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. 2025 Aug 21;20(8):e0330478. doi: 10.1371/journal.pone.0330478

MSC and HUVEC co-cultured fillers overcome intractable fistula in a new mouse model

Soichiro Hirasawa 1,*,#, Kentaro Murakami 1,#, Masayuki Kano 2,#, Satoshi Endo 1,#, Takeshi Toyozumi 1,#, Yasunori Matsumoto 1,#, Ryota Otsuka 1,#, Nobufumi Sekino 1,#, Tadashi Shiraishi 1,#, Takahiro Ryuzaki 1,#, Kazuya Kinoshita 1,#, Takuma Sasaki 1,#, Hisahiro Matsubara 1,#
Editor: Keykavoos Gholami3
PMCID: PMC12370064  PMID: 40839600

Abstract

Anastomotic leakage can lead to intractable fistulae after gastrointestinal surgery in patients with severe comorbidities. In this study, we aimed to devise new intractable fistula mouse models and evaluate the utility of the fillers containing human mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs). After determining the optimal ratio of MSCs to HUVECs as fillers, we created new intractable fistula mouse models and verified the usefulness of the above-mentioned fillers for these fistulas. As the filler containing a 1:1 ratio of MSC: HUVEC showed the highest expression of FGF2 and VEGF among the organization-forming fillers, we determined that this was the optimal ratio. When this filler was transplanted into irradiated and steroid-treated mice with excisional wounds, the skin defects healed significantly faster in the filler-transplanted group than in the non-transplanted group (P < 0.05). Furthermore, we established a new mouse model of a gastrointestinal fistula by securing the cecum to the abdominal wall and puncturing the skin, abdominal wall, and intestinal wall with an indwelling needle. The fistula remained patent for at least seven days and was intractable. Unlike the adhesive group (group 1) (0/5) and the group implanted with fillers containing MSCs (group 2) (1/5), all fistulas were closed in the group implanted with fillers containing MSCs and HUVECs (group 3) (5/5). This study demonstrated that a treatment strategy using HUVEC is advantageous for treating intractable fistulae connected to the gastrointestinal tract. HUVEC should be included when fillers are used to close fistulas.

Introduction

Anastomotic leakage (AL) is one of the most alarming complications of gastrointestinal surgery. Although this incidence has decreased with the advancement of instrumental anastomosis, an analysis of the National Clinical Database (NCD) data, which covers more than 95% of surgical procedures in Japan, shows that anastomotic leakage is observed in 12.6% of esophagectomies [1], 2.3% of distal gastrectomies [2] and 1.8% of right hemicolectomies [3]. It’s still not 0%. Advances in drainage technology and nutritional management have made it possible to cure these conditions in many cases; however, some cases are difficult to treat. In recent years, with the advent of an aging society, there have been opportunities to perform surgery in patients with many comorbidities. Among these conditions, severe arteriosclerosis, diabetes, use of steroids, and radiation therapy can delay wound healing and sometimes lead to intractable fistulas that resist conservative treatment [4]. These fistulas can persist for a long time, and managing them with countermeasures can be challenging.

For intractable fistulas, filling the space with materials other than granulation tissue becomes crucial. Although fibrin glue [5], cyanoacrylate [6], and polyglycolic acid mesh [7] have shown promise, concerns remain regarding their safety and long-term efficacy in the body. Additionally, existing options struggle to adequately fill larger spaces due to limited material availability. Therefore, it is necessary to develop a filler that can safely and reliably close fistulas.

Human mesenchymal stem cells (MSCs) are capable of self-renewal and multilineage differentiation into various mesoderm-derived tissues, such as bone, cartilage, fat, and muscle [8,9]. Furthermore, their paracrine effects, via cytokines and extracellular vesicles (EVs) are involved in immunomodulation, inflammation, and tissue regeneration [10]. Because of these capabilities, stem cells have recently played a central role in regenerative medicine in soft tissues [8,9]. In recent years, the use of mesenchymal stem cells for wound healing has increased rapidly, not only in basic research but also in clinical trials. In 2023, the efficacy of adipose tissue-derived mesenchymal stem cells (darvadstrocel) in the treatment of complicated hemorrhoidal fistulas associated with Crohn’s disease was reported, with a combined remission rate of 59.1% after 24 weeks and 68.2% after 52 weeks [11]. Thus, the efficacy of MSCs alone in refractory wounds is limited, and a combination of genetic modifications of MSCs and other cell-based therapies is required.

In 2013, Takebe et al. reported that they produced functional liver tissue with vascular structures by co-culturing human umbilical vein endothelial cells (HUVECs) with MSCs [12]. HUVECs, first isolated by Jaffe in 1973, are well known for their ability to secrete vascular endothelial growth factor (VEGF) and promote angiogenesis [13,14]. Intractable fistulas caused by anastomotic leakage are often found to have poor blood flow due to microvessel obstruction. Therefore, promoting angiogenesis at the site of the defect is critical for tissue regeneration. Studies have shown that co-cultures of MSCs and HUVECs can generate tissues with significantly higher vascular density than those derived from MSCs alone [15].

