Multipotent Human Neonatal Cardiac‐Derived Mesenchymal Stem Cells Modulate Ileitis In Vivo

Transmural skip lesions are pathognomonic for Crohn's disease (CD). Despite advances toward CD treatment, disease recurrence remains a problem. CD requires novel therapies to modulate localized intestinal inflammation and promote intestinal epithelium healing. Human neonatal cardiac‐derived mesenchymal stem cells (nMSCs) demonstrate immune cell modulation accompanied by improved cardiac function recovery in myocardial infarction models. In the established CD‐like ileitis SAMP mouse model, direct skip lesion injection of nMSCs prevents ileal skip lesion growth and significantly down‐regulates the pro‐inflammatory milieu. Significant reduction in the percentage of skip lesion CD68+ macrophages (M1 Mφ, pro‐inflammatory) accompanied by an increase of CD206+ macrophages (M2 Mφ, anti‐inflammatory/pro‐regenerative) is observed in skip lesions following nMSCs injection compared to non‐injected and placebo controls (p < 0.05). Skip lesion size is significantly reduced along with pro‐inflammatory cytokines IFN‐y and TNF‐α, with an increase in intestinal tissue anti‐inflammatory cytokine IL‐10 production. nMSCs are also retained within skip lesions 5 weeks post‐treatment. nMSC administration promotes wound remodeling by modulating inflammatory immune cells and by increasing small bowel gastrointestinal transit with concomitant decreased segment gross pathology score compared to placebo control. Data from this study demonstrate that direct injection of nMSCs into ileal skip lesions attenuates inflammation and improves intestinal physiology.


Introduction
Crohn's disease (CD) is characterized by chronic inflammation with severe devolving intestinal physiology resulting in increased morbidity and mortality. [1]CD pathogenesis encompasses overactive innate and adaptive immune cell behavior, environmental and socio-economic factors, and an assortment of genetic mutations. [2,3]daptive and innate immune cells release pro-inflammatory cytokines that exacerbate skip lesion growth typically within the small intestine but can affect all aspects of the gastrointestinal tract (GI). [4,5][8] However, the development of treatment insensitivity along with multiple severe side effects due to combinatorial treatments decrease healthrelated quality of life metrics while increasing patient morbidity including the increased risk of developing GI cancers. [7,9,10]he lack of long-longstanding efficacious treatment modalities illustrates the need for novel cell therapeutic approaches to significantly modulate intestinal inflammation and promote wound healing.
[13][14][15][16][17] Adult MSCs are multipotent, self-renewing, non-hematopoietic stem cells that possess immunomodulatory and pro-regenerative characteristics that can be used to treat tissue injuries and autoimmune disorders. [4,18,19]Studies from multiple clinical trials have revealed that allogeneic MSC infusions are safe, effective, and non-immunogenic in treating certain immune disorders. [20][23] Similarly, studies have demonstrated that MSCs modulate macrophages (M; inflammatory M1 M into antiinflammatory, pro-reparative M2 M) and release TGF that also participate in the lesion resolution and tissue repair. [24] A very recent finding revealed that intestinal, intraluminally injected MSCs showed modest efficacy by partially attenuating experimental colitis in murine model.This suggests that alternative MSC cell types are needed to enhance stem cell therapy for CD. [4]28] Utilizing our unique resource of human neonatal (<1 month) cardiac tissue, we have consistently demonstrated that neonatal mesenchymal stem cells (nMSCs) demonstrate efficacy in repairing the injured heart, compared to their respective adult counterparts. [29]In direct comparison testing of wellcharacterized stem/progenitor cells, we demonstrated that nM-SCs outperformed other cell types with regard to cardiac functional outcome recovery following myocardial infarction (MI) in an animal model.However, the therapeutic efficacy of nMSCs in an established CD-like ileitis model remains to be investigated.Thus, within the context of this study, we will evaluate the effectiveness of nMSCs to positively modulate multiple aspects of CD-like ileitis in the pre-clinical SAMP mouse model.

