Human Pluripotent Stem Cells for High-Throughput Drug Screening and Characterization of Small Molecules

Abstract

Human pluripotent stem cells (hPSCs), such as induced pluripotent stem cells (iPSCs), hold great promise for drug discovery, toxicology studies, and regenerative medicine. Here, we describe standardized protocols and experimental procedures that combine automated cell culture for scalable production of hPSCs with quantitative high-throughput screening (qHTS) in miniaturized 384-well plates. As a proof of principle, we established dose-response assessments and determined optimal concentrations of 12 small molecule compounds that are commonly used in the stem cell field. Multi-parametric analysis of readouts from diverse assays including cell viability, mitochondrial membrane potential, plasma membrane integrity, and ATP production was used to distinguish normal biological responses from cellular stress induced by small molecule treatment. Collectively, the establishment of integrated workflows for cell manufacturing, qHTS, high-content imaging, and data analysis provides an end-to-end platform for industrial-scale projects and should leverage the drug discovery process using hPSC-derived cell types.

Keywords: Cell viability; Dose–response curves; Embryonic stem cells; High-throughput screening; Induced pluripotent stem cells; Robotic cell culture; Small molecules; Toxicity.

Figure 1.

Schematic overview of the experimental procedures encompassing robotic culture of hPSCs, preparation and plating cells in 384-well format, addition of chemical compounds using acoustic liquid dispensing, and use of three different assays enabled by instruments for quantitative screening and data acquisition.

Figure 2.

Optimization of various cell culture parameters for screening in 384-well format. (A, B) Comparison between two commonly used coating substrates for hPSC culture, recombinant Vitronectin and Geltrex, on plates sourced from two different vendors. Both Vitronectin and Geltrex show consistent cell attachment and comparable confluency at 24 h post-plating. Results were confirmed with one hESC line (WA09) and one iPSC line were. Cells were stained with Calcein dye for quantification. Scale bars, 400 μm. (C, D) Time-course fluorescent images of hESCs and iPSCs plated at different cell densities. Cell density above 55,000 cells/cm² was found to be ideal for formation of homogeneous cell colonies for screening at 48 h after cell plating. Cells were stained with Calcein dye for quantification. Scale bars, 400 μm. (E) Cell viability at differentiation concentrations of DMSO based on CTG assay. The data was obtained at 24 h following DMSO addition. Cells were stained with Calcein dye for quantification.

Figure 3.

Overview of the drug-screening strategy in miniaturized 384-well assay plates. (A) Timeline demonstrating the 3-day process for drug screening which includes coating plates, cell seeding, media change, administration of compounds, and assay measurement at 1 h and 24 h. Representative bright field images are shown on day 1, 2, and 3. (B) Schematic of plate maps depicting orientation of compound source plate and destination plate containing cells. This arrangement allows dispensing chemical compounds at different concentrations for the investigation of dose-response and cytotoxic effects. (C) Table summarizing the concentrations covered in the prepared compound source plate and destination cell plate. The screening covers eleven three-fold dilutions in quadruplicate beginning at 100 μM as highest concentration. 11 dose-points for each compound as follows: 1.7 nM, 5.1 nM, 15.2 nM, 45.7 nM, 137.2 nM, 411.5 nM, 1.2 μM, 3.7 μM, 11.1 μM, 33.3 μM, and 100 μM.

Figure 4.

Compound dose-response curves for hESC (WA09). n = 4 wells per data point. Data were normalized to DMSO as a negative control (0% response) and highly toxic Tetraoctylammonium bromide as a positive control (100% response). Curve fitting was done with nonlinear regression analysis (24, 25).

Figure 5.

Comparison of dose-response curves for two inhibitors of BMP signaling. hESCs (WA09) were assayed 1 h after compound treatment. Note that LDN-193189 is more toxic at higher concentrations as compared to Dorsomorphin. n = 4 wells per data point.