Based on this evidence, we hypothesized that incorporating both MSCs and HUVECs into a cell-based filler could enhance vascularization and improve healing outcomes for gastrointestinal fistulas. While fillers co-cultured with MSCs and HUVECs have been used in adipose and bone tissue engineering [16,17], to our knowledge, their application in gastrointestinal fistula closure has not been reported.

Moreover, to evaluate the therapeutic potential of such fillers in vivo, a reliable and clinically relevant animal model is essential. Although an enterocutaneous fistula model has been previously reported, it resembles a colostomy [18] and does not replicate the complexity of gastrointestinal fistulas. Thus, a novel model that better mimics clinical presentation is needed.

Therefore, the objective of this study was to evaluate whether fillers co-cultured with MSCs and HUVECs could promote angiogenesis and thereby improve the closure rate of intractable gastrointestinal fistulas using a newly established mouse model.

Materials and methods

2.1 Cell culture and creating fillers

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) were obtained from Lonza (Cat# CC-2501, Basel, Switzerland) and cultured in MSC Growth Medium (MSCGM BulletKit, Cat# PT3001) according to the manufacturer’s instructions. Cells were used between passages 3 and 5. The MSC identity was confirmed by the supplier using established surface marker profiles, including CD73, CD90, CD29, CD105, CD166, and CD44 positivity, and the absence of CD14, CD19, CD34, and CD45, in accordance with ISCT criteria.

Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (Cat# CC-2517, Basel, Switzerland) and cultured in EGM-2 medium (Lonza, Cat# CC-3162). Cells were used between passages 3 and 6. According to the supplier, HUVECs were characterized based on endothelial marker expression and angiogenic capacity.

Six hundred microliters (600 μL) of Matrigel Matrix growth factor-reduced (Corning, Arizona, USA) were used to coat each well of a 12-well plate, followed by a 60-minute incubation at 37°C. Cocultures of MSCs and HUVECs were initiated at five different ratios (0:1, 1:3, 1:1, 3:1, and 1:0) to achieve a total cell count of 1.6 x 107. The co-culture ratios of MSCs to HUVECs were selected based on previously reported studies in which these cell types were combined to fabricate tissue fillers [19]. MSC and HUVEC were maintained in an endothelial cell growth medium. The filler was created by culturing human MSCs and HUVECs on Matrigel under three-dimensional (3D) conditions. This approach resulted in a tissue-like structure, with Matrigel serving as a biological scaffold to support the formation of a compact cellular mixture. No additional biopolymers, such as hyaluronic acid, were used.

2.2 Quantitative reverse-transcription PCR

Co-cultures of MSCs and HUVECs were initiated at three different ratios (MSCs:HUVECs = 1:1, 3:1, and 1:0), with a total of 1.6 × 10⁷ cells per sample. The resulting tissues were homogenized by pipetting, and total RNA was extracted using QIAzol Lysis Reagent (Qiagen, Venlo, Netherlands), following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative RT-PCR was performed in triplicate using a MyiQ™2 Two-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and TB Green Fast qPCR Mix (TaKaRa, Shiga, Japan). Primers for fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) were designed using Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/) and synthesized by Eurofins Genomics (Tokyo, Japan). Detailed primer sequences, annealing temperatures, and product sizes are provided in **Supporting Information Table1. qPCR analyses were independently performed in triplicate (n = 3).

2.3 Immunohistochemistry

Tissues with MSCs and HUVECs in a ratio of 1:1 were fixed in 4% paraformaldehyde for 1 d. The sections were then embedded in paraffin. The cells were permeabilized and blocked for 30 min. CD31 was used as a specific marker for HUVECs and CD44 for MSCs. Primary mouse anti-human CD31 (ab218, Abcam plc, Cambridge, UK, dilution 1:100) or rabbit anti-human CD44 (ab51037, Abcam plc, Cambridge, UK, dilution 1:100) antibodies (S0809, Agilent, CA, USA) were added to the tissue and incubated at 37°C for 60 min. A secondary goat anti-rabbit IgG, Alexa Fluor 488 (A11008, Thermo Fisher Scientific, Massachusetts, United States, 4 µg/mL) and goat anti-mouse IgG, Alexa Fluor 594 (A11005 Thermo Fisher Scientific, Massachusetts, United States, 5 µg/mL) were applied in antibody diluent and incubated in the dark at room temperature for 60 min. The cell nuclei were counterstained by DAPI (P36935, Thermo Fisher Scientific, Massachusetts, United States, 1 µg/mL) solution for five minutes. The fluorescent staining was recorded by a Fluorescence microscope (BZ-X700, Keyence, Osaka, Japan).

2.4 Transplantation of filler to subcutis

Male BALB/cAJcl-nu/nu mice (ages 6–8 ages weeks old) were used in this study. The mice were anesthetized using a triple anesthetic (medetomidine (Nippon Zenyaku Kogyo, Hukushima, Japan), butorphanol tartrate (Meiji Seika, Tokyo, Japan), and midazolam (Sandoz, Tokyo, Japan)). They were transplanted with a MSCs filler: HUVECs at a 1:1 or 1:0 ratio. On day seven, the mice were sacrificed by cervical dislocation, and the skin of the transplanted area was resected. The tissues were fixed in paraformaldehyde and stained with hematoxylin. Capillary densities of the fillers were determined using a microscope (BZ-9000, Keyence, Osaka, Japan). Capillary density analysis in the subcutaneous filler transplantation model was performed in triplicate (n = 3).

