Facilitating islet transplantation using a three-step approach with mesenchymal stem cells, encapsulation, and pulsed focused ultrasound

Abstract

Background: The aim of this study was to examine the effect of a three-step approach that utilizes the application of adipose tissue-derived mesenchymal stem cells (AD-MSCs), encapsulation, and pulsed focused ultrasound (pFUS) to help the engraftment and function of transplanted islets.

Methods: In step 1, islets were co-cultured with AD-MSCs to form a coating of AD-MSCs on islets: here, AD-MSCs had a cytoprotective effect on islets; in step 2, islets coated with AD-MSCs were conformally encapsulated in a thin layer of alginate using a co-axial air-flow method: here, the capsule enabled AD-MSCs to be in close proximity to islets; in step 3, encapsulated islets coated with AD-MSCs were treated with pFUS: here, pFUS enhanced the secretion of insulin from islets as well as stimulated the cytoprotective effect of AD-MSCs.

Results: Our approach was shown to prevent islet death and preserve islet functionality in vitro. When 175 syngeneic encapsulated islets coated with AD-MSCs were transplanted beneath the kidney capsule of diabetic mice, and then followed every 3 days with pFUS treatment until day 12 post-transplantation, we saw a significant improvement in islet function with diabetic animals re-establishing glycemic control over the course of our study (i.e., 30 days). In addition, our approach was able to enhance islet engraftment by facilitating their revascularization and reducing inflammation.

Conclusions: This study demonstrates that our clinically translatable three-step approach is able to improve the function and viability of transplanted islets.

Keywords: Diabetes; Encapsulation; Islets transplantation; Mesenchymal stem cells; Pulsed focused ultrasound.

Fig. 1

Experimental overview and characterization of islets encapsulated with AD-MSCs: a Schematic representation of our three-step approach: step 1: AD-MSC coating, step 2: encapsulation, and step 3: pFUS treatment; characterization of b–e AD-MSCs (b confocal image and c–e FACS analysis), f–i alginate capsule (photographic, SEM images, and XPS scans), and j–n confocal images of an j islet, k AD-MSCs, l islet coated with AD-MSCs, and m–n encapsulated with alginate followed by pFUS treatment. Blue: live cells stained with Hoechst. Green: AD-MSCs stained with FDA

Fig. 2

In vitro analysis of islet survival and function in normal conditions: a Representative confocal images, and results of b live/dead, c MTT, and d GSIS assays of tested groups (i.e., Islets, e-Islets, m-Islets, em-Islets, and emf-Islets). Confocal images and live/dead assay have been performed at days 1 and 7, and MTT and GSIS assays at day 7, in culture. Blue: live cells stained with Hoechst. Red: dead cells stained with PI. Green: AD-MSCs stained with FDA. Scale bar = 50 μm. Significant differences: b–d ^aP < 0.05: Islets vs. m-Islets or em-Islets or emf-Islets; ^bP < 0.05: m-Islets vs. em-Islets or emf-Islets; ^dP < 0.05: em-Islets vs. emf-Islets (one-way ANOVA post hoc Tukey test). b *P < 0.05: day 1 vs. day 7; d *P < 0.05: high glucose vs. low glucose (two (b, d) or one (c)-way ANOVA post hoc Tukey test)

Fig. 3

In vitro analysis of islet survival and function following exposure to pro-inflammatory cytokines: a representative confocal images and results of b live/dead, c MTT, and d high-glucose-stimulated insulin secretion assays of tested groups (i.e., Islets, e-Islets, m-Islets, em-Islets, and emf-Islets). Blue: live cells stained with Hoechst. Red: dead cells stained with PI. Green: AD-MSCs stained with FDA. Scale bar = 50 μm. Significant differences: b–d ^aP < 0.05: Islets vs. m-Islets or em-Islets or emf-Islets; ^bP < 0.05: m-Islets vs. em-Islets or emf-Islets; ^dP < 0.05: em-Islets vs. emf-Islets (one-way ANOVA post hoc Tukey test)

