Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential

Abstract

Introduction: Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank.

Methods: The BM-MSCs, AT-MSCs and UC-MSCs were cultured and evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. Additionally, MSCs were assessed for CD105, CD44, CD34, CD90 and MHC-II markers by flow cytometry, and MHC-II was also assessed by immunocytochemistry. To interpret the flow cytometry results, statistical analysis was performed using ANOVA.

Results: The harvesting and culturing procedures of BM-MSCs, AT-MSCs and UC-MSCs were feasible, with an average cell growth until the third passage of 25 days for BM-MSCs, 15 days for AT-MSCs and 26 days for UC-MSCs. MSCs from all sources were able to differentiate into osteogenic (after 10 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs), adipogenic (after 8 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs) and chondrogenic (after 21 days for BM-MSCs, AT-MSCs and UC-MSCs) lineages. MSCs showed high expression of CD105, CD44 and CD90 and low or negative expression of CD34 and MHC-II. The MHC-II was not detected by immunocytochemistry techniques in any of the MSCs studied.

Conclusions: The BM, AT and UC are feasible sources for harvesting equine MSCs, and their immunophenotypic and multipotency characteristics attained minimal criteria for defining MSCs. Due to the low expression of MHC-II by MSCs, all of the sources could be used in clinical trials involving allogeneic therapy in horses. However, the BM-MSCs and AT-MSCs showed fastest ''in vitro'' differentiation and AT-MSCs showed highest cell growth until third passage. These findings suggest that BM and AT may be preferable for cell banking purposes.

Figure 1

MSCs from BM, AT and UC during cell culture showing ≥80% confluence. MSCs from BM (A), AT (B) and UC (C) with approximately 80% confluence after 11, 7 and 15 days of culture, respectively. (A) and (B) 100x magnification and (C) 200x magnification. AT, adipose tissue; BM, bone marrow; MSCs, mesenchymal stem cells; UC, umbilical cord.

Figure 2

Differentiation potential of MSCs from equine BM, AT and UC. Differentiation of MSCs from BM (A, D, G, J), AT (B, E, H, K) and UC (C, F, I, L) into three mesenchymal lineages during the third passage. (A-C) MSCs showing intracytoplasmic lipid droplets confirming the adipogenic lineage. (D-F) MSCs stained with Alizarin Red showing matrix calcium formation. (G-L) MSCs after chondrogenic differentiation stained with Alcian Blue (G-I) and toluidine blue (J-L) showing hyaline matrix. 100x (D-L), 200x (B) and 400x (A, C) magnification. AT, adipose tissue; BM, bone marrow; MSCs, mesenchymal stem cells; UC, umbilical cord.

Figure 3

Expression of cell surface markers by flow cytometry of MSCs from BM, AT and UC. Histograms representing the profile of BM-MSC, AT-MSC and UC-MSC samples analyzed using flow cytometry during the third passage, evaluating the cell surface markers CD90, CD44, CD105, CD34 and MHC-II. MSCs were positive for CD90, CD44 and CD105, were negative for CD34 and showed low expression of MHC-II. AT, adipose tissue; BM, bone marrow; MSCs, mesenchymal stem cells; UC, umbilical cord.

Figure 4

Evaluation of MHC-II expression in MSCs from BM, AT and UC using the immunocytochemistry technique. Immunocytochemistry of MSCs from equine BM (A), AT (B) and UC (C) during the third passage to evaluate the expression of the MHC-II cell surface marker. MSCs from the sources analyzed did not express MHC-II. 200x (A, B) and 100x (C) magnification. AT, adipose tissue; BM, bone marrow; MSCs, mesenchymal stem cells; UC, umbilical cord.