Frontotemporal dementia with parkinsonism-17 is a neurodegenerative disease characterised by pathological aggregation of the tau protein with the formation of neurofibrillary tangles and subsequent neuronal death. The inherited form of frontotemporal dementia can be caused by mutations in several genes, including the MAPT gene on chromosome 17, which encodes the tau protein. As there are currently no medically approved treatments for frontotemporal dementia, there is an urgent need for research using in vitro cell models to understand the molecular genetic mechanisms that lead to the development of the disease, to identify targets for therapeutic intervention and to test potential drugs to prevent neuronal death. Analysis of exome sequencing data from a 46-year-old patient with a clinical diagnosis of Parkinson’s disease revealed the presence of the pathological variant c.2013T>G (rs63750756) in the MAPT gene, which is associated with frontotemporal dementia with parkinsonism-17. By reprogramming the patient’s peripheral blood mononuclear cells, we obtained induced pluripotent stem cells (iPSCs). Two iPSC lines were characterised in detail. Reprogramming was performed by transfection with non-integrating episomal vectors expressing the OCT4, SOX2, KLF4, LIN28, L-MYC and mp53DD proteins. The iPSC lines ICGi052-A and ICGi052-B proliferate stably, form colonies with a morphology characteristic of human pluripotent cells, have a normal diploid karyotype (46,XX), express endogenous alkaline phosphatase and pluripotency markers (OCT4, NANOG, SSEA-4 and TRA-1-60) and are able to differentiate into derivatives of three germ layers: ento-, ecto- and mesoderm. The iPSC lines obtained and characterised in detail in this work represent a unique tool for studying the molecular genetic mechanisms of the pathogenesis of frontotemporal dementia with parkinsonism-17, as well as for testing potential drugs in vitro.

Parkinson’s disease is a multifactorial disease; both genetic predisposition (5% of all cases), environmental factors and age-related changes in the brain and other body systems contribute to its etiology. For the diagnosis and study of the pathology of the development of the disease, it is important to search for new polymorphisms associated with hereditary forms of the disease. We analyzed the clinical exome of a 55-year-old patient with Parkinson’s disease and identified a single nucleotide polymorphism in the GLUD2 gene (c.1492TG). This genetic variant is pathogenic according to the ClinVar database, but the mechanism of pathogenesis is still poorly understood. In addition, there are currently no relevant models based on human cells, which is of great interest. We generated induced pluripotent stem cells (iPSCs) from patient peripheral blood mononuclear cells using non-integrating episomal vectors expressing OCT4, KLF4, L‑MYC, SOX2, LIN28, and p53 shRNA. The obtained iPSC lines (ICGi044-B and ICGi044-C) demonstrate typical ESC-like morphology, normal karyotype (46,XY), express pluripotency markers (OCT4, SOX2, NANOG, SSEA4, TRA-1-60) and are able to give derivatives of three germ layers. The iPSC lines ICGi044-B and ICGi044-C, as well as their neural derivatives, represent an unique in vitro cell model for studying the pathogenetic mechanisms of the development of Parkinson’s disease associated with the c.1492TG mutation in the GLUD2 gene.

The prospect of using LRRK2 inhibitors as treatment strategy for Parkinson’s disease (PD) associated with mutations in the gene GBA1 (GBA-PD), encoding the lysosomal enzyme glucocerebrosidase (GCase) is currently being discussed. We assessed the effectiveness of the LRRK2 kinase activity inhibitor MLi-2 in restoring GCase functions and the effect on the activity of other lysosomal enzymes in cell lines of patients with GBA-PD, LRRK2-PD.

Parkinson’s disease is a neurodegenerative disease, and genetic variants are known in only 5% of cases. Analysis of clinical exome of a patient with a family history of Parkinsonism revealed polymorphisms in the LRRK2 and PINK1 genes. The patient’s mononuclear blood cells are reprogrammed to a pluripotent state using episomal vectors expressing pluripotency factors. The line of induced pluripotent stem cells (iPSCs) demonstrated the typical morphology of human pluripotent cells, had a normal karyotype, expressed OCT4, NANOG, SOX2, and TRA-1-60 and gave derivatives of three germ layers during spontaneous differentiation in vitro. The resulting iPSCs line is a valuable tool for studying the contribution of polymorphic variants of the LRRK2 and PINK1 genes to the pathogenesis of Parkinson’s disease.

