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. 2024 Mar 3;38(2):546–558. doi: 10.21873/invivo.13473

Investigation of Expanded Human Adipose-derived Stem Cell Dosage and Timing for Improved Defecation Function

RYOTA MORI 1, NORIKATSU MIYOSHI 1,2, SHIKI FUJINO 1,3, TSUNEKAZU MIZUSHIMA 1,4, RYOHEI YUKIMOTO 1, TAKAYUKI OGINO 1, HIDEKAZU TAKAHASHI 1, MAMORU UEMURA 1, YUICHIRO DOKI 1, HIDETOSHI EGUCHI 1
PMCID: PMC10905476  PMID: 38418103

Abstract

Background/Aim

Although certain treatment options exist for intestinal incontinence, none are curative. Adipose-derived stem cells (ADSCs) have emerged as promising therapeutic agents, but most preclinical studies of their effectiveness for anal function have used autologous or allogeneic ADSCs. In this study, the effectiveness, timing of administration, and required dosage of human ADSCs were investigated for clinical application.

Materials and Methods

A 10-mm balloon catheter was used to induce anal sphincter injury in immunodeficient mice in the following experimental groups (n=4 per group): ADSC (injected ADSCs after injury), PBS (injected phosphate-buffered saline after injury), and control (uninjured). The effects of different timing (immediately after injection and 30 days following injury) and number of human ADSCs administered was compared among groups based on defecation status and pathological evaluation.

Results

In terms of defecation status, groups receiving ≥1×104 human ADSCs after injection showed improvement. Pathological images showed that compared to the PBS group, the thinnest part of the sphincter was thicker for animals that received ≥1×104 human ADSCs, and fibrosis of the sphincter was notable in those treated with 1×103 human ADSCs or PBS. Furthermore, defecation status was improved by administration of human ADSCs, not only immediately after injury, but also at 30 days following injury.

Conclusion

Human ADSC administration in a mouse model of anal sphincter injury was effective. Injection of ≥1×104 human ADSCs was the amount necessary to improve defecation status, an effect detected in both the acute and chronic phases.

Keywords: Adipose derived stem cell, anal sphincter injury, intestinal incontinence, ARS, defecation function

Intestinal incontinence is a common condition defined as the uncontrolled passage of feces or gas for at least one month and has a negative impact on quality of life (1). Although the main causes of defecation problems have been age-related weakness of defecation-related muscle groups, the number of patients who develop the condition after surgery has increased. Chemoradiation and surgical techniques have improved remarkably in treating rectal cancer, and more patients are undergoing anus-preserving surgery, such as low anterior resection (LAR) or intersphincteric resection (ISR). Although the advantage of these procedures is preservation of the anus, many patients develop severe bowel dysfunction, resulting in incontinence for flatus and feces, urgency, and frequent bowel movements (2). These symptoms are defined as LAR syndrome (LARS) and have a negative impact on quality of life (3,4). The various treatment options for LARS include medication, surgery, sacral neuromodulation, and biofeedback, but they only relieve symptoms and are not curative.

Cell transplantation recently has become a focus in the field of regenerative therapy because of the self-renewal capacity and differential potential of stem cells. Rapid advances in this field over the past two decades have suggested the possibility of curing previously incurable diseases. In particular, adipose-derived stem cells (ADSCs) are promising therapeutic agents because of their ease of collection and expansion, low immunogenicity, and high ability to differentiate into multiple lineages, including muscle cells (5). These cells also have paracrine functions and are associated with immunomodulation, cytokine secretion, and angiogenesis (6,7).

Initially, autologous cell transplantation was introduced into clinical practice because of safety and ethical concerns. The autologous approach, however, carries some disadvantages, including time-consuming culture methods and difficulty maintaining uniformity. Considering these problems, suitable methods need to be investigated for introducing appropriate cells into clinical treatment. Allogeneic transplantation offers one potential candidate approach.

The European Commission has approved allogeneic, expanded human (h)ADSCs (darvadstrocel; Takeda Pharmaceutical, Tokyo, Japan) for treatment of refractory complex perianal fistulas in patients with Crohn’s disease (8). Clinical trials for hADSC administration to patients with anorectal fistulae unrelated to Crohn’s disease are currently underway (9).

