Culturing of Human Mesenchymal Stem Cells as Three-dimensional Aggregates Induces Functional Expression of CXCR4 That Regulates Adhesion to Endothelial Cells

Ira S Cohen,Sergey V Doronin,Irina A Potapova,Peter R Brink

doi:10.1074/jbc.m800184200

Abstract

Culture-expanded human mesenchymal stem cells (hMSCs) are increasingly used in a variety of preclinical and clinical studies. However, these cells have a low rate of engraftment to bone marrow or damaged tissues. Several laboratories have shown that during isolation and subculturing mesenchymal stem cells quickly lose the expression of CXCR4, the key receptor responsible for lymphocytes and hematopoietic stem cell homing. Here we show that culturing of hMSCs as three-dimensional aggregates (hMSC spheroids) restores CXCR4 functional expression. Expression of CXCR4 inversely correlates with the secretion of SDF-1 by hMSCs. Cells from hMSC spheroids up-regulate expression of CD49b, the alpha2 integrin subunit, and suppress the expression of CD49d, the alpha4 integrin subunit. Transfer of cells from the spheroids back to a monolayer suppresses the expression of CXCR4 and CD49b and restores the expression of CD49d. Treatment of cells from the spheroids with SDF-1 leads to CXCR4 internalization and activation of ERK-1,2. Adhesion of hMSCs to human umbilical vein endothelial cells (HUVECs) was investigated. SDF-1, AMD-3100, or exposure of HUVECs to hypoxia did not affect adhesion of hMSCs from a monolayer to HUVECs. Adhesion of cells from hMSC spheroids to HUVECs was stimulated by SDF-1, AMD-3100, or by exposure of HUVECs to hypoxia. Stimulatory effects of hypoxia and addition of SDF-1 or AMD-3100 were not additive. Overall, our data indicate that the expression of CXCR4 by hMSCs regulates hMSC adhesion to endothelial cells.

Highlights

Non-mesenchymal lineages [2, 3]
Cell Culture— human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) were purchased from Cambrex BioScience. hMSCss were cultured at 37 °C in a humidified atmosphere of 5% CO2 in mesenchymal stem cell growth medium (Lonza)
We tested expression of 19 markers commonly used to characterize hMSCs by flow cytometry. hMSCs from a monolayer and the spheroids were positive for CD29, CD44, CD54, CD55, CD73, CD90, CD105, CD166, and HLA-I

Summary

EXPERIMENTAL PROCEDURES

Cell Culture— hMSCs and HUVECs were purchased from Cambrex BioScience. hMSCss were cultured at 37 °C in a humidified atmosphere of 5% CO2 in mesenchymal stem cell growth medium (Lonza). Bovine serum albumin was removed, slides were washed again with PBS, and cells were stained overnight at 4 °C with 1 ␮g/ml anti-human CXCR4 antibody (R&D Systems; catalog number MAB172). Slides were washed three times with PBS ated using 0.25% trypsin-EDTA solution (Lonza) for 15 min in and mounted on coverslips in VectaShield mounting medium the case of hMSC monolayers or for 90 min in the case of the containing 4,6-diamidino-2-phenylindole Isolated cells were washed with PBS, Immunofluorescence was analyzed by deconvolution labeled with 2 ␮g/ml Calcein AM in HBSS for 45 min at 37 °C in microscopy using an Axiovert 200 M fluorescence microscope a humidified atmosphere of 5% CO2, washed with HBSS, and hMSC Adhesion to Endothelial Cells resuspended in DMEM supplemented with 5% fetal bovine serum. Data from five independent preparations of hMSC spheroids and four independent preparations of hMSCs grown as a monolayer were statistically processed with the SigmaStat software package (Sigma)

RESULTS

C F-Actin

DISCUSSION