2.5 Creation of mouse model with intractable enterocutaneous fistula

Male BALB/cAJcl-nu/nu mice (ages 8−10 weeks old) were anesthetized (n = 5). The abdomen, abdominal wall, and cecum were fixed using two stitches of 6−0 nylon (Bear Medic, (Ibaraki, Japan)). The skin, abdominal wall, and intestines were punctured using a 10-gauge indwelling needle (BD Angiocath, BD, New Jersey, USA). Only the outer tube was implanted. Three days later, the outer tube was removed creating an enterocutaneous fistula. In response to weight loss, the mice were supplemented with saline (Otsuka Pharmaceutical Factory, Tokushima, Japan). Over seven days, the enterocutaneous fistula was monitored. On the seventh day, the area was sampled and stained with hematoxylin and eosin (H&E).

2.6 Transplantation of filler to mouse model with intractable enterocutaneous fistula

An enterocutaneous fistula mouse model was established using the method above. Three days after removing the outer tube, the mice were divided into three groups: an adhesive group coated with only physiological tissue adhesive (Group1: n = 5), a transplanted group with fillers containing MSCs (Group2: n = 5), and a transplanted group with fillers containing HUVECs and MSCs in a ratio of 1:1 (Group3: n = 5). Mice in the adhesive group received a coating of physiological tissue adhesive directly onto their fistulae. The filler-transplanted group, on the other hand, had the fillers applied to their fistulae, followed by application of a physiological tissue adhesive to secure the filler and fix it in place. Both groups had their fistulae monitored for seven days, and in the filler-transplanted group, the area around the filler was then sampled and stained with (H&E). For the in vivo intractable fistula model, five mice were used per group (n = 5). Due to the substantial cost and technical difficulty associated with both the creation of the fistula model and the preparation of the cell-based fillers, a larger sample size was not feasible.

2.7 Animals

This study was approved by the Chiba University Animal Experiment Committee (approval number: 4–157), and all procedures were conducted in accordance with the Chiba University Institutional Guidelines for the Use of Laboratory Animals. Mice were housed in individually ventilated cages under specific pathogen-free (SPF) conditions, with a 12-hour light/dark cycle, ambient temperature maintained at 22 ± 2 °C, and relative humidity at 55 ± 10%. Food and water were available ad libitum. Anesthesia was induced using a triple-anesthetic combination consisting of medetomidine, butorphanol tartrate, and midazolam. Animals were monitored postoperatively to minimize suffering. Humane endpoints were defined, and euthanasia was performed by cervical dislocation under deep anesthesia, following institutional guidelines.

2.8 Statistical analysis

Data were analyzed using a Student’s t-test conducted using Excel software (Microsoft, Redmond, WA, USA). Statistical significance was set at P < 0.05.

Results

3.1 Preparation of MSC-HUVEC fillers

Co-cultures of MSCs and HUVECs were initiated in five different ratios (MSCs: HUVECs 0:1, 1:3, 1:1, 3:1, and 1:0) to achieve a total cell count of 1.6 x 107 (Fig 1a). Co-cultures of MSCs and HUVECs at three ratios (MSCs: HUVECs 1:1, 3:1, and 1:0) formed organized structures.

Fig 1. Preparation of MSC and HUVEC filler.

Fig 1

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(a) Co-cultures of MSCs and HUVECs in five ratios. (b) mRNA expression of FGF2 and VEGF in the co-cultured tissues. (c) HE and immunostaining of the co-cultured tissues (MSC: Green, HUVEC: Red). Scale bar=50µm. The error bars represent mean ± standard deviation. These data were analyzed for statistical significance using Student’s t-test: *P<0.05. Abbreviations: MSC, mesenchymal stem cell; HUVEC, human umbilical vein endothelial cell; FGF2, fibroblast growth factor2; VEGF, vascular endothelial growth factor.

3.2 Quantification of angiogenesis markers

Tissues with MSCs: HUVECs ratios of 1:1, 1:3, and 1:0 were homogenized and subjected to quantitative reverse-transcription PCR to examine the expression of marker genes involved in angiogenesis (Fig 1b). Tissues with an MSCs: HUVECs ratio of 1:1 showed the highest expression of FGF2 and VEGF. The full qPCR dataset, including Ct values, ΔCt/ΔΔCt calculations, and expression ratios for each sample and group, are available in S1(FGF2), S2(VEGF) Data. Based on these results, we determined that a 1:1 ratio was optimal for the filler.

