CD146⁺ perivascular cells but not MSCs sustain functional HSPCs with engraftment potential and self-renewal ability. (A) CFU assay revealed significantly higher number of CFUs in CD146⁺ cell cocultures after 1, 2, 4, and 6 weeks of coculture as compared with MSC cocultures (n = 3 independent experiments, each experiment was performed in triplicate; *P < .05, ***P < .001). (B) Representative flow cytometry analysis for the detection of human CD45⁺HLA⁺ cells in bone marrow of NSG mice 6 weeks posttransplantation with phosphate-buffered saline, or with the same number of CD45⁺ cells (10⁵) harvested after 2 weeks of CB CD34⁺ cell coculture with MSCs or CD146⁺ cells. (C) All mice injected with CD45⁺ cells obtained from CD146⁺ cell cocultures showed human engraftment whereas no engraftment was ever detected (ND) in mice that received MSC cocultures (n = 3 independent experiments, n = 11 mice per group; ***P < .0001). (D) Frequency of CD34⁺ progenitors, CD19⁺ lymphoid, and CD14⁺ myeloid cells within the CD45⁺HLA⁺ population of cells in the bone marrow of chimeric mice. (E) Human CD45⁺HLA⁺ hematopoietic cells were also detected 6 weeks posttransplantation in the contralateral tibia of mice injected with HSPCs cocultured with CD146⁺ perivascular cells. (F-I) Representative flow cytometry analysis of secondary host bone marrow. (F) Bone marrow from primary hosts transplanted with MSC coculture was injected in secondary hosts as a negative control. (G) Human engraftment was observed 4 weeks after secondary transplantation of bone marrow from chimeric mice transplanted with CD146⁺ cell coculture (n = 3 engrafted mice of 4). (H) Both CD19⁺ lymphoid and CD33/CD14/CD15⁺ myeloid cells were detectable within the human CD45⁺ engrafted hematopoietic cells in secondary hosts. (I) Quantification of the level of chimerism in secondary mice. All data are presented as mean ± SEM.