Results from animal models of defecation disorder point to improvement in anal function following ADSC administration, but some limitations need to be addressed before clinical application. First, in these studies, ADSCs were administered immediately after anorectal injury, which does not closely reflect chronic defecation dysfunction in clinical practice. Since it is not feasible to administer ADSCs immediately after surgery in all cases of rectal surgery, further practical animal studies are needed. Second, defecation function was measured only by anal pressure in most studies, although some groups have found no correlation between anal pressure and defecation function (10-12). Therefore, in this study, the effect of ADSCs was evaluated based on defecation status, which has been previously reported by this lab to correlate with defecation function. Third, no one has reported on the optimal number of ADSCs to be administered. It is more useful in clinical applications to know the approximate minimum number of units that will be effective. Finally, most studies used autologous or allogeneic ADSCs, and no evaluation of the effectiveness of hADSCs has been published.

Rats typically have been used as a model of defecation disorder (13-15). Mice may have been overlooked because of their small size, which makes it difficult to create an anal sphincter injury model with uniform injury using conventional methods, such as cutting or partial removal of the sphincter muscle. Immunodeficient rats are rare and expensive for research use, however. For this reason, as described in a previous report, a mouse model of anal sphincter injury with dysfunction of defecation was established, using a balloon catheter to create the injury (16). Consequently, experiments using immunodeficient animals could be easily conducted, allowing for studies of hADSCs instead of autologous or allogeneic transplantation, leading to more clinically relevant findings.

The aim of the study was to evaluate in a preclinical model whether hADSCs improve defecation function, the effective dose of hADSCs, and whether hADSCs improve defecation function acutely and long term after injury, better reflecting the clinical situation.

Materials and Methods

Ethics. This study was approved by the Institutional Review Board (Permit number: 19388) and Animal Research Committee (Permit number: 02-031-002) and was conducted in accordance with the protocols approved by the Animal Care and Use Committee of Osaka University.

Animals. Immunodeficient mice (NOD/SCID) were obtained from Nippon Clare (Tokyo, Japan). All animals were bred in temperature- and humidity-controlled rooms with free access to food and water. For these studies, 6- to 8-week-old female mice were used and randomly assigned to the groups (each group consisted of four mice). Mice were bred in cages that were randomized (instead of grouped by treatment) and with their group assignment blinded. Euthanasia was performed by cervical dislocation after inhalation of carbon dioxide (100% concentration of carbon dioxide was administered to displace 30-70% of chamber volume/min in accordance with the AVMA Guidelines 2020).

hADSCs and cell culturing. Two types of expanded hADSCs were used in this study. The cells and the medium (R:stem) were provided by ROHTO Pharmaceutical Co., Ltd. (Osaka, Japan). Normal hADSCs of LONZA (L-ADSCs; PT-5006, Basel, Switzerland) were cultured in R:stem at 37˚C in a 5% CO2 incubator, and the medium was changed every 4 days (17). Accutase (Nacalai Tesque, Kyoto, Japan) was used for cell passaging. Normal hADSCs from ROHTO (R-ADSCs) were provided in a frozen state and used after being thawed and then washed with phosphate-buffered saline (PBS).

Anal sphincter injury mouse model and treatment protocol. A balloon catheter (10 mm diameter) was used to induce anal sphincter injury on the first day only, as previously reported, under general anesthesia using a mixture of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg) (16,18,19). The balloon dilation time was set at 2 min, and dilation was performed twice. The ADSC group received 1000 μl of hADSCs dissolved in PBS immediately after injury at four perianal sites, and the PBS group received 1000 μl PBS as an acute injury comparator; for assessing the effect of hADSCs on chronic defecation disorders, both the balloon catheter and PBS groups received hADSCs at one month after injury. In experiments to confirm the effect of hADSCs, 1×107 hADSCs were administered for the hADSC group, but in a series of studies to identify the optimal dosage, different cell counts were applied (1×103, 1×104,1×105, 1×106, and 1×107). The series of procedures to induce injury to the anal sphincter and the location of the hADSC or PBS injection are shown in Figure 1.

Figure 1. Images taken during and after anorectal injury induced with a balloon catheter in mice. PBS or ADSCs were administered in divided doses at the orange dots after injury. PBS, Phosphate-buffered saline; ADSCs, adipose-derived stem cells.

Figure 1

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As a control group, mice were anesthetized following the same procedure as for animals with induced anorectal injury. After procedures, anesthesia was reversed using atipamezole (1 mg/kg) (19,20).

