[Induction of mouse T lymphocyte differentiation in vitro by thymic organoids]

Objective To induce the differentiation of hematopoietic stem progenitor cells (HSPCs) into T cell by creating thymic organoids and simulating the three-dimensional structure of thymus tissue in vitro. Methods The retroviral vector expressing the DLL1 and Green fluorescent protein (GFP) was constructed, and the OP9-DLL1 cell line was established in OP9 cells with the aid of retroviral infection. The mRNA and protein level of DLL1 in OP9-DLL1 cells was detected by quantitative real-time PCR and Western blot respectively. Immunofluorescence assay was used to detect the DLL1 protein expression and distribution in OP9-DLL1 cells. HSPCs were extracted from E13.5 fetal liver and bone marrow of C57BL/6 mouse, and mixed with OP9-DLL1 cells in an appropriate ratio respectively, then compacted by centrifuging and cultured at the air-liquid interface in medium. Fluorescence microscope was used to observe the growth of thymic organoids. Flow cytometry was used to detect the expression of T cell surface markers, including CD3, CD4, CD8, CD25, CD44, CD45, CD117 and TCRβ. Immunofluorescence cytochemical staining was used to observe the distribution of hematopoietic cells in thymic organoids. Results The retroviral vector expressing DLL1 and GFP was successfully constructed. The OP9 cells were infected with the retrovirus constructed, and OP9-DLL1 cells were obtained by GFP screening. The mRNA and protein level of DLL1 in OP9-DLL1 cells significantly increased, and DLL1 was expressed in the membrane OP9-DLL1 cells. During the 40 days of culture, the thymic organoids remained in good condition and increased gradually in volume. The thymic organoids induced programmed differentiation of T cells, and differentiation of HSPCs into CD3⁺ T cells. Conclusion OP9-DLL1 cells can be used to construct thymic organoids and to induce differentiation of HSPCs into T cells in vitro.