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Robust parameter design of human induced pluripotent stem cell differentiation protocols defines lineage‐specific induction of anterior‐posterior gut tube endodermal cells

ABSTRACT: Tissues and cells derived from pluripotent stem cells (PSC) are likely to become widely used in disease modeling, drug screening, and regenerative medicine. For these applications, the in vitro PSC differentiation process must be elaborately investigated and controlled to reliably obtain the desired end products. However, because traditional experimental methods, such as one factor at a time (OFAT) or brute-force approaches, are impractical for detailed screening of complex PSC cultivation conditions, more strategic and effective screening based on statistical design of experiments (DOE) ought to be indispensable. Among various DOE approaches, we regard robust parameter design (RPD) as particularly suited for differentiation protocol optimization due to its suitability for multi-factorial screening. We confirmed the adaptability of RPD for investigating human induced PSC (hiPSC) lineage specification toward anterior–posterior gut tube endodermal cells and clarified the contribution of each cell signaling pathway and the effect of cell signaling condition alteration on marker RNA expression levels, while increasing the efficiency of the screening in 243-fold (18 vs 4374) compared to that of a brute-force approach. Specific induction of anterior foregut, hepatic, pancreatic, or mid-hindgut cells was achieved using seven iPSC strains with the optimal culture protocols established on the basis of RPD analysis. RPD has the potential to enable efficient construction and optimization of PSC differentiation protocols, and its use is recommended from fundamental research to mass production of PSC-derived products.

ORGANISM(S): Homo sapiens

PROVIDER: GSE163148 | GEO | 2021/01/06

REPOSITORIES: GEO

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Project description:Background: With the growing availability of entire genome sequences, an increasing number of scientists can exploit oligonucleotide microarrays for genome-scale expression studies. While probe-design is a major research area, relatively little work has been reported on the optimization of microarray protocols. Results: As shown in this study, suboptimal conditions can have considerable impact on biologically relevant observations. For example, deviation from the optimal temperature by one degree Celsius lead to a loss of 44% of differentially expressed genes identified. While genes from thousands of Gene Ontology categories were affected, transcription factors and other low-copy-number regulators were disproportionately lost. Calibrated protocols are thus required in order to take full advantage of the large dynamic range of microarrays. For an objective optimization of protocols we introduce an approach that maximizes the amount of information obtained per experiment. A comparison of two typical samples is sufficient for this calibration. We ensure, however, that optimization results are independent of the samples and the specific measures used for calibration. Both simulations and spike-in experiments confirm an unbiased determination of generally optimal experimental conditions. Conclusions: Well calibrated hybridization conditions are thus easily achieved and necessary for the efficient detection of differential expression. They are essential for the sensitive profiling of low-copy-number molecules. This is particularly critical for studies of transcription factor expression, or the inference and study of regulatory networks. Supporting material, including source code and data, is available at http://bioinf.boku.ac.at/pub/optMA2010/. Optimization of hybridization temperature via an assessment of differential expression between two samples (male vs female Drosophila melanogaster) in 6 technical replicates (3 regular + 3 dye-swaps) for hybridizations at different temperatures (in two batches of 50, 52, 54, and 56; and 47, 49, 50, and 51 degree Celsius, with the repeated hybridization at 50 degree Celsius serving to demonstrate batch-to-batch stability).

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Robust parameter design of human induced pluripotent stem cell differentiation protocols defines lineage‐specific induction of anterior‐posterior gut tube endodermal cells

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