Notch signalling is absolutely required for the maturation of thymus-seeding hematopoietic precursor cells into T cells. Through the interplay between the Notch receptor and its ligands (such as DLL1, DLL4 and JAG2) a T cell specifying programme is induced which direct these precursors to become a T cell. Even though the Notch pathway is well conserved, some major differences exist in its transcriptome dynamics between human and mouse. Therefore, we aim to unravel the role of the Notch pathway in normal T cell development, as well as its deregulation in T cell acute lymphoblastic leukemia.

Notch signalling drives human T cell development and is absolutely essential for T lineage specification.
Notch ligands, expressed by retrovirally transduced OP9 feeder cells, are capable of directing CD34+CD38-Lin- CB precursors towards the T cell lineage although at a differential efficiency.

Human embryonic stem cells (hESC) are pluripotent stem cells capable of generating each cell of the three germ layers. The main advantage of applying hESCs in the field of T cell research, is the capability of these cells to self renew rendering a unlimited pool of pluripotent stem cells which can be genetically manipulated in the lab. A major challenge in the field of hESC is the establishment of specific and effective differentiation protocols that directs hESC down a particular lineage fate. So far, attempts to generate T cells from hESC in vitro were mainly unreproducible. In this work, we show that T cells can be generated in vitro from hESC-derived hematopoietic precursor cells present in hematopoietic zones (HZs). These zones are morphologically similar to the in vivo mesoderm originating blood islands during embryonic development, and are formed when hESC are cultured on OP9 stromal cells. Upon subsequent transfer of these HZs on OP9 cells expressing high levels of Delta-like 1 and in the presence of growth factors, cells expand and differentiate towards T cells. Furthermore, we show that T cells derive exclusively from a CD34highCD43low population, further substantiating the notion that hESC-derived CD34highCD43low cells are formed in HZs and are the only population containing multipotent hematopoietic precursor cells which originate from the hemogenic endothelium. In addition, other more efficient protocols for the in vitro generation of T cells out of hESCs are being developed in the lab in order to study the molecular mechanisms driving T cell differentiation using these models.

Schematic approach for the generation of T cells from hESCs.
Pluripotent hESCs are kept in an undifferentiated state on MMEFs (Step 1) whereafter colonies are fragmented and cultured on OP9 cells to allow hematopoietic maturation in hematopoietic zones as a primary cocuture (Step 2). After 10-12 days of culturing on the OP9 feeder, these cultures can be transferred onto an OP9-DLL1 layer capable of generating T cell precursors (Step 3). These secondary cocultures can be weekly analyzed to visualize the development of CD34+CD43- hematopoietic precursors towards more mature T cell subsets (Step 4).

Hematopoietic stem cells (HSCs) originate in the bone marrow and migrate through the blood stream to the thymus where they will develop towards mature T cells. Notch plays an important role during this process as it is needed to block the development of other hematopoietic lineages in favor of T cell development. Next to T cell specification and commitment, Notch also regulates the differentiation towards T cell receptor (TCR)αβ and -γδ mature T cells. However, most underlying transcriptional programs regulating the differentiation from CD34+ HSCs towards mature T cells need further elucidation, especially the developmental decision between TCRαβ and -γδ T cells in human. Therefore, we performed deep total RNA sequencing on 12 discrete developmental stages of human T cell development from two separate healthy thymus donors to obtain both the protein coding as the long non-coding RNA (lncRNA) landscape of human thymopoiesis. Even though lncRNA expression in murine T cell development has been studied extensively, it is not yet clear how lncRNAs behave during human T cell development. Considering the lack of an extensive overview of the full transcriptional network and the species specific expression of lncRNAs, such information in human is of great importance.

Heatmap of differentially expressed coding and non-coding genes between αβ and γδ T cells.
Immature single positive CD28 positive T cells (ISP 28+), together with both early and late double positive T cells (DP 3- and DP 3+) and CD4 and CD8 single positive T cells were compared to immature (γδ 3+1-) and mature (γδ 3+1+) γδ T cells. The differentially expressed genes can be divided into groups of αβ and γδ specific genes, and genes specific to T cell maturation.

The gradual reprogramming of haematopoietic precursors into the T-cell fate is characterized by at least two sequential developmental stages. Following Notch1-dependent T-cell lineage specification during which the first T-cell lineage genes are expressed and myeloid and dendritic cell potential is lost, T-cell specific transcription factors subsequently induce T-cell commitment by repressing residual natural killer (NK)-cell potential. How these processes are regulated in human is poorly understood, especially since efficient T-cell lineage commitment requires a reduction in Notch signalling activity following T-cell specification. For instance, we showed that GATA3, in contrast to TCF1, controls human T-cell lineage commitment through direct regulation of three distinct processes: repression of NK-cell fate, upregulation of T-cell lineage genes to promote further differentiation and restraint of Notch activity.

GATA3 restrains Notch activation at the T-lineage commitment stage. GATA-3 transduced CD34+CD1a- uncommitted intrathymic precursors show an accelerated development toward the CD4+CD8b+ DP stage in both the presence and absence of Notch signaling which highlights the role of GATA3 during T cell commitment following Notch-controlled T lineage specification.