Figure 2. BM endothelial cells promote expansion of hematopoietic progenitor cells by a Notch dependent mechanism.
(A) CD34+ cells were grown in liquid culture on plastic alone in the presence of cytokines (CO) or on BMEC monolayers, in the absence or presence of GSI (2uM). After 5 days, cells were harvested, washed and plated in quadruplicate, at a density of 5,000 cells/ml, in methylcellulose supplemented with IL-3, SCF and EPO. Bar values indicate the average of 3 experiments. CFCs are expressed as number of colonies per 1000 cells seeded. Error bars represent standard error. Differences between the populations (BMEC vs CO; BMEC vs BMEC+GSI) are statistically significant p < 0.02. (B) CD34+ cells cocultured with BMEC-GFP or BMEC-J2 in the presence or absence of GSI (2uM) were harvested at day 5 of co-culture and plated in triplicate at the density of 5000 cells/ml in methylcellulose, as described above. Bars represent the average of 4 experiments. CFCs are expressed as number of colonies per 1000 cells seeded. Error bars represent standard error. Differences between populations (BMEC-J2 vs BMEC-GFP and BMEC-J2+GSI) are statistically significant p < 0.001; (C) RNAs from CD34+ cells cultured alone or in the presence of BMEC and BMEC-J2, in the presence or absence of GSI, were used for RT-PCR amplification of the Hes1 gene. The two blots are representative of two independent experiments. (D–E) CD34+ cells where incubated with Fc fragment (control), Dll4-Fc or anti-N1 antibody for one hour prior to seeding into BMEC monolayers. (D) Expression of activated Notch in CD34+ progenitors. CD34+ cells in the different conditions were harvested after 24 hours of coculture. Cell extracts were analyzed by immunoblot by using a Notch antibody detecting the activated form of Notch. Bar graphs represent ImageQuant densitometric analysis of Notchic protein level. Values indicate percentage of Notchic detected following incubation with N1ab or Dll4-Fc relative to the Fc control (100%). Notch densitometric values were normalized with β-actin levels. (E) CD34+ cells were harvested at 72 hours of co-culture and seeded at 3000/ml in methylcellulose. Bar graphs represent two independent experiments. Bars values are average of 4 wells and represents number of colonies, CFCs, per 1000 cells seeded. Error bars represent standard error.