Derivation of autism spectrum disorder-specific induced pluripotent stem cells from peripheral blood mononuclear cells

Abstract

Induced pluripotent stem cells (iPSCs) hold tremendous potential both as a biological tool to uncover the pathophysiology of disease by creating relevant cell models and as a source of stem cells for cell-based therapeutic applications. Typically, iPSCs have been derived by the transgenic overexpression of transcription factors associated with progenitor cell or stem cell function in fibroblasts derived from skin biopsies. However, the need for skin punch biopsies to derive fibroblasts for reprogramming can present a barrier to study participation among certain populations of individuals, including children with autism spectrum disorders (ASDs). In addition, the acquisition of skin punch biopsies in non-clinic settings presents a challenge. One potential mechanism to avoid these limitations would be the use of peripheral blood mononuclear cells (PBMCs) as the source of the cells for reprogramming. In this article we describe, for the first time, the derivation of iPSC lines from PBMCs isolated from the whole blood of autistic children, and their subsequent differentiation in GABAergic neurons.

Figure 1

Derivation of ASD-specific iPSCs from PBMCs. Transgenic overexpression of OCT3/4, SOX2, KLF4 and c-MYC from doxycycline-inducible lentiviral-based vectors reprogrammed PBMCs derived from individuals with an ASD into iPSC lines. These iPSC colonies adopted the characteristic spherical colonies when grown on mitomycin C-treated MEFs similar to a well characterized control iPSC line. (A) Brightfield images of representative colonies from iPSC derived from two ASD individuals and the control iPSC. The ASD-specific iPSC lines stained positive for several markers of pluripotency (Nanog, Oct3/4, Sox2 and SSEA4) (B) and were alkaline phosphatase positive (C). (D) Quantitative real-time PCR confirmed the expression of pluripotency markers (Nanog, OCT3/4, KLF4, SOX2) and stem cell-associated transcripts (DPPA5 and ZFP42) from representative iPSC lines derived from ASD13 (cl1), ASD26 (cl1) and ASD49 (cl1-3).

Figure 2

ASD iPSC-derived Embryoid Bodies from PBMCs express markers for all three germ layer. A) Phase contrast images of representative EBs derived from the ASD 26 iPSC and ASD49 iPSC. B) Gene expression analysis for ectodermal (Nestin and NCAM), mesodermal (RUNX1 and Brachyury), and endodermal markers (GATA4).

Figure 3

Differentiation of ASD-specific iPSCs into GABAergic neurons. ASD-specific GABAergic neurons were derived from ASD26 and ASD49-specific iPSC lines. The ASD-specific iPSC-derived GABAergic neurons stained positively for both neuronal- (NCAM, MAP2 and β-tubulin III) and GABAergic (VGAT)-specific markers. Representative images from GABA-ergic neurons derived from ASD26 (A) and ASD49 (B) iPSCs are shown. C) Reverse transcription PCR analysis from the ASD26 and ASD49-specific iPSC-derived GABAergic neuronal cultures showed strong expression of the neuronal markers (NCAM and Nestin), mature neuronal markers (NeuroN, Synapsin, and MAP2), GABAergic neuron-specific markers (GAD67 (posterior) and VGAT (anterior)) and the positional markers (SIX3 and HoxB4). The anterior positional marker OTX2 was not expressed in the culture suggesting its expression is unresponsive to RA stimulation. Heterogeneity of the neuronal culture was indicated by expression of the immature neuronal marker DCX. D) Quantitation of the VGAT/NCAM double positive cells in the ASD26 and ASD49-specific iPSC-derived GABAergic neuronal cultures relative to the total number of cells.