Combinational Treatment Involving Decellularized Extracellular Matrix Hydrogels With Mesenchymal Stem Cells Increased the Efficacy of Cell Therapy in Pancreatitis

Abstract

Cell transplantation using mesenchymal stem cells (MSCs) has emerged as a promising approach to repairing and regenerating injured or impaired organs. However, the survival and retention of MSCs following transplantation remain a challenge. Therefore, we investigated the efficacy of co-transplantation of MSCs and decellularized extracellular matrix (dECM) hydrogels, which have high cytocompatibility and biocompatibility. The dECM solution was prepared by enzymatic digestion of an acellular porcine liver scaffold. It could be gelled and formed into porous fibrillar microstructures at physiological temperatures. MSCs expanded three-dimensionally in the hydrogel without cell death. Compared to the 2-dimensional cell culture, MSCs cultured in the hydrogel showed increased secretion of hepatocyte growth factor (HGF) and tumor necrosis factor-inducible gene 6 protein (TSG-6), both of which are major anti-inflammatory and anti-fibrotic paracrine factors of MSCs, under TNFα stimulation. In vivo experiments showed that the co-transplantation of MSCs with dECM hydrogel improved the survival rate of engrafted cells compared to those administered without the hydrogel. MSCs also demonstrated therapeutic effects in improving inflammation and fibrosis of pancreatic tissue in a dibutyltin dichloride (DBTC)-induced rat pancreatitis model. Combinational use of dECM hydrogel with MSCs is a new strategy to overcome the challenges of cell therapy using MSCs and can be used for treating chronic inflammatory diseases in clinical settings.

Keywords

hydrogel extracellular matrix mesenchymal stem cells pancreatitis transplantation

Introduction

Mesenchymal stem cells (MSCs) are a type of somatic stem cells found in bone marrow, adipose tissues, and umbilical cord blood, among other sites¹. In addition to their self-renewal and differentiation potential, MSCs have a secretory profile of diverse bioactive molecules². They include hepatocyte growth factor (HGF), tumor necrosis factor-inducible gene 6 protein (TSG-6), leamine 2, 3 deoxygenase (IDO), and vascular endothelial growth factor (VEGF), which are known to exert anti-inflammatory, immunomodulatory, anti-fibrotic, or angiogenic effects. As these beneficial effects can facilitate regenerative medicine, animal experiments, and preclinical studies on MSC-based cell therapy for inflammatory diseases and injured organs have gained momentum worldwide³. Chronic pancreatitis, one of the common inflammatory diseases, is a multifactorial inflammatory syndrome wherein recurring pancreatitis results in extensive fibrotic tissue replacement⁴. Due to progressive and irreversible pancreatic degeneration, the long course of this disease is associated with pancreatic exocrine and endocrine insufficiency and malignancy⁵. Several reports have shown that MSCs can be used for ameliorating pancreatitis and reducing fibrosis, as well as improving metabolism^6–8. However, clinical applications of MSC transplantation remain limited to date^3,9.

The major barriers to MSC transplantation therapy are poor cell survival and retention in the transplanted host tissue^10,11. Most of the engrafted MSCs disappear due to apoptosis, shear stress, or being phagocytosed by immune cells such as macrophages. Systemic administration of MSCs via intravenous infusion, for example, causes accumulation in the lungs and clearance within 24 h¹². Consequently, less than 5% of all MSCs are reported to survive in the tissue after transplantation, which limits their functional effect after transplantation^13,14. Besides, intravenous administration of large amounts of cells carries the risk of vascular embolism¹⁵. An alternative strategy is to administer MSC-derived bioactive molecules since the therapeutic efficacy of MSCs is believed to be mainly due to their paracrine effect¹⁶. However, it remains challenging to achieve a sustained impact of the administration of MSC-derived factors since such administered molecules are rapidly metabolized systemically¹⁷.

While intravascular cell administration has been attempted in the past as a less invasive technique, recent progress in endoscopic and laparoscopic procedures has laid the foundation for minimally invasive, direct cell delivery to the targeted organs^18,19. However, the lack of a clinically feasible scaffold, which can envelop a large number of cells and support immediate engraftment of those cells when implanted into the body, is the next challenge²⁰. Therefore, it is essential to provide a suitable scaffold for MSCs that ideally enhances the therapeutic functions of the MSCs. Currently, only 6% of clinical trials are estimated to combine MSCs and biomaterials³, suggesting that cell delivery systems using biocompatible materials, which have great potential as a novel technology, are still under development.

Here, we focused on decellularized extracellular matrix (dECM) hydrogel as a candidate effective biomaterial that can facilitate the anti-inflammatory and anti-fibrotic effects of MSCs. The dECM hydrogel is derived from enzymatic digestion of the extracellular matrix scaffold obtained by removing cellular components from native tissues²¹. The dECM scaffold reportedly mimics the native pericellular microenvironment and can provide a supportive niche for encapsulated stem cells²². The dECM hydrogel undergoes gelation at physiological temperature, thus, preventing dispersion and diffusion of transplanted cells after injection, resulting in efficient local delivery and retention in vivo. In addition, the proteoglycans and glycosaminoglycans (GAGs), as well as the non-fibrous proteins in the dECM hydrogels, bind to the paracrine factors derived from MSCs and can release them in a sustained manner¹⁷. Based on these characteristics of dECM hydrogels, they could serve as potentially useful carriers in MSC transplantation.

The aim of this study was to investigate the efficacy of the combinational use of dECM hydrogel and MSCs in a rodent model of pancreatitis. We investigated the benefits of functional enhancement and local retention of engrafted MSCs by encapsulation in dECM hydrogels. We generated a rat model of pancreatitis by administering dibutyltin dichloride (DBTC). MSCs embedded within dECM hydrogels were implanted in the rat pancreas. Our findings reveal the anti-inflammatory and anti-fibrotic effects of this combinational treatment, thus, highlighting its potential as a novel direct-cell therapy strategy for pancreatitis.

