Elsevier

Biomaterials

Volume 277, October 2021, 121067
Biomaterials

Patch grafting, strategies for transplantation of organoids into solid organs such as liver

https://doi.org/10.1016/j.biomaterials.2021.121067Get rights and content

Abstract

Epithelial cell therapies have been at an impasse because of inefficient methods of transplantation to solid organs. Patch grafting strategies were established enabling transplantation of ≥107th organoids/patch of porcine GFP+ biliary tree stem/progenitors into livers of wild type hosts. Grafts consisted of organoids embedded in soft (~100 Pa) hyaluronan hydrogels, both prepared in serum-free Kubota's Medium; placed against target sites; covered with a silk backing impregnated with more rigid hyaluronan hydrogels (~700 Pa); and use of the backing to tether grafts with sutures or glue to target sites. Hyaluronan coatings (~200–300 Pa) onto the serosal surface of the graft served to minimize adhesions with neighboring organs. The organ's clearance of hyaluronans enabled restoration of tissue-specific paracrine and systemic signaling, resulting in return of normal hepatic histology, with donor parenchymal cells uniformly integrated amidst host cells and that had differentiated to mature hepatocytes and cholangiocytes. Grafts containing donor mature hepatocytes, partnered with endothelia, and in the same graft biomaterials as for stem/progenitor organoids, did not engraft. Engraftment occurred if porcine liver-derived mesenchymal stem cells (MSCs) were co-transplanted with donor mature cells. RNA-seq analyses revealed that engraftment correlated with expression of matrix-metalloproteinases (MMPs), especially secreted isoforms that were found expressed strongly by organoids, less so by MSCs, and minimally, if at all, by adult cells.

Engraftment with patch grafting strategies occurred without evidence of emboli or ectopic cell distribution. It was successful with stem/progenitor organoids or with cells with a source(s) of secreted MMP isoforms and offers significant potential for enabling cell therapies for solid organs.

Introduction

There has long been a desire to treat diverse conditions and diseases of solid organs using cell therapies. The greatest hope is to be able to deliver stem cells and/or early progenitors to relevant target tissues to overcome genetic defects or aberrations from chronic diseases or infections [1]. The well-known proliferative capacity and multipotency of stem/progenitor cells are factors in their favor in cell therapies. What has constituted an impasse is the poor ability to transplant cells, especially stem/progenitors, into solid organs [2]. The exception is for skin for which there are well established grafting methods in which cells are mixed with grafting biomaterials and applied directly to the skin surface [3,4]. This is not an option for internal organs, where grafts are easily damaged by the jostling of organs and tissues against each other.

By contrast routine transplantation of hemopoietic cells or mesenchymal stem cells (MSCs) is achieved by delivery into a blood vessel and is dependent on micro-environmental signaling that triggers activation of adhesion molecules binding the donor cells to relevant target sites in a process referred to as “homing” [5,6].

Transplantation of epithelial cells from internal solid organs have been delivered traditionally via a vascular route or by direct injection. The intravascular approach is not logical, since adhesion molecules on these cells are always activated and result in rapid (within seconds) cell aggregation that can generate life-threatening emboli. Even if emboli are managed successfully to minimize health risks, the efficiency of cell engraftment is low, only ~10–20% for adult cells, and even lower, <5%, for stem/progenitors [2,7]. Most transplanted cells either die or are transported to ectopic sites, where they can live for months and where they can result in adverse clinical effects [[7], [8], [9], [10]]. The small percentage of cells that engraft into target sites slowly integrate, requiring weeks to reconstitute a significant portion of the tissue. Direct injection can disrupt the integrity of a solid organ through adverse mechanical effects. If not finely controlled, it can result also in hemorrhaging or in cells dying, because they are in a micro-environment not ideal for gas and nutrient exchange.

Recent efforts by a small but growing number of investigators have been focused on grafting strategies for internal organs or eyes [[11], [12], [13], [14], [15], [16], [17], [18]]. Examples include plating cells, such as mesenchymal stem cells (MSCs), on amnions and attaching the amnions to the target site [16]; the MSCs provide paracrine signaling that foster improvement in the diseased state of the organ or tissue. Another is cell sheet engineering [17], in which epithelial cells (hepatocytes) were prepared ex vivo on thermolabile dishes as cell sheets with well-formed gap junctions and lateral junctions; the sheets are released from the dishes by a temperature change; and the floating sheets are transferred and attached to the surface of the organ (e.g. liver). The cell sheets have been shown to provide critical functions needed to overcome a deficit(s) in the organ if diseased [12,17].

Yet another approach has been to incorporate growth factors and other signaling molecules into hyaluronan or fibrin hydrogels that are injected or grafted onto a target site [19,20]. Hyaluronans also help with transplantation of cells; if added to the medium for infusion of cells into the liver; engraftment is mediated by the organ's well-known ability for clearance of hyaluronans [21]. A variation of this proved useful for transplantation of hepatic stem cells that were suspended in a medium containing a thiol-form of hyaluronan that upon direct injection into the liver was triggered to form a hydrogel with PEGDA [22]. This method, referred to as “injection grafting”, resulted in localization of stem/progenitors to the liver and an avoidance of ectopic cell delivery.

