Integration-Free Induced Pluripotent Stem Cells Model Genetic and Neural Developmental Features of Down Syndrome Etiology

Abstract

Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild-type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress-induced apoptosis, and this could be prevented by the antioxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform.

Molecular Mechanisms Regulating Impaired Neurogenesis of Fragile X Syndrome Human Embryonic Stem Cells

“…To test this hypothesis, we knocked down SOX2 expression and overexpressed SOX9 in FX-hNPCs (Fig. 5C) and measured their neural status by analyzing the relative expression of GFAP and MAP2, as previously described [17,28,44]. Our results show that SOX2 inhibition and SOX9 overexpression in FX-hNPCs resulted in a significant increase in both MAP2 and GFAP levels, reflecting a progression of these hNPCs from a primitive to a more advanced neural status.…”

“…We thus subjected hNPCs to 30 days of neuronal differentiation in the presence of Wnt signaling modulators: a GSK3b inhibitor (CHIR99021; ''CHIR''), which reduces GSK3b-mediated phosphorylation of b-catenin, thus increasing Wnt/b-catenin activity [48], and a specific tankyrase inhibitor (XAV939, ''XAV''), which stabilizes Axin levels in the cells, thus decreasing Wnt/b-catenin activity in a GSK3b-independent manner [49]. The MAP2/GFAP bioassay was implemented to measure the efficiency of neuronal neurogenesis, as has been done previously by us and others [17,28,44]. In control cultures (ie, no CHIR or XAV added), the efficiency of neuronal differentiation was significantly lower for FX lines than for HUES lines, as expected (Fig.…”

Deficits in human trisomy 21 iPSCs and neurons

“…OS was detectable in Ts21 differentiated neuronal cultures but not Ts21 iPSCs. Therefore, a unique mechanism may be in play in Ts21 cells during early developmental stages, where compensatory changes in OS genes allow for nearly normal cell proliferation and differentiation but cells remain highly susceptible to insults later in development (38,(47)(48)(49)(50). This mechanism may exacerbate the consequences of APP overexpression that predispose DS individuals to develop Alzheimer's disease pathology (51)(52)(53)(54).…”

The potential of induced pluripotent stem cells in models of neurological disorders: implications on future therapy

“…By comparison, disease-specific iPSCs provide new prospects for disease-related R&D by enabling screening for genes and disease processes potentially modifiable by drugs identified through in vitro screening. Consequently, iPSCs have been successfully derived from patients with NDv disorders including schizophrenia [3][4][5][6][7][8][9][10][11], Down's syndrome [12][13][14][15][16][17][18][19][20][21], autism spectrum disorders (ASDs) including fragile X, Rett and Timothy syndromes [22][23][24][25][26][27][28][29][30][31][32][33][34][35], and epilepsy [36][37][38][39], as well as NDg disorders such as Alzheimer's disease [40][41][42][43][44][45][46][47][48], Parkinson's disease [49][50][51][52]…”