Tissue repair and donor-dependent immune responses after allogeneic skeletal muscle-derived cell sheet transplantation in a rat gastric ulcer model

1. Introduction

There have been remarkable advances in medical devices and pharmaceutical development; however, unmet medical needs—conditions that cannot be resolved by existing medical approaches—still remain. To address these challenges, the advancement of tissue engineering has enabled the use of viable cells as a therapeutic modality.

Cell-based therapies have recently gained increasing attention and are expected to be effective against a variety of diseases. However, many clinical trials currently in the spotlight use materials such as induced pluripotent stem cells,^1–6 organoids, ⁷ and exosomes.^8,9 For approaches relying on allogeneic cells to be widely implemented in clinical practice, it is necessary to establish a consensus on the optimal cell source and delivery strategy. ¹⁰

Autologous cells, which are derived from a patient’s own tissue, offer safety advantages because they are theoretically unrejected and do not become cancerous. Many real-world cell therapies use autologous cells, and local or systemic injections of cell suspensions have shown effectiveness so far. ¹¹

Cell sheet therapy represents a unique cell delivery strategy that is expected to broaden therapeutic applications. Using this technology, the product can be harvested as a sheet-like structure without the use of enzymes, allowing a larger number of cells to be transplanted into the target site. In addition, intact cell–cell junctions and the associated extracellular matrix facilitate rapid tissue regeneration and functional recovery through the paracrine effect.^12,13 Various benefits of cell sheet transplantation have been reported and promising results from several clinical trials for treating tissue and organ damage have been published.^14–17

We previously reported the endoscopic transplantation of autologous cell sheets to prevent scar-related strictures after esophageal endoscopic submucosal dissection (ESD).^18,19 Furthermore, to expand the use of cell sheets into the abdominal cavity, we conducted an investigator-initiated clinical trial. ²⁰ In this trial, we demonstrated that the transplantation of myoblast cell sheets could prevent delayed perforation after duodenal ESD. Although autologous cell sheet transplantation has shown effectiveness in previous clinical trials, disadvantages such as the invasiveness of obtaining a cell source, the time required for culture, and the cost of quality inspection must also be considered. In addition, to industrialize cell sheet therapy and make it accessible for general medical practice in the future, applications using allogeneic cells are essential.

The primary aim of this study was to determine whether allogeneic myoblast sheet transplantation can promote early tissue repair without inducing overt acute immune activation. In addition, we explored whether donor–recipient strain compatibility influences local immunological and transcriptional responses after transplantation.

2. Materials and methods

2.1.1. Ethics for clinical samples

This study was conducted in compliance with the Declaration of Helsinki and was approved by the Clinical Research Review Board at Nagasaki University (approval Nos. 17101611, 20081724 and 21101907). All human muscle tissues used in this study were collected and scheduled for disposal after standard surgery and were not collected specifically or solely for this study. Written informed consent was obtained from all participants.

2.1.2. Culture of skeletal muscle-derived cells

Human skeletal muscle-derived cells (SMDCs) were obtained from surplus skeletal muscle tissue collected during anterior cruciate ligament reconstruction surgery at Nagasaki University, following the acquisition of informed consent. The isolation protocol has been described in our previous publication. ²⁰ The growth medium included 0.08% Orgadron, 1 µg/mL epidermal growth factor, 4% L-glutamine, 20% fetal bovine serum (FBS), and MCDB131. Unless otherwise specified, cells at passage 3 were used for all experiments.

2.1.3. Preparation of peripheral blood mononuclear cells (PBMCs)

Peripheral blood mononuclear cells (PBMCs) were purchased from HemaCare (catalog No. PB009C-50). The culture medium consisted of 30 U/mL interleukin-2 (IL-2), 10% FBS, and RPMI-1640. The PBMCs were thawed in a 37 °C water bath and resuspended in 10 mL of washing medium (10% FBS in PBS) per vial (1.5 × 10⁶ cells). The suspension was centrifuged at 400 × g for 10 min at 4 °C, and the supernatant was discarded. The cell pellet was then resuspended in PBMC culture medium and seeded into a 24-well culture plate for floating culture (IWAKI) at a density of 1 mL per well, corresponding to approximately 1.0 × 10⁵cells/cm². The plates were incubated overnight at 37 °C. Following incubation, cell counting was performed, and the cells were centrifuged again at 400 × g for 10 min at 4 °C. After removing the supernatant, the cells were used for subsequent experiments.

