Induced pluripotent stem (iPS) cells can be derived from a wide range of somatic cells via overexpression of a set of specific genes. With respect to their properties, iPS cells closely resemble embryonic stem cells. Because of their main property, pluripotency, iPS cells have excellent prospects for use in substitutive cell therapy; however, the methods of directed differentiation of iPS cells have not been yet sufficiently elaborated. In this work, we derived human iPS cells from fetal neural stem (FNS) cells by transfection with a polycistronic plasmid vector carrying the mouse Oct4, Sox2, Klf4, and c-Myc genes or a plasmid expressing the human OCT4 gene. We have shown that human FNS cells can be effectively reprogrammed despite a low transfection level (10%–15%) and that the use of 2-propylvaleric (valproic) acid and BIX-01294 increases the yield of iPS cell clones to ∼7-fold. Further, transient expression of OCT4 alone is sufficient for reprogramming. The iPS cells obtained express all the major markers of embryonic stem cells and are able to differentiate in vitro into ectodermal, mesodermal, and endodermal derivatives. In addition, we have found that the human iPS cells derived from FNS cells can be successfully subjected to in vitro directed chondrogenic differentiation to form functional cartilaginous tissue.

GBA variants increase the risk of Parkinson’s disease (PD) by 10 times. The GBA gene encodes the lysosomal enzyme glucocerebrosidase (GCase). The p.N370S substitution causes a violation of the enzyme conformation, which affects its stability in the cell. We studied the biochemical characteristics of dopaminergic (DA) neurons generated from induced pluripotent stem cells (iPSCs) from a PD patient with the GBA p.N370S mutation (GBA-PD), an asymptomatic GBA p.N370S carrier (GBA-carrier), and two healthy donors (control). Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), we measured the activity of six lysosomal enzymes (GCase, galactocerebrosidase (GALC), alpha-glucosidase (GAA), alpha-galactosidase (GLA), sphingomyelinase (ASM), and alpha-iduronidase (IDUA)) in iPSC-derived DA neurons from the GBA-PD and GBA-carrier. DA neurons from the GBA mutation carrier demonstrated decreased GCase activity compared to the control. The decrease was not associated with any changes in GBA expression levels in DA neurons. GCase activity was more markedly decreased in the DA neurons of GBA-PD patient compared to the GBA-carrier. The amount of GCase protein was decreased only in GBA-PD neurons. Additionally, alterations in the activity of the other lysosomal enzymes (GLA and IDUA) were found in GBA-PD neurons compared to GBA-carrier and control neurons. Further study of the molecular differences between the GBA-PD and the GBA-carrier is essential to investigate whether genetic factors or external conditions are the causes of the penetrance of the p.N370S GBA variant.

The derivation of stable multipotent trophoblast stem (TS) cell lines from preimplantation, and early postimplantation mouse embryos has been reported previously. FGF4, and its receptor FGFR2, have been identified as embryonic signaling factors responsible for the maintenance of the undifferentiated state of multipotent TS cells. Here we report the derivation of stable TS-like cell lines from the vole M. rossiaemeridionalis, in the absence of FGF4 and heparin. Vole TS-like cells are similar to murine TS cells with respect to their morphology, transcription factor gene expression and differentiation in vitro into derivatives of the trophectoderm lineage, and with respect to their ability to invade and erode host tissues, forming haemorrhagic tumours after subcutaneous injection into nude mice. Moreover, vole TS-like cells carry an inactive paternal X chromosome, indicating that they have undergone imprinted X inactivation, which is characteristic of the trophoblast lineage. Our results indicate that an alternative signaling pathway may be responsible for the establishment and stable proliferation of vole TS-like cells.

Huntington’s disease (HD) is a severe neurodegenerative disorder caused by a CAG triplet expansion in the first exon of the HTT gene. Here we report the introduction of an HD mutation into the genome of healthy human embryonic fibroblasts through CRISPR/Cas9-mediated homologous recombination. We verified the specificity of the created HTT-editing system and confirmed the absence of undesirable genomic modifications at off-target sites. We showed that both mutant and control isogenic induced pluripotent stem cells (iPSCs) derived by reprogramming of the fibroblast clones can be differentiated into striatal medium spiny neurons. We next demonstrated phenotypic abnormalities in the mutant iPSC-derived neural cells, including impaired neural rosette formation and increased sensitivity to growth factor withdrawal. Moreover, using electron microscopic analysis, we detected a series of ultrastructural defects in the mutant neurons, which did not contain huntingtin aggregates, suggesting that these defects appear early in HD development. Thus, our study describes creation of a new isogenic iPSC-based cell system that models HD and recapitulates HD-specific disturbances in the mutant cells, including some ultrastructural features implemented for the first time.

Mutations in the GBA1 gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), cause Gaucher disease (GD) and are the most common genetic risk factor for Parkinson’s disease (PD). Pharmacological chaperones (PCs) are being developed as an alternative treatment approach for GD and PD. To date, NCGC00241607 (NCGC607) is one of the most promising PCs. Using molecular docking and molecular dynamics simulation we identified and characterized six allosteric binding sites on the GCase surface suitable for PCs. Two sites were energetically more preferable for NCGC607 and located nearby to the active site of the enzyme. We evaluated the effects of NCGC607 treatment on GCase activity and protein levels, glycolipids concentration in cultured macrophages from GD (n = 9) and GBA-PD (n = 5) patients as well as in induced human pluripotent stem cells (iPSC)—derived dopaminergic (DA) neurons from GBA-PD patient. The results showed that NCGC607 treatment increased GCase activity (by 1.3-fold) and protein levels (by 1.5-fold), decreased glycolipids concentration (by 4.0-fold) in cultured macrophages derived from GD patients and also enhanced GCase activity (by 1.5-fold) in cultured macrophages derived from GBA-PD patients with N370S mutation (p < 0.05). In iPSC-derived DA neurons from GBA-PD patients with N370S mutation NCGC607 treatment increased GCase activity and protein levels by 1.1-fold and 1.7-fold (p < 0.05). Thus, our results showed that NCGC607 could bind to allosteric sites on the GCase surface and confirmed its efficacy on cultured macrophages from GD and GBA-PD patients as well as on iPSC-derived DA neurons from GBA-PD patients.