In vivo clonal tracking of human skeletal stem cells with single cell RNA-sequencing readout

10X Genomics single cell RNA-sequencing of freshly isolated human skeletal stem cells from the growth plate or periosteum of 17 weeks gestational age fetal bone tissue were collected. The goal of this work was to trace single cell dynamics of human skeletal stem cells in an in vivo setting. Single barcoded human skeletal stem cells were tracked in the graft tissue they formed after xenotransplanation into mice. Single cell analysis assessed tissue composition and was overlayed with barcode readouts to find contributions of individual clones. Differences in stem cell properties and lineage dynamics were assessed between growth plate and periosteal human skeletal stem cells. 1.5-2x10 5 freshly sorted cells of either growth plate (GP) or periosteal (PE) source tissue were seeded into 12-well plate with 1 mL of Opti-MEM Reduced Serum Media containing lentiviral barcode particles generated from CloneTracker XP™ 10M Barcode-3' Library in pScribe5-Venus-Puro plasmid (Cellecta, Cat#BCXP10M3VP-P). Cells were spun in the well plate in plate carriers at 1,500g for 60 min at 24–27 °C. Directly following that step, 3 mL of fresh culture media was added and cells were incubated at 37 °C for 24 h. At this point, the media was changed and cells allowed to further incubate for three days. Cells were then lifted using collagenase and stained with FACS antibodies. Sorted cells with phenotypic hSSC surface marker profile and simultaneous expression of Venus fluorophore were used for renal transplantation assays into NSG mice. To prevent dilution of clones in final readout, 500 Venus+, barcoded hSSCs were mixed with 5,000 Venus-, non-barcoded hSSC supporter cells of the same source tissue, mixed with 5 µL of Matrigel and transplanted below the renal capsule of mice. Three transplants per hSSC source were conducted. Each mouse was transplanted with hSSCs from the growth plate under the renal capsule of one kidney and with hSSCs from the periosteum contralaterally. After three weeks, kidneys were excised from sacrificed mice and assessed for Venus expression in the graft tissue via microscope. Grafts were then dissected out, pooled by respective hSSC sources and processed for flow cytometric isolation of Venus+ cells. Sorted cells from grafts were processed for 10X genomics single cell RNA-sequencing for interrogation into cell fates and clonal relationships. In addition to 10X libraries (GP, PE) we sequenced specifically amplified barcode regions for GP (GPbc) and PE (PEbc) samples in order to extract clonal barcode information.