Automating Human Induced Pluripotent Stem Cell Culture and Differentiation of iPSC-Derived Retinal Pigment Epithelium for Personalized Drug Testing

Abstract

Derivation and differentiation of human induced pluripotent stem cells (hiPSCs) provide the opportunity to generate medically important cell types from individual patients and patient populations for research and the development of potential cell therapies. This technology allows disease modeling and drug screening to be carried out using diverse population cohorts and with more relevant cell phenotypes than can be accommodated using traditional immortalized cell lines. However, technical complexities in the culture and differentiation of hiPSCs, including lack of scale and standardization and prolonged experimental timelines, limit the adoption of this technology for many large-scale studies, including personalized drug screening. The entry of reproducible end-to-end automated workflows for hiPSC culture and differentiation, demonstrated on commercially available platforms, provides enhanced accessibility of this technology for both research laboratories and commercial pharmaceutical testing. Here we have utilized TECAN Fluent automated cell culture workstations to perform hiPSC culture and differentiation in a reproducible and scalable process to generate patient-derived retinal pigment epithelial cells for downstream use, including drug testing. hiPSCs derived from multiple donors with age-related macular degeneration (AMD) were introduced into our automated workflow, and cell lines were cultured and differentiated into retinal pigment epithelium (RPE). Donor hiPSC-RPE lines were subsequently entered in an automated drug testing workflow to measure mitochondrial function after exposure to “mitoactive” compounds. This work demonstrates scalable, reproducible culture and differentiation of hiPSC lines from individuals on the TECAN Fluent platform and illustrates the potential for end-to-end automation of hiPSC-based personalized drug testing.

Testing Mitochondrial-Targeted Drugs in iPSC-RPE from Patients with Age-Related Macular Degeneration

“…The treatment effect was modest, as only ≈25% of patients showed reduced AMD progression [12]. Similar to reports for patients in the clinical trials, in vitro studies using primary and induced pluripotent stem cell (iPSC)-RPE cultured from AMD donors have shown significant variability and heterogeneity in the responses of individual cell lines after exposure to drugs [13][14][15][16][17]. This variation may arise from multiple etiologies proposed for AMD initiation and progression in the patient population [18] and highlights that a more personalized approach is required to match the optimal intervention to the patient-specific defect that is causing their disease.…”

Analyzing mitochondrial respiration of human induced pluripotent stem cell-derived myeloid progenitors using Seahorse technology

“…Hoechst-positive cells detected by Cytation1 and shown in the Gen5 software 9 , 10 can be directly imported into the corresponding seahorse experiment recorded by Wave software ( Figure 7 A). Subsequently, in the Edit Normalization Mode of the seahorse assay shown in the Wave software, the normalization unit can be set into 10,000 cells and the normalization values are automatedly calculated ( Figure 7 B), while the control wells (without cells) show as blanks.…”

Therapeutic Approaches for Age-Related Macular Degeneration

“…To date, various molecules that promote cell reprogramming have been reported as substitutes for genetic materials. Despite these promising results, nowadays, one of the major problems is the risk of reprogrammed stem cells developing into tumors, with this being the major obstacle to overcome [ 71 , 72 , 73 ].…”

“…The first one involves the regeneration or replacement of the RPE, whereas the second is a trophic role, in which they produce growth factors and cytokines, that have a supportive paracrine effect on local structures within the macula. Currently, there are two medium-term trials supporting the safety and survival of the cells transplanted and the effectiveness of the technique on the improvement of the macular degeneration up to a time period of 22 months [ 66 , 71 , 74 , 75 ]. Thus, long-term trials are still needed to assess the safety of the technique over the time, thus further studies needed to clarify safety and efficacy.…”