A high-content growth factor screen in human CB progenitors identifies CCL28 as potent stimulator of HSPC proliferation. (A) Outcome of the primary screen. Proliferation of cells cultured in S10 was set to 1 and relative fold increase of CD34⁺ cells was determined after 7 days of culture. The mean values from 2 screens are shown. The experimental design of the primary screen is shown as an overlay. (B) Outcome of the validation. Plotted are screening and validation results from 36 selected candidate factors (n = 4). Shown are floating bars from the minimum to the maximum, the line indicates the mean. (C) CB CD34⁺ cells were cultured in S10 or S100 together, with increasing concentrations of TPO or CCL28, and analyzed for proliferation and CD34 expression by FACS at day 7 (n = 3-4). After titration, CCL28 was used at 500 ng/mL. (D) FL- and BM-derived CD34⁺ cells were cultured in S10 and analyzed for proliferation and CD34 expression by FACS at day 7 (n = 2-3). Error bars represent SD. (E) Numbers of total CFCs per an equivalent of 100 input CB CD34⁺ cells at day 0 (n = 3). (F-G) Proliferation (F; n = 7) and CFC (G; n = 5) potential of CB progenitors cultured in single cytokine stimulation for 7 days. (H) Gene set enrichment analysis results for cell cycle and cell death signatures in SCF vs SCF + CCL28 conditions. NES, normalized enrichment score; NOM P, nominal P value; FDR, false discovery rate. (I-J) Representative cell cycle (I) and apoptosis (J) FACS plots of CD34⁺-gated cells. (K-L) Quantification of cell cycle (K) and apoptosis (L) data (n = 5). The dashed lines in figures (C), (D), and (F) indicate input cell numbers; black and gray bars represent CD34⁺ and CD34^– cells, respectively. *P < .05; **P < .01; ***P < .01; ns, not significant; na, not applicable.