A human thymic epithelial cell culture system for the promotion of lymphopoiesis from hematopoietic stem cells

Abstract

Objective A human thymic epithelial cell (TEC) line expressing human leukocyte antigen (HLA)-ABC and HLA-DR was engineered to overexpress murine Delta-like 1 (TEC-Dl1) for the purpose of establishing a human culture system that supports T lymphopoiesis from hematopoietic progenitor cells (HPCs). Materials and Methods Cord blood (CB) or bone marrow (BM) HPCs were co-cultured with either the parental TEC line expressing low levels of the Notch ligands, Delta-like 1 and Delta-like 4 or with TEC-Dl1 to determine if these cell lines support human lymphopoiesis. Results In co-cultures with CB or BM HPCs, TEC-Dl1 cells promote de novo generation of CD7posCD1apos T-lineage committed cells. Most CD7posCD1ahi cells are CD4posCD8pos double positive (DP). We found that TEC-Dl1 cells are insufficient to generate mature CD3hi CD4pos or CD3hi CD8pos single positive (SP) T cells from the CD4posCD8pos DP T cells; however, we detected CD3lo cells within the DP and SP CD4 and CD8 populations. The CD3lo SP cells expressed lower levels of IL-2Rα and IL-7Rα compared to CD3lo DP cells. In contrast to the TEC-Dl1 line, the parental TEC-84 line expressing low levels of human Notch ligands permits HPC differentiation to the B-cell lineage. Conclusions We report for the first time a human TEC line that supports lymphopoiesis from CB and BM HPC. The TEC cell lines described herein provide a novel human thymic stroma model to study the contribution of HLA molecules and Notch ligands to T cell commitment and maturation and could be utilized to promote lymphopoiesis for immune cell therapy.

Adipocyte-Derived Soluble Factor(s) Inhibits Early Stages of B Lymphopoiesis

“…We first showed that the rabbit adipocyte CM inhibited differentiation of human CD34 + HSC-enriched cells into CD79 + B-lineage cells in co-cultures with OP9 cells (data not shown). We then co-cultured human HSCs and OP9 cells with rabbit adipocyte CM and analyzed the cells for CLPs, pre-proB and proB cells (28–30). At day 10, we found that the percentages and absolute numbers of CLPs (CD7 + CD34 + ) were similar in cultures with or without adipocyte CM (Fig.…”

Thymus involution and regeneration: two sides of the same coin?

“…As an alternative to the de novo generation of TEC progenitors, attempts have been made to reconstitute certain features of thymic environments by artificial means, initially in in vitro studies 72,73 and then using in vivo reconstitutions 74 . Collectively, these studies confirmed the decisive role of the Notch 1 ligand delta-like ligand 4 (DLL4) as a key determinant of T cell lineage induction 75,76 .…”

Gene Modification and Three-Dimensional Scaffolds as Novel Tools to Allow the Use of Postnatal Thymic Epithelial Cells for Thymus Regeneration Approaches

“…Indeed, adult TECs lose their ability to aggregate ex vivo and fail to maintain expression of important molecules, such as those of the Notch signaling pathway, when are not supported by a well‐organized 3D environment. Based on this insight, other researchers were able to successfully establish in vitro bone marrow derived cell lines (OP9‐DL1 or MS5‐Dll4 or MS5‐Dll1) or human thymic stromal cell lines (TEC‐Dl1) overexpressing the Notch ligand delta‐like 1 (Dll1) and Notch ligand delta‐like 4 (Dll4) . Even though these experimental systems were able to partially support thymocyte development in vitro, they could not recapitulate the complexity of the 3D thymic microenvironment and were not meant to create a functional approach for in vivo thymic regeneration.…”