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The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34(+)CD73(-)CD184(-) fraction of day 8 embryoid bodies and it undergoes a NOTCH-dependent EHT to generate RUNX1C(+) cells with multilineage potential. Arterial and venous VE progenitors, in contrast, segregate to the CD34(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.

T-cell development follows a defined set of stage-specific differentiation steps. However, molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. To address this, human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T lineage in OP9-DL1 cocultures. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. Quantitative clonal analyses demonstrated that CD34(+)CD38(-) and CD34(+)CD38(lo) subsets of UCB contain a similarly high T-lineage progenitor frequency, whereas the frequency in CD34(+)CD38(+/hi) cells was 5-fold lower. Delta-like/Notch-induced signals increased the T-cell progenitor frequency of CD34(+)CD38(-/lo) cells differentiated on OP9-DL1, and 2 distinct progenitor subsets, CD34(+)CD45RA(+)CD7(++)CD5(-)CD1a(-) (proT1) and CD34(+)CD45RA(+)CD7(++)CD5(+)CD1a(-) (proT2), were identified and their thymus engrafting capacity was examined, with proT2 cells showing a 3-fold enhanced reconstituting capacity compared with the proT1 subset. Furthermore, in vitro-generated CD34(+)CD7(++) progenitors effectively engrafted the thymus of immunodeficient mice, which was enhanced by the addition of an IL-7/IL-7 antibody complex. Taken together, the identification of T-progenitor subsets readily generated in vitro may offer important avenues to improve cellular-based immune-reconstitution approaches.

The thymus provides a unique environment for the induction of T-cell lineage commitment and differentiation, which is predicted by specific Notch ligand-receptor interactions on epithelial cells and lymphoid progenitors, respectively. Accordingly, a bone marrow-derived stromal cell line (OP9) ectopically expressing the Notch ligand Delta-like 1 (Dll1) or Dll4 (OP9-DL1 and OP9-DL4, respectively) gains the ability to recapitulate thymus-like function, supporting T-cell differentiation of both mouse and human progenitors. In this study, we extend these findings by demonstrating that, unlike the NIH3T3 cell line, mouse primary fibroblasts made to express Dll4 (mFibro-DL4) acquire the capacity to promote and support T-cell development from hematopoietic stem cells (HSCs) into TCRαβ(+), CD4(+) and CD8(+) T-lineage cells. However, mFibro-DL4 cells showed a lower efficiency of T-cell generation than OP9-DL4 cells did. Nevertheless, progenitor T-cells (CD117(+) CD44(+) CD25(+)) generated in HSC/mFibro-DL4 co-cultures, when intravenously transferred into immunodeficient (Rag2(-/-) γc(-/-)) mice, home to the thymus, undergo differentiation, and give rise to mature T-cells that go on to populate the periphery. Surprisingly, primary human fibroblast cells expressing Dll4 showed very low efficiency in supporting human T-lineage differentiation, which could not be improved by blocking myelopoiesis. Nevertheless, mFibro-DL4 cells could support human T-cell lineage differentiation. Our results provide a functional framework for the induction of T-cell development using easily accessible fibroblasts made to express Dll4. These cells, which are amenable for in vitro applications, can be further utilized in the design of individualized systems for T-cell production, with implications for the treatment of immunodeficiencies.

T cells typically differentiate via a series of coordinated steps within the highly specialized microenvironment of the thymus. Traditionally, human T-lymphopoiesis in vitro has been studied using the hybrid human/mouse fetal thymic organ culture system. Pioneering work by McCune et al. devised a method to examine human T cell development in vivo in relation to HIV-1 using the SCID/hu (thy/liv) model. This was followed by models that better reflected the ability of human hematopoietic cells to home and differentiate within the mouse host without human fetal tissues; however, human T cell development in these animals was poor. In this chapter, we outline a procedure to generate human progenitor T (proT) cells in vitro from umbilical cord blood-derived hematopoietic stem cells using the OP9-DL1 cell system; in addition, we describe the method used to examine the engraftment of in vitro-derived proT cells into immunodeficient mouse strains.

Traditionally, the study of human T cell development has relied on the availability of human and mouse thymic tissue. In this chapter, we outline a simple in vitro protocol for generating large numbers of human T-lineage cells from umbilical cord blood (CB)- derived hematopoietic stem cells (HSCs) using a bone marrow stromal cell line. This protocol is broken into three major steps: (1) the maintenance of a working stock of OP9 bone marrow stromal cells expressing the Notch receptor ligand Delta-like 1 (OP9- DL1), (2) the purification of human HSCs from umbilical CB, and (3) the initiation and maintenance/expansion of OP9-DL1 cocultures over time (see Fig. 1). The use of this system opens avenues for basic research as it equips us with a simple in vitro method for studying human T cell development.

Immunodeficient individuals are susceptible to opportunistic infection. While stem cell transplantation can restore a functional immune system, T cells are slow to recover and limited in eliciting adaptive immune responses. Approaches to selectively enhance T cell function have focused on boosting thymopoiesis to generate new T cells or expanding existing T cells. By taking advantage of the role of Notch signaling in T cell development, we have developed an in vitro system able to generate large numbers of progenitor T cells from human hematopoietic stem cells. Here, we discuss this in vitro system and its implications for the potential treatment of T cell immunodeficiency.