Highlights from the Journal of Huntington’s Disease
Induced pluripotent stem cells in HD research
By: Kimberly Kegel-Gleason, PhD
Original article: Tousley A, Kegel-Gleason KB. Induced Pluripotent Stem Cells in Huntington’s Disease Research: Progress and Opportunity. J Huntingtons Dis. 2016 Jun 28;5(2):99-131. doi: 10.3233/JHD-160199.
Although many cell types are affected in HD, the impact of the disease on CNS neurons is remarkable. Neurons of the cortex and striatum degenerate and eventually die, causing the majority of HD symptoms. Until recently, it has been difficult to study human CNS neurons because of ethical considerations – it is not ethical to perform a biopsy on human patients to obtain brain cells for research. Embryonic stem cells (ESCs) are pluripotent, meaning that they have the ability to become neurons; however, the use of ESCs to obtain neurons is also fraught with ethical challenges, including the destruction of an embryo. Enter induced pluripotent stem cells (iPSCs). Skin or blood cells from controls and HD patients can be made into iPSCs by the introduction of just a few factors. iPSCs are very similar to ESCs and can be differentiated to resemble CNS neurons or other CNS cell types in order to study disease mechanisms, and to screen compounds that might be developed into new therapies.
A major advantage to iPSCs is that normal and mutant proteins are expressed at endogenous levels just as they are in the human patient; furthermore, the effects of varied genetic backgrounds on the behavior of the mutant protein can be assessed. Studies with iPSCs from patients with neurodegenerative diseases other than HD have provided new insights previously not found using animal models. For instance, using three-dimensional cultures of CNS neurons from iPSCs from human Alzheimer disease (AD) patients, intracellular tangles, which are major feature of AD, were observed.1 Intracellular tangles had never been recapitulated in mouse models of AD, and the results pointed to a particular protein only found in humans as a major target of pathology.
As with research in other neurodegenerative diseases, iPSCs from controls and HD patients have been in development to uncover previously unknown human-specific pathological mechanisms, to validate phenotypes identified in animal models, and for compound screening. In our review,2 we characterize the state of the HD iPSC field. We describe the current inventory of cells available to HD researchers, many of which cells are freely available. We also highlight changes that have been identified in HD cells compared to controls, in pathways, individual gene changes, functional phenotypes, and the role of stress and aging. Furthermore, we compare results obtained with various neuronal differentiation protocols.
Table: Summary of major progress and opportunities for expanded research using HD IPSCs
|Numerous HD iPSCs created||Increase the number of HD and control lines used within each study|
|A few genetically corrected, isogenic iPSCs created||Increase number of genetically corrected, isogenic iPSCs created and available (CRISPR/Cas9)|
|Several studies on neuronal cultures using diverse differentiation protocols||Use of reproducible protocols for comparison of results across laboratories|
|Stress-induced phenotypes identified||Expand the phenotypes identified in the absence of stress|
|SiRNAs and miRNAs targeting alleles with SNPs, Zinc Finger Proteins||Develop additional allele-specific reagents and tools to target mHTT|
|Two studies with iPSC astrocytes||Studies with iPSC glia (astrocytes, oligodendrocytes, microglia)|
|Co-culturing, 3-D cultures, and organoids|
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We also note the pervasive use of stress to unveil functional phenotypes in HD cells. Because stress exacerbates HD symptoms in patients, this may reflect important disease mechanisms. One argument in favor of this approach is that iPSC induced neurons are very immature compared to those in the brain of an adult with HD, so stress might mimic aging to enhance a phenotype. However, more time spent investigating what may be more subtle phenotypes in the absence of stress may lead to a better understanding of underlying pathology that leaves a cell vulnerable to stress and eventually triggers disease.
Although much progress has been made, culturing and differentiating iPSCs is still extremely expensive, time consuming and difficult, thus limiting the number of investigators who can take advantage of this valuable resource. We hope this review will enable those new to the iPSC field to consider and control the inherent problems with iPSC lines, and so enable reproducible research across the field.
One major limitation we found for interpreting data across the HD field is the relative paucity of cell lines used – many times, reports include data from just one cell line. In order to increase the reproducibility of research across the field, we suggest that results from at least three HD cell lines from three individual patients and three cell lines from three individual controls be used for robust phenotypes (six lines total). For more subtle phenotypes it may be necessary to use many more cell lines. For comparison, 10–12 control cell lines are currently being used by investigators in other fields.3 Alternatively, a combination of control cell lines (cell lines from unaffected individuals), and genetically corrected cell lines (using homologous recombination or CRISPR), and use of effective mHTT-lowering reagents such as siRNAs, miRNAs or zinc finger proteins in HD lines could be used. We note that data from genetically corrected cell lines should be interpreted with caution because the cell lines undergo several rounds of selective pressure during their generation that could alter a particular phenotype.
Our review of the field identifies areas of opportunity for which additional research would be of great value. For instance, very few studies used other brain cell types that can be differentiated from iPSCs such as astrocytes and oligodendrocytes, which may also impact HD pathology. The field should welcome more studies using iPSC-derived glial cells.
1. Choi SH, Kim YH, Hebisch M, et al. A three-dimensional human neural cell culture model of Alzheimer’s disease. Nature. 2014;515(7526):274-278.
2. Tousley A, Kegel-Gleason KB. Induced Pluripotent Stem Cells in Huntington’s Disease Research: Progress and Opportunity. J Huntington’s dis. 2016;5(2):99-131.
3. Boulting GL, Kiskinis E, Croft GF, et al. A functionally characterized test set of human induced pluripotent stem cells. Nat Biotechnol. 2011;29(3):279-286.