3.3 Immunostaining of MSCs and HUVECs

Skin tissues co-cultured with MSCs and HUVECs at a 1:1 ratio were stained with fluorescent antibodies specific for MSCs (green) and HUVECs (red) and visualized using immunofluorescence microscopy (Fig 1c). The results showed that MSCs and HUVECs were mixed to form tissues. Immunostaining was used to observe the spatial distribution and interaction of MSCs and HUVECs within the filler, but not for determining the optimal co-culture ratio.

3.4 Transplantation of filler to subcutis

Next, the changes in the prepared filler, as shown in Fig 1, were observed in vivo. H&E staining of the filler with MSCs and HUVECs at a ratio of 1:1 administered to mouse subcutaneous tissue showed that the filler had grown under the skin (Fig 2a). To evaluate the function of HUVECs in angiogenesis, capillary density was determined (Fig 2b). The results showed that the capillary density in a filler with MSCs and HUVECs at a ratio of 1:1 was 422.7/mm2, which was significantly higher than that in a filler at a ratio of 1:0 (53.9/mm2). Raw data and summary statistics for these comparisons are available in Supporting Information S3 Data.

Fig 2. The filler transplanted into the mouse subcutaneous space.

Fig 2

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(a) HE staining of the filler transplanted into the mouse subcutaneous space (granulation formed by the filler: black arrows). (b) Comparison of vascular density of the transplanted fillers mixed with different MSC and HUVEC proportions (vascular: yellow arrows). The error bars represent mean ± standard deviation. These data were analyzed for statistical significance using Student’s t-test: *P<0.05. Abbreviations: MSC, mesenchymal stem cell; HUVEC.

3.5 Mouse model with intractable enterocutaneous fistula

To create a fistula mouse model, the cecum was secured to the abdominal wall, and an indwelling needle was inserted through the skin, abdominal wall, and intestinal wall. Only the outer tube of the indwelling needle remained, creating a permanent opening (fistula) (Fig 3). Three days later, fistula formation was confirmed after removal of the outer tube. Seven days after removal of the indwelling needle, all caecal fistulas (n = 5) remained open. Hematoxylin and eosin staining confirmed fistulous tract formation from the intestine to the subcutis.

Fig 3. Creation of a mouse model of intractable enterocutaneous fistula.

Fig 3

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Gross and microscopic images of the enterocutaneous fistula were shown.

3.6 Transplantation of fillers in a mouse model with intractable enterocutaneous fistula

To evaluate the usefulness of filler implants in enterocutaneous fistulae, the healing process was compared among an adhesive group (Group1), a transplanted group with fillers containing MSCs (Group2), and a transplanted group with fillers containing HUVECs and MSCs (Group3) (Fig 4). In group 1, none of the 5 mice(0/5) achieved fistula closure and continued to experience leakage of intestinal fluid. In group 2, one out of 5 mice (1/5) successfully closed their fistula.

Fig 4. Treatment results with filler in each group.

Fig 4

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Microscopic images of the granulation formed by the filler were shown (granulation formed by the filler: black arrows, vascular: yellow arrows). Abbreviations: MSC, mesenchymal stem cell; HUVEC.

In group 3, all 5 mice (5/5) achieved fistula closure via granulation tissue formation by day 3. Their wounds were completely covered with skin by day seven. H&E staining showed that in group 3, granulation had formed in the area of the fistula, and the injured part of the intestine was closed. Vessel-like structures are observed within the granulation tissue.

Discussion

In this study, we created a new intractable fistula model and evaluated the usefulness of fillers co-cultured with MSCs and HUVECs. Tissues co-cultured with MSCs and HUVECs at a 1:1 ratio showed the highest expression of angiogenic markers and the highest blood vessel density within the tissue; therefore, we decided to use this tissue as a filler. We established a novel mouse fistula model by securing the cecum to the abdominal wall. This model resulted in an intractable fistula that resisted spontaneous healing. Notably, the filler described above demonstrated a high success rate in achieving closure of these fistulas.

To assess the potential of fillers in treating refractory gastrointestinal fistulas (occurring after cancer surgery), we developed a reliable and easily observable mouse model replicating key features of these clinically challenging non-healing fistulas. Although a model similar to a prosthesis has been previously reported, it differs from a fistula that develops after suture failure, which was our target in this study, because the mucosa is directly continuous with the skin [13]. Because there have been no adequate reports to date, we devised a new fistula model at the beginning of this study. First, a fistula connecting the gastrointestinal tract to the skin was created for ease of observation. However, fistulas in the intestinal tract often result in death due to the massive leakage of intestinal fluid. Therefore, we focused on the mouse cecum. Because the mouse cecum is long and does not produce much stool, the general condition of the mice with a fistula connecting the cecum to the skin was stable, and the fistula did not close spontaneously. We speculate that this model will be useful for future research on fistulas.

Recently, tissue regenerative medicine for enterocutaneous fistulae using MSC has been expected, such as MSC sheets, [9] and sutures made from MSC [10]. However, these all have a small amount of tissue, and cannot close fistulae with large spaces. A major cause of failure in transplantation of regenerated tissue is inadequate vascularization of the tissue [11]. Enterocutaneous fistulae are often associated with poor blood flow; thus the tissue needs to be vascularized and viable. Similarly, in this study, the complete healing of refractory fistulas created in mice using stem cells alone was difficult to achieve.