Fig. 4

In vivo analysis of islet survival and function (metabolic analysis): a islet transplantation using a kidney subcapsular approach; pFUS treatment on the kidney transplanted with islets; transplanted kidney with different experimental groups (i.e., Islets, e-Islets, m-Islets, em-Islets, and emf-Islets) at the time of euthanasia (black arrows indicate transplanted islets); results of b–e BG measurements, f–i normoglycemia percentage, j–m IPGTT, n–q area under the IPGTT curve (AUC_0-120min), r–u BG clearance rates calculated from slope of IPGTT curves from 30 to 90 min and v–y body weight of mice post-transplant measured at various time points over 30 days. Results shows the effect of b, f, j, n, r, v islet encapsulation by comparing Islets with e-Islets, c, g, k, o, s, w AD-MSC coating on islets by comparing Islets with m-Islets, d, h, l, p, t, x encapsulating islets with AD-MSCs by comparing e-Islets with em-Islets, e, i, m, q, u, y and pFUS treatment by comparing em-Islets with emf-Islets groups. Significant differences: b–y ^aP < 0.05: e-Islets vs. Islets; ^bP < 0.05: m-Islets vs. Islets; ^cP < 0.05: m-Islets vs. e-Islets; ^dP < 0.05: em-Islets vs. e-Islets; ^eP < 0.05: em-Islets vs. m-Islets; ^fP < 0.05: em-Islets vs. Islets; ^gP < 0.05: emf-Islets vs. em-Islets; ^hP < 0.05: emf-Islets vs. m-Islets; ⁱP < 0.05: emf-Islets vs. e-Islets; ^jP < 0.05: emf-Islets vs. Islets; *P < 0.05: baseline vs. all other time-points (two (b, h, n, t, d, j, p, v, g, m, s, y) or one (k, q, w, l, r, x)-way ANOVA post hoc Tukey test or unpaired Student’s t test (e, f))

Fig. 5

In vivo analysis of islet survival and function (histological analysis): a representative images following H&E, insulin, and vWF immunohistochemical staining of islets in tested groups (i.e., Islets, e-Islets, m-Islets, em-Islets, and emf-Islets) that were transplanted under the kidney capsule. Black arrows indicate islets present within the representative images and red arrows indicate blood vessels within the islets; b quantification of surface area occupied by islets; c–e quantification of positive c insulin and d vWF staining. Results were analyzed with at least 15–20 islets from 5 different sections through the kidney of each animal. Significant differences: ^aP < 0.05: Islets vs. e-Islets or m-Islets or em-Islets or emf-Islets; ^bP < 0.05: e-Islets vs. m-Islets or em-Islets or emf-Islets; ^cP < 0.05: m-Islets vs. em-Islets or emf-Islets; ^dP < 0.05: em-Islets vs. emf-Islets (one-way ANOVA post hoc Tukey test)

Fig. 6

In vivo analysis of islet survival and function (molecular analysis): the level of insulin within a the kidney and b blood serum of mice transplanted with islets (measured with insulin ELISA). The c cytokine expression profile in the kidney of mice transplanted with islets (measured with multiplex ELISA) in following tested groups: Islets, e-Islets, m-Islets, em-Islets, and emf-Islets. Significant differences: ^aP < 0.05: Islets vs. e-Islets or m-Islets or em-Islets or emf-Islets; ^bP < 0.05: e-Islets vs. m-Islets or em-Islets or emf-Islets; ^cP < 0.05: m-Islets vs. em-Islets or emf-Islets; ^dP < 0.05: em-Islets vs. emf-Islets (one-way ANOVA post hoc Tukey test)

Fig. 7

a Representative images following TNF-α immunohistochemical staining and H&E staining of islets that were transplanted under the kidney capsule. Black arrows indicate islets present within the representative images; quantification of b positive TNF-α staining, c inflammatory cell infiltrate; and d the cytokine expression profile in the kidney of mice transplanted with islets (measured with multiplex ELISA) in following tested groups: Islets, e-Islets, m-Islets, em-Islets, and emf-Islets. Significant differences: b–d ^aP < 0.05: Islets vs. e-Islets or m-Islets or em-Islets or emf-Islets; ^bP < 0.05: e-Islets vs. m-Islets or em-Islets or emf-Islets; ^cP < 0.05: m-Islets vs. em-Islets or emf-Islets; ^dP < 0.05: em-Islets vs. emf-Islets (one-way ANOVA post hoc Tukey test)