Phenotypic Characterization of Adherent Cells Population CD34+ CD90+ CD105+ Derived from Wharton's Jelly

Abstract

BACKGROUND Mesenchymal stromal cells, MSCs, show expression of specific antigens on their surface. The aim of the study is to assess the phenotype of stem cells like isolated from the umbilical cord with respect to the presence of surface antigens CD34, CD90, and CD105 and differences in the expression of surface antigens in cells isolated from freshly sampled material in comparison with the phenotype of cells from in vitro culture. MATERIAL AND METHODS Stem cells collected from the umbilical cord from healthy patients and then cultured in vitro. To assess the phenotype of stem cells, cytometric analysis was carried out. To assess the phenotype of cells we used fluorescently labelled monoclonal antibodies: APC Mouse anti-human CD34, PC5 Mouse anti-human CD90 and PE Mouse anti-human CD105. RESULTS In the case of cells from the umbilical cord and then cultured in vitro for the period of 10-14 days CD34 expression is lower (69,5%) in comparison with the group of cells not cultured. Not cultured cells were demonstrated 37% of cells co-expression of antigens CD34 and CD105, over 21% of CD34/CD90 cells and over 24% of CD105/CD90. Cultured cells group was showed higher percentage of CD90, CD105, CD34/CD105, CD34/CD90, CD105/CD90 in comparison with not cultured cells. CONCLUSIONS Our reults suggested that adherent cells population from umbilical cord, demonstrate CD34 expression In vivo. Moreover, the phenotype of MSCs, mainly in the context of CD34 expression, may vary depending on the place of collection of cells and the length of growing the cell culture.

Figure 1

(A) Microscopic image of sample 01WJC-CC from Wharton’s jelly. The photograph was taken on day 9 (magnification 200×, eyepiece 10×, lens 20×) using an Olympus CKX 41 inverted microscope and Olympus XC50 camera. (B) Microscopic image of sample 02WJC-CC from Wharton’s jelly. The photograph was taken on day 10 (magnification 200×, eyepiece 10×, lens 20×) using an Olympus CKX 41 inverted microscope and Olympus XC50 camera. (C) Microscopic image of sample 04WJC-CC from Wharton’s jelly. The photograph was taken on day 14 (magnification 200×, eyepiece 10×, lens 20×) using an Olympus CKX 41 inverted microscope and Olympus XC50 camera.

Figure 2

Dot-plot charts of cell morphology and distribution of cells in terms of intensity of expression of surface antigens CD34, CD105, and CD90 in the analyzed 01WJC-CC sample on day 10 of cell culture – cells isolated from Wharton jelly and then cultured in vitro – test group WJC-CC. (A) Dependence of the analyzed cells on their shape and granularity: FSC-Forward Scatter/SSC-side scatter. (B) CD105 vs. SSC: R49 CD105+. (C) CD34 vs. SSC: R1 CD34+. (D) CD90 vs. SSC: R50 CD90+. (E) CD90 vs. CD105: R38 CD90−/CD105+; R39 CD90+/CD105+; R41 CD90+/CD105−, R41: CD90−/CD105−. (F) CD34 vs. CD105: R2 CD34−/CD105+; R3: CD34+/CD105+; R5: CD34+/CD105−; R4: CD34−/CD105− (G) CD90 vs. CD34: R30 CD90−/CD34+; R31 CD90+/CD34+; R33 CD90+/CD34−; R32 CD90−/CD34−. The analysis and chart were made using Summit™ Software on a digital flow.

Figure 3

Dot-plot charts of cell morphology and distribution of cells in terms of intensity of expression of surface antigens CD34, CD105, and CD90 in the analyzed 06WJC– cells isolated from Wharton’s jelly – test group WJC. (A) Dependence of the analyzed cells on their shape and granularity: FSC-Forward Scatter/SSC-side scatter. (B) CD34 vs. SSC: R1: CD34+. (C) CD34 vs. CD105: R2 CD34−/CD105+; R3: CD34+/CD105+; R5: CD34+/CD105−; R4: CD34−/CD105−. (D) CD90 vs. CD34: R30 CD90−/CD34+; R31 CD90+/CD34+; R33 CD90+/CD34−; R32 CD90−/CD34−. (E) CD90 vs. CD105: R38 CD90−/CD105+; R39 CD90+/CD105+; R41 CD90+/CD105−, R41: CD90−/CD105−. The analysis and chart were made using Summit™ Software on a digital flow cytometer with a MoFlow XDP Beckman Coulter cell sorter.

Figure 4

(A) Frequency distribution of CD34, CD105, and CD90 on the cell surface, depending on the analyzed group (WJC, WJC-CC). The graph shows the average percentage of (±SD) cells demonstrating CD34, CD105, and CD90 expression in the test group. (B) Mean percentage of (±SD) cells demonstrating co-expression of CD34 and CD105, CD34 and CD90, CD90 and CD105 in the test group (n=5; * p<0,05; ** p<0.01; *** p<0.001 WC vs. CC, t test; Pearson’s r correlation coefficient).

Figure 5

(A) Photographs of single samples of cells from the WJC group, presenting microscope image (BF) and fluorescence in channels, showing the expression of studied antigens. SSC-side scatter – assessment of cell morphology. The analysis and photographs were taken using the FlowSight f. Amnis flow cytometer. (B) Photographs of single samples of cells from the analyzed WJC-CC group, illustrating microscope image (BF) and fluorescence in channels showing the expression of the studied antigens. SSC-side scatter – assessment of cell morphology. The analysis and photographs were made using the FlowSight f. Amnis flow cytometer. (C) Photographs of single samples of cells from negative control from the analyzed WJC group, presenting microscope image (BF) and fluorescence in channels showing the expression of studied antigens. SSC-side scatter – assessment of cell morphology. The analysis and photographs were made using the FlowSight f. Amnis flow cytometer. (D) Photographs of single samples of cells from negative control from the analyzed WJC-CC group, illustrating microscope image (BF) and fluorescence in channels showing the expression of studied antigens. SSC-side scatter – assessment of cell morphology. The analysis and photographs were made using the FlowSight f. Amnis flow cytometer.