Flow cytometric analysis. The expression of surface proteins by the hADSCs was examined using flow cytometry. Relative fluorescence intensities were measured using a BD FACS Aria II (BD Biosciences, San Jose, CA, USA), with the following antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-human CD90 (328107; BioLegend, San Diego, CA, USA), PE/Cyanine (Cy)7-conjugated anti-human CD34 (343515; BioLegend), allophycocyanin (APC)-conjugated anti-human CD73 (344005; BioLegend), APC/Cy7-conjugated anti-human CD45 (368515; BioLegend), and brilliant violet (BV) 421-conjugated anti-human CD105 (323219; BioLegend). As isotype controls for these antibodies, the following antibodies were used: FITC-conjugated anti-mouse IgG1, k isotype Ctrl (555748; BD Biosciences), PE/Cy7-conjugated anti-mouse IgG1, k isotype Ctrl (557872; BD Biosciences), APC-conjugated anti-mouse IgG1, k isotype Ctrl (554681; BD Biosciences), APC/Cy7-conjugated anti-mouse IgG1, k isotype Ctrl (557873; BD Biosciences), and BV421-conjugated anti-mouse IgG1, k isotype Ctrl (562438; BD Biosciences). 7-aminoactinomycin D (420403; BioLegend) was used to exclude any dead and damaged cells. Data were analyzed using the FlowJo 10.6.1 software program (FlowJo LLC, Ashland, OR, USA).

Evaluation of defecation function. Defecation function was evaluated by defecation status rather than by anorectal pressure. Fecal weight per stool for 24 h was normally measured on day 0 (before anal sphincter injury) and on days 1, 8, 15, 22, and 29 after anal sphincter injury, as previously reported by this group (16). In the experiments that involved administering hADSCs at one month after injury (day 31), defecation status was measured on day 31 (before hADSC administration) and on days 38, 45, 52, and 59, because the perioperative period is generally considered to fall within 30 days after surgery. Animal group assignment was blinded during the defecation status checks.

Histological examination. The rectum, anal sphincter, and surrounding connective tissue were resected under anesthesia for subsequent examination and fixed in 10% formalin, with care taken not to introduce deformities. After dehydration with an ethanol concentration series, samples were embedded in paraffin and the paraffin blocks sectioned at 3-5 μm onto slides. Sections were stained with hematoxylin and eosin or Masson’s trichrome stain for detection of collagen deposits (TRM-1; Cosmo Bio, Tokyo, Japan). Group status was blinded for assessment of pathological images.

Immunohistochemistry. For immunohistochemistry, paraffin blocks were deparaffinized and sectioned at 3 μm onto slides. Each slide was boiled for 15 min and then immersed in methanol-hydrogen peroxide solution for 25 min at room temperature to quench endogenous peroxidase. Slides then were blocked with blocking serum for 30 min at room temperature and incubated overnight with primary antibody, followed by a 30-min incubation with secondary antibody at room temperature. To counterstain DNA, slides were mounted with ProLong® Glass Antifade Mountant with NucBlue® Stain (P36983; Thermo Fisher Scientific, Waltham, MA, USA). The following primary antibodies were used for immunofluorescence studies: rabbit anti-HLA antibody (1:200, ab52922; Abcam, Cambridge, MA, USA), rabbit anti-Ki67 antibody (1:400, ab15580; Abcam), rabbit anti-CD206 antibody (1:200, 18704-1-AP; Proteintech Group Inc, Rosemont, IL, USA), and rat anti-F4/80 antibody (1:1000, MCA497RT; Bio-Rad Laboratories Inc, Hercules, CA, USA). The following secondary antibodies were used: anti-rabbit IgG (1:1000, A11008; Alexa Fluor 488, Thermo Fisher Scientific), and anti-rat IgG (1:400, 4418; Alexa Fluor 647, Cell Signaling Technology, Danvers, MD, USA). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stain was performed using the MEBSTAIN Apoptosis TUNEL Kit Direct (8445; MBL, Aichi, Japan), following the manufacturer’s instructions.

Imaging measurements and analyses. All images were captured with BIOREVO BZ-X710 (Keyence, Tokyo, Japan). Measurements of cross-sectional areas of the rectal lumen and anal sphincter and thickness of the anal sphincter were made using image-processing software (ImageJ, National Institutes of Health, Bethesda, MD, USA). The number of cells stained with fluorescent antibodies was automatically counted with a BR-Z analyzer (KEYENCE).

