Overcoming the Variability of iPSCs in the Manufacturing of Cell-Based Therapies.

Various factors are known to contribute to the diversity of human induced pluripotent stem cells (hiPSCs). Among these are the donor's genetic background and family history, the somatic cell source, the iPSC reprogramming method, and the culture system of choice. Moreover, variability is seen even in iPSC clones, generated in a single reprogramming event, where the donor, somatic cell type, and reprogramming platform are the same. The diversity seen in iPSC lines often translates to epigenetic differences, as well as to differences in the expansion rate, iPSC line culture robustness, and their ability to differentiate into specific cell types. As such, the diversity of iPSCs presents a hurdle to standardizing iPSC-based cell therapy manufacturing. In this review, we will expand on the various factors that impact iPSC diversity and the strategies and tools that could be taken by the industry to overcome the differences amongst various iPSC lines, therefore enabling robust and reproducible iPSC-based cell therapy manufacturing processes.

Fluid shear stress promotes embryonic stem cell pluripotency via interplay between ß-catenin and vinculin in bioreactor culture.

The expansion of pluripotent stem cells (PSCs) as aggregates in stirred suspension bioreactors is garnering attention as an alternative to adherent culture. However, the hydrodynamic environment in the bioreactor can modulate PSC behavior, pluripotency and differentiation potential in ways that need to be well understood. In this study, we investigated how murine embryonic stem cells (mESCs) sense fluid shear stress and modulate a noncanonical Wnt signaling response to promote pluripotency. mESCs showed higher expression of pluripotency marker genes, Oct4, Sox2, and Nanog in the absence of leukemia inhibitory factor (LIF) in stirred suspension bioreactors compared to adherent culture, a phenomenon we have termed mechanopluripotency. In bioreactor culture, fluid shear promoted the nuclear translocation of the less well-known pluripotency regulator ß-catenin and concomitant increase of c-Myc expression, an upstream regulator of Oct4, Sox2, and Nanog. We also observed similar ß-catenin nuclear translocation in LIF-free mESCs cultured on E-cadherin substrate under defined fluid shear stress conditions in flow chamber plates. mESCs showed lower shear-induced expression of pluripotency marker genes when ß-catenin was inhibited, suggesting that ß-catenin signaling is crucial to mESC mechanopluripotency. Key to this process is vinculin, which is known to rearrange and associate more strongly with adherens junctions in response to fluid shear. When the vinculin gene is disrupted, we observe that nuclear ß-catenin translocation and mechanopluripotency are abrogated. Our results indicate that mechanotransduction through the adherens junction complex is important for mESC pluripotency maintenance.

Botulinum hemagglutinin-mediated in situ break-up of human induced pluripotent stem cell aggregates for high-density suspension culture.

Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high-density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break-up using botulinum hemagglutinin (HA), which specifically bound with E-cadherin and disrupted cell-cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E-cadherin-mediated cell-cell connections to facilitate the break-up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 106 cells ml-1 was obtained by aggregate break-up into small ones, which was three times higher than that with the conventional culture without aggregate break-up. Therefore, the temporary activity of HA for disrupting E-cadherin-mediated cell-cell connection was key to establishing a simple in situ method for hiPSC aggregate break-up in bioreactors, leading to high cell density in suspension culture.

A retinoid analogue, TTNPB, promotes clonal expansion of human pluripotent stem cells by upregulating CLDN2 and HoxA1.

Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine passage leads to massive cell death. Although the Rho-associated protein kinase inhibitor, Y-27632 can enhance hPSC survival and proliferation at high seeding density, dissociated single cells undergo apoptosis at clonal density. This presents a major hurdle when deriving genetically modified hPSC lines since transfection and genome editing efficiencies are not satisfactory. As a result, colonies tend to contain heterogeneous mixtures of both modified and unmodified cells, making it difficult to isolate the desired clone buried within the colony. In this study, we report improved clonal expansion of hPSCs using a retinoic acid analogue, TTNPB. When combined with Y-27632, TTNPB synergistically increased hPSC cloning efficiency by more than 2 orders of magnitude (0.2% to 20%), whereas TTNPB itself increased more than double cell number expansion compared to Y-27632. Furthermore, TTNPB-treated cells showed two times higher aggregate formation and cell proliferation compared to Y-27632 in suspension culture. TTNPB-treated cells displayed a normal karyotype, pluripotency and were able to stochastically differentiate into all three germ layers both in vitro and in vivo. TTNBP acts, in part, by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. By promoting clonal expansion, TTNPB provides a new approach for isolating and expanding pure hPSCs for future cell therapy applications.

Overview of the Therapeutic Applications of Stem Cell-Derived Exosomes: A Research and Commercial Perspective.