Even with the best of these methods, the effectiveness is limited by the numbers of cells that can be transplanted, by the slowness in integration of donor cells into the target organ, and for sheet grafting, by the fact that grafts remain at the surface of the organ. The restriction of the donor cells to a surface graft site limits interactions with the community of cells and the matrix and soluble signals inherent in the tissue-specific microenvironment of an organ or tissue.

We demonstrate a new strategy, “patch grafting”, that works well for any cells, whether stem/progenitors or mature cells, and even for organoids, aggregates of epithelial stem/progenitors partnered with early lineage stage mesenchymal cells (ELSMCs) comprising angioblasts and precursors to endothelia and to stellate cells. In this report we demonstrate patch grafting using organoids of porcine biliary tree stem cells (BTSCs), precursors to both liver and pancreas, partnered with ELSMCs, and found in the biliary trees of all mammals tested [23]. We refer to these organoids as BTSC/ELSMCs. They are isolated from the biliary trees, where these cells are found in niches, peribiliary glands (PBGs), located throughout the biliary tree, especially at the branching points; the largest numbers of them are in the large, intrahepatic bile ducts and in the hepato-pancreatic common duct [[23], [24], [25]]. The biliary tree stem cells are immediate precursors to hepatic stem cells, and these to hepatoblasts, located in or near the canals of Hering [26,27], and to pancreatic stem cells, located in the PBGs of the hepato-pancreatic common duct and these to the pancreatic ductal progenitors in the pancreatic duct glands within the pancreas [28].

Recent analyses on their genetic signatures have characterized the signaling controlling the cells through the maturational lineage stages from BTSCs to hepatic lineage stages (hepatic stem cells to hepatoblasts to adult hepatocytes and cholangiocytes) and, in parallel, to pancreatic ones (pancreatic stem cells to pancreatic ductal progenitors to islets and acinar cells) [24,25,[28], [29], [30], [31], [32], [33], [34]].

As a demonstration of the new grafting strategies, organoids of porcine BTSCs/ELSMCs, prepared from GFP + transgenic pigs, have been transplanted by patch grafting onto livers of wild type pigs. The hosts have normal livers and are devoid of any disease or a state fostering a regenerative demand, indicating that successful patch grafting does not require those conditions to enable engraftment to occur. In addition, we show that the strategies work also for mature, adult epithelia, though there are distinctions in how patch grafting must be done.

Section snippets

Materials and methods

All methods are provided in the online supplement and include tables of key resources.

Biliary tree, a source of co-hepato/pancreatic stem progenitors, that can be used as donor cells

A flowchart for the grafting strategies is given in Fig. 1A. Donor cells consisted of biliary tree stem cells (BTSCs), present in peribiliary glands throughout the biliary tree of all mammals analyzed [23,31,35] and here were isolated from porcine biliary trees. These have been extensively characterized for their location in situ, their genetic signatures, and for proof ex vivo and in vivo that they are precursors to both liver and to pancreas [23,31,35].

Those from porcine tissues have now been

Discussion

Grafting strategies have been explored by which to overcome the long-standing impasse of transplantation of epithelial cells into solid organs such as liver. The standard methods have been direct injection or delivery into a vascular connection into the organ, most commonly in the case of liver, the portal vein for mature cells such as hepatocytes, or the hepatic artery for small cells (under 12 μm), such as stem/progenitors. The past methods are inefficient and result in serious, potentially

Conclusions

Patch grafting was found to be safe and effective for transplantation of organoids of stem/progenitors into the liver and, with distinct variations in the strategies, are applicable also to adult donor cells [59]. The cells engrafted, migrated throughout the organ, and then matured into or were stabilized as fully functional adult cells, all occurring within 2 weeks. Patch grafting provides far greater potential than existing transplantation methods (direct injection, delivery by a vascular

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this paper.

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. A grant on the H2B-GFP pigs (NIH HL051587) was awarded to J.A. Piedrahita, PhD at NCSU (Raleigh, NC). Jeremy Cribb, PhD, from the UNC Department of Physics and Astronomy, provided assistance in defining the rheological properties of the 3 versions of hyaluronan hydrogels used for transplantation of cells. The studies with him were supported by an

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    Coequal Second Authors.

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    Senior Authors contributing to experimental design and management.

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    Senior authors overall, contributing to experimental design, management and to writing of the drafts of the manuscript.

    4

    Current contact information for Dr. Lozoya: Translational Science and Innovation Laboratory, IQVIA/Q2 Solutions, Durham, NC 27703.

    5

    Current contact information for Dr. Adin: Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida.

    6

    Current contact information for Dr. He: Dr. Zhiying He, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200123, China.

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