Establishment of an adherent cell feeder layer from human umbilical cord blood for support of long-term hematopoietic progenitor cell growth.

Abstract

Previous attempts to establish a stromal cell feeder layer from human umbilical cord blood (HUCB) have met with very limited success. It has been suggested that there is an n ient number of stromal precursor cells in HUCB to form a hematopoletic-supporting feeder layer in primary cultures. The present study shows that HUCB does contain a significant accesory cell population that routinely develops into a confluent, adherent cell layer under defined primary culture conditions. HUCB-derived adherent layers were shown to support long-term hematopoletic activity for an average of 4 months. This was achieved by using a customized coverslip with a modified surface structure as the cell attachment substratum and using a specializ culture feeding regime. We have characterized the various cell types (including fibroblasts, macrophages, and endothelial cells) and extracellular matrix proteins (including fibronectin, collagen m, and laminin) that were present in abundance in the HUCB-derived adherent cell layer. In contrast, oil red O-staining fat cells were rarely detected. ELISA and bioassays showed that stem cell factor and interleukin 6 were produced by the HUCB stromal cell cultures, but interleukin 3 or granulocyte/macrophage colonystimulating factor was not detected. Application of this hemapoloetic culture system to transgenic and gene therapy studies of stem cells is dis s.

Establishment of an adherent cell layer from human umbilical cord blood

“…This finding differs from that reported by Ye et al (1994), although the reasons for this discrepancy are unclear. Although, with advancing age, marrow stromal cells can differentiate along an adipogenesis pathway, the exact function of these cells is still not clear (Gimble et al, 1995).…”

Recent advances into the understanding of mesenchymal stem cell trafficking

“…Wang et al (2006), on the other hand, reported a reduction in the circulating pool of MSC in patients 1-week after MI and suggested that the MSC may have been recruited to the damaged myocardium. There are also several reports of the derivation of MSC from circulating fetal blood or umbilical cord (Ye et al, 1994;Campagnoli et al, 2000Campagnoli et al, , 2001Erices et al, 2000), and of the presence of MSC in the peripheral blood of other species (Huss et al, 2000;Wu et al, 2003). The two studies by Mansilla et al (2006) and Wang et al (2006) suggest that injury/trauma might evoke the release of MSC; thus systemic administration of MSC might enhance the presence of the circulating MSC, which may ultimately lead to an enhanced therapeutic response if the cells can be efficiently targeted to the damaged tissues.…”

The hematopoietic stroma

“…The existence of stromal precursors in cord blood under normal conditions is the subject of intense discussion (58)(59)(60)(61)(62). A representative recent study (58), for instance, reports deficient myeloid progenitor cell growth in LTC of umbilical cord blood, suggesting that this is due to the impaired development of an adherent layer.…”

“…A representative recent study (58), for instance, reports deficient myeloid progenitor cell growth in LTC of umbilical cord blood, suggesting that this is due to the impaired development of an adherent layer. Under special conditions, Ye et al (59) and Nieda et al (60) reported the establishment of an adherent stromal layer (using a special substratum) and of an endothelial layer (in the presence of IL-2 and conditioned medium from the 5637 carcinoma cell line), respectively. We have recently described the establishment, under normal long-term culture conditions, of an adherent layer from human umbilical cord blood capable to support the proliferation of hematopoietic clonogenic cells (63; Alfonso ZZC, Forneck ED and Nardi NB, unpublished results).…”