Abstract
Self renewal and differentiation of hematopoietic stem cells (HSC) are governed by interaction with the supportive microenvironment of the bone marrow. Secreted factors as well as specific cell adhesion proteins are involved in this interaction. As an in vitro model system, the hematopoietic microenvironment can be mimicked by supportive mesenchymal stromal cells (MSC).
We have compared the supportive potential of human MSC from bone marrow (BM) isolated under two different culture conditions (BM-MSC-M1 and BM-MSC-M2), from adipose tissue (AT) and umbilical cord blood (CB) that were all cultivated as described before (Wagner et al. Exp Hematol.2005;11:1402–1416.). As controls we have used the human fibroblast cell line HS68 and the murine fetal liver cell line AFT024. CD34+ cells were isolated from human cord blood and cultured in direct contact with irradiated stromal cells. After four, seven and twelve days the immunophenotype of the hematopoietic cells was analyzed by flow cytometry. Many progenitor cells cultured on BM-MSC or AFT024 maintained a primitive phenotype of CD34+/CD38- cells whereas the proportion of these cells was reduced upon cultivation with CB-MSC and cells cultured on AT-MSC and HS68 displayed a significantly higher expression of CD38 and lower expression of CD34. Furthermore, long term culture initiating-cell (LTC-IC) assays were performed on the different feeder layer. LTC-IC frequency was significantly higher on BM-MSC that were isolated under the two different culture conditions (BM-MSC-M1 1,15 ±0.11%; BM-MSC-M2 1.14±0.08%) and on CB-MSC (1.10±0.13%) compared to AT-MSC (0.32±0.09%) and HS68 (0.67±0.12%). We have compared gene expression profiles of BM-MSC-M1, BM-MSC-M2, CB-MSC, AT-MSC and HS68 by cDNA microarray analysis (51,144 different cDNA clones of the RZPD3 Unigene Set). Differential expression of various genes correlated with the observed differences in supportive potential. Among these were adhesion proteins like N-cadherin, cadherin11, fibronectin1, various integrins (ITGA1, ITGA5 and ITGB1) and VCAM1 as well as secreted proteins including osteonectin, CTGF and SDF1. Westerblot analysis verified on protein level that cadherin11, N-cadherin, and ITGB1 were highly expressed on BM-MSC as compared to AT-MSC and HS68 fibroblasts.
In conclusion MSC from human bone marrow or from umbilical cord blood support to a significantly higher degree the maintenance and proliferation of primitive hematopoietic progenitors than MSC derived from adipose tissue. This affinity correlated with up-regulation of cadherin11, N-cadherin and intergrin-beta1 on BM-MSC and CB-MSC.
MiRNA Profiles of Extracellular Vesicles Secreted by Mesenchymal Stromal Cells—Can They Predict Potential Off-Target Effects?