nMSCs Injection Reduces Ileal Lesions and Alleviates Tissue Pathogenesis
We analyzed the effect of nMSCs to attenuate lesion progression by comparing pre-treatment SAMP mice at 10 weeks of age to an-imals at 15 weeks of age (5 weeks post-treatment) via evaluation of lesion size and infiltration of inflammatory immune cells.The skip lesions in the ileal tissue were measured prior to the injection of nMSCs and placebo (saline-injected SAMP mice) control in 10-week-old mice (Figure 1a).At pre-injection, ileal lesion sizes (5 lesions/animal represented as mm 2 ) were comparable across all the conditions and between the groups (placebo 4.2 ± 0.4; nM-SCs 4.6 ± 0.5; p = 0.734) (Figure 1a).Macroscopic evaluation at 5 weeks post-treatment showed a significant reduction in ileitis as assessed by lesion size in nMSCs treated mice compared to the placebo control (placebo 5.4 ± 0.1 versus nMSCs 3.3 ± 0.7; p = 0.001) (Figure 1a,b).Histological staining on ileal lesions showed that nMSCs treatment markedly reduced infiltration of inflammatory cells and increased epithelial cells adjacent to the lesions compared to the placebo control (Figure 1c).

nMSCs Modulate Macrophage Sub-Types in Ileal Lesions
Histological examination of ileal tissue showed a statistically significant reduction of CD68 + M within lesions following nMSC treatment compared to placebo control at post-5-week treatment [NI (no injection) 66.0 ± 2.9, placebo 66.  2c,d).

Therapeutic Efficacy of nMSCs on Small Bowels and Intestinal Pathology
To further investigate GI transit and a segment gross pathology score (SGPS) following nMSCs treatment, we performed an established fluorescein isothiocyanate (FITC)-dextran motility assays. [30]The GI tract was divided into 15 segments (S1 to S15; Sections 1-10 are small bowel and 11-15 are cecum and large intestine) and the percent total fluorescence in each segment was examined to create an SGPS.The appearance of increased proximal distribution of FITC-dextran is indicative of delayed GI transit and enhanced gross diseases pathology between SB6 AND SB10.The SAMP mice injected with nMSCs showed reduced distribution of FITC dextran from segment SB6 up to distal direction suggesting reduced ileitis and histology scores (reduced lesions and strictures) (Figure 4).nMSCs: SB6 6.4 ± 0.1, SB7 5.3 ± 0.9, SB8 4.7 ± 1.2, SB9 5.5 ± 2.0, SB10 9.7 ± 1.2.Conversely, the placebo control showed the presence of significantly increased proximal distributions peaks indicating delayed GI transit due to severe lesions and ileitis.Placebo control: SB6 9.9 ± 2.6, SB7 11.8 ± 2.2, SB8 14.8 ± 1.7, SB9 3.3 ± 1.1, SB10 4.2 ± 1.1.The occurrence of the heightened peak in the placebo groups is suggesting the development of increased strictures in the small bowel segments and pathology score.Conclusively, the direct injection of nMSCs mitigated small bowel motility dysfunction and intestinal obstruction compared to placebo controls suggesting that, nMSCs injection promotes the regeneration of epithelial cells to improve the GI transit in SAMP mice.

Discussion
CD is comprised of chronic inflammation in the proximal, distal small intestine and in the mucosa of the colon resulting in destructive intestinal epithelium due to dysregulated immune cell activation. [31]34][35] Recent studies have also shown that steroids and thiopurine can  modulate the inflammation and promote mucosal healing in the chemically induced murine colitis model. [10]However, the complications of the aforementioned therapies include immune reaction, development of skin lesions, peri-operative morbidity, postmanagement infections, and increased risk for malignancies, which warrants safe and efficient therapy for CD. [10,35,36]Also, the non-specific immune suppression and its side effects warrant ef-ficient therapy not only to suppress systemic inflammation but to also promote epithelial wound healing applicable animal models that will advance treatment options for the millions afflicted with CD.
