Significant Expansion of the Donor Pool Achieved by Utilizing Islets of Variable Quality in the Production of Allogeneic “neo-islets”, 3-D Organoids
Anna M. Gooch, Sabiha S. Chowdhury, Ping M. Zhang, Zhuma M. Hu, Christof Westenfelder
Abstract
Novel biotherapies for Type 1 Diabetes that provide a significantly expanded donor pool and that deliver all islet hormones without requiring anti-rejection drugs are urgently needed. Scoring systems have improved islet allotransplantation outcomes, but their use may potentially result in the waste of valuable cells for novel therapies. To address these issues, we created “Neo-Islets” (NIs), islet-sized organoids, by co-culturing in ultralow adhesion flasks culture-expanded islet (ICs) and Mesenchymal Stromal Cells (MSCs) (x 24 hrs, 1:1 ratio). The MSCs exert powerful immune- and cyto-protective, anti-inflammatory, proangiogenic, and other beneficial actions in NIs. The robust in vitro expansion of all islet hormone-producing cells is coupled to their expected progressive de-differentiation mediated by serum-induced cell cycle entry and Epithelial-Mesenchymal Transition (EMT). Re-differentiation in vivo of the ICs and resumption of their physiological functions occurs by reversal of EMT and serum withdrawal-induced exit from the cell cycle. Accordingly, we reported that allogeneic, i.p.-administered NIs engraft in the omentum, increase Treg numbers and reestablish permanent normoglycemia in autoimmune diabetic NOD mice without immunosuppression. Our FDA-guided pilot study (INAD 012–0776) in insulin-dependent pet dogs showed similar responses, and both human- and canine-NIs established normoglycemia in STZ-diabetic NOD/SCID mice even though the utilized islets would be scored as unsuitable for transplantation.
Introduction
A globally available therapeutic that re-establishes insulin independence in patients with Type 1 Diabetes (T1DM) while also not requiring potentially toxic antirejection drugs is urgently needed in order to improve the quality of life and prognosis of millions of insulin-dependent diabetic patients worldwide. Effective current treatments are allogeneic pancreas or islet transplants, both of which can provide endogenous insulin replacement and normalized glycemic control. However, the limited availability of donors, the need for repeated islet transplants, and the reliance on often toxic antirejection drugs limits the safety and use of such therapies [1, 2]. Because both allogeneic islet and pancreas transplants, and several experimental, allogeneic, insulin producing, stem- or precursor cell-based therapies still depend on the life-long use of potentially harmful antirejection drugs [3–6], auto- and allo-immune isolation of beta-like cells is being tested with various encapsulation technologies. Unfortunately, some of these are still prone to foreign body reactions, amyloid accumulation, release of alloantigens and consequently have frequently failed [7–11]. To address these issues, we created “Neo-Islets” (NIs), islet-sized organoids, by co-aggregating culture-expanded islet cells (ICs) and Mesenchymal Stromal Cells (MSCs) in a 1:1 ratio. The MSCs exert in the microenvironment of the engrafted NIs powerful immune- and cyto-protective, anti-inflammatory, proangiogenic, and other beneficial actions.
Materials and methods
Cell culture
Islet cell culture.
Research grade human islets from 6 non-diabetic human donors (see Table 1 for demographics and Islet characteristics) were purchased in lots of ~5,000 Islet Equivalents from Prodo Labs (Aliso Viejo, CA). Islet cells derived from this inhomogeneous group of islet donors were expanded by culturing whole islets in tissue culture flasks, using RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA) + 10% human Platelet Lysate (hPL; Cell Therapy and Regenerative Medicine, University of Utah, Salt Lake City) + 1 x L-Glutamine-Penicillin-Streptomycin solution (GPS; Sigma G1146) until ~90% confluent. For passaging, cells were released with 2x Trypsin (Sigma, St. Louis, MO), pelleted by centrifugation at 600x g for 5 min., washed with DMEM 5 mM glucose (Gibco) + 10% hPL + GPS (complete medium), and reseeded at a density of 2x10e5 cells into T75 flasks in complete medium. hICs were characterized by rtPCR for expression of IC specific genes. Human ICs (hICs) and canine ICs (cICs) doubling times and population doublings (PDLs) were calculated by standard methods (see below).
