Abstract
SummaryReprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost.
Highlights
A crucial problem in both the analysis of many human diseases and the development of effective therapies to treat disease is the incomplete understanding of the role played by human genetic variation in their development
We describe a cost-effective, high-throughput suite of these methods including flow cytometry using fluorescent cell barcoding (FCB), qPCR for expression analysis, and SNP arrays for digital karyotyping (Figure 1), which will facilitate the characterization of the large numbers of induced pluripotent stem cells (iPSCs) lines currently being generated in individual laboratories as well as in biobanks to examine human diseases
We included fibroblasts from two individuals with a familial Alzheimer’s disease (FAD) mutation in the amyloid b precursor protein (APP), two non-demented control (NDC) individuals, three individuals with sporadic Alzheimer’s disease (SAD), and one individual with hippocampal sclerosis (Table 1) to ensure that our methods would be applicable for analysis of cell lines regardless of disease status
Summary
A crucial problem in both the analysis of many human diseases and the development of effective therapies to treat disease is the incomplete understanding of the role played by human genetic variation in their development. Pluripotent stem cells can provide disease-relevant cell types to model human diseases. Many cell types have been derived from pluripotent cell lines, and exciting advances in disease modeling and drug screening have been published (Avior et al, 2016; Brennand et al, 2011; Israel et al, 2012; Itzhaki et al, 2011; Mertens et al, 2015). A current limitation to using induced pluripotent stem cells (iPSCs) to model human disease is the time-inefficiency and cost of standard characterization methods required after reprogramming. To model certain diseases, hundreds of patient-specific pluripotent lines are necessary to be adequately powered to test the relationship of genetic variants with cellular phenotypes and disease development
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