Single Cell Transcriptome Mapping of Human Thymopoiesis Reveals a Continuum of Cell States During T-Lineage Specification and Commitment

Single cell studies elucidating the transcriptional continuum underpinning lineage commitment in the human bone marrow (BM) have advanced the understanding of hematopoiesis beyond the classic hierarchical model. However, T-lineage commitment, which occurs in the thymus remains incompletely defined. We mapped single cell transcriptomes spanning post-natal human thymopoiesis including the earliest progenitors. Instead of previously described discrete populations, transcriptional and lineage assay data resolved a continuum of novel cell states within CD34+ thymic progenitor cells. The initial stages of thymopoiesis involve multilineage priming of single cells followed by a gradual transition to T-commitment, a continuum previously undescribed outside the BM. Early progenitors express a hematopoietic stem cell (HSC) like profile but unlike HSC show initiation of T-priming. Species related differences exist in the earliest progenitor cells. Overall design: We performed single cell RNA-Seq on CD34+CD4-CD8-GlyA- progenitor (CD34+) and CD34-CD45+GlyA- differentiated (CD34-) cells isolated from human post-natal thymus by magnetic column separation followed by fluorescence activated cell separation (FACS). In the case of thymus 1, the CD34+ cells used for library preparation also included 2.5% (~125 cells) separately sorted CD34+CD7-CD1a-CD4-CD8-GlyA- ( a subset enriched for the earliest thymic progenitor cells) cells that had been added to the sorted CD34+CD4-CD8-GlyA- cells . In the case of thymus 4, FACS isolation of CD34+ cells was done by index sorting. Index sorting parameters were set to include at least 40 CD34+CD7-CD1a-CD4-CD8-GlyA- (Thy1) and 40 CD34+CD7+CD1a+CD4-CD8-GlyA- (Thy3, T-committed progenitor subset) cells among the 192 sorted cells. Cell type information (Thy1, Thy2 (CD34+CD7+CD1a-CD4-CD8-GlyA-) or Thy3) based on facs is provided as a csv file. Cell barcodes used for sequencing are provided as csv files.