Flow cytometry sorting of TAS2R46 positive human bone marrow mononuclear cells.

<p>(A) Approximately 10 milliliters of fresh human bone marrow was subject to Ficoll separation. The mononuclear cells were stained with rabbit anti-TAS2R46 and appropriate secondary antibody. TAS2R46 high sorted cells (roughly 705,000 cells) were placed into hMSC growth medium and incubated at 37C, 5% O₂, 5% CO₂. The resulting hMSC culture from donor 1 expanded within 1 week post-sort and shown in A. The TAS2R46 negative/low fraction did not result in any hMSC outgrowths. Crystal violet staining of hMSC was performed 2 weeks after isolation. Tri-differentiations were performed three weeks after isolation and stained appropriately. Scale bars represent 100 microns. (B) Bone marrow mononuclear cells were isolated and costained for TAS2R46 and classical markers of MSC: CD105, CD90, and CD73. Expression of each of these markers is shown with respect to prior gating on TAS2R46 high versus TAS2R46 negative/low expression. (C) Example of sorting strategy into populations of high to low TAS2R46 expression for two other bone BMMC donors. (D) Enumeration of MSC counted at day 7 of culture expansion normalized to the input number of sorted BMMC. Note P1 of all three donors gave rise to no MSC.