Anti-CD48 monoclonal antibody, clone C444 (6-5.9)

Transforming growth factor beta (TGF-beta) is well known for its important function in hematopoietic stem cell (HSC) quiescence. However, the molecular mechanism underlining this function remains obscure. Endoglin (Eng), a type III receptor for the TGF-beta superfamily, has been shown to selectively mark long-term HSCs; however, its necessity in adult HSCs is unknown due to embryonic lethality. Using conditional deletion of Eng combined with serial transplantation, we show that this TGF-beta receptor is critical to maintain the HSC pool. Transplantation of Eng-deleted whole bone marrow or purified HSCs into lethally irradiated mice results in a profound engraftment defect in tertiary and quaternary recipients. Cell cycle analysis of primary grafts revealed decreased frequency of HSCs in G(0), suggesting that lack of Eng impairs reentry of HSCs to quiescence. Using cytometry by time of flight (CyTOF) to evaluate the activity of signaling pathways in individual HSCs, we find that Eng is required within the Lin(-)Sca(+)Kit(+)-CD48(-) CD150(+) fraction for canonical and non-canonical TGF-beta signaling, as indicated by decreased phosphorylation of SMAD2/3 and the p38 MAPK-activated protein kinase 2, respectively. These findings support an essential role for Eng in positively modulating TGF-beta signaling to ensure maintenance of HSC quiescence.

WM-68 cell line has been shown to produce anti-CD48 antibody - highly specific to B cell malignancies. For their clinical usage, cultivation of WM68 cells in animal serum pose a technical and safety disadvantage. Therefore, the present study was aimed to establish an adaptation protocol for WM68 hybridoma cell line - from high to a low serum concentration and finally into a serum-free culture condition. The WM68 hybridoma cell line cultured in 10% FBS exhibited excellent cell culture profiles cell viability, density and antibody production. Similar trend was observed when WM68 cell line was cultured in 0.5% FBS. However, in 0.5% FBS, data revealed poor antibody production in the stationary phase compared to 10% FBS. Finally, WM68 cell line cultured in 0.5% FBS, was passaged and plated in a serum free condition. Unfortunately, cells didn't survive past day one and manifested obvious signs of apoptosis. Taken together, these data suggested that 10% FBS and 0.5% FBS are suitable culture conditions for the propagation of WM68 hybridoma cell line and to generate antibodies. Thus, employing additional media factors and avant-garde cell culture techniques can enhance our understanding of the WM68 cell line. culture requirements in a serum free media.