Methods

Preparation of Decellularized Extracellular Matrix Solution

We used the liver as a source of extracellular matrix solution due to its excellent yield for ECM and its constituent elements. The liver is the largest parenchyma organ, which makes it possible to obtain a large amount of ECM by decellularization. The components of the ECM are reportedly similar to those of the pancreas and are rich in collagen type 1²³, a standard MSC culture substrate. Göttingen miniature pig livers used for the dECM solution were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). They were stored at -80°C and gradually thawed for 2 days at 4°C before decellularization. Livers were then rinsed with phosphate-buffered saline (PBS; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) by continuous perfusion through the portal vein until the drainage was transparent. The following day, 0.5% sodium dodecyl sulfate (SDS; Fujifilm Wako Pure Chemical Corp.) was continuously perfused for 6 h to wash out cellular components. After being thoroughly washed with PBS, they were subject to further decellularization with 0.5% TritonX-100 (Sigma-Aldrich Co., Tokyo, Japan) with 0.05% ethylene glycol-bis (β-aminoethyl ether)-N, N, N’,N’-tetraacetic acid (EGTA; Dojinkagaku, Ltd. Tokyo, Japan) and 2 mM 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS; Dojinkagaku, Ltd.) for 3 h. The scaffolds were washed with PBS again and cut into 10 mm-sized pieces. The pieces of decellularized liver were stirred with PBS containing 25 μg/ml 1× antibiotic-antimycotic (Thermo Fisher Scientific K.K., Tokyo, Japan) and 300 μg/ml sodium colistin methanesulfonate (Fujifirm Wako Pure Chemical Co.) for 1 h. They were then stirred with PBS containing 25 μg/ml 1× antibiotic-antimycotic and 200 μg/ml gentamicin sulfate (Nacalai Tesque Co., Ltd., Tokyo, Japan) for 1 h. Pieces were lyophilized for 3 days and milled to obtain liver dECM powder. The powdered dECM was sterilized with 3 kGy of γ-ray (RADIA INDUSTRY Co., Ltd., Gunma, Japan). In all, 25 mg of the liver dECM powder was then enzymatically digested with 25 mg pepsin (Nacalai Tesque Co.) and dissolved in 25 ml of 0.01 M HCl for 72 h at room temperature. Digested dECM solution was neutralized to pH 7.4 with 0.1 N NaOH. Finally, 8 mg/ml of dECM solution was derived from porcine liver and stored at 4°C until use.

DNA Quantification

DNA from native liver tissue and dECM hydrogel was extracted using Trizol (Thermo Fisher Scientific K.K.), according to the manufacturer’s protocol. Briefly, samples weighing 100 mg were homogenized in 1 ml of Trizol using a Micro Smash MS-100 (TOMY Ltd., Tokyo, Japan). The homogenate was treated with 200 μl of chloroform and centrifuged at 12,000 × g for 15 min at 4°C. After discarding the supernatant, 300 μl of 100% ethanol was added to the pellet and incubated for 3 min at room temperature, followed by centrifugation at 2,000 × g for 5 min. The DNA pellet was washed twice with 0.1 M citrate buffer and resuspended with 1.5 ml of 75% ethanol. After 20 min of incubation at room temperature, the samples were centrifuged at 2,000 × g for 5 min at 4°C. The pellet was dried for 15 min and dissolved in 8 mM NaOH. DNA content was quantified using NanoDrop OneC (Thermo Fisher Scientific K.K.) and normalized to the dry weight of each sample.

Glycosaminoglycan and Collagen Quantification Assay

Sulfated GAGs contained in native liver tissues and dECM hydrogels were quantified using the Blyscan Sulfated Glycosaminoglycan Assay kit (Biocolor Ltd. Newtownabbey, UK). Samples were lyophilized and weighed, then incubated in 25 mg/ml of papain extraction reagent (Sigma-Aldrich Co.) at 65°C overnight. Papain-digested samples were centrifuged at 10,000 × g for 10 min. The supernatants were assayed according to the manufacturer’s protocols. For collagen quantification assay, the QuickZyme Total Collagen Assay kit (QuickZyme Biosciences B.V., South Holland, Netherlands) was used. Collagen content was determined by quantifying hydroxyproline produced by hydrolysis of lyophilized samples in 6 M HCl at 95°C for 20 h. GloMax® Discover (Promega Co., WI, USA) was used to measure the absorbance of samples in both assays.

Rheological Analysis

Viscoelasticity was measured with a PZ-RHEO NDS-1000 (TAISEI Co., Tokyo, Japan). The dECM solutions at different concentrations (2, 4, 6, and 8 mg/ml) were prepared, placed in molds, and gelated in an incubator at 37°C for 30 min before measurement. The thickness of the samples and measurement frequency was fixed to 5 mm and 3 Hz, respectively.

Scanning Electron Microscopy (SEM)

The dECM hydrogel with or without embedded MSCs was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 16 h at 4°C. Thereafter, samples were fixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 2 h at 4°C. Samples were serially dehydrated with 50%, 70%, 80%, 90%, and 100% ethanol for 30 min each and dried by supercritical drying method using EM CPD2000 (Leica microsystems Co., Tokyo, Japan). Dried samples were coated using an osmium coater (Neoc-ST; meiwafosis Co., Tokyo, Japan) and visualized using a SU6600 low-vacuum analytical SEM (Hitachi High-Technologies Co., Tokyo, Japan).