[39][40] MSCs are non-hematopoietic, self-renewing, hypoimmunogenic stem cells and have been shown to attenuate inflammation in chemically induced experimental mouse and rat colitis models [25,41] MSCs including adipose-derived MSCs (AD-MSCs) [12,42] umbilical cord-derived MSCs (UC-MSCs) [14] dental follicle derived MSCs (DF-MSCs) [43] and bone marrow-derived MSCs (BM-MSCs). [25,5]The aforementioned cell types have been shown to possess therapeutic efficacy in mitigating inflammation in experimental colitis models.A recent study identified that a single dose of intraperitoneal administered AD-MSCs attenuated intestinal inflammation and restored epithelial barrier integrity in the DSS-induced experimental colitis mice model. [44]Similarly, a study by Gonzalez et al. also demonstrated that AD-MSC injections markedly ameliorated clinical and histopathologic severity of colitis and inflammation in TNBS-induced colitis in murine models. [15]Although MSCs have therapeutic potential in the pre-clinical experimental colitis model, the efficacy of MSCs in treating IBD patients in clinical trials remains modest.Also, the chemical-induced experimental model is not an ideal model to mimic pathology observed in CD patients.Therefore, in this study, we employed a mouse model that develops CD-like ileitis that resembles intestinal lesions of CD patients in clinical settings.
The immune modulation and wound-healing potential of MSCs are highly influenced by the inflammatory milieu of injured tissues. [45]MSCs isolated from adult donors have reduced proliferation rates and increased immune rejections and thus moderate efficacy in treating tissue injuries. [46]Similarly, our recent findings demonstrated that the therapeutic efficacy of MSCs is highly influenced by the chronological age of the donors (29).In this regard, the adult MSCs administration showed poor engraftment, low survival, limited paracrine effect, and reduced cardiac function outcome in the rat MI model. [29]Studies have identified that the tissue infiltration of inflammatory immune cells and accumulation of inflammatory cytokines increases hypoxic and apoptotic stimuli that may induce injected MSCs clearance. [47,48]Recent findings from our group demonstrated that cardiac-derived neonatal MSCs (nMSCs) showed superior cardiac functional outcome compared to adult-derived MSCs (aMSCs) due to their increased retention and their sustained paracrine secretion in the rat MI model. [29,49]In this study, we investigated the therapeutic efficacy of nMSCs to protect intestinal epithelium by modulating immune cells and inflammation in the SAMP mice.Consistent with previous findings on MSCs as cellular therapy in the experimental colitis mouse model, histological examination of colon tissue showed that a single dose of direct injection of nMSCs in the ileitis lesions attenuated the transmural inflammation and promoted epithelial cells regeneration in the ileum compared to no-injection and placebo controls in the CD-like ileitis mouse model.
Macrophages (M) are a known driver of inflammation in the GI tract in CD patients.Pre-clinical studies on experimental colitis in the M deficient mice model confirm that M plays a key role in promoting intestinal lesions and obstructions. [50,51][54][55] To define the innate immune modulation potential of nMSCs, we investigated the level of inflammatory M1 M (CD86 + ) and pro-regenerative anti-inflammatory M2 M (CD206 + ) in SAMP mice.Intra-lesional administration of nMSCs modulated inflammation by increasing M2 M resulting in reduced inflammatory M1 M in the ileal skip lesions.Significant increases in anti-inflammatory cytokine IL-10 following nMSCs administration suggests that nMSCs modulate inflammatory M1 M into pro-reparative M2 M and the expansion of T-regulatory cells phenotypes resulting in the attenuation of inflammation and regeneration of intestinal epithelial cells.