2.1.4. T cell proliferation suppression assay

The assay was performed as previously described. ²¹ SMDCs were seeded at a density of 5 × 10⁵cells/well in 1 mL of medium into 24-well plates 48 h prior to the assay. T cell proliferation was assessed using the CellTrace™ Far Red Cell Proliferation Kit (#C34572, Thermo Fisher Scientific). Washed PBMCs were stained with a 1:1000 dilution of Far Red dye in PBS at 37 °C for 20 min, followed by incubation in five volumes of PBMC culture medium at room temperature for 5 min. The cells were then washed with PBS prior to co-culture. The assay was performed by adding fresh SMDC culture medium and seeding allogeneic PBMCs at a density of 1 × 10⁵cells/well. PBMCs were activated using Dynabeads (#DB11344, VERITAS) at a bead-to-cell ratio of 0.5:1. After 5 days of co-culture, T cells were gently collected by pipetting, and the extent of proliferation was analyzed using flow cytometry. All experiments were conducted with a single biological replicate and technical replicate (n = 1).

2.1.5. Antigen analysis

SMDCs prepared according to the protocol described in Section 2.1.2 were cryopreserved, thawed, and resuspended in skeletal muscle cell growth medium at a final concentration of 4.0 × 10⁵cells/mL. For the monoculture, 10 mL of the cell suspension was seeded in a T-175 flask (#159910, Thermo Fisher Scientific) and cultured at 37 °C for 4 days. For the co-culture experiments, SMDCs were cultured for 1 day under the same conditions. After removing the culture supernatant, PBMCs prepared according to the protocol described in Section 2.1.3 were resuspended in skeletal muscle cell growth medium at a final concentration of 2.67 × 10⁵cells/mL. A total of 15 mL of the PBMC suspension was added to the flask containing SMDCs, and the co-culture was maintained at 37 °C for 4 days.

Adherent SMDCs were detached using TrypLE Select (catalog No. 12604021, Gibco) at 37 °C. After washing and cell counting, the cells were incubated with antibodies at the indicated dilutions for 30–60 min in the dark. After two washing steps, the fluorescence intensity was measured using a flow cytometer (CytoFLEX, Beckman Coulter) with one biological replicate and one technical replicate (n = 1 for each). The antibodies and their respective dilutions used in this study were as follows: CD40 (BioLegend, #334305, 1:20), CD80 (BioLegend, #305205, 1:5), CD86 (BioLegend, #374203, 1:5), PD-L1 (BioLegend, #374509, 1:20), PD-L2 (BD, #569833, 1:20), ICAM-1 (CD54) (BioLegend, #353107, 1:20), VCAM-1 (Thermo, #A15439, 1:10), CD58 (Thermo, #MA1-19575, 1:5), CD155 (BioLegend, #337627, 1:20), HLA-G1 (Thermo, #MA1-19610, 1:10), HLA-E (Thermo, #12-9953-42, 1:20), HLA-ABC (Nippon Becton Dickinson Co., Ltd., #557348, 1:15), and HLA-DR (Nippon Becton Dickinson Co., Ltd., #560944, 1:15).

2.1.6. qPCR analysis of cytokine expression

After removing the culture supernatant from the 24-well plates containing adherent SMDCs, each well was washed with PBS. PBMCs were then seeded at a concentration of 6.6 × 10⁵cells/mL, corresponding to a seeding density of 3.5 × 10⁴cells/cm². These cells were cocultured for 48 h in an incubator at 37 °C with 5% CO₂.

Following incubation, the SMDCs adherent to the culture plate were washed with PBS and lysed using a lysis buffer composed of PureLink™ RNA Mini Kit Lysis Buffer (Thermo Fisher Scientific) supplemented with 2-mercaptoethanol.

Total RNA was extracted from the cell lysates using the PureLink™ RNA Mini Kit (Thermo Fisher Scientific), and complementary DNA (cDNA) was synthesized using the SuperScript™ IV VILO Master Mix (Thermo Fisher Scientific). Quantitative real-time PCR was performed using the TaqMan® Gene Expression Assay and TaqMan® Fast Advanced Master Mix (both from Life Technologies) to quantify cytokine gene expression levels. The assay IDs and corresponding target genes used in the TaqMan® Gene Expression Assays are listed in Table 1. Experiments were performed using a single biological replicate and three technical replicates. Error bars were generated from the technical replicates (n = 3).

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Table 1.

TaqMan gene expression assays were used in this study.

Fluorescent probe assay ID	Target gene
Hs00174114_m1	IL-2
Hs00985639_m1	IL-6
Hs01003716_m1	IL-15
Hs00989291_m1	IFN-γ
Hs00174128_m1	TNF-α

2.1.7. Ethics of animal experimentation

Animal experimental procedures were performed in accordance with the Guidelines for Animal Experimentation of Nagasaki University with the approval of the Institutional Animal Care and Use Committee (approval No. 2305301868). All the rats were purchased from Nippon SLC Co., Ltd. (Shizuoka, Japan). The rats were maintained in an environment with a temperature of 22–26 °C and a humidity of 30–70%; they were allowed ad libitum access to chow (Oriental Yeast, Tokyo, Japan) and autoclaved water at the Research Center for Biomedical Models and Animal Welfare, Nagasaki University Graduate School of Biomedical Sciences.