Cell Viability Assay

Rat adipose mesenchymal stem cells (MSA01C; Cosmo Bio Co., Ltd. Tokyo, Japan) were used in this study. The cells used in the experiment were passed once after seeding. The proliferation of MSCs embedded in dECM hydrogels of different concentrations was quantified using the RealTime-Glo™ MT Cell Viability Assay (Promega Co., WI, USA). The dECM solutions of 2, 4, and 6 mg/ml containing 1.0 × 10⁴ MSCs in 100 µl were seeded to 96-well plates. As a control, 1.0 × 10⁴ MSCs without embedding in the dECM solution were also prepared. Incubation at 37°C for 30 min allowed the dECM solution-MSC mixtures to be gelatinized, and 100 µl of MesenPRO RS™ Medium (Thermo Fisher Scientific K.K.) with Zell shield (Minerva Biolabs Inc., Berlin, Germany) was added to each well. After 24 h of incubation, the culture medium was changed to that containing the Realtime-Glo reagent prepared according to the manufacturer’s protocol. Absorbance was measured repeatedly every 12 h up to 48 h using GloMax® Discover (Promega Co.). For Live/Dead imaging analysis, 300 µl of 4 mg/ml dECM hydrogel containing 1.0 × 10⁵ MSCs were seeded in the same manner in 24-well plates and cultured with 500 µl of medium for 4 days. The cells were stained with a live/dead cell imaging kit (Thermo Fisher Scientific K.K.) according to the manufacturer’s protocol and imaged using a BZ-X800 fluorescence microscope (Keyence Corp., Osaka, Japan). The intensity of fluorescence emitted from live or dead cells was measured using GloMax® Discover (Promega Co., WI, USA). For observation of cell migration encapsulated in the dECM hydrogel, the 3D CELL CULTURE CHIP (AIM BIOTECH PTE. Ltd., Nucleos, Singapore) was used.

RNA Extraction and Quantitative Real-Time PCR

Total RNA was extracted from cultured cells and rat pancreatic tissues with TRIzol (Thermo Fisher Scientific K.K.) according to the manufacturer’s instructions. Rat pancreatic tissues and MSCs embedded in dECM gel were homogenized using Micro Smash MS-100R (TOMY Ltd.) under 3,500 rpm with zirconia beads (ZSB-10, TOMY Ltd., Tokyo, Japan). Complementary DNA was synthesized using Prime Script RT Master Mix (Takara Bio Inc., Shiga, Japan). Quantitative real-time PCR with SYBR Master Mix (Thermo Fisher Scientific K.K.) was performed using QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific K.K.). The primer list is presented in Supplementary Table 1. The relative expression level of each target was analyzed using the comparative computed tomography (CT) method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control.

Functional Analysis of MSCs Embedded in dECM Hydrogel

The MSCs were seeded in 12-well plates at a density of 1 × 10⁵ MSCs/well with or without embedding in 600 µl of 4 mg/ml dECM solution. The dECM solution-MSC mixture suspended in plates was incubated at 37°C for 30 min for gelation. After confirming gelation, 500 µl of MesenPRO RS™Medium (Thermo Fisher Scientific K.K.) with Zell shield (Minerva Biolabs, Inc.) was added to each well and incubated for 24 h. The culture medium was then replaced with 500 µl of culture medium with or without 10 ng/ml of recombinant rat TNFα protein (R&D Systems, Inc., MN, USA). Following 48 h of cell culture, TSG-6, and HGF secreted in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA), using rat tumor necrosis factor-inducible gene 6 protein (TSG-6) ELISA kit (WUHAN HUAMEI BIOTECH Co., Ltd, Wuhan, China) and rat HGF Quantikine ELISA kit (R&D Systems, Inc., Minneapolis., MN, USA) according to the manufacturer’s instructions. The absorbance was measured by GloMax® Discover (Promega Co.). Normalization was performed with the estimated number of live cells calculated based on the absorbance values from the live/dead staining assay. Cultured cells were collected for quantitative Real-Time PCR analysis.

Animals

Eight-week-old male Sprague-Dawley (SD) rats (CLEA Japan, Inc, Tokyo, Japan) were used in this study. The experimental procedures and protocols were approved by the Animal Ethics Committee of Keio University Tokyo, Japan (approval number: A2022-044), and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA).

In vivo Imaging

The MSCs were seeded in 10-cm dishes. After reaching 80% confluency, the culture medium was replaced with a fresh culture medium containing 0.2 mg/ml of live cell fluorescent nanoparticles supplied with LuminiCell Tracker™ 670- Cell Labeling Kit (Sigma-Aldrich Co.). The particles are designed to emit fluorescence in a highly aggregated state in live cells and are useful for MSC tracking in vivo²⁴. Then, the cells were incubated for 1 h. After excess nanoparticles were rinsed with PBS, the cells were collected. Three rats each were anesthetized, and their pancreas was exposed by surgical incision. Several 5.0 × 10⁵ labeled MSCs in 0.5 ml of PBS or 4.0 mg/ml dECM solution were injected under the anterior pancreatic fascia. The fluorescence intensity of the labeled cells in the pancreas was observed over 2 weeks using IVIS Spectrum CT (excitation, 500 nm; emission, 680 nm).

DBTC-Induced Rat Pancreatitis Model

The dibutyltin dichloride (DBTC; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) was dissolved in a 2:3 mixture of 100% ethanol (Sigma-Aldrich Co.) and glycerol (FUJIFILM Wako Pure Chemical Co.) and adjusted to 8 mg/ml. Rats were anesthetized with continuous inhalation of 2% isoflurane and received an infusion of 8 mg/kg DBTC solution via the tail vein, as previously reported²⁵. Rats that changed body weight within ±5% after 1 week of DBTC injection were used for the experiment to standardize the degree of DBTC-induced injury (Supplementary Figure 1A). The changes in body weight and progressive histological alterations of these candidate rats were consistent with a previous report²⁶, whereby a rat model of pancreatitis was generated by the same procedure (Supplementary Figure 1B). The DBTC-induced pancreatitis model mimics the course of chronic pancreatitis, with progressive pancreatic devastation due to persistent inflammation induced by a single dose of DBTC via the tail vein. In this experiment, pancreatitis during the transition from the acute to the chronic phase was targeted. Two weeks after the DBTC injection, six rats were used for each of the following four groups: DBTC-, Gel-, MSC-, and MSC in Gel-group. Each group of rats was injected with 1 ml of the following solutions, respectively, under the pancreatic fascia: PBS, 4.0 mg/ml dECM hydrogel, PBS containing 1.0 × 10⁶ MSCs, and 4.0 mg/ml dECM hydrogel containing 1.0 × 10⁶ MSCs. Rats that had not received DBTC and had an injection of PBS under the pancreatic fascia were used as controls. The pancreas was sampled at 4 weeks after DBTC administration. The extent of inflammation in pancreatitis was quantified by a pancreatic histological scoring system based on the extent of inflammatory cells and pancreatic edema, vacuolization, and cell necrosis, as previously reported²⁷ (Supplementary Table 2).