Progress in extracellular vesicle (EV) research over the past two decades has generated significant interest in using EVs in the biomedical field. Exosomes are a subgroup of EVs that comprise endocytic membrane-bound nanovesicles of 40 to 160 nm diameter. These vesicles have been shown to facilitate intercellular communication via the delivery of cellular molecules. There are currently several exciting applications for exosomes being developed in therapeutics, diagnostics, drug delivery, and cellular reprogramming. Stem cell-derived exosomes present the opportunity to harness the power of stem cells while circumventing several of the risks associated with their use. This review summarizes the recent developments in exosome technology and lends a prospective view to the future of exosome use and application in research and medicine. Through a review of relevant patent filings, recent literature, and ongoing clinical trials, a valuable overview of the field of exosomes is provided. © 2021 Wiley Periodicals LLC.

Cell-Based Therapy Manufacturing in Stirred Suspension Bioreactor: Thoughts for cGMP Compliance.

Cell-based therapy (CBT) is attracting much attention to treat incurable diseases. In recent years, several clinical trials have been conducted using human pluripotent stem cells (hPSCs), and other potential therapeutic cells. Various private- and government-funded organizations are investing in finding permanent cures for diseases that are difficult or expensive to treat over a lifespan, such as age-related macular degeneration, Parkinson's disease, or diabetes, etc. Clinical-grade cell manufacturing requiring current good manufacturing practices (cGMP) has therefore become an important issue to make safe and effective CBT products. Current cell production practices are adopted from conventional antibody or protein production in the pharmaceutical industry, wherein cells are used as a vector to produce the desired products. With CBT, however, the "cells are the final products" and sensitive to physico- chemical parameters and storage conditions anywhere between isolation and patient administration. In addition, the manufacturing of cellular products involves multi-stage processing, including cell isolation, genetic modification, PSC derivation, expansion, differentiation, purification, characterization, cryopreservation, etc. Posing a high risk of product contamination, these can be time- and cost- prohibitive due to maintenance of cGMP. The growing demand of CBT needs integrated manufacturing systems that can provide a more simple and cost-effective platform. Here, we discuss the current methods and limitations of CBT, based upon experience with biologics production. We review current cell manufacturing integration, automation and provide an overview of some important considerations and best cGMP practices. Finally, we propose how multi-stage cell processing can be integrated into a single bioreactor, in order to develop streamlined cGMP-compliant cell processing systems.

Post-Passage rock inhibition induces cytoskeletal aberrations and apoptosis in Human embryonic stem cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are prone to anoikis after single cell dissociation. The small molecule, Y-27632 is known to increase survival of hESCs and hiPSCs by inhibiting the Rho-associated protein kinase (ROCK). However, the underlying mechanisms are still unclear. Here, we thoroughly screened small molecules to investigate the adhesion and survival of hESCs in adherent culture. Y-27632 provided higher adhesion and survival of hESCs by increased cell migration and preventing cell blebbing in single dissociated cells. The combination of Matrigel with poly-d-lysine increased the attachment and survival of dissociated cells via actin filament and microtubule spreading in Y-27632-treated cells. Although Y-27632 prevented apoptosis by suppressing actin filament contraction, microtubule bundling, and blebbing, prolonged Y-27632 treatment presented a different morphology in the attached growing hESC colony. It induced apoptosis of cells by promoting cytoplasmic spread, E-cadherin structural change, and increased detachment. It also induced actin cytoskeleton disruption, combined with microtubule and intermediate filament elongation. For optimal hPSC culture, our research suggests that Y-27632 should be removed shortly after passaging.

Size- and time-dependent growth properties of human induced pluripotent stem cells in the culture of single aggregate.

Aggregate culture of human induced pluripotent stem cells (hiPSCs) is a promising method to obtain high number of cells for cell therapy applications. This study quantitatively evaluated the effects of initial cell number and culture time on the growth of hiPSCs in the culture of single aggregate. Small size aggregates ((1.1 ± 0.4) × 101-(2.8 ± 0.5) × 101 cells/aggregate) showed a lower growth rate in comparison to medium size aggregates ((8.8 ± 0.8) × 101-(6.8 ± 1.1) × 102 cells/aggregate) during early-stage of culture (24-72 h). However, when small size aggregates were cultured in conditioned medium, their growth rate increased significantly. On the other hand, large size aggregates ((1.1 ± 0.2) × 103-(3.5 ± 1.1) × 103 cells/aggregate) showed a lower growth rate and lower expression level of proliferation marker (ki-67) in the center region of aggregate in comparison to medium size aggregate during early-stage of culture. Medium size aggregates showed the highest growth rate during early-stage of culture. Furthermore, hiPSCs proliferation was dependent on culture time because the growth rate decreased significantly during late-stage of culture (72-120 h) at which point collagen type I accumulated on the periphery of aggregate, suggesting blockage of diffusive transport of nutrients, oxygen and metabolites into and out of the aggregates. Consideration of initial cell number and culture time are important to maintain balance between autocrine factors secretion and extracellular matrix accumulation on the aggregate periphery to achieve optimal growth of hiPSCs in the culture of single aggregate.