DL4-μbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system

T cells are pivotal effectors of the immune system and can be harnessed as therapeutics 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 T-lineage cells from CD34+cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-μbeads, along with lymphopoietic cytokines, induce an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+αβ T cells. Single-cell RNA sequencing of human PSC-derived proT cells reveals 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 demonstrates 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.


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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Juan Carlos Zuniga-Pflucker May 31, 2021 Flow cytometry data was obtained using BD Biosciences LSR II. Single-cell RNA sequencing was performed using 10X Genomics.
Flow cytometry data was analyzed using FlowJo 9.9.6. Statistical analysis was performed using Prism 6 software. Progenitor frequencies were determined by the method of maximum likelihood applied to the Poisson model using the following software: http:// bioinf.wehi.edu.au/software/elda/. Single-cell RNA sequencing data was analyzed using R software 4.0.2 with the package Seurat (version 4.0).
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All methods for each particular experiment were performed in a similar fashion, including the same materials used. Three to four independent experiments were performed (which were reliably reproduced).
Mice were age matched (3-5 days neonatal) for each particular experiment.
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NSG and C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME), housed, bred and maintained in the Comparative Research Facility of the Sunnybrook Research Institute, under specific pathogen-free conditions. 3-5 day neonatal mice of both male and females were used.
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