Porcine embryonic stem cells: An alternative solution for the shortage of human islets to treat type 1 diabetes?

Transplantation of human embryonic stem cell (ESC) derived beta‐like cells and xenotransplantation of porcine islets are two potential cures for type 1 diabetes (T1D). We have previously reported the isolation of porcine ESCs and have also separately shown that islets from genetically modified pigs can effectively cure diabetes in a preclinical baboon model. Here we discuss the possibility of combining these technologies to produce gene‐edited porcine ESC‐derived islets as an alternative for treating T1D, which may offer several advantages compared with these approaches.


CURRENT APPROACHES TO SOLVING THE SHORTAGE OF HUMAN ISLETS
Two approaches currently lead the way in the quest to solve the shortage of human islets for treating type 1 diabetes (T1D) by transplantation.The first involves the differentiation of human embryonic stem cells (ESCs) to beta-like cells. 1,2While this approach is promising, a recent consensus statement concluded that there is a need to develop simpler and more efficient methods which ideally produce all the major islet endocrine cell types. 3The second approach is xenotransplantation, requiring genetic modification of the donor pig to overcome the rejection of pig islets.Xenotransplantation has undergone a resurgence in recent times with the advent of gene editing, which has allowed multiple knockouts and knock-ins to be performed in a single generation rather than taking years to achieve by breeding. 4Gene editing has also advanced the prospect of blastocyst complementation to generate human islets in pigs.This strategy proposes knocking out the essen- tial pancreatic development gene PDX-1 in the developing pig embryo and injecting human ESCs to produce the missing pancreas. 5While these approaches hold promise, arguably the most advanced-the differentiation of human ESCs to beta-like cells-is still only in early-phase clinical trials after more than two decades of research. 2

PORCINE ESCs-A THIRD ALTERNATIVE TO SOLVING THE SHORTAGE OF ISLETS?
7][8] This work was initially aimed at facilitating the production of genetically modified pigs containing multiple modifications because the limited life of fibroblasts in vitro, limited the number of modifications that could be made in a single generation using animal cloning. 9However, this work was put on hold with the advent of gene editing, which we used to produce the first knock-in pigs for xenotransplantation Xenotransplantation. 2024;31:e12840.
wileyonlinelibrary.com/journal/xen 1 of 3 https://doi.org/10.1111/xen.12840purposes. 107][8] In characterizing these cells, we have also produced chimaeric pigs, 7 confirming their pluripotency and highlighting that they are closer to naive than primed ESCs. 11Since our initial work, an increasing number of porcine ESC cell types have been isolated which could also be used 12 , ideally once their pluripotency has been confirmed in chimaera studies.
In contrast, it is now generally accepted that the majority of existing human ESCs (as well as induced pluripotent stem cells; IPSCs) are closer to mouse epiblast stem cells derived from the post-implantation epiblast. 11While so-called primed ESCs can be differentiated to various cell lineages, primed mouse ESCs do not readily make chimaeras when injected into blastocysts, suggesting that they are not as pluripotent as per the original definition. 11We have differentiated our porcine ESCs to PDX-1-expressing cells. 7,8More recently, we have also shown that they can be differentiated in two steps to beta-like cells (unpublished results).In contrast, existing protocols to differentiate primed human ESCs into beta-like cells use five to seven steps. 1 Although preliminary, these results suggest that naïve ESCs may have an enhanced differentiation capacity possibly because of their earlier origin, which may address the shortcomings of primed ESCs for treating T1D. 3 In particular, the need for more efficient differentiation protocols and which ideally produce all major endocrine cell types. 3While naive human ESC's have been isolated which could answer this question, 11 there are no reports comparing the differentiation of naïve and primed human ESCs to beta-like cells.Although it is notable that naïve human ESCs produce more definitive endoderm and pancreatic progenitors than primed cells in teratoma studies. 13 parallel to our work with porcine ESCs, we have demonstrated long term (>600 days) function of GTKO/CD55-CD59-HT porcine islets in diabetic baboons. 14More complex modifications have been achieved by other groups using gene editing and we eagerly await the results of similar preclinical trials.Our ability to differentiate porcine ESCs to beta-like cells, together with our demonstration that genetically modified pig islets can effectively cure T1D, suggests that gene editing and differentiation of porcine ESCs may provide a hybrid approach to treating T1D.

ADVANTAGES OF PORCINE ESCs
In proposing such an alternative, the question that needs to be asked is what advantages could gene-edited porcine beta-like cells provide compared with the approaches currently being trialed?Compared with porcine islet xenotransplantation, the most obvious advantage is that there would be no need to maintain hundreds if not thousands of pigs in expensive defined pathogen-free facilities. 15Instead, cell lines could be maintained in relatively small cryobank facilities located across the world and expanded as required.Furthermore, the use of cell lines rather than animals would facilitate the standardization of the islet product, which could be rigorously tested in vitro and in vivo before transplantation. 15e second major advantage of porcine ESCs is that they avoid the ethical complications surrounding the use of human embryos to derive ESC lines.This is important because ESCs accumulate chromosomal abnormalities over time, ultimately requiring new lines to be derived. 16,17This is problematic given the relatively limited supply of human embryos, the majority of which are donated because they are deemed unsuitable for transfer.In contrast, in vivo-derived as well as in vitro-produced pig embryos from abattoir-sourced ovaries provide a virtually unlimited supply of viable embryos. 6,18ne editing also now makes it possible to introduce multiple modifications into porcine ESC lines in a single generation, allowing rapid testing of strategies aimed at overcoming rejection including novel approaches such as local immunosuppression. 4,10All of this can be done in the pig without the ethical considerations surrounding the genetic manipulation of human embryos and ESC lines.While human IPSCs may overcome both issues they are nevertheless primed, which as mentioned earlier may not be the best cell type for islet transplantation and cell therapy in general.Although the development of various strategies to convert these to a naïve cell type may overcome any deficiencies. 11

BACK TO THE FUTURE
In conclusion, gene edited porcine ESCs have the potential to provide a stand-alone therapy for T1D, with several advantages compared with porcine islets and human ESCs.At the very least, this approach may provide an interim solution before these other therapies come into their own.The various pluripotent cells which continue to be isolated for the pig 12 would also provide a muchneeded large animal model for this and cell therapy applications in general.In proposing this alternative, it has not escaped our attention that such an approach is very much "back to the future," as pig insulin was used to treat T1D for decades before the development of recombinant human insulin in the 1980s. 19Furthermore, the recent production of gene-edited pigs in which the pig proinsulin gene was replaced by the human proinsulin gene and which expressed human insulin and C-peptide 19 , makes this even more attractive and a step closer to producing a bio-inspired cell delivery system for T1D. 20

ACKNOWLEDGMENTS
Open access publishing facilitated by The University of Adelaide, as part of the Wiley -The University of Adelaide agreement via the Council of Australian University Librarians.

CONFLICT OF INTEREST STATEMENT
Mark B. Nottle is Owner/Director of ICMStemcell Pty Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
© 2024 The Authors.Xenotransplantation published by John Wiley & Sons Ltd.