Artificial thymic organoids generated with peripheral blood CD34⁺ cells represent a quick and powerful tool to determine the nature of severe T cell lymphopenia

Abstract The study of early T-cell development in humans is challenging because of the scarce availability of thymic samples and limitations of in vitro T-cell differentiation assays. We have previously shown that artificial thymic organoids (ATOs), generated by aggregating DLL4-expressing stromal cells with CD34 +cells isolated from bone marrow (BM) or mobilized peripheral blood (MPB), are a reliable tool to determine the hematopoietic-intrinsic or extrinsic nature of severe T-cell lymphopenia. However, BM or MPB samples are not often available and delays in obtaining these samples might dangerously postpone the patient’s clinical management decision. We thus set out to evaluate whether we could obtain reliable results from ATOs generated with CD34 +cells isolated from PB. The main challenge of using CD34 +cells from un-mobilized PB is that the frequency of these cells is much lower than that found in BM or MPB. For the generation of ATOs with BM or MPB CD34 +cells we used a minimum of 5000 cells/ATO, but these numbers would not be feasible for PB samples. We thus generated ATOs with decreased numbers of CD34 +cells, and we determined that 1000 cells/ATO was the minimum necessary to achieve efficient T-cell differentiation. Additionally, we slightly modified the experimental protocol and added SCF for the first 3 weeks of culture, in addition to IL7 and FLT3L, which are present throughout the T-cell differentiation assay. We also established that we could efficiently generate ATOs when starting from fresh blood (up to 3–4 days) and a minimum amount of 5–7 ml, for infants and early pediatric samples. Reduced efficiency in ATO generation was however obtained when isolating CD34 +cells from PB drawn more that 5–6 days earlier or from frozen samples. This work was supported by the Division of Intramural Research, NIAID, NIH.