Statistical analysis. The results were analyzed with the appropriate respective tests using JMP pro14.0.0 (SAS Institute Inc, Cary, NC, USA), and all data are presented as means±standard error of the mean (SEM). The Mann-Whitney U-test was used to compare the differences between the PBS group and the other groups to examine the effect of ADSCs depending on the dose. For comparisons among three groups, one-way analysis of variance (ANOVA) was used, followed by Tukey’s post hoc test. A p-value lower than 0.05 was considered statistically significant.

Results

R-ADSCs and L-ADSCs had the same cell surface phenotype and morphology. Two different hADSCs, R-ADSCs and L-ADSCs, were analyzed by flow cytometry to assess for any differences in properties. Both types were positive for CD73, CD90, and CD105, which are considered positive markers for hADSCs, and negative for CD45 and CD34, which are considered negative markers (Figure 2A). The morphology of R-ADSCs and L-ADSCs was confirmed by microscopy as similar to that of typical ADSCs (Figure 2B).

Figure 2. Surface marker analysis of hADSCs using representative positive and negative ADSC markers was performed using flow cytometry. (A) CD73, CD90, and CD105 served as positive markers and CD45 and CD34 as negative markers. The results in the upper row were obtained from R-hADSCs, and those in the lower row were from L-hADSCs. Grey shadings indicate isotype control. (B) Representative images of R-hADSCs and L-hADSCs, cultured in vitro. R-hADSCs, Normal human adipose-derived stem cells from ROHTO; L-hADSCs, normal human adipose-derived stem cells of LONZA.

Figure 2

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hADSCs healed damaged anal sphincter muscles and promoted the recovery of defecation function in a mouse model of anal sphincter injury. NOD/SCID mice were divided into four groups (n=4 per group): no anal sphincter injury (controls), injured animals administered 1×107 R-ADSCs, injured animals administered 1×107 L-ADSCs, and injured animals administered only PBS. Fecal weight per stool was measured on day 0 (before anal sphincter injury) and on days 1, 8, 15, 22, and 29 after anal sphincter injury in each group to evaluate defecation status. Then, on day 29, the mice were sacrificed and their anuses removed along with the surrounding connective tissue for pathological evaluation. A scheme of the experimental schedule is shown in Figure 3A.

Figure 3. Examination of ADSC effectiveness with administration in the acute phase. ADSCs were administered in a mouse model of anal sphincter injury. (A) Experimental schedule. (B) Defecation status of the four groups. Each bar represents the mean±SEM of quadruple measurements of weight per piece of stool (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Body weight of the four groups. Each bar represents the mean±SEM of quadruple measurements (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (D) Representative photomicrographs of histological sections of the anal sphincter and surrounding connective tissue on day 29 with hematoxylin and eosin staining (ADSC, PBS, and control groups). (E) Representative photomicrographs of histological sections of the anal sphincter on day 29 with Masson’s trichrome staining (ADSC, PBS, and control groups). (F) Bar plot of pathological evaluation of the anal sphincter on day 29 (ADSC, PBS, and control groups). Crosssectional area of the rectal lumen and anal sphincter and thickness of the anal sphincter were used as criteria for pathological evaluation. Each bar represents the mean±SEM of quadruple measurements (*p<0.05, one-way ANOVA followed by the Tukey’s post hoc test). ADSCs, Adiposederived stem cells; PBS, phosphate-buffered saline; SEM, standard error of mean; n.s., not significant.

Figure 3

Figure 3

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Defecation status in both groups treated with hADSCs improved from day 8, and differences from the control group disappeared from day 22; however, the PBS group showed no improvement (Figure 3B, one-way ANOVA followed by Tukey’s post hoc test). There was no difference in body weight among the four groups up to one month (Figure 3C, one-way ANOVA followed by Tukey’s post hoc test). All mice included in this experiment survived in a healthy state one month after the start of the experiment. Because the L-ADSC and R-ADSC groups did not differ, only R-ADSCs were used as the ADSC group in subsequent experiments.

Histological imaging showed that the injured anus was noticeably deformed, with fibrosis and tearing in the PBS compared with the ADSC and control groups (Figure 3D and E). When the pathological images were analyzed, the PBS group had less sphincter area in cross section and less thickness in the thinnest part of the sphincter compared with the ADSC and control groups. The ADSC and control groups did not differ (Figure 3F, one-way ANOVA followed by Tukey’s post hoc test).

hADSCs differentiated into muscle and surrounding connective tissue, showed increased M2 macrophages, promoted cell division, and inhibited cell necrosis. HLA expression in the ADSC groups was analyzed by immunohistochemistry. HLA-positive cells, which are only expressed in cells of human origin, were observed in the muscle tissue and surrounding connective tissue in the ADSC group (Figure 4A), suggesting the potential of ADSCs to differentiate in the injured anal sphincter muscle and the surrounding connective tissue.

