DL4-ubeads Induce T Cell Differentiation from Stem Cells in a Stromal Cell-Free System

T cells serve as pivotal effectors of the immune system and can be harnessed as therapeutic agents for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and mature T cells from CD34+ cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-μbeads, along with lymphopoietic cytokines, induced an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+ T cells. Single-cell RNA sequencing of human PSC-derived proT cells revealed a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34+CD7+ proT cells into immunodeficient mice demonstrated efficient thymic engraftment and functional maturation of peripheral T cells. DL4-μbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources. Single-cell RNA sequencing of 2,761 human iPSC-derived progenitor T cells using 10x Genomics platform and using the Illumina NovaSeq 6000.