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. 2023 Feb 27;12(5):760.
doi: 10.3390/cells12050760.

Human Bone Marrow-Derived Mesenchymal Stromal Cells Reduce the Severity of Experimental Necrotizing Enterocolitis in a Concentration-Dependent Manner

Livia Provitera  1 , Andrea Tomaselli  1   2 , Genny Raffaeli  1   2 , Stefania Crippa  3 , Cristina Arribas  4 , Ilaria Amodeo  1 , Silvia Gulden  5 , Giacomo Simeone Amelio  1 , Valeria Cortesi  1   2 , Francesca Manzoni  1   2 , Gaia Cervellini  1   2 , Jacopo Cerasani  1   2 , Camilla Menis  1   2 , Nicola Pesenti  1   6   7 , Matteo Tripodi  1 , Ludovica Santi  3 , Marco Maggioni  8 , Caterina Lonati  9 , Samanta Oldoni  9 , Francesca Algieri  10 , Felipe Garrido  4 , Maria Ester Bernardo  3   11   12 , Fabio Mosca  1   2 , Giacomo Cavallaro  1
Affiliations

Human Bone Marrow-Derived Mesenchymal Stromal Cells Reduce the Severity of Experimental Necrotizing Enterocolitis in a Concentration-Dependent Manner

Livia Provitera et al. Cells. .

Abstract

Necrotizing enterocolitis (NEC) is a devastating gut disease in preterm neonates. In NEC animal models, mesenchymal stromal cells (MSCs) administration has reduced the incidence and severity of NEC. We developed and characterized a novel mouse model of NEC to evaluate the effect of human bone marrow-derived MSCs (hBM-MSCs) in tissue regeneration and epithelial gut repair. NEC was induced in C57BL/6 mouse pups at postnatal days (PND) 3-6 by (A) gavage feeding term infant formula, (B) hypoxia/hypothermia, and (C) lipopolysaccharide. Intraperitoneal injections of PBS or two hBM-MSCs doses (0.5 × 106 or 1 × 106) were given on PND2. At PND 6, we harvested intestine samples from all groups. The NEC group showed an incidence of NEC of 50% compared with controls (p < 0.001). Severity of bowel damage was reduced by hBM-MSCs compared to the PBS-treated NEC group in a concentration-dependent manner, with hBM-MSCs (1 × 106) inducing a NEC incidence reduction of up to 0% (p < 0.001). We showed that hBM-MSCs enhanced intestinal cell survival, preserving intestinal barrier integrity and decreasing mucosal inflammation and apoptosis. In conclusion, we established a novel NEC animal model and demonstrated that hBM-MSCs administration reduced the NEC incidence and severity in a concentration-dependent manner, enhancing intestinal barrier integrity.

Keywords: apoptosis; caspase 3; human bone marrow mesenchymal stromal cells; inflammation; interleukin 1b; mouse model; necrotizing enterocolitis; neonate; zonula occludens-1.

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Conflict of interest statement

The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Timeline of NEC induction protocol. NEC, necrotizing enterocolitis; PBS, phosphate-buffered saline; hBM-MSCs, human bone marrow-derived mesenchymal stromal cells; LPS, lipopolysaccharide; i.p., intraperitoneal.
Figure 2
Figure 2
Histological grading of NEC severity in a NEC neonatal mouse model. Pups were subjected to NEC induction protocol and euthanized after 72 h or earlier in case of distress symptoms. Intestines were harvested, fixed in 10% formalin, and stained with hematoxylin–eosin. The severity of experimental NEC was classified from 0 to 4 according to a previously validated scoring system [66], as follows: (A) grade 0: intact villi; (B) grade 1: superficial epithelial cell sloughing; (C) grade 2: partially compromised villi structure, basal cells still present; (D) grade 3: complete villous necrosis, with basal cells still appreciable; (E) grade 4: transmural necrosis, basal membrane destroyed. Samples with histological scores of 2 or higher were considered positive for NEC. An injury score of 3 or 4 defines severe NEC. NEC: necrotizing enterocolitis. Image magnification 100×.
Figure 3
Figure 3
Term infant formula feedings, hypoxic–ischemic insults, and enteral LPS administration induced NEC in a neonatal mouse model. (A) NEC histological grading and incidence in control (n = 16) and NEC (n = 26) groups. (B) Representative histological sections from each group (hematoxylin/eosin staining). Image magnification: upper panels, 40×; lower panels, 100×. LPS: lipopolysaccharide; NEC: necrotizing enterocolitis. *** p ≤ 0.001 (Fisher’s exact test).
Figure 4
Figure 4
hBM-MSCs reduced NEC incidence and severity in a concentration-dependent manner in a neonatal mouse NEC model. (A) NEC incidence in all the experimental groups based on NEC histological grade (Control, n = 16; NEC + PBS, n = 15; NEC + hBM-MSCs (0.5 × 106), n = 23; NEC + hBM-MSCs (1 × 106), n = 15). (Control vs. NEC + PBS; NEC + PBS vs. NEC + hBM-MSCs (1 × 106); NEC + hBM-MSCs (0.5 × 106) vs. NEC + hBM-MSCs (1 × 106)). (B) Representative histological sections from each treatment group (hematoxylin/eosin staining). Image magnification: upper panels, 40×; lower panels, 100×. hBM-MSCs: human bone marrow-derived mesenchymal stromal cells; NEC: necrotizing enterocolitis; PBS: phosphate-buffered saline. *** p < 0.001 (Fisher’s Exact test).
Figure 5
Figure 5
hBM-MSCs reduced apoptosis and stimulated tissue regeneration in a neonatal mouse NEC model. (A) Representative Caspase-3 staining on histological intestinal sections from mice belonging to the NEC + PBS and NEC + hBM-MSCs (1 × 106) experimental groups. Scale bar: 100 µm. (B,C) Representative Ki67 staining and its relative quantification on histological intestinal sections from mice belonging to the NEC + PBS and NEC + hBM-MSCs (1 × 106) experimental groups (n = 5) Scale bar: 200 µm. (D) Relative gene expression of the anti-apoptotic gene Bcl2 and the pro-inflammatory cytokine IL-1B. (E) Relative gene expression of the tight junction component ZO-1 in the four indicated experimental groups. Bcl2: B-cell lymphoma 2; hBM-MSCs: human bone marrow-derived mesenchymal stromal cells; IL-1B: Interleukin 1b; NEC: necrotizing enterocolitis; PBS: phosphate-buffered saline; ZO-1: zonula occludens-1. * p < 0.05; ** p < 0.01.
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