Generation of Isogenic Controls for In Vitro Disease Modelling of X-Chromosomal Disorders

Abstract

Generation of proper controls is crucial in induced pluripotent stem cell (iPSC) studies. X-chromosomal disorders offer the potential to develop isogenic controls due to random X-chromosomal inactivation (XCI). However, the generation of such lines is currently hampered by skewed X-inactivation in fibroblast lines and X-chromosomal reactivation (XCR) after reprogramming. Here we describe a method to generate a pure iPSC population with respect to the specific inactivated X-chromosome (Xi). We used fibroblasts from Rett patients, who all have a causal mutation in the X-linked MeCP2 gene. Pre-sorting these fibroblasts followed by episomal reprogramming, allowed us to overcome skewness in fibroblast lines and to retain the X-chromosomal state, which was unpredictable with lentiviral reprogramming. This means that fibroblast pre-sorting followed by episomal reprogramming can be used to reliably generate iPSC lines with specified X-chromosomal phenotype such as Rett syndrome. Electronic supplementary material The online version of this article (10.1007/s12015-018-9851-8) contains supplementary material, which is available to authorized users.

Quantitative proteomic alterations of human iPSC-based neuronal development indicate early onset of Rett syndrome

“…RTT patient and iCTR fibroblasts were reprogrammed into iPSCs via electroporation of reprogramming plasmids as published before [24]. Pluripotency was confirmed using the PluriTest analysis ( Fig.…”

“…So far, only a few proteomic studies have been performed on RTT human derived tissue [19, 30, 31]. The current study provides mass spectrometry-based quantitative proteomic data, depth of about 7000 proteins, using an earlier developed iPSC-based models involving RTT patient cells and isogenic controls [24]. We showed that changes in dendrite morphology or synaptic defects, previously associated with RTT [22, 32], already become apparent at early developmental stages.…”

“…To generate pure RTT fibroblast lines and isogenic controls cells were detached from cell culture plate and single fibroblasts were seeded in wells of a 96-well plate. Cells were further expanded and characterized on their MeCP2 state by immunocytochemistry and PCR [24]. All of our experiments were exempt from the approval of the institutional review board.…”

Quantitative proteomic analysis shows alterations in patient Rett syndrome iPSC cultures at early neuronal progenitor stages

“…cells expressing the healthy X-chromosome, fibroblasts were detached from cell culture plate and single fibroblasts were seeded in a 96-well plate. Cells were further expanded and characterized for their MeCP2 state by immunocytochemistry and PCR [24]. All of our experiments were exempt from the approval of the institutional review board.…”

“…RTT patient and iCTR fibroblasts were reprogrammed into iPSCs via electroporation of reprogramming plasmids [24]. Pluripotency was confirmed using classic assays, including immunocytochemistry (Additional file 1: Figure S1a) and RNA expression (Additional file 1: Figure S1b).…”