Histological Analysis

Rat pancreatic tissues were fixed with 4% paraformaldehyde for 2 days. The fixed samples were then embedded in paraffin and subsequently sectioned to 0.5 µm thickness. Sections were subjected to hematoxylin and eosin (H&E) and AZAN staining as previously described²⁸. In an immunohistological analysis, the sections were deparaffinized and rehydrated, and the antigens were retrieved. The primary and secondary antibodies used for staining Collagen I were anti-Collagen I (ab90395; Abcam Inc.) and donkey anti-mouse IgG Alexa Fluor 488 (ab150105, Abcam Inc.). The immunostaining of αSMA was performed using anti-α-SMA (A5228, Sigma-Aldrich Co.) by horseradish peroxidase-diaminobenzidine (HRP-DAB) method. Similarly, serial sections were stained with anti-CD31 (ab24590, Abcam, Inc.) and used to recognize vascular structures. Mounting was done in ProLong™ Diamond Antifade Mountant with DAPI (P36971, Invitrogen Inc.). Each section was imaged using a BZ-X800 fluorescence microscope (Keyence Corp., Osaka, Japan). The area of fibrosis and αSMA-positive areas in individual pancreatic samples of each group were calculated as the average of five random fields of view in a 4x bright field using ImageJ Fiji²⁹. The areas of vascular structures were excluded in the calculation of αSMA-positive areas because these were also stained by αSMA.

RNA-Sequence Transcriptomic Analysis

Before alignment, FastQC (Version0.11.7) was used to assess the quality of the raw paired-end sequence reads generated by the Illumina NovaSeq 6000. Low-quality (<20) bases and adapter sequences were trimmed by Trimmomatic software (Version 0.38), and trimmed reads were aligned to the reference genome using RNA-seq aligner HISAT2 (Version 2.1.0). The abundance of uniquely mapped reads was estimated with featureCounts (Version 1.6.3). The raw read counts were normalized with transcripts per million (TPM). The samples were clustered with the Wald method based on Euclidean distances of the normalized counts using the R packages, stats (Version 3.6.1), and gplots (Version 3.0.1.1). The raw read counts were normalized by relative log normalization (RLE) and differential expression analysis was conducted with DESeq2 (Version 1.24.0). Differentially expressed genes (DEGs) were detected with the thresholds of|log2FC (Fold Change)| > 1 with a p-value adjusted to < 0.05 by the Benjamini and Hochberg method. Gene ontology (GO) enrichment analysis was performed using the DAVID 2021 functional annotation tool (https://david.ncifcrf.gov/).

Statistical Analysis

Acquired data are presented as mean ± standard deviation and are obtained from at least triplicates for each data point. The student’s t-test was used for comparison between the two groups. Differences among multiple groups were analyzed by Kruskal-Wallis and Dunn’s comparison test for the two groups of interest using SPSS version 26.0.

Results

Preparation and Characteristics of dECM Hydrogels

The dECM solution derived from the porcine liver was prepared by performing decellularization using detergent washes and enzymatic digestion (Fig. 1A). Wang et al³⁰. examined the efficiency of decellularization of the porcine liver using several detergents and reported that SDS was the most efficient in removing cellular components, while Triton-X resulted in a DNA residue. To avoid excessive damage and loss of ECM components due to the use of strong ionic detergents³¹, we optimized our decellularization protocol to minimize the use of SDS and sequential use of other nonionic detergents. In H&E staining, our decellularized liver tissue showed no cellular components, and the extracellular scaffold was well-preserved. Immunostaining images of decellularized liver tissue also showed the absence of nuclei and were positive for Collagen I, a major component of the extracellular scaffold (Supplementary Figure 2). The viscous dECM solution obtained after pepsin digestion and neutralization stabilized the gelation reaction for 30 min at 37°C. As shown in the H&E staining and immunostaining images, DNA quantification confirmed that the amount of DNA was significantly reduced in the dECM solution by more than 99.5% (Fig. 1B). Meanwhile, collagen content by weight in dECM solution was not different compared to that from native liver tissue (7.4 ± 1.1 vs. 7.2 ± 0.7 μg/mg dry weight, P = 0.74) (Fig. 1C). The sulfated glycosaminoglycan (S-GAG) content by weight was slightly reduced in dECM solution (1.7 ± 0.1 vs. 1.1 ± 0.1 μg/mg dry weight, P < 0.01), which may be due to the presence of some GAGs in cell components such as cell surface³² (Fig. 1D). The gelation capacity of dECM solutions adjusted to 2, 4, 6, and 8 mg/ml was evaluated by incubation at 37°C for 30 min (Fig. 1E). The dECM hydrogel at 2 mg/ml was visually fluid and could not retain its shape; at concentrations above 4 mg/ml, the dECM hydrogel was stable and solidified further as the concentration increased. Rheological analysis showed that the storage modulus (E’) of the dECM hydrogel increased with concentration and was greater than the loss modulus (E ”), while E’ and E ” of the 2 mg/ml dECM hydrogel were comparable and had more liquid-like properties (Fig. 1F). SEM of 4 mg/ml hydrogels revealed a randomly oriented fibrous network forming a microenvironment that allows the cellular exchange of gas and nutrients as well as cell migration (Fig. 1G).

Figure 1.

Preparation and characteristics of liver decellularized extracellular matrix (dECM) hydrogels. (A) The process of preparing the dECM solution is shown. The porcine liver was decellularized by flushing detergents via the portal vein to remove cellular components. After decellularization, the liver scaffold was minced and lyophilized. Lyophilized dECM was milled and enzymatically digested under stirring. After neutralization, the dECM solution was obtained. (B) Quantification of DNA content in the native liver tissues and dECM hydrogel. (C) Quantification of collagen content of native liver tissues and dECM hydrogel. (D) Quantification of glycosaminoglycan (GAG) content of native liver tissues and dECM hydrogel. Values were normalized to the initial dry weight of each sample and expressed as mean ± SD in Fig. 1B–D, n = 4, **P < 0.01. (E) The appearance of dECM hydrogel in different concentrations (2, 4, 6, and 8 mg/ml). (F) Storage modulus (E’) and loss modulus (E’’) of dECM hydrogels in different concentrations (mean ± SD, n = 4). (G) Scanning electron microscope image of 4 mg/ml dECM hydrogel. Scale bar = 1 µm. dECM = decellularized extracellular matrix; GAG = glycosaminoglycan.