HUVEC, isolated from the umbilical vein by Jaffe in 1973, secretes VEGF and promotes angiogenesis. Several studies have reported good results using fillers with HUVECs alone or HUVEC-derived exosomes [20], but the method of making fillers has not been established, as it differs in each study. In this study, VEGF and FGF2 were selected as representative markers of angiogenesis, which is a critical factor for the engraftment and functional integration of the filler. Both genes are well-established proangiogenic factors that directly contribute to vascular network formation. Moreover, these genes have frequently been used in previous studies evaluating MSC and HUVEC co-cultures, especially when assessing optimal cell ratios for tissue integration and angiogenic potential [21,22]. Based on this rationale and our own observations, we considered VEGF and FGF2 appropriate indicators of tissue organization capacity in our filler model. When MSCs and HUVECs were mixed in a 1:1 ratio, the tissue with the highest VEGF expression was formed. Previous reports have shown that tissues with a 1:1 or 1:3 ratio of MSCs to HUVECs have the highest expression of angiogenic markers, and the results of this study are consistent with those of previous reports [19,21]. Furthermore, filler-derived granulation tissue prepared at this ratio had a significantly higher vascular density and histopathological angiogenesis than granulation tissue without HUVECs. Direct or indirect interactions between MSCs and HUVECs, as well as VEGF secreted by HUVECs, have been suggested as reasons why combination therapy with these cells may favor wound healing. Beloglazova et al. reported that MSCs support the formation of EC tubular networks (ETNs) via the urokinase-type plasminogen activator (uPA) system when co-cultured with ECs [22]; Chance et al. reported that adipose-derived EVs promote HUVEC tube formation, and fat-derived EVs promote HUVEC tube formation [23].

Based on these results, we implanted fillers in our newly created mouse model of intractable fistulas and found that fillers with MSCs alone failed to achieve complete healing. In contrast, fillers co-cultured with HUVECs and MSCs achieved healing in all cases. The relatively low efficacy of MSC-only fillers observed in this study may be due to the challenging microenvironment of the gastrointestinal fistula model, which simulates clinical enterocutaneous fistulas. In such conditions, MSCs alone may not sufficiently promote tissue integration. However, co-administration of HUVECs likely enhanced angiogenesis, thereby supporting better engraftment of the filler. To the best of our knowledge, this is the first time that a filler co-cultured with MSCs and HUVECs is effective in closing refractory fistulas involving the gastrointestinal tract. Recently, the function of this filler mixture in the repair of myocardial and cerebral infarctions has been analyzed [24,25] and it is expected to be useful in many areas of regenerative medicine. In the future, this mixed filler is expected to benefit patients undergoing gastrointestinal surgery.

However, this study has two limitations. First, using immunosuppressed mice meant that the assessment of potential rejection, an important factor in clinical applications, was not possible. While MSCs generally have low immunogenicity [26], evaluating the immunogenicity of HUVECs in this context is crucial. Utilizing HUVECs derived from autologous induced pluripotent stem cells could address potential immune-related concerns. Second, we were unable to identify the mechanism of interaction between MSCs and HUVECs. Once this is clarified, it may be possible to develop the filler to a higher standard.

Conclusions

In conclusion, using a realistic and observable model, we demonstrated the superiority of fillers co-cultured with MSCs and HUVECs. As this has not yet been verified in a realistic clinical model, we report that using HUVECs is advantageous for treating gastrointestinal fistulas. Therefore, HUVECs should be added to MSCs when fillers are used to close refractory gastrointestinal fistulas.

Supporting information

S1. Data qPCR FGF2.

(XLSX)

pone.0330478.s001.xlsx (10KB, xlsx)
S2. Data qPCR VEGF.

(XLSX)

pone.0330478.s002.xlsx (10KB, xlsx)
S3. Data.

(XLSX)

pone.0330478.s003.xlsx (9.5KB, xlsx)

Acknowledgments

We would like to thank Keiko Iida for their help with this research.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was supported in part by a Grant-in-Aid for Scientific Research (KAKENHI: 20K17673) from the Japan Society for the Promotion of Science.

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Decision Letter 0

Keykavoos Gholami

27 May 2025

plosone@plos.org

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: No

**********

Reviewer #1: I have reviewed the manuscript entitled “MSC and HUVEC co-cultured fillers overcome intractable fistula in a new mouse model.” The study addresses an important clinical issue, namely the treatment of intractable fistulae after gastrointestinal surgery, by introducing a novel mouse model and evaluating the therapeutic potential of fillers containing human MSCs and HUVECs. The findings are promising and could have meaningful translational implications. However, several methodological and reporting concerns should be addressed before the manuscript can be considered for publication.

Methodological Concerns

Cell Identity and Characterization:

The manuscript does not provide sufficient detail on the identity and source of the cells used (MSC and HUVEC). For reproducibility and validation, please include information on how these cells were characterized (e.g., surface marker profiles, passage numbers).

Provide catalog numbers or supplier information for both HUVECs and MSCs to enhance transparency.

