/− embryos show a lethality that starts around E13.5, while Kras4A −/− embryos develop normally, with no detectable abnormalities. In contrast, Kras −/− embryos displayed a dual phenotype affecting both the heart and placenta, whereas Kras4B −/− embryos exhibited only the placental phenotype. The cardiac phenotype was complex, combining ventricular non-compaction, ventricular septal defects, double outlet right ventricle, and overriding aorta, likely resulting from impaired cardiac precursor proliferation. The placental phenotype was characterized by reduced placental size, and a marked decrease in glycogen trophoblast cells, correlating with hypoglycemia and hypoxia in Kras −/− and Kras4B −/− embryos. Thus, our findings confirm the predominant role of KRAS4B in KRAS-mediated developmental functions, but also suggest hidden functions of KRAS4A. Importantly, this study is the first to identify KRAS as a key regulator of a specific cell differentiation process and to characterize the biological defects caused by its loss.

Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5 GFP reporter cardiac fibroblasts. We first established a new NKX2.5 GFP reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5 GFP hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5 GFP cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.

Direct cardiac reprogramming has emerged as a novel therapeutic approach to treat and regenerate injured hearts through the direct conversion of fibroblasts into cardiac cells. Most studies have focused on the reprogramming of fibroblasts into induced cardiomyocytes (iCMs). The first study in which this technology was described, showed that at least a combination of three transcription factors, GATA4, MEF2C and TBX5 (GMT cocktail), was required for the reprogramming into iCMs in vitro using mouse cells. However, this was later demonstrated to be insufficient for the reprogramming of human cells and additional factors were required. Thereafter, most studies have focused on implementing reprogramming efficiency and obtaining fully reprogrammed and functional iCMs, by the incorporation of other transcription factors, microRNAs or small molecules to the original GMT cocktail. In this respect, great advances have been made in recent years. However, there is still no consensus on which of these GMT-based varieties is best, and robust and highly reproducible protocols are still urgently required, especially in the case of human cells. On the other hand, apart from CMs, other cells such as endothelial and smooth muscle cells to form new blood vessels will be fundamental for the correct reconstruction of damaged cardiac tissue. With this aim, several studies have centered on the direct reprogramming of fibroblasts into induced cardiac progenitor cells (iCPCs) able to give rise to all myocardial cell lineages. Especially interesting are reports in which multipotent and highly expandable mouse iCPCs have been obtained, suggesting that clinically relevant amounts of these cells could be created. However, as of yet, this has not been achieved with human iCPCs, and exactly what stage of maturity is appropriate for a cell therapy product remains an open question. Nonetheless, the major concern in regenerative medicine is the poor retention, survival, and engraftment of transplanted cells in the cardiac tissue. To circumvent this issue, several cell pre-conditioning approaches are currently being explored. As an alternative to cell injection, in vivo reprogramming may face fewer barriers for its translation to the clinic. This approach has achieved better results in terms of efficiency and iCMs maturity in mouse models, indicating that the heart environment can favor this process. In this context, in recent years some studies have focused on the development of safer delivery systems such as Sendai virus, Adenovirus, chemical cocktails or nanoparticles. This article provides an in-depth review of the in vitro and in vivo cardiac reprograming technology used in mouse and human cells to obtain iCMs and iCPCs, and discusses what challenges still lie ahead and what hurdles are to be overcome before results from this field can be transferred to the clinical settings.

The success of cell therapy for the treatment of myocardial infarction depends on finding novel approaches that can substantially implement the engraftment of the transplanted cells. In order to enhance cell engraftment, most studies have focused on the pretreatment of transplantable cells. Here we have considered an alternative approach that involves the preconditioning of infarcted heart tissue to reduce endogenous cell activity and thus provide an advantage to our exogenous cells. This treatment is routinely used in other tissues such as bone marrow and skeletal muscle to improve cell engraftment, but it has never been taken in cardiac tissue. To avoid long-term cardiotoxicity induced by full heart irradiation we developed a rat model of a catheter-based heart irradiation system to locally impact a delimited region of the infarcted cardiac tissue. As proof of concept, we transferred ZsGreen+ iPSCs in the infarcted heart, due to their ease of use and detection. We found a very significant increase in cell engraftment in preirradiated rats. In this study, we demonstrate for the first time that preconditioning the infarcted cardiac tissue with local irradiation can substantially enhance cell engraftment.

Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5 reporter cardiac fibroblasts. We first established a new NKX2.5 reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5 hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5 cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.

Background: While pancreatic cysts have been described in syndromic ciliopathies, the pancreas is not commonly recognized as a target organ. However, several ciliary gene knockout mouse models develop a pancreatic phenotype combining acinar atrophy and adipocyte accumulation, hereby called adipopancreatosis, suggesting a link between ciliary dysfunction and pancreatic disease. Objective: We investigated whether mutations in ciliopathy-associated genes are linked to pancreatic dysfunction in humans. Design: We analyzed a cohort of 341 patients with pediatric-onset pancreatic anomalies and characterized the pancreatic phenotype of new mouse models with conditional Nphp3 inactivation or bearing Nphp3 mutations recapitulating human mutations. In patients, pancreatic fat content was quantified using Dixon-MRI. Results: Mutations in the cilium-related HNF1B and NPHP3 were identified in patients presenting with both renal and pancreatic dysfunction. Nphp3 mutant mice developed acinar atrophy, adipopancreatosis, and moderate inflammation. Adipocytes in the pancreas exhibited a white adipocyte-like profile and likely originated from mesothelial-derived fibroblasts. Reduced numbers and altered length of ductal cilia were monitored. Interestingly, secretory canaliculi, typically unnoticed structures found within and between acinar cells and connected to the acinar lumen, exhibited a microcystic morphology. Consistent with the mouse phenotype, Dixon-MRI revealed significantly increased pancreatic fat content in patients with HNF1B and NPHP3 mutations. Conclusion: We describe a previously unrecognized pancreatic manifestation of ciliopathies, which we name ciliogenic pancreatopathy. Patients with known ciliopathy-causing mutations should be evaluated for this pancreatic condition, particularly those with kidney disease, as concomitant exocrine pancreatic insufficiency may further compromise renal function or the outcome of kidney graft.Competing Interest StatementThe authors have declared no competing interest.