[54][55] Intraperitoneal injections of UC-MSCs increased the level of IL-10 and infiltration of regulatory T cells (T-regs) cells to modulate the inflammation in the DSS and TNBS-induced colitis mice model. [14]Similarly, systemic infusion of AD-MSCs attenuated the inflammation by increased IL-10 releasing M2 M and T-regs to suppress Th1 effector cell responses in vitro and in vivo. [15]Consistent with the aforementioned findings, in this study, we have identified a significant increase in IL-10 levels in the lesions administered with nMSCs which confirms that nMSCs increase IL-10 producing M2 M and T-regs in the inflammatory milieu.We next examined the half-life of injected nMSCs in the lesion site by targeting HLA.Histological analysis for nMSCs retention revealed that nMSCs can be detected even after 5 weeks of injection which confirms that nMSCs are immunologically tolerant against host immune responses.Interestingly, we found significantly increased M2 M in the nMSCs injected area suggesting that nMSCs modulate inflammatory M 1M into anti-inflammatory M2 M to attenuate the inflammation in ileitis lesions.These results indicate that the intralesional administration of nMSCs is well tolerated against host immune rejection and thus nMSCs can be an ideal stem cell type for a future clinical application to treat CD patients.
Dysregulated immune cell activation releases increased inflammatory cytokines TNF- and IFN- which exacerbates the inflammatory conditions in CD patients. [13]Several studies have identified that the accumulation of inflammatory cytokines including TNF- and IFN- increases intestinal small bowel tissue inflammation and iliac epithelial cells degenerations. [4,16,42]irect injection of UC-MSCs attenuated inflammation by decreasing inflammatory cytokines, TNF- and IFN-, in the experimental colitis model. [14]Additionally, UC-MSCs improved macroscopic, histological scores and reduced symptoms associated with colitis.Similarly, AD-MSCs injection reduced the expression of TNF- and IFN- in the colitis model in vivo. [15]erein, we investigated the immune modulatory effect of nM-SCs by assessing the presence of inflammatory cytokines associated with CD-like ileitis.Histological evaluation of lesion tissue showed that nMSCs administration significantly reduced pro-inflammatory mediators TNF- and IFN- suggesting that nMSCs attenuate inflammatory cytokine-producing cells such as macrophages, dendritic cells, and cytotoxic T cells.Our results are consistent with previous reports showing the significant reduction in inflammatory cytokines TNF- and IFN- in the mice model of ileitis and their immune modulation potential makes nMSCs a putative stem cell that may be used to treat CD in clinical patients.
FITC-dextran distribution assay was employed to assess the GI transit delay and segment pathology score that are indicative of inflammatory lesions and colon strictures. [30]Small bowel GI transit and segment gross pathology analyzed by the FITCdextran distribution assay revealed that nMSCs injection showed significantly improved GI transit and reduced segment pathology in the ileitis mice.We speculate that these findings suggest that the injected nMSCs promoted the wound healing of epithelial layers and submucosa by vasculogenesis and differentiation of nMSCs into epithelial cells which leads to the improvement in intestinal physiology.Our results not only demonstrated the inflammation resolution by nMSCs, but also showed that the administration of nMSCs promotes mucosal healing through their wound-healing capabilities in vivo.
Although our results demonstrate significant improvement with regard to intestinal physiology and short-term resolution of inflammation, our study has certain limitations.The direct lesion injection of nMSCs may not be ideal in a patient setting where open surgery would be required for the pinpoint delivery of nMSCs to skip lesions.Surgery in this setting is typically reserved to resect irreversibly damaged tissue.As an alternative strategy, nMSCs could potentially be delivered via endoscopy-guided approaches to areas of inflammation without the invasiveness of an open surgery.Alternatively, a non-invasive delivery platform of the nMSCs could bypass this issue.MAdCAM-1 is the ligand for 47 integrin as well as l-selectin (CD62L).58] Our preliminary data demonstrate that our nMSCs express 47 integrin and l-selectin (Figure 1d).The blockage of in vivo 47 integrin/MAdCAM-1 or l-selectin/MAdCAM-1 interactions through intravenous nMSC delivery may significantly reduce and subsequently prevent ileitis.Delivery can also prevent guttropic inflammatory 47+CD4 T-cells and other l-selectin+ leukocytes from exacerbating localized inflammation as nMSCs will compete with adaptive immune cells for MAdCAM-1 binding.Thus, nMSCs can be used to directly target MAdCAM-1 expressed on HEVs during ileitis via a non-invasive, intravenous injection.Finally, our investigation demonstrated that nMSCs promote epithelial cell layer reformation in the ileum.However, whether nMSCs could be able to differentiate into epithelial cells and relevant colonic cells in the SAMP model is still unclear.The mechanism for increased nMSCs retention in the inflammatory milieu at post-5-week treatment remains unknown as does the safety profile that also needs to be further investigated.With the resolution of the aforementioned issues, nMSCs may be a feasible alternative to current cellular therapeutics used to treat CD.