Proliferation and Viability of MSCs Cultured in dECM Hydrogels

The MSCs embedded within dECM hydrogels were evaluated for their proliferation and viability. MTT assays were performed on cells embedded in 2, 4, and 6 mg/ml hydrogels, and the outcome was compared with that from assays on 2D cell culture (Fig. 2A). The luminescence intensity of each hydrogel-embedded cell was low immediately after starting the cell culture, which might be due to the time lag until the substrate reached the cells embedded in the hydrogel. After 12 h of incubation with substrates, luminescence from the hydrogel-embedded cells was observed in all hydrogels with different concentrations. Notably, the emission intensity was higher for the lower concentrations of dECM hydrogel. As suggested in Fig. 1E, the 2 mg/ml dECM hydrogel could not maintain its shape and peeled off from the bottom of the wells due to traction caused by cell growth. Based on these results, 4 mg/ml dECM hydrogel was considered suitable for MSCs culture and was used in the subsequent experiments. The porous fibrous structure of the dECM hydrogel allows migration and proliferation of the encapsulated cells (Supplementary Figures 3A, B). The MSCs cultured in 4 mg/ml dECM hydrogel expanded three-dimensionally and showed high viability in Live/Dead assay (Fig. 2B, C, and Supplementary Figure 4). The viability of the cells remained more than 99.5% on day 4 after seeding. SEM images showed that MSCs released numerous vesicles and extended pseudopodia into the fiber structure of hydrogel to make intercellular contact (Fig. 2D). Compared to 2D cell cultures, the hydrogel-embedded MSCs exhibited significantly higher RNA expression levels of pluripotency markers (Nanog, SOX2, Oct4, and CXCR4³³ with 11.4 ± 3.5-, 1.9 ± 0.4-, 3.1 ± 1.5-, and 3.6 ± 1.2-fold increases, respectively) (Fig. 2E). These results suggest that the cultural environment provided by dECM hydrogel enhanced MSCs’ stemness.

Figure 2.

Proliferation and viability of MSCs cultured in dECM hydrogels. (A) Cell proliferation assay performed on MSCs cultured in 2, 4, and 6 mg/ml dECM hydrogels and those on the dish (0 mg/ml); the hydrogel detached from the bottom of the dish and shrank within 12 h in culture using 2 mg/ml hydrogel (black squares). (B) The live/dead assay for MSCs cultured in 4 mg/ml dECM hydrogel. The fluorescence intensity emitted by live and dead cells was measured over 4 days. (C) Brightfield images of MSCs cultured on the dish (On dish) and in 4 mg/ml hydrogel (In gel). Scale bar = 100 µm (D) Scanning electron microscopy image of MSCs cultured in 4 mg/ml hydrogel. MSCs released numerous microvesicles (left) and extended their pseudopodia inside the fibrous structure of the dECM hydrogel (right). Scale bar = 2.5 µm (E) Relative gene expression levels of stemness markers in MSCs cultured on dish versus those cultured in 4 mg/ml dECM hydrogel. (mean ± SD, n = 4), *P < 0.05. MSC: mesenchymal stem cells; dECM: decellularized extracellular matrix.

Inflammatory Response of MSCs Cultured in dECM Hydrogel

MSCs cultured in dECM hydrogels (In gel) and on 2D dishes (On dish) were stimulated with TNF-α, a typical cytokine released from injured tissues³⁴. Then, the RNA expression and secretion levels of HGF and TSG-6, which play a central role in the anti-inflammatory, anti-fibrotic, and immunomodulatory effects of MSCs³⁵, were investigated in both culture conditions (Fig. 3A). Without TNFα stimulation, the RNA expression level of TSG-6 in-gel culture was not increased compared to that from the on-dish culture, while HGF significantly increased 2.2 ± 0.4-fold (P < 0.01). With TNFα stimulation, increased RNA expression of both TSG-6 and HGF was observed, which was significantly higher in the in gel culture than in the on-dish culture (28.6 ± 7.4 vs. 2.9 ± 1.0, P < 0.01 and 9.4 ± 3.3 vs. 5.1 ± 1.1, P = 0.02, respectively) (Fig. 3B). Next, ELISA was performed to validate the secreted levels of both factors. They showed a similar trend to the results of RNA expression levels, confirming that the secretion of both the anti-inflammatory factors under inflammatory conditions was significantly increased to approximately threefold by culturing in the dECM hydrogels (Fig. 3C).

Figure 3.

Inflammatory response of MSCs cultured in dECM hydrogel. (A) Schema of experiments on the response of MSCs for inflammatory stimulus induced by TNFα. (B) Relative mRNA expression levels of tumor necrosis factor-stimulated gene-6 (TSG-6) and hepatocyte growth factor (HGF) in MSCs cultured on the dish or in dECM hydrogel, and with or without TNFα stimulation (mean ± SD, n = 6), *P < 0.05. **P < 0.01. (C) Comparison of TSG-6 and HGF secretion levels of MSCs cultured on the dish or in dECM hydrogel, and with or without TNF-α stimulation (mean ± SD, n = 8) *P < 0.05. MSC = mesenchymal stem cells; dECM = decellularized extracellular matrix; TSG-6: tumor necrosis factor-inducible gene 6 protein; TNFα: Tumor necrosis factor α; HGF: hepatocyte growth factor.

