CCL28 directly stimulates putative human HSCs and improves the long-term repopulating activity. (A) Representative histogram plots for cell surface expression of CCR3 and CCR10 in different fractions of CB (n = 5). FMO, fluorescence minus one; MNC, mononuclear cells. (B-C) CD34-enriched CB cells were plated in limiting dilutions either at day 0 or day 7 after culture in S10 and the indicated cytokines. Shown are LTC-IC numbers (B) and frequencies (C) as calculated by L-Calc (n = 3). (D-F) CB CD34^hiCD38^loCD90⁺CD45RA^– cells were cultured in S10 and the respective cytokines and monitored for proliferation and progenitor activity. (D) Representative bright-field microscopy images (i) and FACS plots (ii) of the progeny from 1 × 10² CD34^hiCD38^loCD90⁺CD45RA^– cells at day 10. (E-F) Total cell number (E) and frequency (F) of CD34^–, CD34⁺CD90^–, and CD34⁺CD90⁺ cells as determined by FACS analysis at days 10 to 13 (n = 3). (G) Numbers of differential CFCs per an equivalent of 100 input CD34^hiCD38^loCD90⁺CD45RA^– cells at day 0 (n = 4). Statistical significance was calculated on total CFCs. Delta (Δ) indicates the difference between TPO and CCL28 treatment. (H-K) CD34^hiCD38^loCD90⁺CD45RA^– cells were transplanted either directly or after 7 days of culture with the indicated cytokines into sublethally irradiated NSG recipients. Engraftment levels of human cells in peripheral blood after 1 (H) and 4 (I) months are shown. The dashed line in (H) marks the 1% cutoff for positive engraftment. Each data point represents an individual mouse; shown is data from 3 independent experiments with 3 to 5 recipients per group. Experiment 1: 1 × 10³ IEM; experiments 2 and 3: 2 × 10³ input equivalents per mouse. (J) Collated data showing recipient means across all experiments. (K) Lineage distribution in peripheral blood of NSG recipients 4 months post transplantation. *P < .05; **P < .01, ***P < .001.