Abstract: Epithelial cell therapies have been at an impasse because of inefficient methods of transplantation to solid organs. We established patch grafting strategies enabled transplantation of ≥10^8th (pigs) or ≥10^6th-7th (mice) organoids/patch of biliary tree stem/progenitors into livers of hosts. Grafts consisted of organoids prepared in serum-free Kubota’s Medium and embedded in soft (~100 Pa) hyaluronan hydrogels; placed against target sites; covered with a backing of silk impregnated with more rigid hyaluronan hydrogels (~700 Pa); and use of backing to tether grafts with sutures or glue to target sites. A hyaluronan coating (~200 Pa) onto the serosal surface of the graft served to minimize adhesions with neighbouring organs. Graft conditions fostered expression of multiple matrix-metalloproteinases (MMPs) resulting in transient loss of the organ’s matrix-dictated histological features and correlated with rapid integration of organoids throughout the organs and without emboli or ectopic cell distribution. Within another week, there was clearance of graft biomaterials, muted expression of MMPs, restoration of matrix-dictated histology, and maturation of donor cells to functional adult fates. The ability of patch grafts of organoids to rescue hosts from genetic-based disease states was demonstrated with grafts on livers, able to rescue NRG/FAH-KO mice from type I tyrosinemia. With the same grafts, if on pancreas, they were able to rescue NRG/Akita mice from type I diabetes. Utilizing the same grafting biomaterials and the same mesenchymal stem/progenitors, varying only the choice of epithelial stem cell populations in the organoids, the patch grafting provides a generic method of delivering cells into target organs.

Keywords: patch grafting, liver, pancreas, biliary tree, stem/progenitors, organoids, tyrosinemia, diabetes, cell therapies

Acknowledgements:
A sponsored research grant from Vesta Therapeutics (Bethesda, MD) to L. M. Reid at UNC (Chapel Hill, NC) provided most of the funding for the studies. Vesta Therapeutics is a wholly owned subsidiary of Toucan Capital (Bethesda, MD). A grant on the H2B-GFP pigs (NIH HL051587) was awarded to J.A. Piedrahita at NCSU (Raleigh, NC). John Cullen, DVM (now retired) and Stephany Montgomery, DVM, PhD (UNC at Chapel Hill) provided guidance in early stages of the project.
Patents have been filed on various aspects of the technologies including for grafting strategies for solid organs. All are owned by UNC. The clinical uses in humans of the IP are licensed to Vesta Therapeutics (Bethesda, MD), and the clinical uses in non-humans (veterinary uses) and the non-clinical, commercial uses are licensed to PhoenixSongs Biologicals (Branford, CT). None of the authors have equity in Vesta, nor have a position within the company, and none have been provided payments for consulting. LMR is one of the founders of PhoenixSongs Biologicals and has an equity position in the company but does not receive salary nor consulting nor research funds from the company.
Bioinformatic analysis and genetic signature study were supported by This work was funded by Major Program of National Key Research and Development Project (2020YFA0112600, 2019YFA0801502), National Natural Science Foundation of China (82173019, 82270638, 82203741), Shanghai Pujiang Program (21PJD059), the Project of Shanghai Science and Technology Commission (19140902900, 22ZR1451100, 22Y11908500), Program of Shanghai Academic/Technology Research Leader (20XD1434000), Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai, Jiangxi Provincial Natural Science Foundation (20212ACB206033), and Shanghai Engineering Research Center of Stem Cells Translational Medicine (20DZ2255100).
Discounted rates for using core services were provided by federal funding of the cores. These included: a histology core funded by the Center for Gastrointestinal and Biliary Disease Biology via an NIDDK Grant (DK34987); the Lineberger Cancer Center grant (NCI grant # CA016086) with Shelly Earp, PhD being the director; the Carolina Center for Genome Sciences (Katherine Hoadley, director) and the UNC Center for Bioinformatics (Hemant Kelkar, director).

Related publications:
1. Zhang W, Lanzoni G, Hani H, Overi D, Cardinale V, Simpson S, Pitman W, Allen A, Yi X, Wang X, Gerber D, Prestwich G, Lozoya O, Gaudio E, Alvaro D, Tokaz D, Dominguez-Bendala J, ¹Adin C, ² Piedrahita J, Mathews K, ³Sethupathy P, Carpino G, ⁴He Z, Wauthier E, and ⁵Reid LM. Patch grafting, strategies for transplantation of organoids into solid organs such as liver. Biomaterials. 2021 Oct. http://doi.10.1016/j.biomaterials.2021.121067. Bolded: senior authors. Corresponding senior authors on ¹veterinary issues; ²porcine genetics; ³genetic signatures, ⁴bioinformatics, and on ⁵stem cells, matrix biology/chemistry and overall.

2. Zhang W, Wauthier E, Lanzoni G, Hani, H, Yi X, Overi D , Lei Shi L, Simpson S, Allen A, Suitt C, Ezzell J.A., Alvaro D, Cardinale V, Gaudio E, Carpino G, Prestwich G, Dominguez-Bendala J, Gerber D, Matthews K, ¹Piedrahita J, ²Adin C, ³Sethupathy, P, ⁴He Z, and ⁵Reid L.M. Patch grafting of organoids of stem/progenitors into solid organs can correct genetic-based disease states. Biomaterials. 2022. https://doi.org/10.1016/j.biomaterials.2022.121647. Bolded: Senior authors. Corresponding senior authors on ¹porcine genetics; ²veterinary issues; ³genetic signatures, ⁴bioinformatics, and on ⁵stem cells, matrix biology/chemistry and overall.

Biography: Dr. Wencheng Zhang is a current research associate professor at Shanghai East Hospital Affiliated to Tongji University. He went to the Northwest Agriculture and Forestry University for his undergraduate with a major of bioscience from 2003 to 2007. Then, he got his PhD in regenerative medicine from the Academy of Military Medicine Science in 2012. He went to the University of North Carolina at Chapel Hill for his postdoctoral training with Prof. Lola M. Reid for studying the endodermal stem cells and developing the patch grafting strategy. And later, he worked as a research associate in Prof. Reid`s and Prof. Jian Liu`s labs, respectively, for investigating the biological effects of heparin sulfate glycosaminoglycans. At current, Dr. Zhang`s research has been supported by four national or provincial grants. He has 20 publications on the journal such as Cell Stem Cell, Journal of Hepatology, Hepatology, and Biomaterials, etc. with a total impact factor 178.838; total citation on Google Scholar 208; he also is an inventor on 9 major patents including being a lead inventor on patents on patch grafting strategies. His current research interests include hepatic-maturation of stem cells post-grafting, as well as glycosaminoglycans in stem cell expansion and cell fate determination.