Figure 2.

CD146^hiCD73^hi Mesenchyme Supports Ex Vivo Maintenance of Clonogenic, Engraftable, and Self-Renewing HSPCs

CD146^hiCD73^hi and CD146^loCD73^lo mesenchymal cells generated from H1-derived hEMP were isolated and co-cultured as monolayers with CB CD34⁺ cells in 5% serum without added cytokines. After 2 weeks, hematopoietic cells were assayed (A–G) in vitro and (H–J) in vivo.

(A) Total number of live (DAPI^–) CD45⁺ cells recovered from co-cultures, shown as cell yield normalized to 1,000 input CD34⁺ cells.

(B) Representative FACS analysis of CD45⁺ gated cells from co-cultures.

(C–G) Cell yield of CD34⁺, CD34⁺ Lin⁻, CD14⁺ myeloid, CD10⁺/CD19⁺ B lymphoid cells, and CFU (after re-plating) recovered from 2-week CD146^hiCD73^hi co-cultures, normalized to CD146^loCD73^lo co-cultures.

(A and C–G) n = 16 independent experiments, each in duplicate. Shown are mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001; unpaired two-tailed Student's t test.

(H) Flow cytometry showing gating for the detection of human cells (hCD45⁺HLA ABC⁺) and multi-lineage reconstitution after primary transplant. PBS control with no cells injected.

(I) Proportion of mice with engraftment, i.e., detection of >1% human cells after primary transplant (1°) with 1 × 10⁵ CD45⁺ cells obtained either from fresh CB CD34⁺ cells (CB w/o culture), or 2 weeks after co-culture on CD146^hiCD73^hi or CD146^loCD73^lo.

(J) Engraftment (percent of human cells) of mice 6 weeks after primary and secondary transplants (n = 5 independent experiments, total 9–14 mice per group; ^∗∗∗p <.0001; ns, not significant).

Error bars represent SEM. See also Figure S3.