Conclusion
Within the context of this study, we have demonstrated for the first time that direct injection of human cardiac-derived nM-SCs could alleviate systemic inflammation and recover intestinal physiology in a murine model of CD-like ileitis.The underlying possible mechanism is that the nMSCs secretome including paracrine factors and exosomes might modulate the inflammatory immune cells into pro-reparative anti-inflammatory immune cells.Similarly, paracrine-released molecules by nMSCs may serve as the basis to promote intestinal physiology.Our data demonstrate that nMSC therapy attenuated the inflammation and induced wound healing in the intestinal tissues that can be taken into consideration to treat CD patients in the clinical settings.

Experimental Section
SAMP1/YitFcsJ Animal Model: The SAMP1/YitFcsJ ("SAMP"; Jackson Laboratories) mouse model exhibited CD-like ileitis and mimicked aspects of human CD and thus can be extrapolated to the human condition.It was an accepted pre-clinical model of CD-like ileitis. [30]Gross observations/clinical symptoms of SAMP mice included luminal blockage, skin crypt elongation, lesions, intestinal perforations, villus loss, transluminal inflammation, fissure and fistula formation, impaired nutritional uptake, and delayed small bowel transit at the age of 10 weeks.At the microscopic level, epithelial hyperplasia of Paneth and goblet cells became evident along with disproportionate villus/crypt ratios within the ileum.Mice approached 100% penetrance at nearly 10 weeks of age.Protracted expression of TNF- and IFN-y was also evident at early stages of life and gradually accrued overtime and disease progression.All animal procedures were performed under the oversight and guidance of the Northwestern University Institutional Animal Care and Use Committee (IACUC) through an approved animal protocol (#IS00018168).SAMP mice were divided into three groups: i) no-injection, ii) placebo (saline injection), and iii) nMSC injection group.
Small Intestinal nMSC Lesion Direct Injection: SAMP mice (≈15-25 grams; 10 weeks of age; female) were provided an ad libitum liquid diet (Jevity; 1 cal Isotonic Nutrition and Fiber) for 24 h prior to surgery and nMSC small intestinal injections.Immediately prior to surgery, 2% isoflurane with O 2 anesthesia was administered and a midline abdominal incision (1.0-1.5 cm) was made at the linea alba to gain access to the abdominal cavity and to identify the small intestines.Following skip lesion identification on the small intestine, animals were either injected with nMSCs (20 million per kg in 30 μL of media into the skip lesions (n = 5 lesions/mouse; n = 3 mice), or injected with saline (30 μL; termed as placebo controls; n = 5/mouse, n = 4 mice) by the handler utilizing a 27 gauge needle.Noninjected mice served as unmanipulated controls (n = 3).The injected areas were marked with India ink and then identified using measurements from the cecum.Organs and intestines were relocated to their original anatomical positions following the procedure and the intestines were gently rinsed with sterile saline prior to return to the peritoneal cavity.The abdomen was closed in 2 layers that included the muscle and skin with 4.0 PDS, and 4.0 Ethilon used for skin closure.Following recovery, animals were placed back into their respective cages with non-edible bedding.They were provided ad lib water and liquid diet for the first 7 post-operative days following recovery.Animals were returned to regular chow 7 days post-op and monitored on a regular basis.