2.1.8. Autologous skeletal muscle-derived cell sheet fabrication

In this study, commercially available cell lines syngeneic to the recipient rats were used to mimic autologous SMDCs. Rat SMDC L8 cells (American Type Culture Collection, Manassas, VA, USA; catalog No. 95102434), established from Wistar rats, was used as a syngeneic cell-line comparator (L8-derived syngeneic comparator) rather than a true primary autologous SMDC sheet. L8 cells were seeded in temperature-responsive 6-well culture plates (UpCell 6 Multi-well Plate; CellSeed, Tokyo, Japan; catalog No. CS3004), at a density of 1 × 10⁵cells/well. The cells were seeded in high-glucose DMEM (Thermo Fisher Scientific, Waltham, MA, USA; catalog No. 11995065) supplemented with 10% FBS (Thermo Fisher Scientific; catalog No. 1606731) and 10 μg/mL gentamicin sulfate (Fuji Pharma Co., Ltd., Tokyo, Japan) at 37 °C and 5% CO₂. The medium was replaced twice a week with fresh medium. After 9 days of culture, confluent SMDCs on temperature-responsive dishes were transferred and left at 20 °C for approximately 5 h to allow spontaneous detachment of the SMDC sheets. The SMDC sheets were then washed thrice with ice-cold Hanks’ balanced salt solution containing calcium chloride and magnesium sulfate (HBSS+; Thermo Fisher Scientific; catalog No. 14025092).

2.1.9. Allogenic skeletal muscle-derived cell sheet fabrication

The experiment was performed using two different strains of rats (BN/SsN Slc and DA/Slc) as donors. Four-week-old male rats were euthanized using carbon dioxide and skeletal muscles were harvested from both thighs. The muscles were shredded and digested at 37 °C in a shaker bath with TrypLE Express (Thermo Fisher Scientific; catalog No. 12604021) containing collagenase type I (Thermo Fisher Scientific; catalog No. 17100017), gentamicin sulfate (Fuji Pharma Co., Ltd.), and Fungizone^® (amphotericin B; Bristol Myers Squibb, NY, USA; catalog No. 279112053) for 140 min. The isolated cells were collected by centrifugation (5 min at 800 × g), resuspended in low-glucose DMEM (Thermo Fisher Scientific; catalog No. 11885084) supplemented with 10% FBS and 10 μg/mL gentamicin sulfate, and seeded into non-coated flasks. After 4 h at 37 °C and 5% CO₂, the cell suspensions were replated into new flasks double-coated with poly-L-lysine (Sigma-Aldrich Co. LLC, MO, USA; catalog No. P4707-50ML) and laminin (Sigma-Aldrich; catalog No. L2020-1MG). The medium was replaced with one containing 3 μg/mL dexamethasone sodium phosphate (MSD K.K., Tokyo, Japan) after 24 h. After culturing for 11 days, the cells were reseeded in an UpCell 6 Multi-well Plate at a density of 8 × 10⁵cells/well on the day before transplantation. Allogeneic SMDC sheets were prepared in the same manner as the autologous sheets.

2.1.10. Gastric ulcer induction and the skeletal muscle-derived cell sheet transplantation

Gastric ulcers were induced in rats using a modified version of previously reported methods.^22,23 Male Slc:Wistar rats aged 8–12 weeks were anesthetized by isoflurane inhalation (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan; catalog No. 099-06571). The abdomen was swabbed with 70% ethanol and a midline incision was made. The stomach was exposed, and the anterior and posterior walls of the antrum were clamped with ring forceps (outer diameter 8 × 10 mm); 100 µL of 40% acetic acid solution was injected into the clamped lumen using a 26-gauge needle attached to a 1-mL syringe. The injected acid was then removed after 30 s. In the sheet transplantation group, SMDC sheets were transpelanted using a round coverslip (diameter 20 mm) on the serosal side of the gastric anterior wall at the injection site. Three types of transplanted SMDC sheets were used: L8 cell sheets mimicking autologous SMDCs (Syn-L8), allogeneic SMDCs derived from BN/SsN Slc rats (Allo-BN), and allogeneic SMDCs derived from DA/Slc rats (Allo-DA). In the control group, no SMDC sheet transplantation was performed and the abdomen was immediately closed. To account for the possibility that drug-induced ulcers might not be successfully created because of technical issues, as well as for unexpected rat loss, the experiment was initiated with 10 rats in each group.

