A 3D culture platform enables development of zinc-binding prodrugs for targeted proliferation of β cells

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Abstract

Interrogating zinc-binding prodrug in a 3D-reconstructed β cell environment.

Abstract

Advances in treating β cell loss include islet replacement therapies or increasing cell proliferation rate in type 1 and type 2 diabetes, respectively. We propose developing multiple proliferation-inducing prodrugs that target high concentration of zinc ions in β cells. Unfortunately, typical two-dimensional (2D) cell cultures do not mimic in vivo conditions, displaying a markedly lowered zinc content, while 3D culture systems are laborious and expensive. Therefore, we developed the Disque Platform (DP)—a high-fidelity culture system where stem cell–derived β cells are reaggregated into thin, 3D discs within 2D 96-well plates. We validated the DP against standard 2D and 3D cultures and interrogated our zinc-activated prodrugs, which release their cargo upon zinc chelation—so preferentially in β cells. Through developing a reliable screening platform that bridges the advantages of 2D and 3D culture systems, we identified an effective hit that exhibits 2.4-fold increase in β cell proliferation compared to harmine.

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Generation of functional human pancreatic β cells in vitro.

Felicia W Pagliuca, Jeffrey Millman, Mads Gürtler … (2014)

The generation of insulin-producing pancreatic β cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide β cells. Here, we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive β cells from hPSC in vitro. These stem-cell-derived β cells (SC-β) express markers found in mature β cells, flux Ca(2+) in response to glucose, package insulin into secretory granules, and secrete quantities of insulin comparable to adult β cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice. Copyright © 2014 Elsevier Inc. All rights reserved.

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Five-year follow-up after clinical islet transplantation.

Edmond Ryan, Breay Paty, Peter A. Senior … (2005)

Islet transplantation can restore endogenous beta-cell function to subjects with type 1 diabetes. Sixty-five patients received an islet transplant in Edmonton as of 1 November 2004. Their mean age was 42.9 +/- 1.2 years, their mean duration of diabetes was 27.1 +/- 1.3 years, and 57% were women. The main indication was problematic hypoglycemia. Forty-four patients completed the islet transplant as defined by insulin independence, and three further patients received >16,000 islet equivalents (IE)/kg but remained on insulin and are deemed complete. Those who became insulin independent received a total of 799,912 +/- 30,220 IE (11,910 +/- 469 IE/kg). Five subjects became insulin independent after one transplant. Fifty-two patients had two transplants, and 11 subjects had three transplants. In the completed patients, 5-year follow-up reveals that the majority ( approximately 80%) have C-peptide present post-islet transplant, but only a minority ( approximately 10%) maintain insulin independence. The median duration of insulin independence was 15 months (interquartile range 6.2-25.5). The HbA(1c) (A1C) level was well controlled in those off insulin (6.4% [6.1-6.7]) and in those back on insulin but C-peptide positive (6.7% [5.9-7.5]) and higher in those who lost all graft function (9.0% [6.7-9.3]) (P < 0.05). Those who resumed insulin therapy did not appear more insulin resistant compared with those off insulin and required half their pretransplant daily dose of insulin but had a lower increment of C-peptide to a standard meal challenge (0.44 +/- 0.06 vs. 0.76 +/- 0.06 nmol/l, P < 0.001). The Hypoglycemic score and lability index both improved significantly posttransplant. In the 128 procedures performed, bleeding occurred in 15 and branch portal vein thrombosis in 5 subjects. Complications of immunosuppressive therapy included mouth ulcers, diarrhea, anemia, and ovarian cysts. Of the 47 completed patients, 4 required retinal laser photocoagulation or vitrectomy and 5 patients with microalbuminuria developed macroproteinuria. The need for multiple antihypertensive medications increased from 6% pretransplant to 42% posttransplant, while the use of statin therapy increased from 23 to 83% posttransplant. There was no change in the neurothesiometer scores pre- versus posttransplant. In conclusion, islet transplantation can relieve glucose instability and problems with hypoglycemia. C-peptide secretion was maintained in the majority of subjects for up to 5 years, although most reverted to using some insulin. The results, though promising, still point to the need for further progress in the availability of transplantable islets, improving islet engraftment, preserving islet function, and reducing toxic immunosuppression.

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Spheroid-based drug screen: considerations and practical approach.

