|A Brief History of Pluripotent Cells for Huntington’s Disease|
Huntington’s Disease (HD) is an autosomal-dominant progressive neurodegenerative disease, characterized by movement, cognitive, and emotional disorders caused by an expanded CAG repeat in exon 1 of the Huntingtin (HTT) gene resulting in an expanded polyglutamine stretch known as the polyQ region or stretch. Generally, fewer than 36 CAG repeats in the polyQ region is observed in healthy patients but the presence of more than 40 CAGs invariably causes disease onset within a normal lifespan, and longer repeats predict younger disease presentation. The classic clinical features, typically of adult onset, are progressive motor impairment, cognitive decline, chorea and seizures caused by neural degeneration, particularly of striatal medium spiny neurons (MSNs) expressing dopamine- and cAMP-regulated phosphoprotein (DARPP-32). Animal studies (Heng et al and Davies et al) have not been found to reflect all HD pathologies adequately and thus so far have not led to successful humantherapies.Therefore, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) represent the best resources for modelling HD both for understanding disease mechanisms and uncovering potential therapeutic targets.
Pluripotent cells carrying the HD mutation were first demonstrated through the derivation of an hESC line from embryos donated after in vitro fertilisation (IVF) and preimplantation genetic diagnosis (PGD) (Mateizel et al, Niclis et al, Verlinsky et al 2005, and Verlinskey et al 2006). Mateizel et al found the HD-hESC line generated to express genes typical of pluripotent cells but was not definitively characterised as being fully pluripotent. The line still carried the abnormal CAG repeat and indeed had increased to carry 44 CAG repeats, which represented a small enlargement in comparison with the 42 repeats of the affected father. Niclis et al generated and characterised two HD-hESC linescarrying mutant alleles with 37 and 51CAG repeats respectively and, in addition, showed these lines were capable of differentiation into neural lineages that were affected by the disease process. However, one line carried a trisomy of chromosome 12 and both CAG repeats were relatively short. A further 4 HD-hESC lines were generated in two related studies (Verlinsky et al 2005, and Verlinskey et al 2006), with the resultant hESC lines characterised through pluripotency-associated gene expression, alkaline phosphatase activity and cell surface marker expression. Bradley et al generated 4 HD-hESC lines with between 40 and 50 CAG repeats with sibling-matched controls for one line and reported that all lines were deemed pluripotent and able to generate MAP2+ neuronal cells. Finally, a recent study (Feyeux et al) studied the impact of mutant HTT on signalling pathways in the pre-symptomatic period through deriving HD-hESCs from affected blastocysts and applying differential transcriptomics. They found that HTT itself was downregulated in neural cell derived from HD-hESCs, alongside the downregulation of CHCDH2 (mitochondrial function), TRIM4 and PKIB (PKA-dependent pathway regulation), with a similar dysregulation observed previously for CHCDH2 and TRIM4 in HD patients blood samples.
The first HD-hiPSC line generated came in 2008 in an ambitious piece of work which sought to generate hiPSC from patients with a variety of genetic diseases with either Mendelian or complex inheritance (Park et al). The HD-hiPSC lines generated from a single patient were pluripotent and contained 72 CAG repeats in onehuntingtin gene allele and 19 repeats in the other. Soon after another report came from a group that had previously generated a transgenic monkey model of human Huntington’s disease and who had derived and characterisation iPSCs from this model (rHD-iPSCs) (Chan et al) in the hope that this could be a powerful platform for investigating HD pathogenesis and the evaluation of human HD therapies in a closely-related disease model. Pluripotent rHD-iPSCs could differentiate into neuronal cell types and developed cellular features comparable to HD, including the accumulation of mutant huntingtin (Htt) aggregate and the formation of intranuclear inclusions (NIs). Next, a previously generated HD-hiPSC line (Chan et al) was characterised for its ability to generate useful neural cell types (Zhang et al). Embryoid body formation allowed the generation of neural stem cells (NSCs) and subsequent differentiation strategies led to the production of DARPP-32 expressing MSNs containing the same CAG expansion as the mutation in the HD patient from where the hiPSC line was derived. Interestingly HD-hiPSC-derived NSCs showed enhanced Caspase activity relative to NSCs derived from wild type hiPSCs or hESCs.
2012 then led to a surge of publications on HD-hiPSCs. Camnasio et al generated multiple human HD-hiPSC lines including two rare homozygous genotypes, finding that CAG repeats did not alter in length and that HD-hiPSCs were similar to a wild type hiPSC line with regards to reprogramming efficacy, growth rate, caspase activation (contrary to Zhanget al) and neuronal differentiation. One interesting novel difference noted in this study was the increased lysosomal activity in HD-hiPSCs during self-renewal and differentiation and an increased number of subcellular acidic compartments suggestive of highly active protein degradation. Differences in the lysosomal pathway were also noticed in a related article by Castiglioni et al during their derivation and characterisation studies of mouse HD-iPSCs generated from fibroblasts of R6/2 mice, genetically altered to carry a mutant Htt gene. Another notable difference was the misexpression of genes in the cholesterol biosynthesis pathway, know to be altered in HD. An interesting study by Juoppere et al derived iPSCs from a patient with adult onset HD (50 CAG repeats) and his daughter with juvenile HD (109 CAG repeats). The major finding in this study was the appearance of cytoplasmic, electron-clear vacuoles in astrocytes from both HD-hiPSC lines but which were more pronounced in the cells derived from the daughter. This phenotype had been previously observed in peripheral blood lymphocytes from individuals with HD, suggesting that vacuolation may be a common HD phenotype. Jeon et al also studied the juvenile form of HD in HD-hiPSCs (72 CAG repeats), and their studies suggest that these cells show a lower propensity for initial neural induction, but could still differentiate into GABAergic striatal neurons. They also went on to transplant HD-hiPSC-derived neural precursors (HD-iPSC-NPC) into a rat model of HD demonstrating a significant behavioural recovery, although at 33 weeks post transplant, clear signs of HD pathology were still observed.
More recent studies have addressed proteomic differences in HD-iPSCs (Chae et al), CAG-repeat expansion in iPSCs (The HD iPSC Consortium) and genetic correction of mutant Htt (An and Zhang et al). Chae et al performed comparative proteomic analysis between hESC, hiPSCs and HD-hiPSCs under self-renewing conditions finding that oxidative-stress related proteins were majorly dysregulated, alongside other proteins which affect apoptosis and neural differentiation, confirming previous data connecting such differences with HD disease pathology. A study from the Hd iPSC Consortium, a collection of laboratories directed toward studying HD, reported the derivation and characterisation of 14 HD-hiPSC lines with controls, finding that gene expression profiles could distinguish between CAG-repeat length and early onset versus late onset HD, a potentially useful diagnostic tool, and also that neural cells derived from HD-iPSCs had alterations in their electrophysiology, metabolism, cell adhesion and survival linked to CAG-repeat length. The authors note that this range of iPSCs could represent a useful resource in the screening of new therapeutics. Finally, and perhaps most excitingly, is the study from An and Zhang et al which reports the replacement of the expanded CAG repeat in human HD-hiPSCs lines (generated by Park et al and characterised by Zhang et al) with a normal repeat through homologous combination and its persistence in neurally differentiated cells in vitro and in vivo. Further, this correction led to the reversal of pathway alterations and disease phenotypes associated with HD in NSCs, a thrilling finding for those who hope that iPSCs can be used for patient specific therapy.
STEM CELLS correspondent Stuart P. Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.