Genes & Cells

The utilization of technology for the generation of induced pluripotent stem cells (iPSCs) and their subsequent differentiation is a promising approach for the study of disease pathogenesis and development of methods for treating optical neuropathies and retinopathy, which are the most common types of visual pathologies, in which retinal ganglion cells degenerate (consequently, optic nerve atrophy) or pigment epithelial cells and photoreceptors are affected, respectively. The prospect of patient-specific iPSCs has become a powerful alternative tool for discovering novel disease-causing mutations, studying genotype–phenotype relationships, screening therapeutic toxicity, and developing personalized cell therapy for optical neuropathies and retinopathies.

Numerous studies have demonstrated the possibility of creating different types of retinal cells from iPSCs, which provides a rapid development of the research area of human diseases for which no relevant animal models are available or access to primary human tissues and cells is limited.

This review presents various protocols for generating iPSCs from somatic cells and their subsequent differentiation, with an emphasis on the observed biological effects of the resulting cell cultures, including organoids, and discusses the prospects of using such models. The article may be useful to researchers studying the pathogenesis of various hereditary forms of blindness and developers of approaches for the treatment of these diseases who need a relevant cellular model.

BACKGROUND: Human genotype is a factor that determines the severity of COVID-19. Previously, a large-scale whole-genome association study of the COVID-19 Host Genetics Initiative (2021) investigated the association of genetic variants at multiple loci with COVID-19 severity. The genetic variants that have the greatest effect on COVID-19 severity are expected to have a low frequency in the population. Therefore, the study of rare variants may provide additional insights into the disease pathogenesis and thus help in the development of prevention and treatment options.

AIM: To search for genes enriched for rare genetic variants associated with COVID-19 severity in the Russian population by replication analysis.

METHODS: The clinical exome of a Russian cohort of patients was sequenced based on the St. Petersburg State Budgetary Institution “City Hospital No. 40” and St Petersburg University. The study used biomaterial from patients hospitalized at City Hospital No. 40 diagnosed with COVID-19 and healthy individuals (population control group). The severity of the course of COVID-19 was determined according to the results of lung computed tomography. The list of genes for subsequent replication was generated by a literature review. Burden test methods were used for the replication analysis of genes associated with COVID-19 severity.

RESULTS: In total, 701 clinical exomes were sequenced from 263 individuals with severe COVID-19 and 438 healthy individuals. In the literature review, 18 genes associated with severe COVID-19 were included in the replication analysis. The replication analysis did not identify any genes whose association with severe COVID-19 was confirmed in the study cohort.

CONCLUSION: The replication analysis did not identify any genes that showed a significant association between the functional variant enrichment and COVID-19 severity. However, the direction of the correlation was consistent with the findings of previous studies. Expanding the study cohort would increase the power of the tests and allow us to detect additional rare variants that influence the severity of COVID-19 progression.

BACKGROUND: Decreased skeletal muscle mass and properties lead to the development of various pathologies and increased risk for injuries. Studies of the molecular mechanisms of skeletal muscle adaptation to resistance training to increase muscle mass and strength appear imperative for medicine and sports.

AIM: To assess changes in the proteomic profile (quantitative panoramic mass spectrometric analysis) of skeletal muscles and the correlation of these changes with the expression of the corresponding mRNAs (RNA-sequencing) before and after 12 weeks of strength training and changes in the transcriptome 8 and 24 h after an acute resistance exercise with one leg.

METHODS: Ten untrained men (aged 23 [20.8–25.9] years; body mass index, 22 [20.9–25.1] kg/m²) performed a two-legged seated platform press for 12 weeks (3 times/week, 50–75% of maximum voluntary contraction [MVC]). After training, the volunteers performed an acute strength exercise with one leg. Before and after 12 weeks of training, the MVC and volume of the quadriceps femoris muscle were assessed. Before and after training, as well as 8 and 24 h after the acute resistance exercise, a biopsy of the vastus lateralis muscle was performed from the loaded and contralateral limbs for immunohistochemical, proteomic (high-performance liquid chromatography-tandem mass spectrometry), and transcriptomic (RNA-sequencing) analyses.

RESULTS: The 12-week strength training increased the MVC by 19%, quadriceps femoris volume by 12%, cross-sectional area of type 2 (fast) fibers by 29%, minimum Feret diameter of type 2 fibers by 10%, and type 1 (slow) fibers by 13%. Of the 1174 detected proteins, 24 increased, and 83 decreased in content. Strength training resulted in an increase in the expression levels of 142 and a decrease in 65 of the 12,112 mRNAs detected, with enrichment for the functional terms of the extracellular environment, matrix, basement membrane, etc. Changes in the contents of 433 mRNAs after 8 h and 639 mRNAs after 24 h were found when comparing the once-loaded muscle with the contralateral one (genes associated with contractile activity). Changes in the content of only a small part of proteins (5–9 out of 107) correlated with the changes in the corresponding mRNAs.

CONCLUSION: Proteomic analysis showed that the 12-week resistance training had little effect on the relative abundance of high-abundance proteins in muscles. The increase in muscle mass induced by training appears to be explained by a similar change in the synthesis/degradation rates of the detected proteins. In the comparison of proteomic data with changes in mRNA expression after 12 weeks of training and 8 and 24 h after a single load (gene response specific to contractile activity), changes in protein contents caused by strength training were regulated mainly at the post-transcriptional level.

BACKGROUND: The UBE2A protein belongs to the E2 family of ubiquitin-binding enzymes involved in the ubiquitination of substrate proteins. UBE2A mutations lead to congenital X-linked mental retardation syndrome-type Nascimento. How UBE2A participates in the central nervous system development is still unknown.

AIM: To establish a cell model based on induced pluripotent stem cells (iPSCs) to study the molecular and cellular functions of UBE2A in neurogenesis.

METHODS: Using genomic CRISPR-Cas9 editing and lentiviral transduction, a cell model based on iPSCs from two healthy donors was designed. This cell model includes isogenic iPSCs with knockout and inducible hyperexpression of UBE2A. In addition, iPSCs were obtained by reprogramming peripheral blood mononuclear cells of a patient diagnosed with X-linked mental retardation of Nascimento type, which has a deletion spanning the whole UBE2A locus.

RESULTS: The obtained iPSCs demonstrate an ESC-like morphology. They express pluripotent cell markers OCT4, SOX2, SSEA-4, and TRA-1-81 and have normal karyotypes. iPSCs with UBE2A knockout or hyperexpression had significantly increased nuclei size compared with the isogenic control.

CONCLUSION: The developed iPSC-based cell model can be used for fundamental studies of the functions of UBE2A in neurogenesis.