PARP1 Gene Knockout Suppresses Expression of DNA Base Excision Repair Genes.

The effect of PARP1 knockout in HEK293 cells on the gene expression of DNA base excision repair (BER) proteins was studied. It was shown that the expression of all differentially expressed genes (DEGs) of BER was reduced by knockout. The expression of the DNA glycosylase gene NEIL1, which is considered to be one of the common "hubs" for binding BER proteins, has changed the most. The expression of genes of auxiliary subunits of DNA polymerases δ and ε is also significantly reduced. The PARP1 gene knockout cell line obtained is an adequate cell model for studying the activity of the BER process in the absence of PARP1 and testing drugs aimed at inhibiting repair processes. It has been found for the first time that knockout of the PARP1 gene results in a significant change in the level of expression of proteins responsible for ribosome biogenesis and the functioning of the proteasome.

Identification of specific gene methylation patterns during motor neuron differentiation from spinal muscular atrophy patient-derived iPSC.

Spinal muscular atrophy is a progressive motor neuron disorder caused by deletions or point mutations in the SMN1 gene. It is not known why motor neurons are particularly sensitive to a decrease in SMN protein levels and what factors besides SMN2 underlie the high clinical heterogeneity of the disease. Here we studied the methylation patterns of genes on sequential stages of motor neuron differentiation from induced pluripotent stem cells derived from the patients with SMA type I and II. The genes involved in the regulation of pluripotency, neural differentiation as well as those associated with spinal muscular atrophy development were included. The results show that the PAX6, HB9, CHAT, ARHGAP22, and SMN2 genes are differently methylated in cells derived from SMA patients compared to the cells of healthy individuals. This study clarifies the specificities of the disease pathogenesis and extends the knowledge of pathways involved in the SMA progression.

Generation of an induced pluripotent stem cell line ICGi030-A from a Wilson's disease patient carrying a frameshift mutation p.Lys1013fs and missense mutation p.H1069Q in the ATP7B gene.

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.

Generation of an induced pluripotent stem cell line, ICGi028-A, by reprogramming peripheral blood mononuclear cells of a patient suffering from hypertrophic cardiomyopathy and carrying a heterozygous p.E510Q mutation in HADHA.

Hypertrophic cardiomyopathy (HCM) is a frequent cardiovascular pathology caused by a huge number of mutations in sarcomere-associated proteins. This genetic diversity leads to differences in pathogenetic mechanisms and hampers HCM therapy. Cardiomyocytes derived from patient-specific induced pluripotent stem cells give new opportunities for studying underlying HCM mechanisms. We generated an iPSC line from peripheral blood mononuclear cells of an HCM patient with a heterozygous p.E510Q mutation in HADHA using non-integrating episomal vectors. The iPSC line showed typical morphology, expression of pluripotency markers, capacity to be differentiated into derivatives of three germ layers, and presence of the patient-specific mutation.

Generation of an induced pluripotent stem cell line, ICGi029-A, by reprogramming peripheral blood mononuclear cells of a patient suffering from hypertrophic cardiomyopathy and carrying a heterozygous p.N515del mutation in MYBPC3.

Hypertrophic cardiomyopathy (HCM) is a common cardiovascular disease. However, effective methods of its therapy have not been developed so far. To date patient-specific induced pluripotent stem cell-derived cardiomyocytes are supposed to be a useful tool for studying HCM molecular mechanisms and to help find new approaches to HCM therapy. Using non-integrating episomal vectors, we generated an iPSC line from peripheral blood mononuclear cells of an HCM patient carrying a heterozygous p.N515del mutation in MYBPC3. The iPSC line expressed pluripotency markers, gave rise to derivatives of three germ layers during spontaneous differentiation, had normal karyotype, and retained the patient-specific mutation.

Generation of a spinal muscular atrophy type III patient-specific induced pluripotent stem cell line ICGi003-A.

Spinal muscular atrophy (SMA) is a genetic disease, which characterized by the degeneration of motor neurons in the spinal cord and further striated muscle atrophy. The research of the processes in diseased neurons is complicated due to the impossibility of obtaining them safely from patients. Thus, we generated SMA type III induced pluripotent stem cell lines via using non-integrated episomal plasmid vectors. The resulting cell line expresses the major pluripotency markers and can differentiate in vitro into derivatives of three germ layers. The iPSC line can be used for further studies by providing in vitro the relevant cell types.

