Milk Derived Mesenchymal Stromal Cells: new insights for clinical applications

Breastfeeding is the first choice for neonatal nutrition and represents the best solution to ensure child health and development thanks to its nutritional properties. It has been described that human milk, whose composition is complex and comprehend nutritional and non-nutritive bioactive factors, participate to the fetal immune-system maturation, neurodevelopment and glucose tolerance. Recently, breast milk was identified as a rich source of stem cells such as Mesenchymal Stem Cells (MSCs), a unique cellular population with immunomodulatory, neuromodulatory and glucose modulatory properties. To date, MDMSCs have not yet been well characterized and their role in fetal health and development is still controversial. Therefore, in this study we characterized and investigated MDMSCs contribution in immune-response modulation, neonatal neurodevelopment and glucose homeostasis through the analysis of TNF-a, IDO1, OPN, BDNF, NT3, NT4, GLUT1 and GLUT4 gene expression levels by Real Time PCR and compared these potential abilities between MDMSCs isolated from colostrum and from transitional milk. Colostrum and transitional milk samples were collected from 21 volunteers. MDMSCs were isolated and cultured in Dulbecco’s modified Minimum Essential Medium (DMEM) supplemented with 10% Fetal Bovine Serum in flasks pre-coated with 5µg/cm2 fibronectin. MDMSCs characterization was performed by flow cytometry assessing the expressions of CD105, CD90 and CD73, main MSCs surface markers, and of HLA-DR (Human Leucocyte Antigen DR), receptor responsible of non-self antigens presentation to immunological system. Finally, MDMSCs were processed for mRNA isolation and gene expression of stemness makers Oct-4 and NANOG, immunomodulatory markers TNF-, OPN, IDO-1, neurotrophic factors BDNF, NT-3, NT-4 and glucose transporters GLUT-1 and GLUT-4 were assessed by Real Time PCR. All MDMSCs lines investigated were positive for CD105, CD90 and CD73 surface antigens, while they were negative for HLA-DR. Moreover, all MDMSCs lines properly expressed both Oct4 and NANOG mRNA and showed an adequate fibroblast-like morphology. TNF-a, IDO1 and OPN gene expression levels where higher in MDMSCs compared to PDMSCs and their expression was increased in colostrum compared to transitional milk. Additionally, only BDNF, and NT-4 were expressed in MDMSCs and in a decreased way compared to PDMSCs. Moreover, BDNF, and NT-4 expression is higher in colostrum compared to transitional milk. GLUT-1 and GLUT-4 gene expression levels were decreased in MDMSCs compared to PDMSCs. However, GLUT-1 was more expressed in transitional milk while GLUT-4 was more expressed in colostrum milk. Finally, GLUT-1 and GLUT-4 expression resulted decreased in MDMSCs isolated from women with GDM compared to MDMSCs isolated from healthy women. In conclusion, we described for the first time to our knowledge human milk as an alternative source of Mesenchymal Stromal Cells (MDMSCs) and we demonstrated the direct involvement of this unique cellular population in fetal immune-system maturation, neurodevelopment and glucose homeostasis. The absence of HLA-DR on MDMSCs paves the way for their potential use in allogenic cell therapy without risk of rejection. However, further studies are requested to deepen the knowledge on this cellular population.