(237f) Bioprocessing to Fabricate Stem Cell-Derived ? Cells for Diabetes Cell Replacement Therapy

INTRODUCTION: A major goal of cellular engineering is to produce mature, functional cells for therapeutic applications. Human pluripotent stem cells (hPSCs) are incredibly useful for this goal, as they have the potential to become any cell type found in the body if given the correct signals from the surrounding environment. Generation of insulin-producing β cells from hPSCs is one such application of this technology for the treatment of patients with diabetes [1]. We have developed a multi-faceted strategy involving modulating the physical microenvironment through the cytoskeleton and three-dimensional cell arrangement in addition to targeted activation and inhibition of specific cellular signal transduction networks to produce stem cell-derived β (SC-β) cells capable of rapidly reversing diabetes in mice.

RESULTS: We found that conditions that promote a disorganized actin cytoskeleton to be a strong inducer of pancreatic endocrine, including three-dimensional cell arrangement, cellular attachment onto soft surfaces, or treatment with the compound latrunculin A that depolymerizes actin. Specifically, these conditions induce the expression of the transcription factor NEUROG3, which is the master regulator of endocrine cell fate during development. We also found that combination of these conditions to modulate the actin cytoskeleton with Alk5 inhibition, NOTCH inhibition, and thyroid hormone receptor activation to be a strong inducer of SC-β cell differentiation [2]. These SC-β cells expressed many markers found in real β cells from donors, such as insulin, PDX1, and NKX6-1. They were able to function, undergoing glucose-stimulated insulin secretion, including dynamically responding to high glucose levels with both first and second phase insulin secretion kinetics. We have scaled this process to make over 100 million cells per batch with over 90% of cells characterized as pancreatic endocrine. Single-cell RNA sequencing (scRNA-seq) revealed not only SC-β cells but also other endocrine cell types found in the pancreas, including α-like and δ-like cells expressing glucagon and somatostatin, respectively. Upon transplantation into mice with severe pre-existing diabetes (>500 mg/dL blood glucose), SC-β cells rapidly restored normoglycemia in 2 weeks and could maintain blood glucose control for 1 year. The in vivo environment induced further maturation in the gene expression signature of transplanted SC-β cells to more closely resemble β cells from donors, as assessed with scRNA-seq.

CONCLUSIONS: Our approach combines modulation of the insoluble microenvironment or direct cytoskeletal modulation with activation and inhibition of developmental signaling pathways to produce SC-β cells that are functional and capable of reversing pre-existing diabetes in mice [3]. These results demonstrate the potential for SC-β cells to be used in diabetes cell replacement therapy [4].

REFERENCES:

[1] Pagliuca FW, Millman JR, Gürtler M, Segel M, Van Dervort A, Ryu JH, Peterson QP, Greiner D, Melton DA. Generation of functional human pancreatic β cells in vitro. Cell. 2014 Oct 9;159(2):428-39. doi: 10.1016/j.cell.2014.09.040.

[2] Velazco-Cruz L, Song J, Maxwell KG, Goedegebuure MM, Augsornworawat P, Hogrebe NJ, Millman JR. Acquisition of Dynamic Function in Human Stem Cell-Derived β Cells. Stem Cell Reports. 2019 Feb 12;12(2):351-365. doi:10.1016/j.stemcr.2018.12.012.

[3] Hogrebe NJ, Augsornworawat P, Maxwell KG, Velazco-Cruz L, Millman JR. Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells. Nature Biotechnology. 2020 Apr;38(4):460-470. doi:10.1038/s41587-020-0430-6.

[4] Maxwell KG, Augsornworawat P, Velazco-Cruz L, Kim MH, Asada R, Hogrebe NJ, Morikawa S, Urano F, Millman JR. Gene-edited human stem cell-derived β cells from a patient with monogenic diabetes reverse preexisting diabetes in mice. Science Translational Medicine. 2020 Apr 22;12(540):eaax9106. doi: 10.1126