Genetic correction of Huntington's disease phenotypes in induced pluripotent stem cells

Abstract

Huntington's disease (HD) is caused by a CAG expansion in the huntingtin gene. Expansion of the polyglutamine tract in the huntingtin protein results in massive cell death in the striatum of HD patients. We report that human induced pluripotent stem cells (iPSCs) derived from HD patient fibroblasts can be corrected by the replacement of the expanded CAG repeat with a normal repeat using homologous recombination, and that the correction persists in iPSC differentiation into DARPP-32-positive neurons in vitro and in vivo. Further, correction of the HD-iPSCs normalized pathogenic HD signaling pathways (cadherin, TGF-β, BDNF, and caspase activation) and reversed disease phenotypes such as susceptibility to cell death and altered mitochondrial bioenergetics in neural stem cells. The ability to make patient-specific, genetically corrected iPSCs from HD patients will provide relevant disease models in identical genetic backgrounds and is a critical step for the eventual use of these cells in cell replacement therapy.

Figure 1. Targeted Correction of the Expanded Allele in HD-iPSCs

(A) Exon 1 (e1) and surrounding regions of the expanded HD locus of patient-derived HD-iPSCs containing 72 polyglutamine repeats; the designed targeting vector from BAC RP11-866L6 with short and long arms of 4.5 and 10 kb respectively; and the resulting correctly targeted allele with 21 polyglutamine repeats and FLP flanked PGK-neo and EGFP cassettes inserted upstream of the reported promoter region. HindIII sites (H) and resulting targeted fragment sizes using the 300 bp external Southern probe (black bar) are shown. (B) Brightfield (BF) and GFP fluorescent images of HD-iPSCs nucleofected with pEGFP plasmid at 1 day postnucleofection and after 3 weeks of G418 selection. (C) Primary PCR-based screen of G418 resistant clones for loss of the expanded (72 CAG) allele. Targeted clones indicated in green were later verified by Southern blot as correctly targeted alleles. (D) Southern blotting analysis of HindIII digested HD-iPSC clone genomic DNA and control samples. A P32 radiolabeled 300 bp external probe hybridizes with a 10 kb nontargeted (WT) or 5 kb targeted HindIII. Labeled are positive and negative BAC DNA controls for cassette insertion or noninsertion (BAC Rec+ and BAC Rec−, respectively); nontargeted HD-iPSC clones C99 and C112; and correctly targeted HD-iPSC clones C127 and C116. For further Southern blot analysis and sequencing of clones, see Figure S1. (E) Expanded polyglutamine correction revealed by western blot analysis of lysates from HD-iPSCs and corrected clones C116 and C127 using antibodies for huntingtin (Htt2166) and expanded polyglutamine (1C2). A line of normal iPSCs (Park et al., 2008) derived from non-HD patient fibrobasts is included as a wild-type allele control. (F) Summary of the gene targeting experiment and resulting targeting frequencies observed.

Figure 2. Characterization of Corrected HD-iPSC Clones

(A) Corrected HD-iPSC line C127 immunostained for stem cell markers (Oct4, Sox2, Nanog, SSEA4, and TRA-1-60), costained with DAPI. (B) Immunostaining of C127-derived embryoid bodies shows differentiation into ectoderm (Nestin), mesoderm (smooth muscle actin, SMA), and endoderm (Sox17) fates. (C) Karyotyping and G-banding analysis confirms the absence of genetic abnormalities in one line (C127) of corrected HD-iPSC clones. (D) Genes altered as a function of genetic correction in the CAG locus of HTT in pathways and domain interactions. (E) Gene array analysis indicates that the corrected clones remain similar to the HD-iPSC clones, suggesting that the genetic correction did not alter the gene expression profile of the cells. See Figure S2 and Table S1 for microarray analysis. (F) RT-qPCR verification of altered pathways in uncorrected HD-iPSCs compared with corrected HD-iPSCs and normal iPSCs. RNA samples were harvested from HD-iPSCs, normal iPSCs, and two lines of corrected HD-iPSCs. RT-qPCR was performed to show upregulation of cadherin family genes and down-regulation of TGF-β pathway genes. One-way ANOVA is used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3. Corrected HD-iPSC NSCs Have a Phenotype Similar to That of Normal iPSC NSCs

(A and B) TUNEL staining and quantification of NSCs derived from uncorrected HD-iPSCs (HD-NSCs), two lines of corrected HD-iPSCs (C116-NSCs and C127-NSCs), and healthy iPSCs (normal NSCs). (C) NSCs derived from uncorrected HD-iPSCs showed an elevated caspase-3/7 activity upon growth factor withdrawl. +GF, normal culture condition with growth factors; −GF, growth factor deprived condition. We did not detect phenotypes in iPSCs; see Figure S3. (D) NSCs from corrected and uncorrected HD-iPSCs show that BDNF levels in corrected NSCs are consistent with those of a normal phenotype. (E) NSCs derived from corrected cells show higher maximum respiration capacity than NSCs from uncorrected HD-iPSCs. Cells were analyzed on a Seahorse XF24 Extracellular Flux Analyzer for oxygen consumption rate (OCR). The average OCR after addition of FCCP (an uncoupler) reflects mitochondria maximum respiration capacity. Corrected NSCs and normal NSCs showed higher maximum respiration capacity than uncorrected HD-NSCs. Also see Figure S3D. (F) RT-qPCR of TGF-β1 showed a decreased level of TGF-β1 mRNA in HD-NSCs compared with that in corrected and normal NSCs. (G and H) RT-qPCR and western blotting of N-cadherin showed a decreased level of N-cadherin mRNA and protein in HD-NSCs compared with that in corrected and normal NSCs. One-way ANOVA is used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4. Directed Differentiation of Corrected HD-iPSC Clones

(A) NSCs were derived from corrected HD-iPSCs with the EB method and were stained for the NSC marker Nestin. (B) NSCs were further induced to striatal neurons with expression of DARPP-32 and βIII-tubulin. (C) Calbindin and GABA is shown by ICC staining. (D) Two weeks after striatal injection of corrected NSCs (C116-NSCs), immunofluorescence of R6/2 mouse brain sections detects human-specific STEM121 and DAPI. (E–G) STEM121 and MAP2 R6/2 mouse brain sections counterstained with DAPI. (H and I) STEM121 and GABA R6/2 mouse brain sections counterstained with DAPI. (J and K) STEM121 and DARPP-32 R6/2 mouse brain sections counterstained with DAPI. (L) STEM121 and GFAP R6/2 mouse brain sections counterstained with DAPI. For further analysis see Figure S4.