Self-Condensation Culture Enables Vascularization of Tissue Fragments for Efficient Therapeutic Transplantation

Abstract

Clinical transplantation of tissue fragments, including islets, faces a critical challenge because of a lack of effective strategies that ensure efficient engraftment through the timely integration of vascular networks. We recently developed a complex organoid engineering method by "self-condensation" culture based on mesenchymal cell-dependent contraction, thereby enabling dissociated heterotypic lineages including endothelial cells to self-organize in a spatiotemporal manner. Here, we report the successful adaptation of this method for generating complex tissues from diverse tissue fragments derived from various organs, including pancreatic islets. The self-condensation of human and mouse islets with endothelial cells not only promoted functionalization in culture but also massively improved post-transplant engraftment. Therapeutically, fulminant diabetic mice were more efficiently treated by a vascularized islet transplant compared with the conventional approach. Given the general limitations of post-transplant vascularization associated with 3D tissue-based therapy, our approach offers a promising means of enhancing efficacy in the context of therapeutic tissue transplantation.

Keywords: islet transplantation; organoid; tissue engineering; tissue-based therapy; vascularization.

Figure 1.. Self-Condensation into Endothelialized Tissues from Diverse Tissue Fragments

(A) General strategy for creating endothelialized tissue fragments in vitro. (B) The endothelialization technique is adaptable to all tissue types examined in this study, including islets, brain fragments, heart fragments, intestine fragments, kidney fragments, liver fragments, lung fragments, and even spheroids of induced pluripotent stem cells. Scale bars, 1,000 μm. (C) Gross observation of a mini-sized vascularized islet (upper) and an islet alone (lower). Scale bars, 500 μm. (D) Formation of a three-dimensional mini-sized vascularized mouse islet in co-cultures of islets with HUVECs and human MSCs. The arrowheads indicate the blood vessel-like structures. Scale bars, 250 μm. (E) Immunofluorescence staining of the pre-transplantation vascularized islet (upper) and islet alone (lower). GCG, glucagon; hCD31, human CD31; INS, insulin; SST, somatostatin. Scale bars, 100 μm. (F) Hierarchical clustering of the pancreatic lineage 121 genes with the relevance in pancreatic development or β cell function. Each row in the heatmap represents a sample, and each column represents the expression level of a gene. The color scale below the heatmap represents the raw Z score ranging from green (high expression) to red (low expression).

Figure 2.. Vascularized Islet Self-Condensation Enables Efficient Therapeutic Transplantation

(A and B) Brightfield image of the collected vascularized mouse islet (A), islet alone (B), and recipient kidneys after transplantation. Scale bars, 500 μm. (C) Kaplan-Meier survival curves of the diabetic mice. *p < 0.05. (D) Percentage of body weight variations in the diabetic mice. The data represent the mean ± SD. n = 11 vascularized mouse islet, n = 10 mouse islet alone, n = 8 diabetes mellitus (DM) sham mice, and n = 15 DM mice. (E) Blood glucose measurements of the progressively diabetic mice. The glucose measurements were saturated at 600 mg/dL. The data represent the mean ± SD. n = 11 vascularized mouse islet, n = 10 islet alone, n = 8 DM sham mice, and n = 15 DM mice. (F) In vivo human insulin secretion. The data represent the mean ± SD. n = 5 vascularized human islet and n = 8 human islet alone. **p < 0.01; *p < 0.05. (G) Glucose tolerance testing 35 days after transplantation. The data represent the mean ± SD. n = 4 vascularized human islet and n = 4 human islet alone. (H) Area under the curve for (G). The data represent the mean ± SD. n = 4 vascularized human islet and n = 4 human islet alone.

Figure 3.. Rapid Induction of Functional Vascular Networks In Vivo

(A–C) Macroscopic observation of the transplanted mini-sized vascularized islet (n = 19) (A) and islet alone (n = 7) (B) at multiple time points, showing the rapid perfusion of blood vessels (C). Asterisk indicates the transplants. Scale bars, 1,000 μm. ND, not detected. (D) Intravital tracking of a vascularized mouse islet. Islet, green; HUVECs, red; human MSCs, unlabeled; mouse CD31, blue. The dotted area indicates the transplants. Scale bars, 100 μm. (E) Dextran infusion showing recipient circulation. The dotted area indicates the mouse islet. Scale bar, 250 μm. (F and G) Intravital imaging of a vascularized mouse islet (F) and mouse islet alone (G) 7 days after transplantation. Islet, green; HUVECs and human MSCs, unlabeled; dextran, red. Scale bars, 100 μm. (H and I) Intravital imaging of a vascularized human islet (H) and a human islet alone (I) after transplantation. Islet, unlabeled; HUVECs, red; human MSCs, unlabeled; dextran, green; human CD31, blue; mouse CD31, red. The dotted area indicates the islet. Scale bars, 100 μm. (J) Quantification of the dextran-positive area. The data represent the mean ± SD. ***p < 0.001; **p < 0.01 (n = 3, 5, 6, 8, and 5 mouse pancreatic islet, mouse islet alone Tx, vascularized mouse islet Tx, human islet alone, and vascularized human islet, respectively). (K) Visualization of the connections between HUVECs and host vessels at day 14. Mouse CD31, green; HUVECs, red; dextran, blue. Scale bar, 25 μm. (L) Localization of human MSCs at day 14. Arrowheads indicate hMSC localization in perivascular region. Dextran, green; human MSC, red; mouse CD31, blue. Scale bar, 50 μm.

Figure 4.. Preservation of Natural Islet Components in the Vascularized Islet Transplant

(A and B) H&E and immunofluorescence staining of the grafts from a vascularized mouse islet Tx (A) and mouse islet alone Tx (B) at day 35. The dotted line indicates the border on the brain. Col IV, collagen IV; hCD31, human CD31; INS, insulin; LAM, laminin. Scale bars, 50 μm. (C and D) Quantification of the laminin-positive (n = 29) (C), and collagen IV-positive (n = 3) (D) areas. Data represent the mean ± SD. ****p < 0.0001. ns, not significant. (E) Immunofluorescence staining of the grafts at day 35. GCG, glucagon; INS, insulin; SST, somatostatin. Scale bars, 50 μm. (F) Quantification of the endocrine cells in the graft. Mouse islet alone, n = 10; vascularized mouse islet, n = 9. **p < 0.01. (G–I) Quantification of the insulin-positive (G), glucagon-positive (H), and somatostatin-positive (I) areas in the graft. The data represent the mean ± SD. n = 12 mouse pancreatic islet Tx, n = 13 mouse islet alone, and n = 9 vascularized mouse islet Tx. ***p < 0.001; **p < 0.01; *p < 0.05. ns, not significant. (J–L) Electron microscopy of the vascularized mouse islet Tx (J), mouse islet Tx (K), and pancreatic mouse islet (L) at 35 days. α, alpha cell; β, beta cell; δ, delta cell; BV, blood vessel; EC, exocrine cell. Scale bars, 5 μm