One of the causes of diabetes in infants is the defect of the insulin gene (INS). Gene mutations can lead to proinsulin misfolding, an increased endoplasmic reticulum (ER) stress and possible beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. We generated induced pluripotent stem cells (iPSCs) from a patient diagnosed with neonatal diabetes mellitus carrying the INS mutation in the 2nd intron (c.188-31G>A) and engineered isogenic CRISPR/Cas9 mutation-corrected cell lines. Differentiation into beta-like cells demonstrated that mutation led to the emergence of an ectopic splice site within the INS and appearance of the abnormal RNA transcript. Isogenic iPSC lines differentiated into beta-like cells showed a clear difference in formation of organoids at pancreatic progenitor stage of differentiation. Moreover, MIN6 insulinoma cell line expressing mutated cDNA demonstrated significant decrease in proliferation capacity and activation of ER stress and unfolded protein response (UPR)-associated genes. These findings shed light on the mechanism underlying the pathogenesis of monogenic diabetes.

The ability of human pluripotent stem cells for unlimited proliferation and self-renewal promotes their application in the fields of regenerative medicine. The morphological assessment of growing colonies and cells, as a non-invasive method, allows the best clones for further clinical applications to be safely selected. For this purpose, we analyzed seven morphological parameters of both colonies and cells extracted from the phase-contrast images of human embryonic stem cell line H9, control human induced pluripotent stem cell (hiPSC) line AD3, and hiPSC line HPCASRi002-A (CaSR) in various passages during their growth for 120 h. The morphological phenotype of each colony was classified using a visual analysis and associated with its potential for pluripotency and clonality maintenance, thus defining the colony phenotype as the control parameter. Using the analysis of variance for the morphological parameters of each line, we showed that selected parameters carried information about different cell lines and different phenotypes within each line. We demonstrated that a model of classification of colonies and cells by phenotype, built on the selected parameters as predictors, recognized the phenotype with an accuracy of 70–75%. In addition, we performed a qRT-PCR analysis of eleven pluripotency markers genes. By analyzing the variance of their expression in samples from different lines and with different phenotypes, we identified group-specific sets of genes that could be used as the most informative ones for the separation of the best clones. Our results indicated the fundamental possibility of constructing a morphological portrait of a colony informative for the automatic identification of the phenotype and for linking this portrait to the expression of pluripotency markers.

Anti-human immunodeficiency virus (HIV) broadly neutralizing antibodies (bNAbs) offer a promising approach for the treatment of HIV-1. The current paradigm for antibody therapy involves passive antibody transfer, requiring regular delivery of bNAbs in treating chronic diseases such as HIV-1. An alternative strategy is to use AAV-mediated gene transfer to enable in vivo production of desirable anti-HIV-1 antibodies. In this study, we investigated two sets of triple combinations of AAV9-vectors encoding different bNAbs: N6, 10E8, 10-1074 (CombiMab1), and VRC07-523, PGDM1400, 10-1074 (CombiMab2). We used CBAxC57Bl and C57BL/6 mouse models to characterize rAAV-induced antibody expression and to evaluate the neutralization capacity of mouse sera against a global panel of HIV-1 viral strains. rAAV9-mediated IgG expression varied between bNAb clones and mouse strains, with C57BL/6 mice exhibiting higher bNAb titers following rAAV delivery. Although CombiMab2 treatment elicited a higher IgG titer than CombiMab1, both combinations resulted in neutralization of all the viral strains from the global HIV-1 panel. Our data highlight the potential of AAV vectors as a long-term option for HIV-1 therapy.

Diabetes mellitus (DM) is a metabolic disease characterized by persistent hyperglycemia, resulting from defects in insulin secretion or impaired insulin action. In cases of severe pancreatic cell dysfunction and deficiency, the primary treatment remains lifelong insulin injections. A potential alternative is allogeneic pancreatic cell transplantation from a donor, which can stabilize glucose levels. However, the scarcity of donor material and the risk of immune rejection limit the widespread use of this approach. An alternative solution involves using in vitro-derived insulin-producing cells generated through the differentiation of pluripotent stem cells (PSCs), which could overcome the shortage of transplantable material. Furthermore, patient-specific cells—obtained directly from the patient via reprogramming of blood or skin cells into induced pluripotent stem cells (iPSCs)—would avoid immune rejection. Advances in this field have led to the active development and optimization of PSC differentiation into hormone-producing cells worldwide, with more than hundred patients dosed in clinical trials with ESC-derived cells and the single trial of iPSC-derived cells. This review highlights recent progress and prospects in generating insulin-producing cells from human PSCs, their applications in therapy development and disease modeling, as well as the current challenges and potential solutions.

Neonatal severe primary hyperparathyroidism (NSHPT) is a rare autosomal recessive disorder of calcium homeostasis that manifests shortly after birth with hypercalcemia and bone disease. NSHPT, in most cases, is attributed to mutations in the calcium-sensing receptor (CASR) gene. We reprogrammed dermal fibroblasts derived from a patient with NSHPT carrying a compound heterozygous mutation in the CASR gene into induced pluripotent stem cells (iPSCs). The established iPSCs expressed pluripotency markers, maintained normal karyotype and differentiated into all three germ layers. This line is a valuable resource for modeling of hyperparathyroidism related to CASR mutations.

Multiple endocrine neoplasia type 1 (MEN1) is a rare tumor syndrome that manifests differently among various patients. Despite the mutations in the MEN1 gene that commonly predispose tumor development, there are no obvious phenotype–genotype correlations. The existing animal and in vitro models do not allow for studies of the molecular genetics of the disease in a human-specific context. We aimed to create a new human cell-based model, which would consider the variability in genetic or environmental factors that cause the complexity of MEN1 syndrome. Here, we generated patient-specific induced pluripotent stem cell lines carrying the mutation c.1252G>T, D418Y in the MEN1 gene. To reduce the genetically determined variability of the existing cellular models, we created an isogenic cell system by modifying the target allele through CRISPR/Cas9 editing with great specificity and efficiency. The high potential of these cell lines to differentiate into the endodermal lineage in defined conditions ensures the next steps in the development of more specialized cells that are commonly affected in MEN1 patients, such as parathyroid or pancreatic islet cells. We anticipate that this isogenic system will be broadly useful to comprehensively study MEN1 gene function across different contexts, including in vitro modeling of MEN1 syndrome.