Induced pluripotent stem cells derived from the developing striatum as a potential donor source for cell replacement therapy for Huntington disease

Abstract

Background: Cell replacement therapy (CRT) for Huntington disease (HD) requires a source of striatal (STR) progenitors capable of restoring the function lost due to STR degeneration. Authentic STR progenitors can be collected from the fetal putative striatum, or whole ganglionic eminence (WGE), but these tissues remain impractical for widespread clinical application, and alternative donor sources are required. Here we begin exploring the possibility that induced pluripotent stem cells (iPSC) derived from WGE may retain an epigenetic memory of their tissue of origin, which could enhance their ability to differentiate into STR cells.

Results: We generate four iPSC lines from human WGE (hWGE) and establish that they have a capacity similar to human embryonic stem cells with regard to their ability to differentiate toward an STR phenotype, as measured by expression and demethylation of key STR genes, while maintaining an overall different methylome. Finally, we demonstrate that these STR-differentiated hWGE iPSCs share characteristics with hWGE (i.e., authentic STR tissues) both in vitro and following transplantation into an HD model. Overall, iPSCs derived from human WGE show promise as a donor source for CRT for HD.

Keywords: Huntington disease; cell therapy; epigenetic memory; fetal tissue; iPSC; striatal differentiation.

Figure 1

Induced PSCs can be generated from hWGE tissues. (A) Images of hESC and human iPSC colonies. (B) RT-PCR of OCT4, SOX2, NANOG, LIN28 and C-MYC expression in undifferentiated hWGE iPSCs. (C) ICC for OCT4 (green), SOX2 (green) and TRA-1-60 (red) in undifferentiated hWGE iPSCs. (D) ICC for endoderm (vimentin, left, red; AFP, right, red), mesoderm (α-SMA, left, red; desmin, right, green) and ectoderm (nestin, left, green; β-III tubulin, right, green) with Hoechst (blue) in in vitro spontaneously differentiated hWGE iPSCs. (E) Hematoxylin and eosin staining in teratomas from immune-deficient mice following subcutaneous injection of hWGE iPSCs. Arrows indicate adipose and gland-like tissues (endoderm), chondroblasts and smooth muscle fibers (mesoderm) and neural rosettes (ectoderm). RT-PCR, reverse transcription-polymerase chain reaction; ICC, immunocytochemistry staining; AFP, alpha fetoprotein; SMA, smooth muscle actin. (Color version of figure is available online).

Figure 2

Human WGE iPSCs can differentiate toward an STR fate but retain distinct methylation profiles. (A) Human WGE iPSCs undergoing STR differentiation: neuroectodermal progenitors expressing ZO-1 (red) and Nestin (green) (day 12 to day 14); ventral telencephalon progenitors expressing DLX1 (red) and DLX2 (green) (day 14 to day 18); STR progenitors expressing β-III tubulin (red) and ISL1 (green) (day 18 to day 25); MSNs expressing CTIP2 (red) and DARPP-32 (green) (day 35 to day 45); and a phase contrast image showing mature neuronal morphology (see also supplementary Figure 2). (B) Heat map and (C) MDS plot of the 1000 most variably methylated probes between undifferentiated PSCs and differentiated STR cells. (D) Results of gene enrichment analysis of differentially methylated genes between PSC (n = 3) and STR (n = 3) samples, showing the top four most significant associated terms in the Allen Brain Atlas upregulated gene library, adjusted Pvalues and associated genes. MDS, multidimensional scaling. (Color version of figure is available online).

Figure 3

STR-differentiated hWGE iPSCs share characteristics with hWGE. (A) QPCR analysis showing relative gene expression in hWGE iPSC STR cells (n = 5) relative to hWGE (n = 6). (B) Electrophysiological assessment of hWGE iPSC STR cells and hWGE (see also supplementary Table 3). (C) Images of hWGE iPSC STR cells and hWGE grafts: immunohistochemical staining for HuNu (brown) and immunofluorescent staining for HuNu (red) and human-specific DARPP-32 (huDARPP-32, green). (D) Graft volume and counts of DARPP-32 cells per graft. *P< 0.05, **P< 0.01, ***P< 0.001. HuNu, human nuclear antigen; QPCR, quantitative polymerase chain reaction. (Color version of figure is available online).