Single-Cell RNA-Seq Mapping of Human Thymopoiesis Reveals Lineage Specification Trajectories and a Commitment Spectrum in T Cell Development

Abstract

Highlights d CD34 + human thymic progenitors present a spectrum of specification and commitment states d Earliest progenitors are CD7and exhibit stem-cell-like and T-primed transcriptomes d Loss of B-potential precedes that of myeloid and NK potentials during T-commitment d A CD34 + subpopulation of cells is primed for the plasmacytoid dendritic lineage

Processing Human Thymic Tissue for Single Cell RNA-Seq

“…Post-thaw cell recovery will be approximately 30%–50% of the pre-freeze cell count. Cryopreserved and freshly prepared CD34 + cells have yielded similar results in single cell RNA-seq studies done in our lab ( Le et al., 2020 ). CD34 – thymic cells predominantly consist of CD4 + CD8 + (double positive, DP) cells.…”

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Figure 3 Gating Strategy for Sorting CD34 + Lin – and CD34 + CD7 – CD1a – Cells Thy1, CD34 + CD7 – CD1a – ; Thy2, CD34 + CD7 + CD1a – ; Thy3, CD34 + CD7 + CD1a + . Figure reprinted with permission from Le et al. (2020) .

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“…(2020) .

Figure 4 Gating Strategy for Sorting CD34 – CD45 + GlyA – , CD3 + CD4 + CD8 – , and CD3 + CD4 – CD8 + Cells Figure reprinted with permission from Le et al. (2020) .

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“… Thy1, CD34 + CD7 – CD1a – ; Thy2, CD34 + CD7 + CD1a – ; Thy3, CD34 + CD7 + CD1a + . Figure reprinted with permission from Le et al. (2020) .…”

“…CD34 + cells represent <1% of thymic cells, so this protocol uses magnetic- followed by fluorescence-activated cell separation to isolate highly enriched CD34 + cells. For complete details on the use and execution of this protocol, please refer to Le et al. (2020) .…”