Coupling transcriptomic data with cellular immunophenotypes
To evaluate the surface immunophenotypes for Drop-seq clusters, we sequenced the transcriptomes for 96 mast cell progenitors (CD34+ CD117+ FcεRI+) and 1,835 canonically defined LMPPs (CD34+ CD38- CD45RA+) from indexed FACS sorting. The raw data processing and quality control were the same as described for Drop-seq data. To associate FACS-sorted cells with cells profiled with Drop-seq, we leveraged information from the reconstructed hierarchy with micro-clusters by building a random forest classifier. Briefly, we assigned a branch identity to each single cell from Drop-seq, based on its corresponding micro-cluster. A random forest classifier was then trained on these cells using the ranger() function in R with default parameters. We used the 505 ‘branch-dependent’ genes for classifier construction. We then applied the classifier on FACS-sorted cells to reveal the transcriptomic states, and compared protein signals for surface markers with indices recorded on the sorter (CD10, CD52, CSF3R, CD38, CD45RA and CD62L for LMPP, and CD71 for mast cell progenitors).
In vitro differentiation with MS5-MBN assay
Mouse MS5 cells were treated with Mitomycin C for three hours to inhibit mitotic proliferation, and seeded on 0.02% gelatin-coated 96-well cell culture plates 24 hours before human cell sorting to ensure attachment. Approximately 10^4 MS5 cells were plated per well, in H5100 media. Human CD34+ cells were enriched from three separate cord blood units as described above, using Ficoll density centrifugation and magnetic separation. Each cord blood unit was processed individually. 24 hours after seeding MS5 cells, we replaced the media with fresh H5100 media supplemented with cytokines. Enriched CD34+ cells were stained with the following antibodies: CD34-APC, CD38-Alexa Fluor 700, CD45RA-APC Cy7, CD10-BV 421, CD52-FITC and CD114 (CSF3R)-PE, with concentrations determined from titrations, and 250-300 cells (CD34+ CD38- CD45RA+ CD10- CD52+ CSF3R-, or CD34+ CD38- CD45RA+ CD10- CD52- CSF3R+) were sorted into each well containing attached MS5 cells. Cell culture was maintained in CO2 incubator for three weeks, with weekly change of half the cytokine-supplemented media. At the end of the third week, all cells from each well were harvested by pipetting and stained with the following antibodies: human CD45-AF700, CD19-FITC, CD11b-APC Cy7 and CD56-PE, and resuspended in MACS buffer (PBS + 0.2% BSA) with DAPI before flow cytometry. Live human cells were identified as DAPI- hCD45+, and differentiation output were evaluated as CD19+ B cells, CD56+ CD11b- NK cells and CD56- CD11b+ myeloid cells within the live human cell gate.