Primer Information:

The manuscript lists primer sequences within the text. For clarity and ease of reference, I strongly recommend presenting all primer sequences and related details (gene, sequence, annealing temperature, product size) in a table.

Animal Care and Maintenance:

The section describing animal maintenance and welfare procedures is brief. Please expand on husbandry conditions, ethical approval information, and any measures taken to minimize animal suffering.

Results and Data Interpretation

Selection of MSC:HUVEC Ratios:

The rationale for selecting the specific co-culture ratios (1:1, 1:3, and 1:0) of MSCs to HUVECs is unclear. Please elaborate on why these ratios were chosen and whether preliminary experiments or relevant literature informed these choices.

Choice of Gene Expression Analysis:

Only VEGF and FGF2 were analyzed as markers for organization-forming ability. Could the authors clarify why these two genes were chosen? It would strengthen the manuscript to discuss whether other angiogenic or tissue remodeling factors were considered, and provide justification for focusing on these particular genes.

Reviewer #2: Dear Editor,

I am grateful to be considered as a reviewer for the paper entitled "MSC and HUVEC co-cultured fillers overcome intractable fistula in a new mouse model" submitted to PLOS ONE.

This manuscript holds significant translational potential and addresses an essential issue in gastrointestinal surgeries known as intractable fistulae. Although the authors have presented the topic clearly and developed an innovative model with encouraging findings, I recommend addressing several issues to enhance the overall quality of the paper.

1- The introduction fails to explain the impact and necessity of MSCs in this context. What makes them important for this therapeutic filler, and in what ways do they support HUVECs in healing fistulas?

2- The introduction lacks a clear statement of the study’s final objective. Is it focused on the model’s success rate, fistula closure, inflammation control, angiogenesis promotion, or pain and discharge inhibition?

3- What is the origin of the MSCs employed in this research? Are they xenogeneic, allogeneic, or autologous? Additionally, do they originate from adipose tissue, bone marrow, or another source?

4- Could you clarify the composition of the filler? Does it contain a biopolymer such as hyaluronic acid, or is it simply a mixture of cells? This should be clearly specified in the materials and methods part of the manuscript.

5- On page 7, line 3, details about the microscope need to be added.

6- How many times were the experiments repeated? The statistical analysis part does not mention if the experiments were performed in triplicate.

7- The results do not specify how immunostaining was used to determine that a 1:1 ratio of MSC to HUVEC is optimal.

8- This study would benefit from including an additional experimental group with filler containing only HUVECs to assess their impact independent of MSCs. The inclusion of this data would improve the overall understanding of the findings.

9- The authors need to explain why the efficacy of filler containing only MSCs is significantly lower compared to previous studies.

10- This paper would benefit from English language improvement and a more coherent content organization.

**********

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Reviewer #1: Yes:  Iman Menbari Oskouie

Reviewer #2: No

**********

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PLoS One. 2025 Aug 21;20(8):e0330478. doi: 10.1371/journal.pone.0330478.r002

Author response to Decision Letter 1

23 Jun 2025

Dear Editor and Reviewers,

Thank you very much for your thoughtful and constructive comments on our manuscript entitled "MSC and HUVEC co-cultured fillers overcome intractable fistula in a new mouse model." We appreciate the opportunity to revise and improve our manuscript. Below, we provide point-by-point responses to each of the comments raised by the reviewers and the editorial office. All changes in the revised manuscript are clearly marked for your convenience.

________________________________________

Reviewer #1:

1. Cell Identity and Characterization:

Comment: The manuscript does not provide sufficient detail on the identity and source of the cells used (MSC and HUVEC). For reproducibility and validation, please include information on how these cells were characterized (e.g., surface marker profiles, passage numbers).

Response: We appreciate the reviewer’s comment regarding the need for more detailed information about the source and characterization of the cells used in our study. In response, we have revised the Methods section to include the supplier, catalog numbers, passage numbers, and cell characterization details for both MSCs and HUVECs.

Specifically, human mesenchymal stem cells (MSCs) were purchased from Lonza (Cat# CC-2501, Basel, Switzerland) and cultured using MSCGM BulletKit (Cat# PT3001). Cells were used between passages 3 and 5. According to the manufacturer, MSC identity was confirmed using standard surface marker profiles consistent with ISCT criteria, including positivity for CD73, CD90, CD29, CD105, CD166, and CD44, and negativity for CD14, CD19, CD34, and CD45.

Human umbilical vein endothelial cells (HUVECs) were also obtained from Lonza (Cat# CC-2517, Basel, Switzerland) and cultured in EGM-2 medium (Lonza, Cat# CC-3162). Cells were used between passages 3 and 6. As per the supplier’s certification, HUVECs were characterized based on endothelial marker expression and angiogenic functionality.

This revised information can now be found in the Methods section (page 5, lines 10–20).

2. Primer Information:

Comment: The manuscript lists primer sequences within the text. For clarity and ease of reference, I strongly recommend presenting all primer sequences and related details (gene, sequence, annealing temperature, product size) in a table.

Response: We have added a new table (Table1) listing all primers used, including gene names, sequences, annealing temperatures, and product sizes.