Injection of MSC Suspension Under the Pancreatic Fascia

For long-term cell tracking in vivo, biocompatible organic fluorescent nanoparticles were loaded into MSCs (Fig. 4A, Supplementary Figure 5A). The labeled MSCs were suspended in PBS or 4 mg/ml dECM solution and injected directly under the anterior pancreatic fascia (Fig. 4B). The survival rate of engrafted MSCs was monitored over time using an IVIS imaging system on postoperative days 1, 3, 7, 10, and 14. Serial images showed intense fluorescence from the pancreatic region with both interventions on postoperative day 1, indicating successful cell engraftment. However, MSCs without embedding in hydrogel decreased rapidly to approximately 13% by postoperative day 10 relative to day 1 and became barely detectable. On the other hand, hydrogel-embedded MSCs retained more than 70% survival rate compared to day 1, over 14 days after administration (Fig. 4C, D). Most of the MSCs co-transplanted with the dECM hydrogel were localized on the surface of the pancreatic tissue, and some were found migrating into the internal pancreas along the pancreatic stroma (Supplementary Figure 5B). These results suggested that dECM hydrogel could augment MSCs’ survival rate in vivo and enhance the therapeutic effect of MSCs.

Figure 4.

Assessment of survival rate of engrafted MSCs in vivo. (A) Fluorescence microscopy image of MSCs loaded with organic fluorescent nanoparticles. (Scale bar = 100 µm) (B) Images of the administration of MSCs suspended in 4 mg/ml dECM solution under the anterior pancreatic fascia. The pancreases were exposed by an L-shaped abdominal incision (left) and labeled MSCs were injected with 0.5 ml of PBS or 4.0 mg/ml dECM solution (middle). The referential image shows the injection area visualized by mixing blue dye with the suspension (right). (C, D) Comparison of the survival rate of MSCs alone and hydrogel-embedded MSCs in rats using IVIS imaging system over 2 weeks. MSC = mesenchymal stem cells; dECM = decellularized extracellular matrix; PBS = phosphate-buffered saline.

Therapeutic Efficacy of Co-Transplantation of MSCs and dECM Hydrogel for Pancreatitis

The following five intervention groups were prepared to examine the therapeutic efficacy of co-transplantation of MSCs and dECM hydrogel for DBTC-induced pancreatitis, as shown in Supplementary Figure 6; DBTC-, Gel-, MSC-, and MSC in-gel-groups were injected with PBS, dECM hydrogel, MSCs in PBS, and MSC in dECM hydrogel, respectively, after 2 weeks of DBTC administration. Two weeks after each intervention, the rats were euthanized, and the entire pancreas was sampled. The pancreases were visually atrophic in the DBTC- and the Gel-group (Fig. 5A, top row). In histological analysis, inflammatory cell infiltration and acinar edema were prominent in both groups (Fig. 5A, second row from top). In contrast, these inflammatory changes were not observed in the MSC and the MSC in-gel-group. Inflammation involved in pancreatitis was quantified using the pancreatic histological scoring system, and the results demonstrated the highest therapeutic effect in the MSC in-gel-group; the results were not statistically significantly different from those in the Control-group (2.5 ± 1.2 vs. 0 ± 0, P = 0.24) (Fig. 5B). The fibrotic areas in each group determined by the AZAN staining showed a similar trend to the inflammation scores: the MSC in the gel group demonstrated the most remarkable improvement in fibrosis, comparable to the Control-group (13.5% ± 4.2% vs. 11.3% ± 2.6%, P = 0.61) (Fig. 5A, third row from top and 5C). Pancreatic stellate cells are activated by inflammatory stimuli in tissues and play a key role in the progression of pancreatic fibrosis^36,37. Therefore, we performed immunohistological staining for αSMA, which is a marker of activated pancreatic stellate cells³⁸ (Fig. 5A, bottom row). The least area was accounted for by the number of activated pancreatic stellate cells in the MSC in-gel-group, and the result was comparable with that in the Control-group (0.28 ± 0.14% vs. 0.59 ± 0.16%, P = 0.16) (Fig. 5D). These results suggest that co-transplantation of MSCs and dECM hydrogels exert a high anti-inflammatory effect that suppresses pancreatic stellate cell activation and consequently prevents fibrosis. RNA-sequence analysis revealed DEGs in the Control group versus the DBTC-group and DBTC-group versus MSC in-gel-group as shown in Supplementary Figures 7A and B. In the Control- and DBTC-groups, the upregulated genes included those encoding transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF), which play a central role in the activation of pancreatic stellate cells³⁹, whereas the down-regulated gene included that encoding HGF activator (Hgfac) and amylase alpha 1A (Amy1a), involved in exocrine pancreatic function (Fig. 5E). The top GO categories of the upregulated genes included terms related to ECM production, inflammation, and defense response, while those of the downregulated genes included terms related to metabolic function (Supplementary Figure 7C). These results suggest that DBTC administration induced an inflammatory and fibrogenic response in the pancreatic tissues. In DBTC- and MSC in-gel-groups, the upregulated genes included those encoding Amy1a, as well as fibroblast growth factor 21 (FGF21), which improve various metabolic processes and inhibit fibrogenesis⁴⁰ (Fig. 5F). The down-regulated genes included those encoding TGFβ, PDGF, and Collagen alpha-1 (Col1a), as well as intercellular adhesion molecule-1 (ICAM) and vascular cell adhesion molecule-1 (VCAM), which recruit inflammatory cells into the injured tissues⁴¹, and tissue inhibitor of metalloproteinase (Timp) which is repressive to the anti-fibrotic matrix metalloproteinases⁴². The top GO categories of the up-regulated genes included terms related to metabolic function, while those of downregulated genes included terms associated with ECM production (Supplementary Figure 7D). These results suggest that MSCs co-transplanted with dECM hydrogel inhibit inflammation and fibrogenesis, and prevent decompensation of pancreatic metabolic function.

Figure 5.

Therapeutic effects of co-transplantation of dECM hydrogel and MSCs. (A) Appearance and histological staining images of the pancreas in each intervention group. From left to right: Control-, DBTC-, Gel-, MSC-, and MSC in gel-group. From top to bottom: pancreas appearance, H&E-staining image, AZAN-staining image, αSMA immunostaining image. Scale bar = 100 µm. (B) Pancreatic histological scores for each intervention group. The parameters are as follows: cell necrosis (yellow), vacuolar degeneration (gray), the extent of inflammatory cells (orange), and edema of acinar cells (blue). (C) The ratio of fibrotic areas of pancreatic tissues in each intervention group. (D) The ratio of an α-SMA-positive area in pancreatic tissues in each intervention group. *P < 0.05. (E) Volcano plot of global DEGs in pancreatic tissues between the Control and DBTC groups. Red dots represent significantly up-regulated genes [P < 0.05,—log2(fold-change)—>1]; blue dots represent significantly down-regulated genes [P < 0.05, log2(fold-change) < −1]; gray dots represent insignificantly differentially expressed genes. (F) Volcano plot of global DEGs in pancreatic tissues between the DBTC group and MSC in the gel group. dECM: decellularized extracellular matrix; MSC: mesenchymal stem cells; DBTC: dibutyltin dichloride; αSMA: α smooth muscle actin; DEG: differentially expressed genes.

