Metabolic switching, growth kinetics and cell yields in the scalable manufacture of stem cell-derived insulin-producing cells.

BACKGROUND: Diabetes is a disease affecting over 500 million people globally due to insulin insufficiency or insensitivity. For individuals with type 1 diabetes, pancreatic islet transplantation can help regulate their blood glucose levels. However, the scarcity of cadaveric donor islets limits the number of people that could receive this therapy. To address this issue, human pluripotent stem cells offer a potentially unlimited source for generating insulin-producing cells through directed differentiation. Several protocols have been developed to make stem cell-derived insulin-producing cells. However, there is a lack of knowledge regarding the bioprocess parameters associated with these differentiation protocols and how they can be utilized to increase the cell yield. METHODS: We investigated various bioprocess parameters and quality target product profiles that may influence the differentiation pipeline using a seven-stage protocol in a scalable manner with CellSTACKs and vertical wheel bioreactors (PBS-Minis). RESULTS: Cells maintained > 80% viability through all stages of differentiation and appropriately expressed stage-specific markers. During the initial four stages leading up to the development of pancreatic progenitors, there was an increase in cell numbers. Following pancreatic progenitor stage, there was a gradual decrease in the percentage of proliferative cells, as determined by Ki67 positivity, and a significant loss of cells during the period of endocrine differentiation. By minimizing the occurrence of aggregate fusion, we were able to enhance cell yield during the later stages of differentiation. We suggest that glucose utilization and lactate production are cell quality attributes that should be considered during the characterization of insulin-producing cells derived from stem cells. Our findings also revealed a gradual metabolic shift from glycolysis, during the initial four stages of pancreatic progenitor formation, to oxidative phosphorylation later on during endocrine differentiation. Furthermore, the resulting insulin-producing cells exhibited a response to several secretagogues, including high glucose. CONCLUSION: This study demonstrates process parameters such as glucose consumption and lactate production rates that may be used to facilitate the scalable manufacture of stem cell-derived insulin-producing cells.

The Impact of Different Implantation Sites and Sex on the Differentiation of Human Pancreatic Endoderm Cells Into Insulin-Secreting Cells In Vivo.

Few studies have examined the differentiation of human embryonic stem cell (hESC)-derived pancreatic endoderm cells (PECs) in different implantation sites. Here, we investigate the influence of implantation site and recipient sex on the differentiation of hESC-derived PECs in vivo. Male and female mice were implanted with 5 × 106 hESC-derived PECs under the kidney capsule, in the gonadal fat pad, or subcutaneously within macroencapsulation (TheraCyte) devices. PECs implanted within TheraCyte devices developed glucose-stimulated human C-peptide secretion faster than cells implanted under the kidney capsule or in the gonadal fat pad. Interestingly, hESC-derived PECs implanted under the kidney capsule in females developed glucose-stimulated human C-peptide faster than in males and secreted higher levels of arginine-stimulated glucagon and glucagon-like peptide 1 than other implantation sites. Furthermore, hESC-derived grafts collected from the kidney capsule and gonadal fat pad sites displayed a mix of endocrine and ductal cells as well as contained cysts, whereas TheraCyte device grafts displayed mostly endocrine cells and cysts were not observed. Here we demonstrate that the macroencapsulated subcutaneous site and the female recipient can promote faster differentiation of hESC-derived PECs to endocrine cells in mice. ARTICLE HIGHLIGHTS: Few studies have directly compared the differentiation of human embryonic stem cell-derived progenitors in different implantation sites in male and female recipients. We investigated whether the site of implantation and/or the sex of the recipient influenced the differentiation of pancreatic progenitors in vivo in mice. Mice implanted with cells in macroencapsulation devices contained fewer off-target structures and developed stimulated insulin release faster than other implant sites, while females implanted with cells under the kidney capsule developed stimulated insulin release before males. Macroencapsulation devices reduced the formation of off-target cells from human embryonic stem cell-derived progenitors, a useful characteristic for clinical applications.

Process parameter development for the scaled generation of stem cell-derived pancreatic endocrine cells.

Diabetes is a debilitating disease characterized by high blood glucose levels. The global prevalence of this disease has been projected to reach 700 million adults by the year 2045. Type 1 diabetes represents about 10% of the reported cases of diabetes. Although islet transplantation can be a highly effective method to treat type 1 diabetes, its widespread application is limited by the paucity of cadaveric donor islets. The use of pluripotent stem cells as an unlimited cell source to generate insulin-producing cells for implant is a promising alternative for treating diabetes. However, to be clinically relevant, it is necessary to manufacture these stem cell-derived cells at sufficient scales. Significant advances have been made in differentiation protocols used to generate stem cell-derived cells capable of reversing diabetes in animal models and for testing in clinical trials. We discuss the potential of both stem cell-derived pancreatic progenitors and more matured insulin-producing cells to treat diabetes. We discuss the need for rigorous bioprocess parameter optimization and identify some critical process parameters and strategies that may influence the critical quality attributes of the cells with the goal of facilitating scalable manufacturing of human pluripotent stem cell-derived pancreatic endocrine cells.

Kif5 regulates mitochondrial movement, morphology, function and neuronal survival.

Due to the unique architecture of neurons, trafficking of mitochondria throughout processes to regions of high energetic demand is critical to sustain neuronal health. It has been suggested that compromised mitochondrial trafficking may play a role in neurodegenerative diseases. We evaluated the consequences of disrupted kif5c-mediated mitochondrial trafficking on mitochondrial form and function in primary rat cortical neurons. Morphological changes in mitochondria appeared to be due to remodelling, a phenomenon distinct from mitochondrial fission, which resulted in punctate-shaped mitochondria. We also demonstrated that neurons displaying punctate mitochondria exhibited relatively decreased ROS and increased cellular ATP levels using ROS-sensitive GFP and ATP FRET probes, respectively. Somewhat unexpectedly, neurons overexpressing the dominant negative form of kif5c exhibited enhanced survival following excitotoxicity, suggesting that the impairment of mitochondrial trafficking conferred some form of neuroprotection. However, when neurons were exposed to H2O2, disruption of kif5c exacerbated cell death indicating that the effect on cell viability was dependent on the mode of toxicity. Our results suggest a novel role of kif5c. In addition to mediating mitochondrial transport, kif5c plays a role in the mechanism of regulating mitochondrial morphology. Our results also suggest that kif5c mediated mitochondrial dynamics may play an important role in regulating mitochondrial function and in turn cellular health. Moreover, our studies demonstrate an interesting interplay between the regulation of mitochondrial motility and morphology.