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Sovremennye Tehnologii v Medicine
Article . 2016 . Peer-reviewed
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A Platform for Studying Molecular and Cellular Mechanisms of Parkinson’s Disease Based on Human Induced Pluripotent Stem Cells

Authors: E.V. Novosadova; E.D. Nekrasov; I.V. Chestkov; A.V. Surdina; E.M. Vasina; A.N. Bogomazova; E.S. Manuilova; +10 Authors

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

Abstract

Цель исследования получение индуцированных плюрипотентных стволовых клеток (ИПСК) от пациентов с различными формами болезни Паркинсона (БП) и создание на этой основе платформы для изучения патогенеза болезни на молекулярном и клеточном уровнях с разработкой протокола дифференцировки данных стволовых клеток. Материалы и методы. ИПСК получали из репрограммированных с помощью лентивирусных векторов и на основе вируса Сендай культивированных фибробластов кожи пяти пациентов с различными формами БП (PARK8, PARK2, GBA-ассоциированная и спорадическая формы). Полученные клоны ИПСК культивировали до стадии эмбриоидных тел и после спонтанной дифференцировки окрашивали иммуноцитохимически. Анализ экспрессии генов и нейрональных маркеров в этих линиях ИПСК проводили с помощью полимеразной цепной реакции, сопряженной с обратной транскрипцией. Результаты. Полученные клоны ИПСК имели нормальный кариотип 46 XY, специфично окрашивались антителами на маркеры плюропатентности Oct4, Nanog, TRA-1-81 и SSEA-4 и экспрессировали маркерные гены, отвечающие за поддержание плюрипотентного состояния. В культурах, дифференцированных ИПСК, выявлены клетки, положительно окрашенные антителами на маркеры трех зародышевых листков (эктодермы, мезодермы и энтодермы). Разработан эффективный протокол дифференцировки ИПСК в дофаминергические нейроны, что подтверждено экспрессией маркера этих нейронов фермента тирозингидроксилазы. Заключение. На основе детально охарактеризованных ИПСК от пациентов с различными формами БП и разработанного клеточного протокола создана платформа для изучения патогенеза БП на молекулярном и клеточном уровнях. Получение клеточных культур, обогащенных дофаминергическими нейронами, открывает перспективу их использования для персонализированной клеточной заместительной терапии БП.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 marker tyrosine 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.

Keywords

cell reprogramming; induced pluripotent stem cells; platform for iPSC; fibroblasts; dopaminergic neurons; Parkinson’s disease, КЛЕТОЧНОЕ РЕПРОГРАММИРОВАНИЕ,ИНДУЦИРОВАННЫЕ ПЛЮРИПОТЕНТНЫЕ СТВОЛОВЫЕ КЛЕТКИ,ПЛАТФОРМА ДЛЯ ИПСК,ФИБРОБЛАСТЫ,ДОФАМИНЕРГИЧЕСКИЕ НЕЙРОНЫ,БОЛЕЗНЬ ПАРКИНСОНА

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    popularity
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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
14
Top 10%
Average
Average
gold