Generation of three Duchenne muscular dystrophy patient-derived induced pluripotent stem cell (iPSC) lines ICGi002-A, ICGi002-B and ICGi002-C.

Duchenne muscular dystrophy (DMD) is a severe and rapidly progressive hereditary muscular disease with X-linked recessive inheritance, occurring mainly in males. A complete loss of dystrophin resulted from out-of-frame deletion mutations in the DMD gene leads to Duchenne muscular dystrophy. DMD induced pluripotent stem cells (iPSCs) are a suitable cell model to study muscle development and disease mechanisms underlying muscular dystrophy and to screen novel compounds with potential therapeutic effects. We generated iPSCs from a DMD patient using non-integrating episomal plasmid vectors. The obtained iPSC lines showed ESC-like morphology, expression pluripotency markers, displayed a normal karyotype and possessed trilineage differentiation potential.

Generation of two induced pluripotent stem cell lines from peripheral blood mononuclear cells of a patient with Wilson's disease.

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.

Generation of induced pluripotent stem cell lines ICGi021-A and ICGi022-A from peripheral blood mononuclear cells of two healthy individuals from Siberian population.

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.

Generation of two clonal iPSC lines, ICGi019-A and ICGi019-B, by reprogramming peripheral blood mononuclear cells of a patient suffering from hypertrophic cardiomyopathy and carrying a heterozygous p.M659I mutation in MYH7.

Hypertrophic cardiomyopathy (HCM) is one of the most frequent cardiovascular diseases but no methods to prevent its progression have been developed. Cardiomyocytes derived from patient-specific induced pluripotent stem cells can become a platform to study pathogenesis of the disease and to search for more effective therapy methods. We generated two iPSC lines from peripheral blood mononuclear cells of an HCM patient with heterozygous p.M659I mutation in MYH7 using episomal vectors. The iPSC lines expressed pluripotency markers, demonstrated ability to spontaneously differentiate into derivatives of three germ layers, and retained the mutation.

Studying ALS: Current Approaches, Effect on Potential Treatment Strategy.

Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative diseases, characterized by inevitable progressive paralysis. To date, only two disease modifying therapeutic options are available for the patients with ALS, although they show very modest effect on disease course. The main reason of failure in the field of pharmacological correction of ALS is inability to untangle complex relationships taking place during ALS initiation and progression. Traditional methods of research, based on morphology or transgenic animal models studying provided lots of information about ALS throughout the years. However, translation of these results to humans was unsuccessful due to incomplete recapitulation of molecular pathology and overall inadequacy of the models used in the research.In this review we summarize current knowledge regarding ALS molecular pathology with depiction of novel methods applied recently for the studies. Furthermore we describe present and potential treatment strategies that are based on the recent findings in ALS disease mechanisms.

Generation of induced pluripotent stem cell line ICGi018-A from peripheral blood mononuclear cells of a patient with Huntington's disease.

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.

Generation of an induced pluripotent stem cell line, ICGi014-A, by reprogramming peripheral blood mononuclear cells from a patient with homozygous D90A mutation in SOD1 causing Amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by death of motor neurons. To date, neither etiology nor pathogenesis of ALS are known, which leads to the absence of an effective treatment strategy. ALS patient-specific induced pluripotent stem cells (iPSCs) represent an excellent tool for the disease study. We obtained iPSCs line from peripheral blood mononuclear cells of the patient with homozygous Asp90Ala mutation in the SOD1 gene using non-integrating episomal vectors. The iPSCs line retained pathological genotype and expressed pluripotency markers. It also displayed a normal karyotype and the ability to differentiate into derivatives of three germ layers.

Generation of two iPSC lines, (ICGi015-A and ICGi015-B), by reprogramming peripheral blood mononuclear cells from a patient with Parkinson's disease.

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.

Methods for Correction of the Single-Nucleotide Substitution c.840C>T in Exon 7 of the SMN2 Gene.

