A STUDY IF THE EFFECTS OF EXTRACELLULAR MATRIX ON HEMATOPOIETIC CELLS

Abstract

In this study, some biological characteristics of mononuclear cells (MNCs) from umbilical cord blood, and the role of the extracellular matrix (ECM) in hematopoiesis were studied. The ECM investigated were collagen I, laminin, fibronectin, thrombospondin, chondroitin sulfate, heparan sulfate and hyaluronic acid. Firstly, in the study of the biological characteristics of MNCs, it was found that cryopreservation resulted in a significant loss of total viable and progenitor cells. The frequency of CD34+ cells was found to be 1.2 ± 0.5%, which is comparable to that reported in bone marrow MNCs. Also, when MNCs were cultured in agar with the growth factors interleukin-3 (IL-3), erythropoietin (Epo) and granulocyte-macrophage colony stimulating factor (GM-CSF), 62.7 ± 14.7 colonies/105 MNCs were observed. Secondly, the ability of the ECM to bind growth factors was investigated. Laminin was found to bind stem cell factor (SCF) and IL-3 at 0.3ng growth factor/µg ECM, whilst fibronectin bound SCF and IL-3 at 0.9ng growth factor/µg ECM. None of the ECM studied bound IL-6, GM-CSF and Epo. Thirdly, the effects of the ECM and growth factors on the development of MNCs were studied in semi-solid agar and liquid culture systems. In each system, ECM was presented to the cells by coating onto the culture surface and by incorporating in the media. In agar culture with the ECM coated with and without growth factors, many of the ECM inhibited colony formation. Laminin and fibronectin completely inhibited colony formation at 10 µg/culture and 1 µg/culture respectively. Conversely, when the ECM was incorporated into agar with and without growth factors, there were some enhancement and little inhibition of colony formation. Laminin and fibronectin each at 1 µg/culture stimulated week-I progenitor differentiation by 62% (p < 0.005) and 77% (p<0.025) respectively. In liquid culture with growth factors and with the ECM coated, all the components except laminin, enhanced the proliferation of less mature over the more mature progenitors. Whereas when the ECM was incorporated into media with growth factors, there were some enhancement and little inhibition of the proliferation of both the less and more mature progenitors. In both culture systems, the main effect of the ECM when coated was one of restraining progenitor cell development. Conversely, when the ECM was incorporated in media, some enhancement and little inhibition were observed. We propose that this difference is the result of aggregate formation when the ECM was coated, but not when it was soluble. The ECM aggregates exert their inhibitory effects on cell development by sequestering growth factors at the culture surface, and preventing their availability to cells. When the ECM was free in media, it largely exists as individual molecules which can still bind growth factors, but the ECM-growth factor complex is now able to present itself to cells, resulting in a general stimulation of cell development.