Figure 4. Evaluation of pathological images by fluorescent immunostaining to assess effects of hADSC administration. (A) Representative images of HLA expression (green) with nuclei staining (blue) in the ADSC and PBS groups. (B) Representative images of Ki67 expression (green) with nuclei staining (blue) in the anal sphincter and bar plot of expression rate in ADSC, PBS, and control groups (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Representative images of Ki67 expression (green) with nuclei staining (blue) in the surrounding connective tissue and bar plot of expression rate in the ADSC, PBS, and control groups (*p<0.05, one-way ANOVA followed by the Tukey’s post hoc test). hADSCs, Human adipose-derived stem cells; HLA, human leukocyte antigen; PBS, phosphate-buffered saline; n.s., not significant.

Figure 4

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Ki67 and TUNEL staining to assess the effect of hADSCs on cell division and apoptosis showed no difference between the PBS and control groups in the fraction of cells undergoing cell division, but hADSC treatment promoted cell division (Figure 4B and C, one-way ANOVA followed by Tukey’s post hoc test). The percentage of cells undergoing apoptosis did not differ between the ADSC and control groups, but a higher percentage of apoptotic cells was found in the PBS group compared to the control group (Figure 5A and B, one-way ANOVA followed by Tukey’s post hoc test). To examine the effect of hADSCs on the content of M2 macrophages, which influence wound healing, double immunostaining was performed using F4/80, a marker for macrophages, and CD206, a marker for M2 macrophages. As shown in Figure 5C, the ratio of M2 macrophages to total cell count was significantly higher in the ADSC group compared with the PBS and control groups. In addition, the ratio of M2 to all macrophages was significantly higher in the ADSC and control groups than in the PBS group (Figure 5C, one-way ANOVA followed by Tukey’s post hoc test). These results indicate that M1 macrophages normally increase when tissue damage occurs, but that administration of hADSCs increased macrophage content in the surrounding connective tissue, especially of M2 macrophages.

Figure 5. Evaluation of pathological images by fluorescence immunostaining to assess the effects of hADSC administration on day 29. (A) Representative images of TUNEL staining (green) with nuclei staining (blue) in anal sphincter and bar plot of positive rate on day 29 in ADSC, PBS, and control groups (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (B) Representative images of TUNEL staining (green) with nuclei staining (blue) in the surrounding connective tissue and bar plot of positive rate on day 29 in ADSC, PBS, and control groups (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Examining the effect of hADSCs on M2 macrophages in adipose tissue by double immunostaining using F4/80 (red), a marker for macrophages, and CD206 (green), a marker for M2 macrophages with nuclei staining (blue). Representative images of macrophages and a bar plot showing the percentage of M2 macrophages relative to all cells and the percentage of M2 macrophages relative to all macrophages on day 29 (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). hADSCs, Human adipose-derived stem cells; PBS, phosphate-buffered saline; TUNEL, TdT-mediated dUTP nick-end labeling; DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride; n.s., not significant.

Figure 5

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A population greater than 1×104 hADSCs was required to improve defecation function in a mouse model of anal sphincter injury. Next, the amount of hADSCs that would be needed for a positive effect was examined. For this purpose, four doses of hADSCs (1×106, 1×105,1×104, and 1×103) were administered after anal sphincter injury in NOD/SCID mice (n=4 per dosage group). As shown in Figure 6A, groups receiving ≥1×104 hADSCs showed improvement in defecation status from day 21 (Mann-Whitney U-test). A comparison of these dosage groups with the three groups in the interventional experiments, showed that body weight did not differ among the seven groups at one month (Figure 6B, Mann-Whitney U-test). All mice included in this experiment survived in a healthy state to one month after the start of the experiment. In a comparison of dosage groups with the PBS animals, pathological images showed thinning and fibrosis of the sphincter muscle with the 1×103 hADSC treatment as well as in the PBS group (Figure 6C). Analysis of pathological images showed that the thinnest part of the sphincter was thicker in the group that received ≥1×104 hADSCs compared with the PBS group (Figure 6D). In addition, the sphincter cross-sectional area was greater in animals that received 1×107 hADSCs compared with those administered 1×103 hADSCs or PBS only (Figure 6D). Similar to the results described in the previous section, however, the seven groups did not differ in length of the thickest part of the sphincter or in rectal lumen area (Figure 6D, Mann-Whitney U-test). Considering these findings, 1×104 hADSCs was set as the minimum dosage for improved defecation function in these animals.