Results
It was examined whether demographic differences between human islet donors and the quality of isolated islets can adversely affect the characteristics and formation of hNIs, manufactured by co-aggregation of cultured ICs and MSCs. Accordingly, functional profiles of hICs from 6 demographically different, non-diabetic human donors with different body mass indices and IC viabilities and purities (see details in Table 1 and islet images and human donor and islet characteristics in S1 and S2 Files) were compared to each other by assessing their growth rates, islet-specific endocrine gene expression levels as a function of PDLs, GSIS, and their ability to form NIs when co-cultured with MSCs. Identical tests were carried out with canine ICs and ASCs and obtained results were compared to human cell data. Correlation with PDLs was used because (a) the expansion process and entering of the cell cycle trigger the decrease in endocrine gene and protein expression, (b) this expansion is what allows NI technology to partially overcome the donor shortage issue, (c) predictable production endpoints are critical to manufacture of a product, and (d) it is the endocrine expression capacity of the IC component of NIs that is of critical value in this technology, we examined herein whether there was a consistent and predictable correlation between cell expansion and endocrine gene expression.
Discussion
The present study examined the central question whether donors and their islets that may be judged unsuitable for clinical islet transplants based on the application of the NAIDS and other scoring criteria [37, 38, 41, 45] can instead be used to manufacture NIs that have the capacity to significantly improve insulin needs and glucose levels and to eventually re-establish euglycemia and insulin-independence. Specifically, the impact of demographic differences of human and canine pancreatic islet donors and/or phenotypic variations in islet characteristics such post isolation quality, viability, purity and growth rates were analyzed. For this purpose, the islets from both 6 diverse human and 6 diverse dog islet donors were analyzed in vitro as to their growth characteristics in culture, gene expression profiles (insulin, glucagon, somatostatin, PPY) as a function of their population doubling times. In addition, GSIS and their ability to form NIs with Mesenchymal or Adipose Stromal Cells were examined. Obtained results of culture expanded islet cells were compared to the characteristics of their respective parent cells, i.e., freshly isolated islets.
Conclusion
In conclusion, “poor quality” of freshly isolated human and canine islets that may be judged as unsuited for islet transplantation, appear to possess great utility for the manufacturing of high numbers of NIs, 3-D organoids of Mesenchymal Stromal and Islet Cells that have proven to possess excellent therapeutic efficacy in pre-clinical Type 1 Diabetes studies, thereby identifying this novel, minimally invasive, retrievable and economical stem cell-enabled technology as significant progress for the conduct of clinical diabetes trials and eventual therapy. Taken together, we posit that human-NIs created from diverse, “non-clinical grade” donors have the capacity to greatly expand patient access to this curative therapy, facilitated by the efficient in vitro expansion of islet cells. Furthermore, biotherapy-specific adjustments in the current donor and islet scoring systems may be warranted. In recognition of our data and in preparation for a subsequent clinical trial, the FDA has approved our IND-enabling Proof of Concept study that is currently ongoing.
Acknowledgments
Some of the herein presented data were shown in poster format presentation at the 80th and 82nd American Diabetes Association Annual Sessions.
Citation: Gooch AM, Chowdhury SS, Zhang PM, Hu ZM, Westenfelder C (2023) Significant expansion of the donor pool achieved by utilizing islets of variable quality in the production of allogeneic “Neo-Islets”, 3-D organoids of Mesenchymal Stromal and islet cells, a novel immune-isolating biotherapy for Type I Diabetes. PLoS ONE 18(8): e0290460. https://doi.org/10.1371/journal.pone.0290460
Editor: Giovanni Camussi, Universita degli Studi di Torino, ITALY
Received: December 16, 2022; Accepted: August 9, 2023; Published: August 24, 2023
Copyright: © 2023 Gooch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was funded in its entirety by SymbioCellTech, LLC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal’s policy, and the authors of this manuscript have the following competing interests: CW, AG, SC, PZ, and ZH are full time employees of SymbioCellTech, LLC (SCT). CW and AG are also board members of SCT. CW, AG, PZ, and ZH hold profits interest shares in SCT. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0290460#abstract0
