Stem cells and necrotizing enterocolitis: A direct comparison of the efficacy of multiple types of stem cells

Abstract

Purpose: Necrotizing enterocolitis (NEC) is a leading cause of gastrointestinal morbidity and mortality in premature infants. While studies have shown potential for stem cell (SC) therapy in experimental NEC, no study has compared different SC side-by-side. Our purpose was to determine whether one type of SC may more effectively treat NEC than others.

Methods: Four SC were compared: (1) amniotic fluid-derived mesenchymal SC (AF-MSC); (2) amniotic fluid-derived neural SC (AF-NSC); (3) bone marrow-derived mesenchymal SC (BM-MSC); and (4) neonatal enteric neural SC (E-NSC). Using an established rat model of NEC, pups delivered prematurely received an intraperitoneal injection of SC. Control pups were injected with PBS. Additional controls were breast-fed by surrogates and not subjected to experimental NEC. Intestinal tissue was graded histologically.

Results: NEC incidence was: PBS, 61.3%; breast-fed unstressed, 0%; AF-MSC, 19.1%; BM-MSC, 22.9%; AF-NSC, 18.9%; E-NSC 22.2%. All groups demonstrated statistical significance (p<0.05) compared to controls, and there was no difference between SC groups.

Conclusion: All four SC groups reduced the incidence and severity of experimental NEC equivalently. AF-MSC may be preferable because of availability of AF at delivery and ease of expansion, increasing potential for clinical translation.

Level of evidence: V (Animal study).

Keywords: Amniotic fluid; Mesenchymal; NEC; Necrotizing enterocolitis; Neural stem cells; Stem cells.

Figure 1. Flow cytometric analysis of stem cells

Red lines represent negative controls and blue lines represent populations of interest. (A) AF-MSC, positive for CD29, CD49e, CD90; partially positive for Oct4; negative for CD11 and CD45; (B) BM-MSC, positive for CD90 and negative for CD11 and CD45; (C) AF-NSC, positive for Nestin; (D) E-NSC, positive for Nestin.

Figure 2. Multipotency verification

AF-MSC and BM-MSC (passage 5) were induced along adipogenic and osteogenic differentiation pathways. Shown are representative images of (A) AF-MSC with lipid droplets staining positive with Oil Red O after 7d and (B) AF-MSC staining positive for calcium with Alizarin Red S after 14d. Similar findings were obtained for BM-MSC.

Figure 3. Representative images of histologic injury scores

Sections of small intestine were stained with H&E and graded as follows: (A) grade 0, normal intestine; (B) grade 1, epithelial cell lifting; (C) grade 2, necrosis to mid-villus; (D) grade 3, necrosis of entire villus; (E) grade 4, transmural necrosis. Magnification ×20.

Figure 4. Incidence and severity of NEC

With the exception of rat pups in the breastfed group, all other pups were subjected to experimental NEC. (A) Percentages of pups with histologic injury scores of 2, 3 or 4, consistent with histologic NEC. The white numbers within the colored sections of the bars represent the percent incidence of Grade 2, 3 or 4 NEC, and the bold numbers at the top of each bar represent the total incidence of NEC. (B) Percentages of pups with histologic injury scores of 3 or 4, consistent with severe NEC. The white numbers within the colored sections of the bars represent the percent incidence of Grade 3 or 4 NEC, and the bold numbers at the top of each bar represent the total incidence of severe NEC.