Scalable Bioreactor-based Suspension Approach to Generate Stem Cell-derived Islets From Healthy Donor-derived iPSCs.

IF 5.3 2区医学 Q1 IMMUNOLOGY

Transplantation Pub Date : 2025-01-01 Epub Date: 2024-07-18 DOI:10.1097/TP.0000000000005108

Kevin Verhoeff, Nerea Cuesta-Gomez, Jasmine Maghera, Nidheesh Dadheech, Rena Pawlick, Nancy Smith, Doug O'Gorman, Haide Razavy, Braulio Marfil-Garza, Lachlan G Young, Aducio Thiesen, Patrick E MacDonald, A M James Shapiro

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引用次数: 0

Abstract

Background: Induced pluripotent stem cells (iPSCs) offer the potential to generate autologous iPSC-derived islets (iPSC islets), however, remain limited by scalability and product safety.

Methods: Herein, we report stagewise characterization of cells generated following a bioreactor-based differentiation protocol. Cell characteristics were assessed using flow cytometry, quantitative reverse transcription polymerase chain reaction, patch clamping, functional assessment, and in vivo functional and immunohistochemistry evaluation. Protocol yield and costs are assessed to determine scalability.

Results: Differentiation was capable of generating 90.4% PDX1 + /NKX6.1 + pancreatic progenitors and 100% C-peptide + /NKX6.1 + iPSC islet cells. However, 82.1%, 49.6%, and 0.9% of the cells expressed SOX9 (duct), SLC18A1 (enterochromaffin cells), and CDX2 (gut cells), respectively. Explanted grafts contained mature monohormonal islet-like cells, however, CK19 + ductal tissues persist. Using this protocol, semi-planar differentiation using 150 mm plates achieved 5.72 × 10 4 cells/cm 2 (total 8.3 × 10 6 cells), whereas complete suspension differentiation within 100 mL Vertical-Wheel bioreactors significantly increased cell yield to 1.1 × 10 6 cells/mL (total 105.0 × 10 6 cells), reducing costs by 88.8%.

Conclusions: This study offers a scalable suspension-based approach for iPSC islet differentiation within Vertical-Wheel bioreactors with thorough characterization of the ensuing product to enable future protocol comparison and evaluation of approaches for off-target cell elimination. Results suggest that bioreactor-based suspension differentiation protocols may facilitate scalability and clinical implementation of iPSC islet therapies.

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基于可扩展生物反应器的悬浮法从健康供体iPSCs中生成干细胞衍生的胰岛

背景：诱导多能干细胞（iPSCs）提供了产生自体iPSC衍生的胰岛（iPSC胰岛）的潜力，然而，仍然受到可扩展性和产品安全性的限制。方法：在此，我们报告了基于生物反应器的分化方案产生的细胞的分期表征。使用流式细胞术、定量逆转录聚合酶链反应、贴片夹紧、功能评估、体内功能和免疫组织化学评估来评估细胞特性。评估协议产量和成本以确定可伸缩性。结果：分化能产生90.4%的PDX1 + /NKX6.1 +胰腺祖细胞和100%的c肽+ /NKX6.1 + iPSC胰岛细胞。然而，82.1%、49.6%和0.9%的细胞分别表达SOX9（导管）、SLC18A1（肠色素细胞）和CDX2（肠细胞）。移植的移植物含有成熟的单激素胰岛样细胞，然而，CK19 +导管组织仍然存在。使用该方案，使用150 mm板的半平面分化达到5.72 × 10.4细胞/cm 2（总计8.3 × 10.6细胞），而在100 mL垂直轮生物反应器中完全悬浮分化显著提高细胞产量至1.1 × 10.6细胞/mL（总计105.0 × 10.6细胞），降低了88.8%的成本。结论：本研究为垂直轮生物反应器内iPSC胰岛分化提供了一种可扩展的基于悬浮液的方法，并对随后的产物进行了全面的表征，以便将来对脱靶细胞消除方法进行方案比较和评估。结果表明，基于生物反应器的悬浮分化方案可促进iPSC胰岛治疗的可扩展性和临床实施。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Transplantation 医学-免疫学

CiteScore

8.50

自引率

11.30%

发文量

1906

审稿时长

1 months

期刊介绍： The official journal of The Transplantation Society, and the International Liver Transplantation Society, Transplantation is published monthly and is the most cited and influential journal in the field, with more than 25,000 citations per year. Transplantation has been the trusted source for extensive and timely coverage of the most important advances in transplantation for over 50 years. The Editors and Editorial Board are an international group of research and clinical leaders that includes many pioneers of the field, representing a diverse range of areas of expertise. This capable editorial team provides thoughtful and thorough peer review, and delivers rapid, careful and insightful editorial evaluation of all manuscripts submitted to the journal. Transplantation is committed to rapid review and publication. The journal remains competitive with a time to first decision of fewer than 21 days. Transplantation was the first in the field to offer CME credit to its peer reviewers for reviews completed. The journal publishes original research articles in original clinical science and original basic science. Short reports bring attention to research at the forefront of the field. Other areas covered include cell therapy and islet transplantation, immunobiology and genomics, and xenotransplantation.