The Use of Potent Populations of Expanded Fetal Human Placental Stromal Cells for the Treatment of Dextran Sodium Sulfate-Induced Colitis in a Mouse Model

Abstract

Inflammatory Bowel Disease (IBD) is a multifactorial gastrointestinal condition encompassing two major forms of intestinal inflammation: Crohn's disease (CD) and ulcerative colitis (UC). Both conditions are linked to auto-inflammatory reactions and genetic predispositions. Various drug therapies and biological treatments proposed to reduce IBD-associated inflammation. We induced IBD in a mouse model by stimulating bowel inflammation with an oral dextran sodium sulfate (DSS) beverage. Our novel cell therapy approach for IBD involves intramuscular (IM) and intraperitoneal (IP) delivery of non-matched, expanded, potent xenogeneic fetal human mesenchymal stromal cells (f-hPSCs) in 2 × 10⁶ cell injections. This cell therapy has already been shown previously to induce pro-regenerative and anti-inflammatory effects in different systemic and local disorders, where the injected f-hPSCs were shown to respond to the stress of the host and secrete the adequate secretome in response to this stress. In the current study, the IP-injected f-hPSCs treatment of the DSS-induced IBD enhanced the regenerative processes of the damaged bowel and reduced the inflammatory process. This was associated with rapid regain of the mice's weight and a decrease in inflammation-associated parameters, such as colon edema, bowel shortening, and a threefold increase in bowel mass, as estimated by increased colon weight and reduced length. This ratio best emphasized the induced inflammatory response associated with the decrease in the inflamed colon length with an increase in its mass. Although IM f-hPSCs delivery was somehow effective by a few parameters, the IP delivery produced a superior response. The IP f-hPSCs treated mice lost only ~15% of their weight at the peak of the IBD effect, compared to ~25% in untreated mice. A reduction in the inflammatory response of the gut was also indicated by a decrease in neutrophil infiltration, as assayed by a myeloperoxidase (MPO) assay. Additionally, a significant improvement in the histological score of the gut and faster recovery to 90% of its original size was observed. These findings suggest that f-hPSC treatments could serve as an effective and safe anti-inflammatory and pro-regenerative treatment for IBD.

Keywords: C3H mouse model; adult cell therapy; cell therapy; expanded fetal human placental stromal cells (f-hPSCs); inflammatory bowel disease (IBD).

Figure 1

DSS-induced colitis experiment and f-hPSCs treatment. (A) FACS analysis of the cell surface markers of the isolated and expanded f-hPSCs; the typical mesenchymal markers gating was determined for each of the markers tested (range shown by red arrow range), to be positive above the 95% range of the readings of isotypes controls (range shown by blue arrows). (B) Four experimental arms were tested: 3 groups of mice were exposed to 3.5% DSS in drinking water for 4 days. f-hPSCs were injected either IM or IP into the adequate treated groups on days 4 and 6. (C) At the termination of the experiment, the colon was excised, weighed and its length measured. (D) The records of the colon mass/length ratio at the end of the experiment. (E) Lymphocyte counts in the MLN cells at the end of the experiment. The bars above the columns indicate the significance of the difference between the tested groups. Light gray fill represents naïve controls, dark gray represents the non-treated DSS exposed group and light blue and dark blue represent IM and IV f-hPSCs treated DSS exposed mice, respectively. (* = p < 0.05, ** = p < 0.01, *** = p < 0.005). Except for the group of “Naïve” mice, all other groups were exposed to DSS.

Figure 2

CBC at the end of the experiment. CBC values performed at the termination of the experiment for the different experimental groups tested are presented. Light gray fill represents naïve controls, dark gray represents the non-treated DSS exposed group, while light blue and dark blue represent IM and IV f-hPSCs treated DSS exposed mice, respectively. The results of RBC (A) PLT (B) and WBC are presented (C). The peripheral WBC was found significantly elevated in all the DSS-exposed mice. Differential leukocyte counting (highlighted by arrows) showed a major decrease in lymphocyte number in f-hPSCs DSS exposed IP treated mice (D). Similar significant elevations of monocyte counts were recorded in all DSS-exposed mice (E). The most apparent effect of the f-hPSCs treatment is indicated by a very significant reduction in the granulocyte counts in the f-hPSCs IP-treated mice (F). The bars above columns indicate the significance of the difference between the groups tested. (* p < 0.05, ** p < 0.01). Except for the mice group of Naïve, all other groups were exposed to DSS.

Figure 3

H&E stained Histology of the colon. Small colon samples were collected from the sacrificed mice and were processed in paraffin for histology. Representative H&E stained slides were photographed for each group tested (A–D) with magnification of a selected area (A1,B1,C1,D1). The histological sections were used for scoring the severity of the inflammatory response as presented in Figure 4A. The “Naïve” control mice (A) were not exposed to DSS, and (B–D) groups were mice exposed to DSS.

Figure 4

Tissue inflammation and regeneration. The records of the blind histological score for evaluation of tissue inflammation and regeneration parameters are shown in (A). The results of the MPO activity are shown in (B). The correlation between the severity of the inflammatory response as evaluated by MPO assay for infiltrated neutrophils is presented in (C). The bars above columns indicate significant differences between groups, where applicable, (** = p < 0.01, *** = p < 0.005). Except for the mice group of “Naïve”, all other groups were exposed to DSS. The ratio between the MPO activity and histological inflammation score in all groups tested is shown in (D). In all the graphs presented light gray fill in columns and data points represents naïve controls, dark gray represents the non-treated DSS exposed group, while light blue and dark blue represent IM and IV f-hPSCs treated DSS exposed mice, respectively.

Figure 5

Mϕ infiltration to the crypts of the colon. Immuno-stained histology of Mϕ infiltration in the colon histological sections of the different groups was tested. The sections of the colon of all mice in different arms were immune-stained with CD68 (with hematoxylin background). (A–C) with magnification of a selected area marked as rectangle in the low magnifications micrographs (A1,B1,C1). Counts of Mϕ number per total tissue area in the immune-stained sections for naïve, non-treated, and IP f-hPSCs groups are shown (D) (* = p < 0.05). Except for the mice group of “Naïve”, all other groups were exposed to DSS. Light gray fill represents naïve controls, dark gray represents non-treated DSS exposed group, and dark blue represents IV f-hPSCs treated DSS exposed mice.