2.1.11. Sampling

Rats in all groups were sacrificed by exsanguination under deep anesthesia on days 1, 3, and 7 after the operation, and their stomach and blood were collected. A portion of the ulcerated gastric tissue was taken using a biopsy punch and stored at −80 °C for total RNA extraction. The remaining portions were fixed in 4% paraformaldehyde phosphate buffer (PFA; Fujifilm Wako Pure Chemicals; catalog No. 163-20145) and embedded in paraffin. Serum was separated by centrifuging blood samples at 1670 × g for 10 min and then stored at −80 °C until measurement of C-reactive protein (CRP) to evaluate inflammatory parameters.

3. Results

3.3. No increase in systemic inflammatory reactions to cell sheet transplantation

To assess the systemic inflammatory response associated with sheet transplantation, we measured the serum CRP levels. Table 2 shows the serum CRP levels on POD1 and POD3. Compared to the control group, no significant differences were observed in serum CRP levels after allogeneic or syngeneic SMDC sheet transplantation (Table 2).

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Table 2.

The serum CRP levels (µg/mL) on POD 1 and 3.

​	Control	Syn-L8	Allo-BN	Allo-DA	p-value
POD1	306.6±32.4	267.5±8.7	276.7±32.7	276.1±48.3	n.s.
POD3	268.7±13.5	267.5±30.4	253.6±42.7	286.0±35.2	n.s.

The results were shown as Mean±SD. The biological replicates were as follows: POD1) Control n=5; Syn-L8 n=4; Allo-BN n=5; Allo-DA n=5, POD3) Control n=5; Syn-L8 n=5; Allo-BN n=5; Allo-DA n=5. The p-value according to two-tailed Student's t-test was indicated (compared with the control group). n.s., not significant; Control, the absence of the skeletal muscle-derived cells sheet group; Syn-L8, L8 cell sheet transplantation group; Allo-BN, BN/SsN Slc mice-derived allogenic skeletal muscle-derived cellss sheet transplantation group; Allo-DA, DA/Slc mice-derived allogenic skeletal muscle-derived cellss sheet transplantation group.

3.4. Immune cells were not strongly stimulated to proliferate after transplantation of either allogeneic or syngeneic skeletal muscle-derived cell sheets

To investigate the immunological reaction to SMDC sheet transplantation at the graft site, immunostaining for the macrophage marker Iba-1 and the T cell marker CD3 was performed from the serosal to the submucosal surface, and the number of positively stained cells was measured using a WinROOF image analyzer.

Iba-1 expression showed a trend toward higher numbers in the allogeneic and particularly the DA groups at POD3 (Figure 3(a) and (b), and S3A) and significantly higher numbers at POD7 (Figure 3(c) and (d) and S3B). Regarding CD3 expression, the number of positive cells tended to be higher in the Allo-DA group at POD3 (Figure 4(a) and (b) and S4A), although the difference was not significant. However, these trends were not evident at POD7 (Figure 4(c) and (d) and S4B).OPEN IN VIEWER

Figure 3.

Comparing Iba1-positive cell numbers in the ulcer areas. Immuno-histochemical staining was performed using anti-Iba1 antibodies. (a and b) On POD3. (c and d) On POD7. (a and c) The black square in the low-power field marks the area corresponding to the high-power field of view. The top photographs show Iba1-positive cells as brown spots. The bottom photographs show WinROOF digital images, with green corresponding to the area of Iba1-positive cells. Scale bars, 1 mm (low-power field); 200 μm (high-power field). The example photos are as follows: Upper, Syn-L8 on POD3; Lower, Allo-DA on POD7. (b and d) The box plots show the numbers of the immunopositive cells calculated by WinROOF. The box plots represent the minimum, lower quartile, median, upper quartile, and maximum. The p-value according to the Steel–Dwass test was indicated in comparison with the control group. *p< 0.05, n.s., not significant. The biological replicates were as follows: POD3, Control n = 7; Syn-L8 n = 7; Allo-BN n = 5; Allo-DA n = 3. POD7, Control n = 7; Syn-L8 n = 4; Allo-BN n = 5; Allo-DA n = 4.

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Figure 4.