Discussion

MSC-based therapies have been explored for treating various acute injuries or chronic inflammatory diseases due to their availability, versatility, and expansive potential. However, their clinical application has not yet been generalized because of challenges related to delivery methods, poor cell engraftment, and low survival rate after transplantation. To the best of our knowledge, this is the first study to utilize dECM hydrogel to enhance the therapeutic potential of MSCs and demonstrates its efficacy against pancreatitis. The dECM hydrogel exhibited high cell compatibility, increased the stemness of the encapsulated MSCs, and maintained the anti-inflammatory phenotype of the MSCs. The use of dECM hydrogels for cell delivery also enabled cell delivery in a minimally invasive form of injection, and significantly improved cell survival in vivo by providing the MSCs with a supportive scaffold for engraftment. Furthermore, the dECM hydrogel significantly enhanced the ability to secrete TSG-6 and HGF under inflammatory conditions, showing its efficacy in suppressing inflammatory response and attenuating fibrosis in a rat model of DBTC-induced pancreatitis. These results indicate that the dECM hydrogel is a promising carrier to enhance the efficacy of MSC transplantation therapy and can be used in future clinical applications.

The effect of dECM hydrogels on MSCs remains largely unknown to date; however, material properties of the carriers of MSCs, such as composition, hardness, and viscoelasticity could directly affect their survival and secretion profile³. Recent studies have reported a tight correlation between the secretory activities of MSCs and their surrounding matrix, suggesting that niches that mimic native tissue are essential for the therapeutic function of MSCs^43,44. Collagens and branched GAGs are the major components of ECM and provide adhesion sites necessary for cell survival⁴⁵. In addition, the fibrous structures and pores formed by these proteins contribute to increased cell migration and cell-cell interactions⁴⁶. In our study, collagen, and GAG were well retained in the dECM hydrogels. Numerous other proteoglycans are contained in dECM hydrogels, which promote biological cell activities including growth, function, differentiation, and migration⁴⁷. dECM hydrogels provide niches that mimic the innate in vivo environment, making this its advantage. This broad natural ECM composition profile of decellularized hydrogels is challenging to achieve with synthetic materials or hydrogels derived from other single natural materials such as sodium alginate or collagens. MSC behavior depends on cell-cell interactions as well as cell-ECM interactions. Unlike as single cells, MSCs in multicellular aggregates have been reported to possess enhanced stemness, increased expression of anti-inflammatory genes, and enhanced responses to inflammatory stimuli such as TNFα and IFNγ33,^48–50. This increased reactivity has been observed in both microencapsulated and spheroid forms of MSCs and may be attributed to enhanced cell-cell interaction. In this study, MSCs embedded in dECM hydrogel released numerous vesicles containing signaling molecules⁵¹ and extended pseudopodia for intercellular communication. Moreover, the culture environment provided by dECM hydrogel prevents central necrosis, a disadvantage of multicellular aggregate forms⁵², making it an advantage compared to other 3D culture systems. In this study, enhanced stemness and increased secretion of HGF and TSG-6 under inflammatory stimuli were observed in MSCs embedded in dECM hydrogels. These factors are the major anti-inflammatory cytokines released by MSCs⁵³. In pancreatitis, HGF administration reportedly ameliorates inflammation by decreasing IL-1β and IL-6 production, suppressing leukocyte infiltration into pancreatic tissues, and lowering oxidative stress^54,55. Similarly, TSG-6 can ameliorate pancreatitis by decreasing oxidative stress and changing the polarization of macrophages from pro-inflammatory M1 to anti-inflammatory M2⁵⁶,⁵⁷. Since dECM hydrogel facilitated the enhanced release of key cytokines by MSC, it holds potential for use in future therapeutic strategies.

In this study, we showed that the proliferative activity was higher in 4 mg/ml gels than in gels of higher concentrations. We previously reported that different ECM concentrations change the elasticity of the scaffold and the pore size of the fiber structure²⁸, and these mechanical properties may also serve as external signals to influence cell infiltration and/or metabolism⁵⁸. However, given the multi-component nature of decellularized hydrogels, these mechanical factors are so complex and interdependent that concluding how individual properties may affect the behavior of MSCs was difficult in this study. While this study revealed various beneficial effects of hydrogels on MSCs, the potential mechanisms of these effects will be the focus of future research. Notably, dECM hydrogels can be modified by adjusting their concentration or adding bioactive agents⁵⁹, which can improve MSC function.

