Figure 4. TAT-BMI-1 stimulation of CD34+ cell expansion in vitro.

(A) CD34⁺ cells (1–2 × 10⁴/ml) were plated in StemMACS HSC Expansion Medium with StemMACS HSC Expansion Cocktail. During the first 3 days of culture, TAT-BMI-1 or the control TAT-GFP protein were added 4 times a day at a 10 nM concentration. At the time intervals indicated, the cells were counted and re-plated in fresh medium at equal cell densities. Cumulative cell expansion is shown. All assays were performed in triplicate. (B) Colony-forming cell (CFC) assay was performed on cells from the cytokine-driven cultures at day 4, 11, 16 and 24. 100 (d. 4), 200 (d. 11), 300 (d. 16) or 600 (d. 24) cells/well were plated in triplicate assays in semisolid StemMACS HSC-CFU medium complete with cytokines. Colonies were scored after 2 weeks and total numbers of progenitors was normalized to the total cell number in the relevant culture at the time of plating. (C) Secondary colony assays were performed by collecting and re-plating in complete StemMACS HSC-CFU medium cells from primary colony assays (Figure 4B) set up with 100 cells/well from day 4 of liquid cultures. After 2 weeks colonies were scored for either BFU-E or CFU-GM morphology. A representative image of secondary colonies derived from TAT- GFP or TAT-BMI-1-treated cultures is shown. (D) Long term stromal co-cultures. 4 × 10³ cells/well (in triplicate), collected from cytokine-driven cultures after 3 days of treatment with the recombinant TAT-fusion proteins, were transferred onto monolayers of MS5 stromal cells and cultured in Myelocult medium with 1 μM hydrocortisone. At 13, 20 and 27 days the cultures were demi-depopulated, and the suspension-growing cells counted. The total numbers of cells/culture were calculated. (E) Colony-forming cell (CFC) assay were performed at day 13, 20 and 27 with cells collected from the MS5 stromal co-cultures. Colonies were scored as described above and total numbers of progenitors/culture were calculated based on the cell numbers of the relevant co-cultures. (F) Cobblestone area-forming cells (CAFCs) were scored in the stromal co-cultures after 5 weeks of culture. A cobblestone area (CA) is defined as a cluster of at least five small, non refractile cells that grow underneath or within the stromal layer. A representative image of cobblestone area derived from TAT-BMI-1 treated cells is shown. The average values of triplicate samples of representative experiments are shown in all panels, with the indication of SD values. T-test were performed to assess the statistical significance. *= P < 0.05; **= P < .005; ***=P < 0.0005