Comparing CD3ε-positive cell numbers in the ulcer areas. Immuno-histochemical staining was performed using anti-CD3ε antibodies. (a and b) On POD3. (c and d) On POD 7. (a and c) The black square in the low-power field marks the area corresponding to the high-power field of view. The top photographs show CD3ε-positive cells as brown spots. The bottom photographs show WinROOF digital images, with green corresponding to the area of CD3ε-positive cells. Scale bars, 1 mm (low-power field); 200 μm (high-power field). The example photos are as follows: Upper, Control on POD3; Lower, Syn-L8 on POD7. (b and d) The box plots show the numbers of the immunopositive cells calculated by WinROOF. The box plots represent the minimum, lower quartile, median, upper quartile, and maximum. The p-value according to the Steel–Dwass test was indicated in comparison with the control group. n.s., not significant. The biological replicates were as follows: POD3, Control n = 8; Syn-L8 n = 5; Allo-BN n = 5; Allo-DA n = 3. POD7, Control n = 7; Syn-L8 n = 6; Allo-BN n = 5; Allo-DA n = 4.

These findings imply that even under the same allogeneic transplantation conditions, immune responses may vary depending on the donor–recipient combination and that SMDCs from DA rats may trigger a stronger immune response than those from BN rats in the cell sheet graft area.

3.5. RNA-seq analysis suggested differentially expressed genes caused by skeletal muscle-derived cell sheet transplantation

To investigate the factors that may contribute to ulcer shrinkage following SMDC sheet transplantation, we analyzed differentially expressed genes (DEGs) in ulcerated gastric tissues on POD1 using RNA-seq for each SMDC sheet transplantation group compared to the control group (the absence of SMDC sheets). Six DEGs were upregulated and 85 were downregulated in the Auto group using cutoffs of p< 0.05 and |log₂ fold change (FC)| > 2 (Figure 5(a), left; Table S1–S2). Under the same conditions, 87 genes were upregulated and 240 were downregulated in the Allo-BN group (Figure 5(a), middle; Table S3–S4), while 140 were upregulated and 81 were downregulated in the Allo-DA group (Figure 5(a), right; Table S5–S6). PCA showed that the three SMDC sheet-transplantation groups differed significantly from the control group (Figure 5(b)). Interestingly, the Sin-L8 and Allo-BN groups clustered closely, whereas the Allo-DA group was clearly separated from both. These results suggest that differences in lineage (e.g., haplotypes) may exert a greater influence on gene expression changes induced by SMDC sheet transplantation than a simple distinction between autologous and allogeneic conditions. Figure 5(c) and S5 show a summary of the GO analysis based on the downregulated genes. In particular, we focused on terms that were downregulated in both the Sin-L8 and Allo-BN groups compared to those in the control group (Table 3). These findings suggest that autologous and allogeneic SMDC sheet transplantation may downregulate genes involved in inflammation. Venn’s circular diagram shows overlapping up- or down-regulated DEGs in Sin-L8/Allo-BN/Allo-DA groups. This indicates level of shared pathways between applications and overlapping differentially expressed genes among the groups (Figure S6).OPEN IN VIEWER

Figure 5.

RNA-Seq analysis of ulcerated gastric tissues with/without the skeletal muscle-derived cells sheets transplantation. (a) The top 50 differentially expressed genes (DEGs) in each group compared to the control group were presented in a heatmap (in descending order of log2FC). The number of upregulated genes obtained in the Syn-L8 group was less than 50. Blue and red indicate down- and upregulated genes, respectively. (b) PCA analysis among four groups. (c) Gene ontology (GO) analyses were performed using downregulated genes in each group based on Tables S2, S4, and S6. Trends were confirmed across biological replicates, n = 3.

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Table 3.

The downregulated terms in common to the Syn-L8 and Allo-BN groups.

Term ID	Description	LogP
		Syn-L8	Allo-BN
GO:0030593	neutrophil chemotaxis	-25.44	-13.39
GO:0032103	positive regulation of response to external stimulus	-15.76	-8.14
GO:0038094	Fc-gamma receptor signaling pathway	-12.26	-5.79
GO:0002703	regulation of leukocyte mediated immunity	-9.47	-5.58

4. Discussion

The present study demonstrated that allogeneic cell sheets exhibit tissue repair effects comparable to those of syngeneic cell-derived sheets; however, certain combinations of allogeneic donor cells and recipient strains may trigger immune responses. The novelty of the present study is not limited to the use of an allogeneic cell source or a different anatomical target. Rather, this study evaluates allogeneic SMDC sheets in the context of intraperitoneal gastrointestinal wound repair and suggests that donor–recipient compatibility may influence the local immune and transcriptional responses after transplantation. This study highlights both the usefulness and limitations of allogeneic cells and is meaningful for expanding their applications in clinical practice.