3. Animal Care and Maintenance:

Comment: The section describing animal maintenance and welfare procedures is brief. Please expand on husbandry conditions, ethical approval information, and any measures taken to minimize animal suffering.

Response:

we have revised the Materials and Methods section to provide more detailed information on animal husbandry conditions, ethical approval, and procedures used to minimize animal suffering.

Specifically, we now include the approval number from the Chiba University Animal Experiment Committee, and we describe the housing environment (SPF conditions, temperature, humidity, light cycle), the anesthetic regimen (medetomidine, butorphanol tartrate, and midazolam), and humane endpoints. The revised description can be found in the Materials and Methods section (page 9, lines 6–15).

4.Selection of MSC:HUVEC Ratios:

Comment: The rationale for selecting the specific co-culture ratios (1:1, 1:3, and 1:0) of MSCs to HUVECs is unclear. Please elaborate on why these ratios were chosen and whether preliminary experiments or relevant literature informed these choices.

Response: The co-culture ratios of MSCs to HUVECs were selected based on previously reported studies in which these cell types were combined to fabricate tissue fillers.

In our own preliminary experiments described in Section 2.1, co-cultures at three specific ratios (MSCs:HUVECs = 1:1, 3:1, and 1:0) successfully formed organized tissue-like structures.

Therefore, we adopted the same ratios for subsequent analyses, including qPCR.

This rationale has now been clarified in the Materials and Methods section (page 6, lines2 –4, page7 , lines 11–12).

5. Choice of Gene Expression Analysis:

Comment: Only VEGF and FGF2 were analyzed as markers for organization-forming ability. Could the authors clarify why these two genes were chosen? It would strengthen the manuscript to discuss whether other angiogenic or tissue remodeling factors were considered, and provide justification for focusing on these particular genes.

Response: In this study, we focused on VEGF and FGF2 because they are well-established key regulators of angiogenesis and are directly involved in vascular network formation and tissue integration. Since successful engraftment of the filler depends heavily on the promotion of angiogenesis, we selected these two representative proangiogenic factors for gene expression analysis.

Furthermore, several previous studies that examined the optimal MSC:HUVEC co-culture ratios for tissue engineering applications have also used VEGF and FGF2 as primary markers of angiogenic potential. Therefore, we considered the selection of these genes to be a valid and relevant approach for evaluating the organization-forming ability of our co-culture system.

We have added this rationale to the Discussion section (page 13, lines 16–23).

________________________________________

Reviewer #2:

1. Role of MSCs in Filler:

Comment: 1- The introduction fails to explain the impact and necessity of MSCs in this context. What makes them important for this therapeutic filler, and in what ways do they support HUVECs in healing fistulas.

Response: We have revised the Introduction to clarify the role of mesenchymal stem cells (MSCs) in our therapeutic approach. While MSC alone possess regenerative and immunomodulatory properties through paracrine signaling, several studies have demonstrated that co-culture with HUVEC significantly enhances angiogenesis compared to MSCs alone. For example, Takebe et al. (2013) showed that MSC–HUVEC co-cultures could form functional vasculature in engineered tissues. Similarly, Jinling Ma et al. (2014) reported that tissues formed from MSCs and HUVECs exhibited significantly higher blood vessel density than those formed from MSCs alone.

2. Clarify Study Objective:

Comment: The introduction lacks a clear statement of the study’s final objective. Is it focused on the model’s success rate, fistula closure, inflammation control, angiogenesis promotion, or pain and discharge inhibition?

Response: We agree that the study objective needed clarification. We have revised the final paragraph of the Introduction to clearly state the aim of the study. The primary objective of this work is to improve the fistula closure rate using a cell-based filler composed of MSCs and HUVECs, leveraging their angiogenic potential to enhance tissue regeneration. This statement now appears at the end of the Introduction section (page 5, lines 5–7).

3. Source of MSCs:

Comment: What is the origin of the MSCs employed in this research? Are they xenogeneic, allogeneic, or autologous? Additionally, do they originate from adipose tissue, bone marrow, or another source?

Response: The mesenchymal stem cells (MSCs) used in this study were human bone marrow-derived MSCs, purchased from Lonza (Cat# CC-2501, Basel, Switzerland). These cells are xenogeneic relative to the recipient mice. We have clarified this information in the Materials and Methods section (page 5, lines 11–16).

4. Composition of Filler:

Comment: Could you clarify the composition of the filler? Does it contain a biopolymer such as hyaluronic acid, or is it simply a mixture of cells? This should be clearly specified in the materials and methods part of the manuscript.

Response: We have clarified the composition of the filler in the Materials and Methods section. The filler used in this study consisted of a three-dimensional co-culture of human MSCs and HUVECs on Matrigel, which served as a biological scaffold. No other biopolymers, such as hyaluronic acid, were included in the formulation.(page 6, lines 4–8).

5. Microscope Details:

Comment: On page 7, line 3, details about the microscope need to be added.

Response: We have added the specific model and manufacturer of the microscope used (BZ-X9000, Keyence, Osaka, Japan) to the Methods section (Page 8, Line 1).

