V⁺P⁻ stromal cells promote MK expansion via a contact-dependent mechanism. (A) HSCs (50 SLAM cells) were plated in low-cytokine serum-free medium in the presence of increasing proportions (vol/vol) of conditioned medium from a V⁺P⁻ stromal cell layer. The bar graph represents the number of MKs produced per 50 SLAM cells plated. The mean values ± SD from 2 independent experiments were not statistically different. (B) Coculture of HSC and stromal layers were established in a noncontact setup using Transwell insert (V⁺P⁻/TW) with 0.3-μm pores and analyzed for MK content after 13 days of coculture in comparison with cultures without stroma and cocultures with a V⁺P⁻ layer. Values represent mean ± SD of MKs produced per 50 SLAM cells from 3 independent experiments. *P < .01. (Ci) Phase contrast image of a cluster of large cells adherent on the stromal layers observed only during the coculture of HSC with V⁺P⁻–derived stromal layers. Representative image from a day 9 coculture is presented. Scale bar represents 100 μm. (Cii) Immunofluorescence analysis of the expression of CD42c (red) by the large cells forming clusters on V⁺P⁻ stromal layers identified by the expression of VCAM-1 (green). Scale bar represents 30 μm. (D) Expression of Jag1, Jag2, Dll1, Dll4, TPO, and GAPDH transcripts in fetal liver stromal cells from adherent layers. Figures on the left side of the panel indicate amplicon size.