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

Critical Analysis of cGMP Large-Scale Expansion Process in Bioreactors of Human Induced Pluripotent Stem Cells in the Framework of Quality by Design

“…Although the DoEs and optimization through MVDA for the improvement of hiPSC culture conditions [ 44 – 46 ], downstream concentration [ 35 ], and differentiation [ 47 – 49 ] have been described, examples of employing a QRM and QbD approach for the optimization of hiPSC expansion are limited, likely because ATMPs, such as hiPSCs and their derivatives, are a relatively new category in drug regulation. Indeed, ATMPs were included in the pharmaceutical legislation only in 2009 [ 50 ].…”

Clever Experimental Designs: Shortcuts for Better iPSC Differentiation

“…Fractional factorial screenings have also been used for definitive endoderm differentiation [ 45 ]. Mixed-level orthogonal arrays were used to screen cytokines for choroidal endothelium differentiation [ 46 ] and optimize additive doses for four distinct types of endodermal cell induction [ 47 ]. RSM was used to optimize hydrogel peptide concentrations for neural progenitor maturation [ 48 ], optimize the ECM composition for cardiomyocyte differentiation [ 49 ], and screen nutrient compositions driving trilineage specification from hiPSCs based on the response surface models for endodermal, mesodermal, and ectodermal protein expressions [ 50 ].…”

“…To investigate definitive endoderm differentiation and patterning, we constructed an L 18 array design including whole anterior-posterior endoderm by inputting eight cell signaling modifiers, thereby increasing the screening efficiency 243-fold (18 runs vs. 4374 runs) [ 47 ]. RNA expression of 18 end products seemed like “melting pots”, in which some achieved specific differentiation and others produced mixtures of cell lineages.…”

Human Organoid and Supporting Technologies for Cancer and Toxicological Research

“…Theoretically, researchers can obtain any type of human cell at any developmental stage. Human iPSC technology is mainly used for regenerative medicine and drug screening (Aurora and Spence, 2016;Sekine et al, 2020;Marsee et al, 2021;Yasui et al, 2021). From a cancer biology perspective, human iPSC technology is suitable for obtaining normal, healthy cells in order to study tumor onset (Kim et al, 2013;Sun et al, 2019;Frum and Spence, 2021).…”

“…Regarding the throughput of organoid technology, unlike 2D cultured cells, the low throughput of 3D cultured cells might hinder the screening of chemicals. Advances in the field of robotics may help to improve the handling of thousands of chemicals; therefore, we need to await the maturation of robotic technologies in cell handling (Louey et al, 2021;Ostrop et al, 2021;Yasui et al, 2021). The other limitation is the deficiency of an immune system in the in vitro system.…”