Figure 6. Examination of the number of effective doses of hADSCs. (A) Defecation status of the seven groups. Each bar represents the mean±SEM of quadruple measurements of weight per piece of stool (*p<0.05, Mann-Whitney U-test). (B) Body weight of the seven groups. Each bar represents the mean±SEM of quadruple measurements (*p<0.05, Mann-Whitney U-test). (C) Representative photomicrographs of histological sections of the anal sphincter on day 29 with hematoxylin and eosin staining and Masson’s trichrome staining (1×106, 1×105, 1×104, and 1×103 ADSCs). (D) Bar plot of pathological evaluation of the anal sphincter in the seven groups on day 29. Cross-sectional area of the rectal lumen and anal sphincter and thickness of the anal sphincter were used as criteria for pathological evaluation. Each bar represents the mean±SEM of quadruple measurements (*p<0.05, Mann-Whitney U-test). hADSCs, Human adipose-derived stem cells; PBS, phosphate-buffered saline; SEM, standard error of mean; n.s., not significant.

Figure 6

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Defecation function was improved by administration of hADSCs not only immediately after the injury but also at 30 days following injury. Finally, for examining whether administering hADSCs after injury chronicity affects defecation function, NOD/SCID mice were divided into three groups (n=4 per group): no anal sphincter injury (controls), administration of 1×107 hADSCs at 30 days after injury, and administration of PBS 30 days after injury. Fecal weight per stool was measured on day 30 (before hADSC administration) and on days 38, 45, 52, and 59 after injury in each group to evaluate defecation status. A scheme of the experimental schedule is given in Figure 7A.

Figure 7. Examination of whether hADSCs are effective when administered in the chronic phase. NOD/SCID mice were divided into three groups (n=4 per group): without anal sphincter injury (controls), and those administered 1×107 hADSCs or only PBS 30 days after anal sphincter injury. Fecal weight per stool was measured on day 30 (before hADSC administration) and on days 38, 45, 52, and 59 after injury to evaluate defecation status. On day 59, mice were sacrificed and their anuses removed along with the surrounding connective tissue for pathological evaluation. (A) Experimental schedule. (B) Defecation status of the three groups. Each bar represents the mean±SEM of quadruple measurements of weight per piece of stool (*p<0.05, **p<0.01, one-way ANOVA followed by Tukey’s post hoc test). (C) Body weight of the three groups. Each bar represents the mean±SEM of quadruple measurements (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). hADSCs, Human adipose-derived stem cells; NOD/SCID, non-obese diabetes/severe combined immunodeficient; PBS, phosphate-buffered saline; SEM, standard error of mean; n.s., not significant.

Figure 7

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All mice included in this experiment survived in a healthy state 2 months after the start of the experiment. As shown in Figure 7B, animals with damaged sphincter muscles did not exhibit defecation status recovery at 30 days after anal injury, but defecation function was improved at treatment day 14 with hADSCs (Figure 7B, one-way ANOVA followed by Tukey’s post hoc test). The three groups did not differ in body weight during this period (Figure 7C, one-way ANOVA followed by Tukey’s post hoc test).

Discussion

Colorectal cancer is common globally, with rectal cancer accounting for one-third of cases. Unfortunately, 80%-90% of patients who undergo LAR for rectal cancer experience a change in bowel habits after surgery, and the prevalence of severe LARS has not altered with time (21,22). LARS is said to be caused by direct injury to the anal sphincter muscle and damage to the nerves innervating the rectum and anus (23). This study showed that ADSCs represent effective treatment candidates for LARS in both the acute and chronic phases. In past reports, ADSCs were administered early after injury to the anal sphincter, which means that in actual clinical application, ADSCs must be administered at a time when it is not possible to assess whether defecation problems have appeared (14,15). This is not desirable in terms of healthcare costs, since ADSCs may be administered even to patients who do not have defecation problems, and does not indicate the possibility of efficacy in patients who already have LARS. Therefore, the result that ADSCs were effective even in the chronic phase is important for future clinical application.