Genome editing in human pluripotent stem cells using programmable nucleases makes it possible to create models of hereditary pathologies using directed transgenesis, gene knockout, and replacement of individual nucleotides in DNA sequences. Using CRISPR/SpCas9-mediated homologous recombination at the AAVS1 locus, clones of human induced pluripotent stem cells (iPSCs) ICGi022-A (Malakhova et al., 2020) were obtained, which carry transgenes of two variants of the nuclease AsCas12a (also known as AsCpf1), recognizing different PAM consensuses, and the reverse doxycycline transgene-dependent transactivator M2rtTA. For each AsCas12a variant, the lines ICGi022-A-6 (AsCas12a, PAM 5'-TTTV-3') and ICGi022-A-7 (AsCas12a, PAM 5'-TYCV-3') were obtained. Using Western blot analysis, it was shown that the addition of doxycycline to the culture medium causes activation of the expression of AsCas12a(TTTV) and AsCas12a(TYCV) proteins. The resulting transgenic iPSC clones were subjected to molecular and cytogenetic analysis. Using quantitative PCR and immunocytochemical analysis, it was shown that they have a high level of mRNA expression of gene markers of pluripotent cells, namely OCT4, NANOG , and SOX2 , as well as specific expression of protein markers OCT4, SOX2, SSEA-4, and TRA-1-60. In addition, using iPSCs spontaneous differentiation into embryoid bodies, it was found that transgenic clones can give derivatives of all three primitive germ layers: ectoderm, mesoderm, and endoderm. Cytogenetic analysis showed that transgenic iPSC clones have a normal karyotype, 46,XX.

The search for new polymorphisms associated with hereditary diseases is important for diagnostics and the study of the disease’s development pathology. The authors have analyzed a clinical exome of a Parkinson’s disease patient and identified single-nucleotide variations in the LRRK2 ( c.1000G>A , c.2167A>G ) and PINK1 ( c.1562A>C ) genes. The LRRK2:c.1000G>A mutation has uncertain clinical significance and is interesting for further investigation. We generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) of the patient by nonintegrating episomal vectors. iPSCs demonstrate typical morphology and normal karyotype (46,XY), express pluripotency markers (OCT4, SOX2, NANOG, SSEA4, TRA-1-60), and are able to produce derivatives of three germ layers.

The pathological variant p.G2019S in the LRRK2 gene leads to the occurrence of a hereditary form of Parkinson’s disease (PD) and affects 7% of patients with a familial form of the disease. However, the mechanisms that trigger pathological events during the development of the disease are not yet fully understood. The authors obtained iPSCs (ICGi043-A line) from peripheral blood mononuclear cells of a patient with a hereditary form of PD associated with the genetic variant c.6055G>A (p.G2019S, rs34637584) in the LRRK2 gene using transfection with episomal vectors. iPSCs rapidly proliferate in dense monolayer cell colonies, are positive for endogenous alkaline phosphatase, have a normal karyotype (46,XX), express pluripotency markers (OCT4, SOX2, NANOG, TRA-1-60, SSEA-4), and are capable to differentiate into three germ layers (ecto-, endo-, and mesoderm), which confirms their pluripotent status. Future directed differentiation of the obtained iPSCs into dopaminergic neurons will allow the creation of an in vitro cell model of PD associated with the pathological variant c.6055G>A in the LRRK2 gene and contribute to understanding the pathogenesis of PD.

Wilson's disease is a rare autosomal recessive disorder of copper metabolism. The copper accumulation in the viscera appears due to the functional impairment of copper-transporting ATPase, which is encoded by the ATP7B gene. In this study, PBMCs of a patient with two ATP7B mutations were reprogrammed. The first mutation is a missense mutation p.H1069Q, which is the most frequent mutation in the human population. At the same time, the second one is a frameshift mutation p.Lys1013fs. The generated iPSC line had a normal karyotype, maintained the original genotype, expressed pluripotency markers, and demonstrated the ability to differentiate into derivatives of the three germ layers.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the HTT gene. HD patient-specific induced pluripotent stem cells (iPSCs) represent an excellent model for the disease study. We generated iPSC line from blood mononuclear cells of HD patient with 38 CAG repeats in the HTT exon 1 using integration free episomal plasmids expressing Yamanaka factors. The iPSC line retained the disease causing mutation and expressed pluripotency markers. It also displayed a normal karyotype and the ability to differentiate into derivatives of three germ layers.

Wilson's disease is an inherited disorder associated with copper accumulation in the liver, brain and other vital organs. Wilson's disease is caused by mutations in the ATP7B gene. Over 300 mutations of ATP7B have been described. Despite the disease is autosomal recessive, the patient whose PBMCs were reprogrammed in the study harbours heterozygous mutation c.3207C > A (p.H1069Q). Detailed analysis of the ATP7B complete gene sequencing data has not revealed other known disease associated mutation. The generated iPSC lines maintained the original genotype, expressed pluripotency markers, had normal karyotype and demonstrated the ability to differentiate into derivatives of the three germ layers.

ICGi021-A and ICGi022-A iPSC lines were obtained by reprogramming PBMCs of two healthy women of the Siberian population using episomal non-integrating vectors expressing Yamanaka factors. iPSC lines expressed pluripotency markers, had a normal karyotype and demonstrated the ability to differentiate into derivatives of the three germ layers. Clinical exome sequencing data of the original biosamples of the donors are available in the NCBI SRA database. The generated cell lines are useful as “healthy” control in biomedical studies.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletion or mutation in SMN1 gene. SMA human induced pluripotent stem cells (iPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. We generated iPSCs from a SMA type I patient and SMA type II patient by using non-integrating episomal plasmid vectors. The resulting iPSCs are episomal-free, express pluripotency markers, display a normal karyotype, retain the mutation (homozygous deletion of SMN1) and are able to differentiate into the three germ layers.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by mutation in the HTT gene encoding HTT protein. The mutant protein leads to the neuronal death through dysregulation of multiple cellular processes. HD human induced pluripotent stem cells (iPSCs) represent a useful and valid model for the disease study. iPSC line from HD patient with 47 CAG repeats in HTT was generated from blood mononuclear cells by non-integrating episomal vectors. The iPSC line retained the mutation, expressed pluripotency markers, had a normal karyotype and displayed in vitro differentiation to the three germ layers.

