Double labelling of human umbilical cord mesenchymal stem cells with Gd-DTPA and PKH26 and the influence on biological characteristics of hUCMSCs

Abstract

The aim of this study was to determine whether double labelling of human umbilical cord mesenchymal stem cells (hUCMSCs) with gadolinium-diethylene triamine penta-acetic acid (Gd-DTPA) and PKH26 influences their biological characteristics. A tissue adherence technique was used to separate and purify the hUCMSCs and flow cytometry was performed to detect the surface markers expressed on them. Gd-DTPA and PKH26 were used to label the stem cells and MRI and fluorescence microscopy were used to detect the double-labelled hUCMSCs. A MTT assay was used to delineate the growth curve. Transmission electron microscopy (TEM) and atomic force microscopy were used to demonstrate the ultrastructural features of the hUCMSCs. Flow cytometry showed that hUCMSCs highly expressed CD29, CD90, CD44 and CD105. No expression of CD31, CD34 and CD45 was detected. Very low expression of HLA-DR and CD40 was detected. Atomic force microscopy showed these cells were long, spindle shaped, and the cytoplasm and nucleus had clear boundaries. After double labelling, TEM showed Gd particles aggregated in the cytoplasm in a cluster pattern. The proliferation activity, cell cycle, apoptosis and differentiation of the stem cells were not influenced by double labelling. Thus a tissue adherence technique is helpful to separate and purify hUCMSCs effectively; and Gd-DTPA and PKH26 are promising tracers in the investigation of migration and distribution of hUCMSCs in vivo.

Keywords: Gd-DTPA; PKH26; Tracing; magnetic resonance imaging; mesenchymal stem cells.

Figure 1

Morphology of hUCMSCs. (a) Inverted phase contrast microscopy (40×) 24 h after culture of third generation hUCMSCs; (b) Ultrastructure of third generation hUCMSCs under a transmission electron microscope (4800×); (c) Ultrastructure of third generation hUCMSCs under an atomic force microscope.

Figure 2

Expression of hUCMSC surface markers. hUCMSCs did not express CD31, CD34 or CD45, but had high expression of CD29, CD105, CD44 and CD90. In addition, low expression of HLA-DR and CD40 is detected.

Figure 3

MRI of different numbers of Gd-DTPA-labelled hUCMSCs. 1.5T MRI showed that Gd-DTPA-labelled hUCMSCs had hyperintensity on T1WI and T2WI. MRI showed that the intensity on T1 was reduced with the reduction in cell number. When 5 × 10³ cells were subjected to MRI, hyperintensity was not observed on T1.

Figure 4

MRI of Gd-DTPA-labelled hUCMSCs. (a) and (b) show that the intensity changed with the increase in cell passage in T1WI and T2WI::passage1 (P1), passage2 (P2), passage3 (P3), passage4 (P4), and negative control (NC).

Figure 5

hUCMSCs labelled with PKH26 under a laser scanning confocal microscope. (a) Shows that the intensity of red fluorescence on the cell membrane was gradually reduced and granule-like spots were observed with the growth of hUCMSCs. (b) Shows that the intensity of red fluorescence on the cell membrane was gradually reduced with the prolongation of time to PKH26 staining.

Figure 6

hUCMSCs under a transmission electron microscope. (a) and (b) show that hUCMSCs without labelling or with Gd-DTPA respectively. Arrow: Gd granules (14800).

Figure 7

Analysis of cell cycle activity by flow cytometry. The proportion of cells in S and G2/M phases (G2/M+S%) was more than 30%. The percentage of cells in different phases was similar between unlabelled cells and double-labelled cells.

Figure 8

Analysis of cell apoptosis by flow cytometry. The proportion of apoptotic cells was the same in both unlabelled cells and double-labelled cells.

Figure 9

(a) and (b) Oil red O staining for unlabelled cell and labelled cells after adipogenic induction for 21 days (200×). (c) and (d) alizarin red staining for unlabelled cell and labelled cells after osteogenic induction for 28 days (200×).