Juergen Friedrich Kreusch*, S. C. Seidel, Reinhard Ebner … (2008)

Although used in academic research for several decades, 3D culture models have long been regarded expensive, cumbersome and unnecessary in drug development processes. Technical advances, coupled with recent observations showing that gene expression in 3D is much closer to clinical expression profiles than those seen in 2D, have renewed attention and generated hope in the feasibility of maturing organotypic 3D systems to therapy test platforms with greater power to predict clinical efficacies. Here we describe a standardized setup for reproducible, easy-handling culture, treatment and routine analysis of multicellular spheroids, the classical 3D culture system resembling many aspects of the pathophysiological situation in human tumor tissue. We discuss essential conceptual and practical considerations for an adequate establishment and use of spheroid-based drug screening platforms and also provide a list of human carcinoma cell lines, partly on the basis of the NCI-DTP 60-cell line screen, that produce treatable spheroids under identical culture conditions. In contrast to many other settings with which to achieve similar results, the protocol is particularly useful to be integrated into standardized large-scale drug test routines as it requires a minimum number of defined spheroids and a limited amount of drug. The estimated time to run the complete screening protocol described herein--including spheroid initiation, drug treatment and determination of the analytical end points (spheroid integrity, and cell survival through the acid phosphatase assay)--is about 170 h. Monitoring of spheroid growth kinetics to determine growth delay and regrowth, respectively, after drug treatment requires long-term culturing (> or =14 d).

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Author and article information

Journal

Journal ID (nlm-ta): Sci Adv

Journal ID (iso-abbrev): Sci Adv

Journal ID (publisher-id): SciAdv

Journal ID (hwp): advances

Title: Science Advances

Publisher: American Association for the Advancement of Science

ISSN (Electronic): 2375-2548

Publication date Collection: November 2020

Publication date (Electronic): 18 November 2020

Volume: 6

Issue: 47

Electronic Location Identifier: eabc3207

Affiliations

[1 ]Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA.

[2 ]Center for Nanomedicine, Harvard Stem Cell Institute, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA.

[3 ]Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA.

[4 ]Proteomics Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

[5 ]David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA 02142, USA.

[6 ]Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

[7 ]Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.

[8 ]Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA.

[9 ]Divisions of Renal Medicine and Engineering, Brigham and Women’s Hospital, Boston, MA 02115, USA.

[10 ]Chemical Biology Program, Harvard University, Cambridge, MA 02138, USA.

Author notes

[*]

These authors contributed equally to this work.

[† ]Corresponding author. Email: jeffkarp@ 123456mit.edu (J.M.K.); achoudhary@ 123456bwh.harvard.edu (A.C.)

Author information

Kisuk Yang http://orcid.org/0000-0002-2724-5222

Miseon Lee http://orcid.org/0000-0001-9394-2643

Peter Anthony Jones http://orcid.org/0000-0003-0008-0461

Sophie S. Liu http://orcid.org/0000-0002-6616-1339

Angela Zhou http://orcid.org/0000-0001-9228-5960

Jun Xu http://orcid.org/0000-0002-4477-1670

Vedagopuram Sreekanth http://orcid.org/0000-0002-7065-2675

Jamie L. Y. Wu http://orcid.org/0000-0001-7663-4964

Lillian Vo http://orcid.org/0000-0003-1613-0996

Eunjee A. Lee http://orcid.org/0000-0002-7132-3822

Yuhan Lee http://orcid.org/0000-0002-0616-022X

Amit Choudhary http://orcid.org/0000-0002-8437-0150

Jeffrey M. Karp http://orcid.org/0000-0002-4752-7374

Article

Publisher ID: abc3207

DOI: 10.1126/sciadv.abc3207

PMC ID: 7673808

PubMed ID: 33208361

SO-VID: 80828590-312b-452b-9bba-d7ee5fd9e6eb

Copyright © Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

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This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

History

Date received : 23 April 2020

Date accepted : 07 October 2020

Funding

Funded by: doi http://dx.doi.org/10.13039/100000002, National Institutes of Health;

Award ID: R01 DK113597

Funded by: doi http://dx.doi.org/10.13039/100000002, National Institutes of Health;

Award ID: HIRN-CHIB, UC4DK104165-01

Funded by: doi http://dx.doi.org/10.13039/100000861, Burroughs Wellcome Fund;

Award ID: UC4DK116255

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A 3D culture platform enables development of zinc-binding prodrugs for targeted proliferation of β cells

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Gamma Delta T cells

Most cited references 60

Generation of functional human pancreatic β cells in vitro.

Five-year follow-up after clinical islet transplantation.

Spheroid-based drug screen: considerations and practical approach.

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