The CRISPR/Cas technology has a great potential in the treatment of many hereditary diseases. One of the prospective models for the CRISPR/Cas-mediated therapy is spinal muscular atrophy (SMA), a disease caused by deletion of the SMN1 gene that encodes the SMN protein required for the survival of motor neurons. SMA patients' genomes contain either single or several copies of SMN2 gene, which is a paralog of SMN1. Exon 7 of SMN2 has the single-nucleotide substitution c.840C>T leading to the defective splicing and decrease in the amounts of the full-length SMN. The objective of this study was to create and test gene-editing systems for correction of the single-nucleotide substitution c.840C>T in exon 7 of the SMN2 gene in fibroblasts, induced pluripotent stem cells, and motor neuron progenitors derived from a SMA patient. For this purpose, we used plasmid vectors expressing CRISPR/Cas9 and CRISPR/Cpf1, plasmid donor, and 90-nt single-stranded oligonucleotide templates that were delivered to the target cells by electroporation. Although sgRNA_T2 and sgRNA_T3 guiding RNAs were more efficient than sgRNA_T1 in fibroblasts (p < 0.05), no significant differences in the editing efficiency of sgRNA_T1, sgRNA_T2, and sgRNA_T3 was observed in patient-specific induced pluripotent stem cells and motor neuron progenitors. The highest editing efficiency in induced pluripotent stem cells and motor neuron progenitors was demonstrated by the sgRNA_T1 and 90-nt single-stranded oligonucleotide donors.

A New MicroRNA Cluster Involved in the Reprogramming to a Pluripotent State.

Reprogramming of somatic cells to a pluripotent state is a complex, multistage process that is regulated by many factors. Among these factors, non-coding RNAs and microRNAs (miRNAs) have been intensively studied in recent years. MiRNAs play an important role in many processes, particularly in cell reprogramming. In this study, we investigated the reprogramming of rat fibroblasts with a deleted locus encoding a cluster comprising 14 miRNAs (from miR-743a to miR-465). The deletion of this locus was demonstrated to decrease significantly the efficiency of the cell reprogramming. In addition, the cells produced by the reprogramming differed from rat embryonic and induced pluripotent stem cells, which was an indication that reprogramming in these cells had not been completed. We suggest that this miRNA cluster or some of its members are involved in regulating the reprogramming of rat cells to a pluripotent state.

Applying Patient-Specific Induced Pluripotent Stem Cells to Create a Model of Hypertrophic Cardiomyopathy.

Generation of patient-specific induced pluripotent stem cells (iPSCs) and their subsequent differentiation into cardiomyocytes opened new opportunities for studying pathogenesis of inherited cardiovascular diseases. One of these diseases is hypertrophic cardiomyopathy (HCM) for which no efficient therapy methods have been developed so far. In this study, the approach based on patient-specific iPSCs was applied to create a model of the disease. Genetic analysis of a hypertrophic cardiomyopathy patient revealed R326Q mutation in the MYBPC3 gene. iPSCs of the patient were generated and characterized. The cells were differentiated into cardiomyocytes together with the control iPSCs from a healthy donor. The patient's iPSC-derived cardiomyocytes exhibited early HCM features, such as abnormal calcium handling and increased intracellular calcium concentration. Therefore, cardiomyocytes obtained by directed differentiation of iPSCs from the HCM patient can be used as a model system to study HCM pathogenesis.

A Platform for Studying Neurodegeneration Mechanisms Using Genetically Encoded Biosensors.

Patient-specific induced pluripotent stem cells (iPSCs) capable of differentiation into required cell type are a promising model for studying various pathological processes and development of new therapeutic approaches. However, no conventional strategies for using iPSCs in disease research have been established yet. Genetically encoded biosensors can be used for monitoring messenger molecules, metabolites, and enzyme activity in real time with the following conversion of the registered signals in quantitative data, thus allowing evaluation of the impact of certain molecules on pathology development. In this article, we describe the development of a universal cell-based platform for studying pathological processes associated with amyotrophic lateral sclerosis. For this purpose, we have created a series of plasmid constructs for monitoring endoplasmic reticulum stress, oxidative stress, apoptosis, and Ca2+-dependent hyperexcitability and generated transgenic iPSC line carrying mutation in the superoxide dismutase 1 gene (SOD1) and healthy control cell line. Both cell lines have specific transactivator sequence required for doxycycline-controlled transcriptional activation and can be used for a single-step biosensor insertion.

Generation of two iPSC lines (ICGi008-A and ICGi008-B) from skin fibroblasts of a patient with early-onset Alzheimer's disease caused by London familial APP mutation (V717I).

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.

Generation of induced pluripotent stem cell line, ICGi007-A, by reprogramming peripheral blood mononuclear cells from a patient with Huntington's disease.

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. Resource table.