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.

End-to-End Platform for Human Pluripotent Stem Cell Manufacturing.

Industrialization of stem-cell based therapies requires innovative solutions to close the gap between research and commercialization. Scalable cell production platforms are needed to reliably deliver the cell quantities needed during the various stages of development and commercial supply. Human pluripotent stem cells (hPSCs) are a key source material for generating therapeutic cell types. We have developed a closed, automated and scalable stirred tank bioreactor platform, capable of sustaining high fold expansion of hPSCs. Such a platform could facilitate the in-process monitoring and integration of online monitoring systems, leading to significantly reduced labor requirements and contamination risk. hPSCs are expanded in a controlled bioreactor using perfused xeno-free media. Cell harvest and concentration are performed in closed steps. The hPSCs can be cryopreserved to generate a bank of cells, or further processed as needed. Cryopreserved cells can be thawed into a two-dimensional (2D) tissue culture platform or a three-dimensional (3D) bioreactor to initiate a new expansion phase, or be differentiated to the clinically relevant cell type. The expanded hPSCs express hPSC-specific markers, have a normal karyotype and the ability to differentiate to the cells of the three germ layers. This end-to-end platform allows a large scale expansion of high quality hPSCs that can support the required cell demand for various clinical indications.

Rewinding the process of mammalian extinction.

With only three living individuals left on this planet, the northern white rhinoceros (Ceratotherium simum cottoni) could be considered doomed for extinction. It might still be possible, however, to rescue the (sub)species by combining novel stem cell and assisted reproductive technologies. To discuss the various practical options available to us, we convened a multidisciplinary meeting under the name "Conservation by Cellular Technologies." The outcome of this meeting and the proposed road map that, if successfully implemented, would ultimately lead to a self-sustaining population of an extremely endangered species are outlined here. The ideas discussed here, while centered on the northern white rhinoceros, are equally applicable, after proper adjustments, to other mammals on the brink of extinction. Through implementation of these ideas we hope to establish the foundation for reversal of some of the effects of what has been termed the sixth mass extinction event in the history of Earth, and the first anthropogenic one. Zoo Biol. 35:280-292, 2016. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc.

Expansion of Human Pluripotent Stem Cells in Stirred Tank Bioreactors.

Bioreactor technolology enables the expansion of mammalian cells, which can be translated to processes compatible with Current Good Manufacturing Practice (cGMP) regulations. Cells are introduced to the bioreactor vessel, wherein key parameters such as temperature, pH, and oxygen levels are tightly controlled to facilitate growth over time. Here, we describe the microcarrier-based expansion of human pluripotent stem cells in a 3 L stirred tank bioreactor.

Enabling Allogeneic T Cell-Based Therapies: Scalable Stirred-Tank Bioreactor Mediated Manufacturing.

Allogeneic T cells are key immune therapeutic cells to fight cancer and other clinical indications. High T cell dose per patient and increasing patient numbers result in clinical demand for a large number of allogeneic T cells. This necessitates a manufacturing platform that can be scaled up while retaining cell quality. Here we present a closed and scalable platform for T cell manufacturing to meet clinical demand. Upstream manufacturing steps of T cell activation and expansion are done in-vessel, in a stirred-tank bioreactor. T cell selection, which is necessary for CAR-T-based therapy, is done in the bioreactor itself, thus maintaining optimal culture conditions through the selection step. Platform's attributes of automation and performing the steps of T cell activation, expansion, and selection in-vessel, greatly contribute to enhancing process control, cell quality, and to the reduction of manual labor and contamination risk. In addition, the viability of integrating a closed, automated, downstream process of cell concentration, is demonstrated. The presented T cell manufacturing platform has scale-up capabilities while preserving key factors of cell quality and process control.

Human embryonic stem cells as a cellular model for human disorders.

Human embryonic stem cells (HESCs) are pluripotent cell lines derived from the inner cell mass (ICM) of embryos at the blastocyst stage. These cells possess self renewal capacity and differentiation potential to all three embryonic germ layers. These unique characters made HESCs an attractive research tool for studying early human developmental processes as well as a potential therapeutic tool for various human diseases. Here, we focus on HESCs as a cellular model for human disorders. The advantages of such models as well as the various methodologies to achieve HESCs carrying a genetic defect will be discussed.

A panel of induced pluripotent stem cells from chimpanzees: a resource for comparative functional genomics.

Comparative genomics studies in primates are restricted due to our limited access to samples. In order to gain better insight into the genetic processes that underlie variation in complex phenotypes in primates, we must have access to faithful model systems for a wide range of cell types. To facilitate this, we generated a panel of 7 fully characterized chimpanzee induced pluripotent stem cell (iPSC) lines derived from healthy donors. To demonstrate the utility of comparative iPSC panels, we collected RNA-sequencing and DNA methylation data from the chimpanzee iPSCs and the corresponding fibroblast lines, as well as from 7 human iPSCs and their source lines, which encompass multiple populations and cell types. We observe much less within-species variation in iPSCs than in somatic cells, indicating the reprogramming process erases many inter-individual differences. The low within-species regulatory variation in iPSCs allowed us to identify many novel inter-species regulatory differences of small magnitude.