Comparison of proliferative and multilineage differentiation potentials of cord matrix, cord blood, and bone marrow mesenchymal stem cells

Abstract

Background: Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are the two widely studied and characterized adult stem cells. Thus far, MSCs were obtained from the bone marrow, which is a painful procedure. Therefore, MSCs from less common sources like cord blood, adipose tissue, tooth pulp, and so on, have been the subject of research. The purpose of this study is to explore the possibility of finding MSCs from a less controversial, easy, and abundant source, such as the umbilical cord, for potential regenerative medicine applications.

Study design and methods: Five bone marrow samples (BM), seventy cord blood units (CB), and four umbilical cord matrix (CM) samples have been used for the study. Expanded MSCs were checked for biomarker expression by flow cytometry and were also checked for their differentiation to mesodermal and ectodermal lineages.

Results: MSCs could be isolated from 100% BM and CM samples, as compared to only 6% of CB samples. The fold expansion of the mesenchymal stem cells observed in CB (CB-MSCs) was distinctly higher as compared to BM (BM-MSCs) and CM (CM-MSCs). MSCs isolated from all the three sources expressed a characteristic mesenchymal phenotype of CD45 - /vWF - /CD14 - /CD31 - /CD73 + /CD105 + /SSEA4 + /CD29 + /CD44 + /HLAABC +, whereas, the HLA DR was conspicuously absent in CM-MSCs and CB-MSCs. Although osteogenic, chondrogenic, and neural differentiation was observed in MSCs from all sources, adipogenic differentiation was observed only in BM-MSCs.

Conclusion: CM-MSCs are a dependable source of an unlimited number of MSCs for autologous and allogenic use in regenerative medicine.

Keywords: Mesenchymal stem cells; bone marrow; umbilical cord; umbilical cord blood.

Figure 1

Morphology of the mesenchymal stem cells from different adult sources. (a) BMMSC formed colonies on adhering to the surface; (b) And grew as a monolayer of cells; (c) CBMSCs grew a monolayer of fibroblast like cells; (d) CM-MSC formed a monolayer of cells by day 25

Figure 2

Growth kinetics of the Mesenchymal stem cells from different sources. (a) Fold expansion of MSCs isolated from BM ranged from 1.2 to 4.3 fold; (b) CBMSC showed an expansion of 4 to 22.5 fold and; (c) CM-MSCs showed an expansion of 6.6 to 19.1 fold. Average time between two passages ranged between 7-8 days and was consistent for all the different types of MSCs

Figure 3

Immunophenotyping of the expanded BM-MSCs by flow cytometry. The expanded BM MSCs were negative for haematopoeitc marker and strongly expressed the mesenchymal markers. The phenotype expressed by the expanded cells was CD73 + /CD105 + /CD29 + /CD44 + /SSEA4 + /HLA ABC + /HLA DR + /CD45 − / CD14 − /CD31 − /vWF −

Figure 4

Immunophenotyping of the expanded CB-MSCs by flow cytometry. The expanded CBMSCs were negative for haematopoeitc marker and strongly expressed the mesenchymal markers. The phenotype expressed by the expanded cells was CD73 + /CD105 + /CD29 + /CD44 + /SSEA4 + /HLA ABC + /HLA DR − /CD45−/CD14 − /CD31−/vWF −

Figure 5

Immunophenotyping of the expanded CM-MSCs by flow cytometry. The expanded CM-MSCs were negative for haematopoeitc marker and strongly expressed the mesenchymal markers. The phenotype expressed by the expanded cells was CD73 + /CD105 + /CD29 + /CD44 + /SSEA4 + /HLA ABC + /HLA DR-/CD45-/CD14-/CD31-/vWF-

Figure 6

Differentiation of mesenchymal stem cells to osteocytes. Von kossa staining of (a) BM-MSC; (b) CB-MSC; (c) CM-MSC

Figure 7a

(a) Differentiation of mesenchymal stem cells to chondrocytes. Immunohistochemical staining of pellet cultures from different sources for detection of proteoglycans. (a) Alcian blue staining

Figure 7b

Differentiation of mesenchymal stem cells to chondrocytes. Immunohistochemical staining of pellet cultures from different sources for detection of proteoglycans. (b) Safranin O staining

Figure 8

Differentiation of mesenchymal stem cells to adipocytes. (a) Adipogenesis of BMMSCs was detected by the presence of lipid vacuoles; (b) which stained positive with oil red O

Figure 9

Neuronal differentiation of expanded BM-MSCs. Differentiated BM-MSCs expressed neural specific markers such nestin, beta tubulin, neuN, TH, NF-70

Figure 10

Neuronal differentiation of expanded CB-MSCs. Differentiated CBMSCs expressed neural specific markers such nestin, beta tubulin, NF-70, neuN

Figure 11

Neuronal differentiation of the expanded CM-MSCs. Differentiated CM-MSCs expressed neural specific markers such as beta tubulin, NeuN, GFAP, TH, NF-70

Figure 12

Soft agar assay. MSCs from all the three sources did not form colonies in soft agar assay confirming that they do not possess tumorogenic properties