Autologous cells are commonly used in clinical studies because of their immunological safety and favorable outcomes. We previously demonstrated that transplantation of a myoblast cell sheet fabricated from muscle tissue retrieved from the patient onto the serosal surface could prevent delayed perforation after duodenal ESD. ²⁰ This finding indicates the feasibility of autologous cell sheet therapy for various pathological conditions of the abdominal cavity. However, autologous cells have disadvantages including high costs and the time required for their expansion and quality inspection. Additional surgery to collect tissues for cell expansion and sheet fabrication is also necessary. For patients with cancer, the long culture period required for autologous cells presents a major challenge and hampers their clinical application. Therefore, preservable, quality-tested allogeneic cell sheets are needed for a “ready-to-use” approach. The shift toward using allogeneic cells offers additional advantages, such as reduced costs through mass production and quality standardization. ²⁴

Mesenchymal stem cells (MSCs) are the most widely used allogeneic cells owing to their low immunogenicity and immunomodulatory ability. Numerous clinical studies have reported their immunotolerant and anti-inflammatory effects,^25–29 and MSCs have recently been approved by the U.S. Food and Drug Administration for the treatment of steroid-refractory acute graft-versus-host disease in pediatric patients.^30–32 SMDCs have been reported to express cell surface markers similar to those of MSCs, ²¹ suggesting the potential applicability of allogeneic SMDC sheets. Although MSCs-based products represent established allogeneic cell sources, the present study focused on SMDCs because autologous myoblast sheet transplantation has already been clinically applied in our previous gastrointestinal studies. Thus, our objective was to examine whether this established myoblast sheet strategy could be extended toward an allogeneic, ready-to-use approach. To this end, we investigated the efficacy and immune responses associated with intraperitoneal transplantation of allogeneic cells using both in vitro and in vivo experiments.

We first confirmed in a lymphocyte mixed-culture test that human SMDCs could strongly suppress the proliferation of activated lymphocytes in a dose-dependent manner. Kindler et al. reported that human myoblasts share several characteristics with MSCs, ²¹ including similar phenotypic marker expression and the ability to inhibit T cell proliferation under co-culture conditions. They concluded that human myoblasts have both phenotypic and functional similarities to MSCs and can exhibit potent immunosuppressive properties.

We also showed that SMDCs expressed various molecules involved in T cell interactions. Co-stimulatory molecules such as CD40, CD80, and CD86, which are required for efficient T cell activation, ³³ were not expressed in SMDCs, even when co-cultured with activated PBMCs. In contrast, molecules known to mediate lymphocyte inhibition, such as CD155 and PD-L1, were detected. We also observed the expression of ICAM-1, a critical mediator of MSC-mediated immunosuppression, during coculture with PBMCs. ³⁴ As with MSCs used as allogeneic cell resources, in SMDCs, HLA-ABC, which is MHC class I, was expressed, whereas HLA-DR, which is MHC class II, was not expressed.

MSCs are known to exhibit potent immunomodulatory effects, including the suppression of T cell proliferation and induction of anti-inflammatory pathways through mechanisms such as indoleamine 2,3-dioxygenase (IDO) activity and immune checkpoint signaling. ³⁵ Our finding that SMDCs lack costimulatory molecules while expressing checkpoint ligands suggests that these cells may share functional similarities with MSCs, thereby contributing to reduced immunogenicity, even under allogeneic conditions.

Immunological concerns, including the risk of rejection and its potential impact on the efficacy of cell sheet transplantation, remain major issues in allogeneic cell transplantation into the abdominal cavity. ³⁶ Clinical trials using allogeneic cell sheets inside the body have been conducted so far^37–39; however, their use has been limited to the pleural or articular cavities, and the immunological responses in the abdominal cavity remain unclear. Therefore, for the wide application of allogeneic myoblast cell sheet therapy to intraperitoneal organs, as in our previous clinical study, it is important to evaluate both the efficacy and immunological safety of allogeneic myoblast sheet transplantation within the peritoneal cavity. To address this issue, we examined the tissue-repair effects and immune responses associated with cell sheet transplantation using an artificial rat ulcer model, in which we had previously demonstrated that paracrine mechanisms play a significant role in tissue repair. ²³

In their review of rat liver transplantation models, Wang et al. described that the levels and duration of rejection vary depending on the donor–recipient strain combinations. ⁴⁰ According to their review, when Lewis rats were used as recipients, the livers of DA rats elicited stronger rejection, whereas the livers of BN rats induced tolerance. Based on these findings, we selected several donor–recipient strain combinations. To assess the immune responses to both autologous and allogeneic cells, Wistar rats were used as recipients. L8 cells, which are syngeneic to Wistar rats, were used as autologous cells (L8-derived syngeneic comparator: Syn-L8), whereas BN- and DA-derived cells, which were known to differ in immunogenicity in the aforementioned transplantation studies, were used as allogeneic cells.