ADSCs can differentiate into various cell types, including bone, chondrocytes, muscle, adipocytes, neurons, and hepatocytes, and promote tissue regeneration and cell survival through paracrine secretion (24,25). As demonstrated in a mouse model of anal sphincter injury, ADSCs can differentiate into muscle tissue and surrounding connective tissue, activate cell division, and suppress apoptosis.

It has been reported that the immune response is involved in the healing process after injury to muscle tissue. Early infiltration of injured muscle by neutrophils, which shifts the immune environment to an inflammatory state, is a common and essential response to acute muscle injury (26). Once neutrophil infiltration begins, circulating monocytes and macrophages enter the damaged muscle, and inflammatory cytokines, such as interferon-γ and tumor necrosis factor, become abundant (27,28). Both cytokines trigger a shift in macrophages to the inflammatory M1 macrophage phenotype. These reactions are said to occur within the first 24 hours, after which the inflammatory response attenuates. The macrophage population then shifts from M1 to M2 at around 72 hours, when interleukin-10 is elevated (29-31). In this study, M2 macrophages were observed in large numbers in the connective tissue around the anal sphincter in hADSC-treated mice. Whereas defecation function was fully restored in the acute phase model, the chronic phase model showed improvement but not complete restoration. These results suggest that ADSCs not only promote wound healing but also suppress excessive immune responses during the acute phase, resulting in the preservation of defecation function.

In terms of defecation function, the current results show that administration of 1×104 or more hADSCs immediately after injury improved defecation function. However, in terms of pathological results, 1×107 hADSCs were needed to yield benefit. This finding indicates that recovery of the anal sphincter from injury is not the only factor that improves defecation function and that hADSCs may also have a positive effect on the nervous system. In other recent work from this lab using magnetic resonance imaging, the volume of defecation-related muscle in patients with severe LARS was measured, showing an average volume of 36.5 ml (32). The perianal area in the mice used in the current work was about 20.0 μl. Because the current findings highlighted 1×104 ADSCs as the minimum for defecation function recovery in mice, the extrapolated amount for humans would be about 2×107 ADSCs. This result is meaningful for future clinical applications because previous reports have not shown the minimum required dose of ADSCs. It takes a considerable degree of invasiveness and time to collect such a large amount of cells from the patient and culture them for therapeutic use. Therefore, if translated to the clinic, expanded ADSCs as applied in this study may prove useful.

Study limitations. First, anal pressure was not measured in assessing the function of anal sphincter muscle. However, as noted, studies suggest no correlation between anal pressure and the function of anal sphincter muscle in clinical practice, and defecation status is likely more important for patients. Herein, weight per stool was taken as a reflection of defecation function. Second, the mouse model of anal sphincter injury used here does not perfectly replicate the real-world clinical condition of LARS. Third, the number of mice assigned to each group was small (4 mice per group). Fourth, we attempted to demonstrate that hADSCs differentiated into mouse anal sphincter and surrounding connective tissue using the Transfection DsRed lentiviral vector, but were unable to generate stable transfected ADSCs. Therefore, in this study, we showed differentiation of hADSCs only by immunostaining of HLA.

Conclusion

Administration of expanded hADSCs in a mouse model of anal sphincter injury yielded improvement in defecation status and repair of anal sphincter muscles. In addition, a dosage ≥1×104 hADSCs was necessary to improve defecation status, which was effective in both the acute and chronic phases. The goal of the current approach was to improve LARS, and the results are more clinically relevant than those of previous animal studies of ADSCs, with potential for clinical application. Once treatment with ADSCs is established, patients who would have previously opted for permanent colostomy due to concerns about defecation function may now be able to choose anal preservation surgery. We would like to continue our research in the hope that ADSCs will improve the ADL of all patients undergoing rectal surgery.

Conflicts of Interest

This study was supported by ROHTO Pharmaceutical Co., Ltd., through a research grant and provision of two types of adipose-derived stem cells and culture medium.

Authors’ Contributions

RM and RY collected the data; RM and NM confirm the authenticity of all the raw data; RM drafted the manuscript; SF, NM, and TM helped to finalize the manuscript. TO, HT, MU, YD, and HE proofread the content, and HE gave final approval of the article. All Authors have read and approved the final manuscript.

Acknowledgements

The Authors thank San Francisco Edit for English language editing.

References

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