6. Experiment Repetition and Statistics:

Comment: How many times were the experiments repeated? The statistical analysis part does not mention if the experiments were performed in triplicate.

Response: We have clarified the number of replicates in the Materials and Methods section. Specifically, qPCR experiments and capillary density analysis in the subcutaneous filler transplantation model were performed in triplicate (n = 3).

For the in vivo intractable enterocutaneous fistula model, each group included five mice (n = 5). Due to the substantial cost and technical difficulty involved in both creating the fistula model and preparing the cell-based fillers, further expansion of the sample size was not feasible. We have added this information to Methods section (Page 6, Line 22-23, Page 8 Line 2, Page9, Line1-4).

7. Immunostaining and Ratio Determination:

Comment: The results do not specify how immunostaining was used to determine that a 1:1 ratio of MSC to HUVEC is optimal.

Response: We apologize for the confusion. Immunostaining in this study was not used to determine the optimal MSC:HUVEC ratio. Rather, it was performed to visualize how MSCs and HUVECs mixed and organized within the filler structure. The 1:1 ratio was identified as optimal based on qPCR results (FGF2 and VEGF expression) and capillary density measurements, not by immunostaining. We have clarified this distinction in the revised Results sections.

8. Missing HUVEC-only Group:

Comment: This study would benefit from including an additional experimental group with filler containing only HUVECs to assess their impact independent of MSCs. The inclusion of this data would improve the overall understanding of the findings. Recommend including a group with HUVEC-only filler.

Response: We agree that evaluating the independent contribution of HUVECs could provide valuable insight. However, in our preliminary experiments, we found that fillers composed only of HUVECs were unable to form organized tissue structures when cultured on Matrigel. Unlike MSCs, HUVECs alone did not maintain sufficient viability or structure under the same 3D culture conditions. Therefore, it was not feasible to include a HUVEC-only group in the main experiments, as no viable tissue could be produced.

9. MSC-only Efficacy Compared to Literature:

Comment: The authors need to explain why the efficacy of filler containing only MSCs is significantly lower compared to previous studies.

In our study, the efficacy of the MSC-only filler was lower than reported in some previous studies. One reason for this may be the use of a gastrointestinal fistula model, which more closely mimics clinical enterocutaneous fistula conditions and presents a harsher environment for filler engraftment.

We believe that MSCs alone were not sufficient to promote stable engraftment in this ischemic and inflammatory setting. In contrast, the combination of MSCs and HUVECs promoted angiogenesis, which likely contributed to improved filler survival and integration. This explanation has been added to the Discussion section (page 14, lines 15–20).

10. English and Organization:

Comment: This paper would benefit from English language improvement and a more coherent content organization.

Response: In response, we have thoroughly revised the manuscript to improve clarity, grammar, and overall readability. Particular attention was paid to refining the English language and ensuring logical flow across sections. We believe these improvements have enhanced the manuscript’s coherence and accessibility. If further corrections are required, we would be happy to address them.

________________________________________

Editorial Office Comments:

1. Formatting and Style Requirements:

Response: We have revised the manuscript to comply with PLOS ONE’s formatting and file naming guidelines.

2. Funding Information Mismatch:

Response: We have corrected and confirmed that the funding information is consistent throughout the manuscript.

3. Following financial disclosure

Response: We also added the following statement: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

4. Full Ethics Statement:

Response: A full ethics statement, including the name of the ethics committee and the approval number, has been added to the Methods section.

5. Data Availability Statement:

Response: We confirm that all data required to replicate the results are included in the manuscript or supporting information files. If needed, raw data files have been uploaded as Supporting Information.

6. Abstract Citations:

Response: All citations have been removed from the Abstract to comply with PLOS ONE’s guidelines.

________________________________________

We hope the revised manuscript and our responses fully address the concerns raised. We are grateful to the reviewers and the editorial team for their valuable feedback.

Sincerely,

Soichiro Hirasawa, on behalf of all authors

Attachment

Submitted filename: Response to Reviewers.docx

pone.0330478.s005.docx (24.3KB, docx)

Decision Letter 1

Keykavoos Gholami

4 Aug 2025

MSC and HUVEC co-cultured fillers overcome intractable fistula in a new mouse model

PONE-D-25-19698R1

Dear Dr. Hirasawa,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Keykavoos Gholami

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: all comments have been addressed carefully. I think this manuscript is suitable for publication in PLOS ONE.

Reviewer #2: (No Response)

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

**********

Acceptance letter

Keykavoos Gholami

PONE-D-25-19698R1

PLOS ONE

Dear Dr. Hirasawa,

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on behalf of

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Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1. Data qPCR FGF2.

    (XLSX)

    pone.0330478.s001.xlsx (10KB, xlsx)
    S2. Data qPCR VEGF.

    (XLSX)

    pone.0330478.s002.xlsx (10KB, xlsx)
    S3. Data.

    (XLSX)

    pone.0330478.s003.xlsx (9.5KB, xlsx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    pone.0330478.s005.docx (24.3KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

    Articles from PLOS One are provided here courtesy of PLOS

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