Efforts to derive hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) are complicated by the fact that embryonic hematopoiesis consists of two programs, primitive and definitive, that differ in developmental potential. As only definitive hematopoiesis generates HSCs, understanding how this program develops is essential for being able to produce this cell population in vitro. Here we show that both hematopoietic programs transition through hemogenic endothelial intermediates and develop from KDR+CD34−CD144− progenitors that are distinguished by CD235a expression. Generation of primitive progenitors (KDR+CD235a+) depends on stage-specific Activin-nodal signaling and inhibition of the Wnt-β-catenin pathway, whereas specification of definitive progenitors (KDR+CD235a−) requires Wnt-β-catenin signaling during this same time frame. Together, these findings establish simple selective differentiation strategies for the generation of primitive or definitive hematopoietic progenitors via Wnt-β-catenin manipulation, and in doing so provide access to enriched populations for future studies on hPSC-derived hematopoietic development.

The efficient generation of hematopoietic stem cells from human pluripotent stem cells is dependent on the appropriate specification of the definitive hematopoietic program during differentiation. In this study, we used T lymphocyte potential to track the onset of definitive hematopoiesis from human embryonic and induced pluripotent stem cells differentiated with specific morphogens in serum- and stromal-free cultures. We show that this program develops from a progenitor population with characteristics of hemogenic endothelium, including the expression of CD34, VE-cadherin, GATA2, LMO2, and RUNX1. Along with T cells, these progenitors display the capacity to generate myeloid and erythroid cells. Manipulation of Activin/Nodal signaling during early stages of differentiation revealed that development of the definitive hematopoietic progenitor population is not dependent on this pathway, distinguishing it from primitive hematopoiesis. Collectively, these findings demonstrate that it is possible to generate T lymphoid progenitors from pluripotent stem cells and that this lineage develops from a population whose emergence marks the onset of human definitive hematopoiesis.

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 (EBs) and it undergoes a NOTCH-dependent EHT to generate RUNX1C+ cells with multilineage potential. Arterial and venous VE progenitors, by contrast, segregate to the CD34+CD73medCD184+ and CD34+CD73hiCD184− 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 Tlineage 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 vitrogenerated 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 IntroductionT cells develop within the thymus from bone marrow-derived hematopoietic progenitors, and follow a series of stage-specific differentiation events, which are broadly characterized by the developmentally coordinated expression of CD4 and CD8. 1 The initial stages of human T-cell development include precursors that express the stem cell marker CD34, 2,3 which is also present on hematopoietic stem cells (HSCs) and on multipotent or lineage-specified progenitor cells. Within the known hierarchy of T-cell development, the earliest precursor subset is further defined by the lack of CD3, CD4, CD8, and CD1a expression. 4 Although immature stages of T-cell development are typically delineated as CD34 ϩ CD1a Ϫ and CD34 ϩ CD1a ϩ cells, these populations remain heterogeneous. Of note, CD7 expression is considered to be one of the earliest cell surface markers known to appear during T lymphopoiesis. 2,5 Importantly, the transition from CD34 ϩ CD7 ϩ CD1a Ϫ to CD34 ϩ CD7 ϩ CD1a ϩ by early thymocytes is associated with T-cell commitment, as a small percentage (ϳ 10%) of these cells show rearrangement at the T-cell receptor ␤-chain (TCR␤) locus. 6 In addition, CD34 ϩ CD7 ϩ CD1a ϩ cells appear to be T-lineage restricted, as these cells show low precursor activity toward non-T-cell lineages. 7 Current understanding of the aforementioned early stages has been obtained from analyses of human fetal or adult thymocyte subsets, and by analyzing T-cell development in vitro using xenogeneic engraftment of mouse fetal thymus organ cultures (FTOCs). 8,9 Although these systems have provided important insight into T-cell development, the capacity to evaluate specific progenitor populations has remained difficult to assess given the requirem...

