A Platform for Studying Molecular and Cellular Mechanisms of Parkinson’s Disease Based on Human Induced Pluripotent Stem Cells

Abstract

The aim of the study was to obtain induced pluripotent stem cells (iPSCs) from patients with various forms of Parkinson's disease (PD), and to create on this basis a platform for studying the pathogenesis of the disease at the molecular and cellular level with the development of a protocol of the stem cell differentiation.Materials and Methods. iPSCs were derived from cultured skin fibroblasts, taken from five patients with various forms of PD (PARK8, PARK2, GBA-associated and sporadic forms), and reprogrammed with the help of lentiviral vectors and on the basis of Sendai virus. The obtained iPSCs clones were cultured to the stage of embryonic bodies and, after spontaneous differentiation, stained immunocytochemically. Gene expression and neural markers in these iPSCs lines were analysed using reverse transcription polymerase chain reaction.Results. The obtained iPSCs clones had a normal 46 XY karyotype, stained specifically with Oct4, Nanog, TRA-1-81 and SSEA-4 antibodies, and expressed marker genes responsible for maintaining the pluripotent condition. In the cultures of differentiated iPSCs, cells positively stained for the markers of the three primary germ layers (ectoderm, mesoderm, and endoderm) have been revealed. An effective protocol of iPSCs differentiation into dopaminergic neurons has been worked out, and confirmed by the expression of the specific markertyrosine hydroxylase enzyme.Conclusion. On the basis of explicitly characterized iPSCs from patients with various forms of PD and the developed cellular protocol, a platform for studying the pathogenesis of PD at the molecular and cellular level has been created. Obtaining cell population enriched with dopaminergic neurons opens a perspective for their application for personalized cell replacement PD therapy.

Influence of N-Arachidonoyl Dopamine and N-Docosahexaenoyl Dopamine on the Expression of Neurotrophic Factors in Neuronal Differentiated Cultures of Human Induced Pluripotent Stem Cells under Conditions of Oxidative Stress

“…This work was carried out on the iPSC line (IPSHD1.1S) obtained from skin fibroblasts of a healthy donor using CytoTune ™ -iPS 2.0 Sendai Reprogramming Kit. The reprogramming vector includes the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc, shown to be sufficient for efficient reprogramming [ 24 , 25 ]. The obtained iPSC expressed the essential pattern of specific pluripotency-associated genes, possessed a normal karyotype and were capable of producing the derivatives of three embryonic germ layers [ 25 ].…”

“…iPSC lines were cultured in mTeSR medium (“STEM CELL Technologies” Vancouver, BC, Canada) on Matrigel-coated substrates as previously described. The differentiation of iPSCs into neuronal progenitors was performed as previously described [ 25 ].…”

Current State-of-the-Art and Unresolved Problems in Using Human Induced Pluripotent Stem Cell-Derived Dopamine Neurons for Parkinson’s Disease Drug Development

“…The possibility of using iPS cells for drug screening attracts significant attention in the modern literature with multiple reviews devoted to this issue [ 181 , 182 ], and the general aspects of studying pathogenesis of PD and the development of iPS cell-based novel approaches for its treatment are reviewed in [ 183 ]. Particularly, we previously developed the basic test system employing human iPS-derived NPC and neurons for primary assessment of neuroprotective substances [ 184 ]. Recently, a 3D-culture based high throughput screening system was described that offers improved data reproducibility through standardization of organoid size [ 185 ].…”

A Hybrid Detection Method Based on Peroxidase-mediated Signal Amplification and Click Chemistry for Highly Sensitive Background-free Immunofluorescent Staining

“…Human-induced pluripotent stem (iPS) cell lines IPSHD1.1S and IPSPDSP1S were generated previously by reprogramming the human skin fibroblasts with Oct4, SOX2, Nanog, and Klf4 genes. 7 iPS cells were cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC, Canada) on the plastic Petri dishes (SPL Life Sciences, Pocheon, Gyeonggi, South Korea) coated with Matrigel (BD Biosciences, San Jose, CA, USA).…”