The induced pluripotent stem cell (iPSC) lines ICGi008-A and ICGi008-B were generated from dermal fibroblasts using episomal vectors expressing pluripotency factors. Dermal fibroblasts were obtained from a 55 year old male Сaucasian familial Alzheimer's disease (AD) patient carrying heterozygous V717I mutation in the APP gene. The generated iPSC lines maintained the original APP genotype, expressed pluripotency markers, exhibited a normal karyotype and retained the ability to differentiate into cell types of the three germ layers. The iPSC lines will be useful for the study of the AD molecular and cellular mechanisms and drug screening.

Studying Parkinson's disease (PD), one of the most common neurodegenerative disorders worldwide, requires different model systems, including patient-specific induced pluripotent stem cell lines. With the help of non-integrating episomal vectors the iPSC lines ICGi015-A and ICGi015-B were generated from blood mononuclear cells of PD patient, carrying three SNPs, associated with PD development. The obtained iPSC lines express pluripotency markers and demonstrate the ability to in vitro differentiate into the three germ layers. These cell lines may be useful for studying molecular mechanisms of PD and for drug screening.

Conduction and heart rhythm disorders can be caused by both functional pathology and severe organic lesions of the heart. The possibility of using cell-based replacement cell therapy derived from induced pluripotent stem cells to compensate for lost myocardial tissue or the conduction system is currently being studied. The aim of the work is to study the survival and functional activity of cardiomyocytes differentiated from induced human pluripotent stem cells in intramyocardial and subcutaneous abdominal transplantation in a clots of proteins of the basement membrane matrix Matrigel to the SCID mice. After 2 and 5 weeks after intramyocardial and 2, 7, 14, 21 and 28 days after subcutaneous transplantation, the survival and activity of cardiomyocytes were studied by cytological methods. Human cardiomyocytes were detected in mice for at least 35 days. after transplantation and did not cause ectopic electrical activity of the myocardium. When assessing the functional activity of cardiomyocytes in subcutaneous matrigel plugs using the method of optical mapping of calcium ion currents for 2-28 days. after injection, it was shown that only a small fraction of cardiomyocytes after transplantation was able to spontaneously oscillate the calcium ions. We assume that contractile cardiomyocytes obtained from induced pluripotent human cells lose their ability to spontaneous excitation during in vivo transplantation, and we observe only the activity of pacemaker cardiomyocytes in optical mapping.

Характерный маркер кардиальных стволовых клеток – тиразинкиназный рецептор c-kit. Клетки с фенотипом c-kit+ легко выделяются и культивируются in vitro. Однако истинным кардиомиогенным потенциалом обладают только кардиальные стволовые клетки, полученные из сердца постнатальных животных. Исследователи предполагают две популяции c-kit+ клеток в сердце различного происхождения в эмбриогенезе. Клетки c-kit+, способные дифференцироваться в кардиомиоциты, происходят из клеток первичного кардиального поля и исчезают вскоре после рождения, а те, что имеют проэпикардиальное происхождение, экспрессируют мезенхимальные маркеры и способны к дифференцировке в эндотелиальном, муральном и фибробластном направлениях. Ранее мы описали c-kit+ клетки, выделенные из фрагментов ушка правого предсердия, и исследовали их ангиогенный потенциал in vitro. В этом исследовании мы сравнили поверхностные маркеры мезенхимальных стволовых клеток костного мозга, кардиальных стволовых клеток и фибробластов кожи человека методом проточной цитометрии и полимеразной цепной реакции с обратной транскрипцией. Показали, что культура кардиальных стволовых клеток человека, полученная в результате магнитного сортинга с помощью антител на c-kit-рецептор является гетерогенной. В ней присутствуют клетки, несущие характерный набор поверхностных маркеров мезенхимальных стволовых клеток костного мозга, эндотелиальных и муральных клеток. Иммунофенотип фибробластов кожи также представлен характерным набором маркеров мезенхимальных стволовых клеток, за исключением дополнительного маркера CD10, соответствующего эластазе, нейтральной пептидазе. Кроме того, фибробласты кожи дифференцируются в остеогенном и адипогенном направлениях при использовании индукционных сред. Таким образом, фибробласты кожи человека, полученные согласно стандартным протоколам, являются мезенхимальными стволовыми клетками кожи. В соответствии с иммунофенотипом мезенхимальные стволовые клетки костного мозга, кардиальные стволовые клетки и фибробласты кожи экспрессируют гены паракринных факторов HGF, VEGF, PDGFb, ANG1, ANG2, IGF1, TGFb, обладающих кардиопротекторным и ангиогенным эффектами.