A Simple Method to Isolate and Expand Human Umbilical Cord Derived Mesenchymal Stem Cells: Using Explant Method and Umbilical Cord Blood Serum

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated from umbilical cords and are therapeutically used because of their ability to differentiate into various types of cells, in addition to their immunosuppressive and anti-inflammatory properties. Fetal bovine serum (FBS), considered as the standard additive when isolating and culturing MSCs, has a major limitation related to its animal origin. Here, we employed a simple and economically efficient protocol to isolate MSCs from human umbilical cord tissues without using digestive enzymes and replacing FBS with umbilical cord blood serum (CBS). METHODS AND RESULTS: MSCs were isolated by culturing umbilical cord pieces in CBS or FBS supplemented media. Expansion and proliferation kinetics of cells isolated by explant method in the presence of either FBS or CBS were measured, with morphology and multi-differentiation potential of expanded cells characterized by flow cytometry, RT-PCR, and immunofluorescence. MSCs maintained morphology, immunophenotyping, multi-differentiation potential, and self-renewal ability, with better proliferation rates for cells cultured in CBS compared to FBS supplement media. CONCLUSIONS: We here present a simple, reliable and efficient method to isolate MSCs from umbilical cord tissues, where cells maintained proliferation, differentiation potential and immunophenotyping properties and could be efficiently expanded for clinical applications.

[Multi-differentiation potential for mesenchymal stem cells isolated from human umbilical cord]

Stem cells have unique capability to differentiate into many cell types that can normally replace the loss in some cells of the body due to tissue injury. Umbilical cord blood [UCB] and umbilical cord [UC] are the two main sources for hematopoietic stem cells [HSCs] and mesenchymal stem cells [MSCs], respectively, which constitutes the basis for stem cell banks that have been established worldwide and very recently in Syria. Research in our region has mainly focused on cell storage and freezing protocols, and only few studies were conducted to prove the ability of the stored cells to differentiate into their destined lineages. This study aimed to test the potential of cryopreserved MSCs isolated from an umbilical cord taken from new delivery at Maternity University Hospital in Damascus, to differentiate into various types of cells in response to growth and induction factors specific to cell lineages. after isolation, MSCs proliferated in vitro up to fourth passage and were stored by freezing in liquid N2 for at least 6 months. After re-culturing the stored cells, the viabilities of these cells were between 80-90%, and these cells demonstrated similar proliferative characteristics in comparison to the cells prior to cryopreservation. Five protocols were applied on re-cultured cells to induce their differentiation into adipocytes, osteocytes, myocytes, neurocytes, and insulin producing cells [IPCs]. MSC differentiation was confirmed by studying cell morphologies and applying cell staining protocols, gene expression testing, and enzyme-linked immunosorbent assay [ELISA]. Results showed the multi-differentiation potential of MSCs isolated from umbilical cord after cryopreservation, thereby, confirming the negligible effect of cryopreservation on MSCs differentiation potential to five different cell lineages. These results point to the possible benefits of storing umbilical cords as well as UCB in recently established stem cell banks in Syria to use cord-derived MSCs as successful tools in regenerative medicine, aiming ultimately to implant MSCs in vivo as an alternative therapy to regain the defective function of specific cell and tissue types