Clonal tracking of in vitro differentiation of human cord blood lympho-myeloid progenitors

Clonal tracking of lineage outputs from single cord blood progenitors was performed by fluorescence imaging over a 16-day time course. Twenty μL of lymphoid expansion medium (LEM) containing 160 ng/mL anti-CD34-AF647 (Biolegend), 50 ng/mL anti- CD45RA-BV421 (Biolegend) and 40 ng/mL anti-CLEC12A-PE (eBioscience) filtered through a 0.2 μm filter was dispensed into each well of a Terasaki plate (Greiner). Single input cells (CD45+CD34+CD45RA-CLEC12A-CD14-CD15-CD10-) were sorted into each well of the Terasaki plates preloaded with the LEM culture medium. Each Terasaki plate was then placed inside a 15 cm culture dish together with 4-5 open 35 mm dishes containing sterile water to retain a sufficiently humidified atmosphere within the Terasaki plates. On Day 8, 3.5 μL of filtered fresh media with the same antibodies were added to each well of the Terasaki plates. The plates were imaged on a Nikon A1-Si Confocal microscope every 2 days from Day 2 to Day 16. Brightfield and fluorescent images of each well containing a clone were captured using a 10x objective that covered >95% of the bottom area of the Terasaki wells, with the x, y and z positions on the microscope adjusted for each well to include all of the output cells. Cell numbers of each clone were then counted on the brightfield images using the “Cell Counter” function in ImageJ. The fluorescent signals emitted from each surface antibody were used to define the phenotypes of the clonal outputs.