Tissue Specimen Processing and Histological Staining: As described, [30] skip lesions were identified based on India ink marking and removed from animals following euthanasia at 5 weeks post-injection.Lesion size was determined using Calipers (Mitutoyo Absolute Digital Caliper) were utilized to measure lesion size based on the length and width of the lesions.Individual lesions were fixed in a 10% buffered formalin phosphate, dehydrated with graduated ethanol solutions, and finally embedded in paraffin wax molds.Molds were then sectioned onto glass slides at a 5-7 μm thickness and allowed to air dry.Slides were then stained with an established H&E staining protocol (or used for immunofluorescence studies) and a coverslip was attached using Permaslip (Alban Scientific Inc.). [30]mmunofluorescence Staining: The aforementioned prepared slides were deparaffinized, dehydrated with ethanol, and rehydrated in diH 2 0 before being placed in boiling antigen retrieval buffer (0.01 M citrate, pH 6.0, 0.05% Tween-20) for 15 min, and then allowed to cool to room temperature for 30 min.Following cooling, tissue sections were circled with a hydrophobic pen and blocked with bovine serum albumin (BSA-5 mg mL −1 ) (Sigma Aldrich, MO, USA) for 15 min.Intestinal tissue lesions collected and embedded in paraffin were cut into 5 μm-thick sections and stained with individual primary antibodies targeting IL-10, IFN-y, CD68, CD86, CD206, TNF-, HLA-A [IL-10 at 5 μg mL −1 , IFN-y at 10 μg mL −1 , CD68 at 10 μg mL −1 , CD86 at 5 μg mL −1 , CD206 at 5 μg mL −1 , TNF- at 9 μg mL −1 , HLA-A at 10 μg mL −1 ); secondary antibodies ranged from of 2-4 μg mL −1 ] in 1% BSA and incubated for 1 h at room temperature.Slides were washed 3× with 1× PBS and then incubated with respective secondary antibodies for 30 min at room temperature.Following incubation, slides were washed 3× with PBS.The cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) and the slides were visualized under a Nikon Eclipse 50i microscope and Spot advanced Imaging Software (Diagnostic Instruments, MI, USA).Individual primary antibodies were obtained from Abcam (MA, USA).Secondary antibodies: Goat-Anti-Rabbit 555, Goat-Anti-Mouse 488 diluted 1:400 were obtained from Invitrogen Corp., CA, USA.
Immunofluorescence Quantification: An IF quantification protocol was adapted from the previous study. [30]Quantification of fluorescently stained tissue sections was accomplished using a Nikon Eclipse 50i microscope and Spot Advanced Imaging Software (Diagnostic Instruments, MI, USA).Of the treated or non-treated lesions per animal, 3 images per lesion were taken (a total 12 total images per animal; n = 3 animals/experimental group).The number of percent positive inflammatory cells was determined by manually counting fluorescently stained cells using the ImageJ cell counter plugin function software (National Institutes of Health).The total number of cells in each image was determined by opening the DAPI channel alone and converting the image threshold to only highlight DAPI+ cells.To eliminate stacked cells, the watershed function of ImageJ was used.This was followed by the use of the analyze particle tool to determine total cell count.The size was adjusted to 180 pixels-500 and circularity was set from 0.0-1.0. [30]ntestinal Motility: Prior to motility assay, animals were fasted for 12 h as previously described in detail. [30]Mice received an oral gavage of 100 mg mL −1 FITC-dextran (in Kreb's solution; 44 mg/100 g body weight; 70 000 MW, Sigma Aldrich).At 5 weeks post-treatment and after 90 min post-FITC-dextran treatment, animals were humanely euthanized, and the GI tract was carefully removed and included the stomach through the colon.The small intestine was divided into 10 segments (SB) and the colon (Col) was divided into 3 segments.Each segment was flushed with 1 mL of Kreb's solution and the subsequent wash solution was collected.Following collection, samples were centrifuged, and the supernatant was collected and read on a plate reader at 488 nm/519 nm.The total accumulation of FITC-dextran was determined from individual tissue segments and quantified.