Immunohistochemistry showed that the number of monocytes on POD3 remained low in both the Syn-L8 and Allo-BN groups, whereas marked accumulation was observed in the Allo-DA group, although this difference was not statistically significant. Similarly, in the Allo-DA group, lymphocyte accumulation tended to be higher on POD3. This finding implies that certain haplotype combinations may trigger acquired immune responses because we analyzed monocyte infiltration 7 days after transplantation, whereas T-lymphocyte infiltration into wounds has been shown to occur later in cutaneous ulcers transplanted with allogeneic fibroblast sheets. ⁴¹

These findings suggest that the therapeutic effects may persist for a certain period, and that a delicate balance between the immunogenicity and immunomodulatory properties of myoblasts may also be involved.

Our in vivo study showed that, even with allogeneic cells, the tissue repair effect was similar to that observed with syngeneic cells. Several studies have also reported that the therapeutic effects of allogeneic sheet transplantation are comparable to those of autologous transplantation.^41,42 We have previously reported the mode of action of cell sheets using artificial ulcer models, in which various growth factors contribute to ulcer healing. ²³ Memon et al. reported the paracrine effects of various growth factors secreted from cell sheets in heart transplantation models as a mechanism of tissue repair.^12,43 Furthermore, Toya et al. reported the promotion of liver regeneration and suppression of fibrosis in a mouse model of hepatic fibrosis. They also conducted detailed investigations into changes in gene expression following cell sheet transplantation using spatial transcriptomics analysis. ⁴⁴ Although syngeneic and allogeneic cell sheet transplantations produced similar healing effects in the present study, increases in these growth factors were not detected in the RNA-seq analysis, nor were increases observed using RT-PCR (data not shown). These findings imply that factors other than growth factors may contribute to cell sheet function. Because differences in ulcer size were already apparent one day after surgery, factors related to anti-inflammatory effects that counteract tissue damage caused by the chemical agent may be involved.

The RNA-seq analysis revealed that SMDC sheet transplantation induced distinct transcriptional changes in gastric ulcer tissue depending on the haplotype background of the donor cells. In both the Syn-L8 and Allo-BN groups, GO analysis of downregulated DEGs consistently identified terms related to innate immunity and inflammation, including neutrophil chemotaxis, cytokine-cytokine receptor interactions, Fc-gamma receptor signaling, and leukocyte-mediated immunity (Figure 5(c)). This convergent downregulation of inflammatory pathways was observed regardless of whether the transplanted SMDC sheets were syngeneic or allogeneic, suggesting that SMDC sheet transplantation may contribute to ulcer healing through active suppression of local inflammatory responses at the gastric wound site. These findings are consistent with the equivalent therapeutic effects observed in the Syn-L8- and Allo-BN groups and may partly explain the macroscopic and histological similarities in ulcer shrinkage between these groups.

In contrast, the Allo-DA group showed a markedly different transcriptional profile; downregulated genes in this group were enriched for terms related to extracellular matrix organization, striated muscle tissue development, and structural remodeling, rather than inflammation (Figure 5(c)). This divergence was further supported by the PCA, which demonstrated that the Syn-L8 and Allo-BN groups clustered closely together and away from both the control and Allo-DA groups (Figure 5(b)). Notably, BN and Lewis rats share greater MHC haplotype similarities than DA and Lewis rats, suggesting that donor–host haplotype compatibility may exert a stronger influence on the transcriptional response to SMDC sheet transplantation than simple autologous versus allogeneic distinction. Although the current study was not designed to establish a definitive mechanistic pathway, these RNA-seq data provide a foundation for future investigations. In particular, whether the observed downregulation of neutrophil chemotaxis and cytokine signaling pathways represents a direct paracrine effect on transplanted muscle-derived cells or an indirect consequence of accelerated wound healing warrants further exploration using targeted functional studies.

One of the key findings of this study was that the Allo-BN group showed a transcriptional profile closer to that of the syngeneic comparator, whereas the Allo-DA group showed a distinct profile. These results suggest that donor–recipient compatibility may influence local immune and transcriptional responses after allogeneic SMDC sheet transplantation, and that donor selection may be an important consideration in future development of allogeneic SMDC sheet therapy.