Human peripheral blood NK cells may be divided into two main subsets: CD56 bright CD16À and CD56 dim CD16 1. Since TGF-b is known to influence the development of many leukocyte lineages, its effects on NK cell differentiation either from human CD34

• Intrathymic T-cell regeneration is facilitated by human proTcells generated in vitro.• In vitro-generated human proT-cells home to the thymus, wherein they restore thymic structure.Hematopoietic stem cell transplantation (HSCT) is followed by a period of immune deficiency due to a paucity in T-cell reconstitution. Underlying causes are a severely dysfunctional thymus and an impaired production of thymus-seeding progenitors in the host. Here, we addressed whether in vitro-derived human progenitor T (proT)-cells could not only represent a source of thymus-seeding progenitors, but also able to influence the recovery of the thymic microenvironment. We examined whether co-transplantation of in vitro-derived human proT-cells with hematopoietic stem cells (HSCs) was able to facilitate HSC-derived T-lymphopoiesis posttransplant. A competitive transfer approach was used to define the optimal proT subset capable of reconstituting immunodeficient mice. Although the 2 subsets tested (proT1,1 ) showed thymus engrafting function, proT2-cells exhibited superior engrafting capacity. Based on this, when proT2-cells were coinjected with HSCs, a significantly improved and accelerated HSC-derived T-lymphopoiesis was observed. Furthermore, we uncovered a potential mechanism by which receptor activator of nuclear factor kb (RANK) ligand-expressing proT2-cells induce changes in both the function and architecture of the thymus microenvironment, which favors the recruitment of bone marrow-derived lymphoid progenitors. Our findings provide further support for the use of Notch-expanded progenitors in cellbased therapies to aid in the recovery of T-cells in patients undergoing HSCT. (Blood. 2013;122(26):4210-4219)

BackgroundT cell development occurs within the highly specialized thymus. Cytotoxic CD8 T cells are critical in adaptive immunity by targeting virally infected or tumor cells. In this study, we addressed whether functional CD8 T cells can be generated fully in vitro using human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells.ResultsHSC/OP9-DL1 cocultures supported the differentiation of CD8 T cells, which were TCR/CD3hi CD27hi CD1aneg and thus phenotypically resembled mature functional CD8 single positive thymocytes. These in vitro-generated T cells also appeared to be conventional CD8 cells, as they expressed high levels of Eomes and low levels of Plzf, albeit not identical to ex vivo UCB CD8 T cells. Consistent with the phenotypic and molecular characterization, upon TCR-stimulation, in vitro-generated CD8 T cells proliferated, expressed activation markers (MHC-II, CD25, CD38), secreted IFN-γ and expressed Granzyme B, a cytotoxic T-cell effector molecule.ConclusionTaken together, the ability to direct human hematopoietic stem cell or T-progenitor cells towards a mature functional phenotype raises the possibility of establishing cell-based treatments for T-immunodeficiencies by rapidly restoring CD8 effector function, thereby mitigating the risks associated with opportunistic infections.

• Upon in vitro differentiation, iPSCs obtained from patients with SCID and OS show a similar block in T-cell development.• Presence of unresolved single-strand DNA breaks in developing T cells from OS patient-derived iPSCs affects their differentiation.Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro differentiation of human induced pluripotent stem cells (iPSCs) generated from patients with different recombination-activating gene 1 (RAG1) mutations to assess T-cell development and T-cell receptor (TCR) V(D)J recombination. RAG1-mutants from severe combined immunodeficient (SCID) patient cells showed a failure to sustain progression beyond the CD3residual mutant RAG1 recombination activity from an Omenn syndrome (OS) patient, similar impaired T-cell differentiation was observed, due to increased single-strand DNA breaks that likely occur due to heterodimers consisting of both an N-terminal truncated and a catalytically dead RAG1. Furthermore, deep-sequencing analysis of TCR-b (TRB) and TCR-a (TRA) rearrangements of CD3 2 CD4 1 CD8 2 immature single-positive and CD81 double-positive cells showed severe restriction of repertoire diversity with preferential usage of few Variable, Diversity, and Joining genes, and skewed length distribution of the TRB and TRA complementary determining region 3 sequences from SCID and OS iPSC-derived cells, whereas control iPSCs yielded T-cell progenitors with a broadly diversified repertoire. Finally, no TRA/d excision circles (TRECs), a marker of TRA/d locus rearrangements, were detected in SCID and OS-derived T-lineage cells, consistent with a pre-TCR block in T-cell development. This study compares human T-cell development of SCID vs OS patients, and elucidates important differences that help to explain the wide range of immunologic phenotypes that result from different mutations within the same gene of various patients. (Blood. 2016;128(6):783-793)