We have demonstrated that transplantation in combination with dECM hydrogel improves the survival rate of MSCs in vivo. These findings support that the microenvironment provided by the hydrogel favors MSC survival in the transplanted tissues. Incidentally, the dECM hydrogel itself may play a protective role by significantly reducing oxidative stress and apoptosis of MSCs⁶⁰, which in turn may contribute to improved MSC survival rate. Evaluation of these effects in a rat model of pancreatitis using histological and gene expression analysis showed that co-transplantation of hydrogel and MSCs improved the inflammatory state and suppressed fibrosis. Moreover, the anti-inflammatory and immunomodulatory effects of MSCs enhanced by cell–hydrogel and cell–cell interactions were also demonstrated in vivo. Interestingly, the dECM hydrogel reportedly reduces inflammation by activating anti-inflammatory macrophages^61,62. These findings suggest the possibility of an anti-inflammatory effect on a rat model of pancreatitis; however, administration of the hydrogel alone did not result in significant histological amelioration in this analysis. Nevertheless, in combinational use with MSCs, it may contribute to a delayed therapeutic effect. Decellularized tissues have high biocompatibility and degrade gradually in vivo⁶³. Besides, dECM hydrogel itself can store cytokines and release them gradually¹⁷. dECM hydrogels contain GAGs where growth factors bind, thereby contributing to their stability. Thus, dECM hydrogel prevents the rapid degradation of the cytokines and enables their continuous release for more than 5 days. The characteristic feature was not observed in collagen hydrogel which lacks GAGs⁵⁹. In addition to the elevation of the anti-inflammatory gene expression, the protein stability might contribute to an increase in anti-inflammatory cytokines in the hydrogel-embedded MSC culture shown in this study. Based on these findings, the dECM hydrogel is expected to hold MSCs locally and serve as a functional reservoir of cytokines secreted by encapsulated MSCs and exert a sustained therapeutic effect for the adapted inflammatory region. We confirmed the presence of viable MSCs over 14 days after their delivery to the pancreatic interface and demonstrated infiltration of MSCs into the pancreatic tissue. Uday Chandrika et al⁶⁴. reported that MSCs recellularized in the acellular pancreatic scaffold differentiate into pancreatic islets and pancreatic acinar cells. Therefore, MSCs infiltrating into pancreatic tissues would possibly differentiate into these pancreatic cells. However, we could not reveal their fate after infiltration into pancreatic tissues. Future research should clarify the dynamics of MSCs in vivo for safe adaptation for clinical use in the future.

In conclusion, we have shown that dECM hydrogels are carriers that provide a favorable scaffold for MSCs and enhance their therapeutic utility. Based on these results, we propose a biomaterial-based approach using dECM hydrogels in the therapeutic application of MSC transplantation. Along with the recent development of minimally invasive treatments, this approach would be a promising strategy for future clinical application in chronic inflammatory diseases.

Supplemental Material

sj-tif-1-cll-10.1177_09636897231170437 – Supplemental material for Combinational Treatment Involving Decellularized Extracellular Matrix Hydrogels With Mesenchymal Stem Cells Increased the Efficacy of Cell Therapy in Pancreatitis

Supplemental material, sj-tif-1-cll-10.1177_09636897231170437 for Combinational Treatment Involving Decellularized Extracellular Matrix Hydrogels With Mesenchymal Stem Cells Increased the Efficacy of Cell Therapy in Pancreatitis by Hideaki Kojima, Hiroko Kushige, Hiroshi Yagi, Takayuki Nishijima, Nobuko Moritoki, Narihito Nagoshi, Yutaka Nakano, Masayuki Tanaka, Shutaro Hori, Yasushi Hasegawa, Yuta Abe, Minoru Kitago, Masaya Nakamura and Yuko Kitagawa in Cell Transplantation

Supplemental Material

sj-tif-2-cll-10.1177_09636897231170437 – Supplemental material for Combinational Treatment Involving Decellularized Extracellular Matrix Hydrogels With Mesenchymal Stem Cells Increased the Efficacy of Cell Therapy in Pancreatitis

Supplemental material, sj-tif-2-cll-10.1177_09636897231170437 for Combinational Treatment Involving Decellularized Extracellular Matrix Hydrogels With Mesenchymal Stem Cells Increased the Efficacy of Cell Therapy in Pancreatitis by Hideaki Kojima, Hiroko Kushige, Hiroshi Yagi, Takayuki Nishijima, Nobuko Moritoki, Narihito Nagoshi, Yutaka Nakano, Masayuki Tanaka, Shutaro Hori, Yasushi Hasegawa, Yuta Abe, Minoru Kitago, Masaya Nakamura and Yuko Kitagawa in Cell Transplantation

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sj-tif-3-cll-10.1177_09636897231170437 – Supplemental material for Combinational Treatment Involving Decellularized Extracellular Matrix Hydrogels With Mesenchymal Stem Cells Increased the Efficacy of Cell Therapy in Pancreatitis

Supplemental material, sj-tif-3-cll-10.1177_09636897231170437 for Combinational Treatment Involving Decellularized Extracellular Matrix Hydrogels With Mesenchymal Stem Cells Increased the Efficacy of Cell Therapy in Pancreatitis by Hideaki Kojima, Hiroko Kushige, Hiroshi Yagi, Takayuki Nishijima, Nobuko Moritoki, Narihito Nagoshi, Yutaka Nakano, Masayuki Tanaka, Shutaro Hori, Yasushi Hasegawa, Yuta Abe, Minoru Kitago, Masaya Nakamura and Yuko Kitagawa in Cell Transplantation

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Supplemental material, sj-tif-5-cll-10.1177_09636897231170437 for Combinational Treatment Involving Decellularized Extracellular Matrix Hydrogels With Mesenchymal Stem Cells Increased the Efficacy of Cell Therapy in Pancreatitis by Hideaki Kojima, Hiroko Kushige, Hiroshi Yagi, Takayuki Nishijima, Nobuko Moritoki, Narihito Nagoshi, Yutaka Nakano, Masayuki Tanaka, Shutaro Hori, Yasushi Hasegawa, Yuta Abe, Minoru Kitago, Masaya Nakamura and Yuko Kitagawa in Cell Transplantation

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Footnotes

Acknowledgements

We would thank Editage for English language editing

Author Contributions

Conceptualization, H. Ko and H. Ku.; methodology, H. Ko and H. Ku.; investigation, H. Ko, H. Ku., T. N., N. M., N. Y., T. M., S. H., Y. H., Y. A., and M. K., original draft preparation, H. Ko and H. Ku.; Review and editing, H. Y.; supervision, H. Y., M. N., and Y. K.; funding acquisition, H. Y. All authors have reviewed and approved the final manuscript.

Ethical Approval

All experiments were reviewed and approved by the Animal Ethics Committee of Keio University School of Medicine, Tokyo, Japan (approval number: A2022-044).

Statement of Human and Animal Rights

The experiments in this study were conducted by the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA).

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a grant from the Japan Agency for Medical Research and Development (AMED; 22bm1004003h0003) awarded to H.Y.

ORCID iDs

Hideaki Kojima

Hiroshi Yagi

Supplemental Material

Supplemental material for this article is available online.

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