Previous studies have suggested that cell sheets exert their therapeutic effects mainly through paracrine mechanisms during the early phase after transplantation. ¹² In this context, the difference between autologous and allogeneic sheets may be less important if the transplanted sheets do not persist for a prolonged period. Therefore, we considered it important to determine whether the therapeutic effects observed in our model were mediated by paracrine signaling, by integration of the transplanted cell sheet into host tissue, or by both mechanisms. To directly assess the survival or resorption of the transplanted sheets over time, we initially planned to use labeled cell sheets. However, this approach was not feasible because breeding juvenile GFP rats required for myoblast preparation was difficult under the regulations of our institutional facility, and adult GFP rats did not yield a sufficient number of myoblasts (data not shown). Although we did not directly examine the long-term persistence of the transplanted cells in the present study, our previous work showed that even autologous sheets in this model may be washed out within two weeks after transplantation into the abdominal cavity. ⁴⁵ Taken together, these findings suggest that the therapeutic effects of SMDC sheet transplantation are exerted mainly during the early post-transplantation phase. Accordingly, suppression of acute immune responses during this early period may be sufficient to achieve therapeutic benefit. This temporal feature may also explain why allogeneic transplantation can provide efficacy comparable to autologous transplantation without inducing overt immune rejection. Overall, the mechanism of action appears to depend more on transient paracrine signaling and immunomodulation than on long-term engraftment or structural integration.

Several animal studies have studied the effects of autologous and allogeneic cells.^24,43–52 Nagase et al. reported that both autologous and allogeneic sheet transplantation were equally effective in preventing leakage after esophageal anastomosis in a rat model, although a greater number of autologous cells appeared to remain at the transplantation site than allogeneic cells at 5 days after transplantation.^41,42

For clinical translation, allogeneic SMDC sheets should be developed as a ready-to-use adjunctive regenerative therapy for gastrointestinal wall injury or high-risk ulcerative lesions. The donor-dependent responses observed in this study suggest that donor selection, MHC/HLA compatibility assessment, standardized potency testing, and evaluation of acute immune activation will be important components of future translational development.

This study has several limitations. First, we used outbred Wistar rats as recipients in the transplantation experiments and L8-derived sheet was used as a syngeneic cell-line comparator rather than a true primary autologous SMDC sheet. Because L8 cells are an immortalized cell line, this group is not fully equivalent to primary syngeneic or autologous myoblast sheets. Therefore, direct comparisons between the L8-derived comparator and primary allogeneic SMDC sheets should be interpreted with caution.

In contrast, BN and DA rats, which are widely used inbred strains, were used as donors in the allogeneic transplantation model. Thus, we were able to minimize the variability caused by differences in MHC haplotypes. Secondly, the limited sample size in some of the in vitro experiments is one of the major limitations. This study was conducted as an initial investigation and, in particular, the in vitro experiments were carried out as exploratory assessments, with the results serving only to indicate trends. Additionally, since this is a derivative study from another clinical research project, it was performed with the minimum sample size as a prototype. The number of rats in each experimental group was also small. We initially designed the experiment to include 10 rats per group. However, some animals had to be excluded because of intraoperative or postoperative death, or inadequate gastric ulcer formation. Furthermore, in the allogeneic setting, technical difficulties were encountered during the isolation of primary myoblasts and the preparation of stable cell sheets. Consequently, the final number of rats included in the analysis was smaller than originally planned and the small sample size in several animal subgroups, particularly the Allo-DA group on POD7, limits statistical interpretation. Third, ulcer healing was assessed using the maximum ulcer base length on a single H&E-stained section. Although this approach allowed us to maintain consistency with our previous model and to preserve tissue for additional molecular analyses, it does not fully capture ulcer area, depth, re-epithelialization, or granulation tissue formation. Fourth, the acetic acid-induced gastric ulcer model used in this study does not fully reproduce ESD-related delayed perforation or post-ESD ulcer healing. Rather, it was used as a reproducible gastric injury model to evaluate early tissue repair and local immune responses after serosal SMDC sheet transplantation. The mechanism by which serosally transplanted SMDC sheets influence mucosal-side ulcer healing remains to be clarified, and future studies using spatial cell tracking and molecular localization analyses are required. Finally, the present study did not directly evaluate MHC class I/II expression, regulatory T-cell infiltration, or donor-cell persistence after transplantation. Therefore, the immunological mechanisms underlying the donor-dependent responses remain incompletely defined. Future studies should include MHC class I/II profiling, Treg analysis, and direct in vivo tracking of transplanted SMDC sheets to determine whether the observed responses reflect donor-cell immunogenicity, host immunoregulation, or secondary effects of tissue repair.

5. Conclusion

In this exploratory study, SMDC sheet transplantation promoted early gastric ulcer repair without inducing marked systemic inflammatory responses. However, local immune and transcriptional profiles differed according to the donor strain, suggesting that donor–recipient compatibility may influence the biological response to allogeneic SMDC sheet transplantation. These findings support the potential of allogeneic SMDC sheets as a ready-to-use therapeutic strategy for gastrointestinal tissue repair, while also highlighting the need for further studies using primary syngeneic controls, larger sample sizes, direct donor-cell tracking, and more detailed immunological analyses.

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