Statistical Analysis: The statistical analysis was performed using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA).Comparative analysis for cytokines (IFN-y, TNF- and IL-10), immune cells (CD68 + , CD86 + , CD206 + ), and HLA-A detection among no-injection, placebo, and nMSCs were achieved by one-way analysis of variance with Tukey's multiple comparisons.The intestinal healing following nMSCs injection and placebo control was analyzed by the Mann-Whitney U-test.Small bowel transit delay between nMSCs and placebo control was calculated using one-way ANOVA with Dunnett's post hoc test.* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 was considered statistically significant.

Figure 1 .
Figure 1.Ileitis status following direct injection of nMSCs into SAMP mice: A,B) Representative images of intestinal tissue lesion at 5 weeks following a single injection of nMSCs in SAMP mice.Ileitis lesion wound healing was analyzed by histology following nMSCs administration and compared with non-injections and placebo controls.Data are represented as mean ± SEM (n = 3-4).C) Representative H&E staining images revealed reduced infiltration of inflammatory cells and cryptic damage within the submucosa and lamina propria in nMSC-administered mice compared to no-injection and placebo controls.White arrows depict areas of inflammatory cell infiltrate.nMSC treated animals have a subjectively lower level of immune cell presence in lesion tissue.D) Immunofluorescence staining of nMSCs with anti-47 and l-selectin (CD62L) antibodies demonstrating the expression of these pivotal proteins in gut-immune cell trafficking.Scale bars-10×-200 μm, 40×-50 μm.Statistical significance was analyzed by the Mann-Whitney U-test and p < 0.05 were considered as significant.

Figure 2 .
Figure 2. The percentage of macrophage phenotypes following nMSCs administration: A,B) Representative images show the percentage of CD68, CD86, and CD206 cells measured by immune histochemistry using intestinal lesions following nMSCs treatment in SAMP mice.Magnification 40×, Scale bar 50 um.C) The tissue lesion samples collected from the SAMP mice following direct injections of nMSCs were utilized to evaluate the nMSCs retention by targeting human leukocyte antigen by immune-histology (HLA-A).Magnification 20×, Scale bar 100 μm.D) The frequency of nMSC retention was expressed as a percentage of nMSCs in the lesion area compared to placebo control.3-4 mice were studied in each group.Data are represented as mean and standard error of the mean of the percentage of CD68 and CD206 cells.Statistical significance was analyzed by the Mann-Whitney U-test and p < 0.05 were considered as significant.

Figure 3 .
Figure 3.The level of pro-and anti-inflammatory cytokines in the lesions: A,B) Histological analysis of lesions tissues shows a significant reduction in pro-inflammatory cytokines (IFN-y, TNF-) and elevated anti-inflammatory cytokine IL-10 following nMSC injection compared to no-injection and placebo controls.Magnification 40×, scale bar 50 μm.3-4 mice were studied in each group.Statistical significance was analyzed by the Mann-Whitney U-test and p < 0.05 were considered as significant.

Figure 4 .
Figure 4. Therapeutic efficacy of nMSCs on GI transit delay and segment gross pathology scores in SAMP mice: GI transit was assessed at 5 weeks post-injection (15 W of age) using FITC-dextran distribution across the GI tract which revealed the significant GI transit improvement in small bowel (SB) region in mice injected with nMSCs compared to no-injection and placebo controls.The total of 15 total segments including stomach (11 segments) to colonic regions (4 segments) were measured and each segment was represented as percent of total fluorescence and the distribution weighted toward more proximal segments considered indicative of slowing of transit.The mean fluorescence intensity of SB was peaked at segments SB5-SB7 in no injection and placebo controls compared to nM-SCs treated mice.Conversely, the fluorescence intensity of nMSCs peaked at colonic region (segment SB8 to Col1).The cumulative fluorescence percentage of the SB3-SB8 region was comparatively lower in the nMSCs treated group compared to no injection and placebo.[P-values were calculated using one-way ANOVA with Dunnett's post-hoc test].Values are presented as means ± SEM (n = 3-4 animals per group).p < 0.05 were considered as statistically significant.