Functional definition of a transcription factor hierarchy regulating T cell lineage commitment.

T cell factor 1 (Tcf1) is the first T cell-specific protein induced by Notch signaling in the thymus, leading to the activation of two major target genes, Gata3 and Bcl11b. Tcf1 deficiency results in partial arrests in T cell development, high apoptosis, and increased development of B and myeloid cells. Phenotypically, seemingly fully T cell-committed thymocytes with Tcf1 deficiency have promiscuous gene expression and an altered epigenetic profile and can dedifferentiate into more immature thymocytes and non-T cells. Restoring Bcl11b expression in Tcf1-deficient cells rescues T cell development but does not strongly suppress the development of non-T cells; in contrast, expressing Gata3 suppresses their development but does not rescue T cell development. Thus, T cell development is controlled by a minimal transcription factor network involving Notch signaling, Tcf1, and the subsequent division of labor between Bcl11b and Gata3, thereby ensuring a properly regulated T cell gene expression program.

Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening.

To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds.

The development of T cells from stem cells in mice and humans.

T cells develop from hematopoietic stem cells in the specialized microenvironment of the thymus. The main transcriptional players of T-cell differentiation such as Notch, Tcf-1, Gata3 and Bcl11b have been identified, but their role and regulation are not yet completely understood. In humans, functional experiments on T-cell development have traditionally been rather difficult to perform, but novel in vitro culture systems and in vivo xenograft models have allowed detailed studies on human T-cell development. Recent work has allowed the use of human severe combined immunodeficiency stem cells to unravel developmental checkpoints for human thymocyte development.

The non-canonical Wnt receptor Ryk regulates hematopoietic stem cell repopulation in part by controlling proliferation and apoptosis.

The development of blood and immune cells requires strict control by various signaling pathways in order to regulate self-renewal, differentiation and apoptosis in stem and progenitor cells. Recent evidence indicates critical roles for the canonical and non-canonical Wnt pathways in hematopoiesis. The non-canonical Wnt pathway is important for establishment of cell polarity and cell migration and regulates apoptosis in the thymus. We here investigate the role of the non-canonical Wnt receptor Ryk in hematopoiesis and lymphoid development. We show that there are dynamic changes in Ryk expression during development and in different hematopoietic tissues. Functionally, Ryk regulates NK cell development in a temporal fashion. Moreover, Ryk-deficient mice show diminished, but not absent self-renewal of hematopoietic stem cells (HSC), via effects on mildly increased proliferation and apoptosis. Thus, Ryk deficiency in HSCs from fetal liver reduces their quiescence, leading to proliferation-induced apoptosis and decreased self-renewal.

Aberrant Wnt Signaling in Leukemia.

The Wnt signaling pathway is essential in the development and homeostasis of blood and immune cells, but its exact role is still controversial and is the subject of intense research. The malignant counterpart of normal hematopoietic cells, leukemic (stem) cells, have hijacked the Wnt pathway for their self-renewal and proliferation. Here we review the multiple ways dysregulated Wnt signaling can contribute to leukemogenesis, both cell autonomously as well as by changes in the microenvironment.

High Levels of Canonical Wnt Signaling Lead to Loss of Stemness and Increased Differentiation in Hematopoietic Stem Cells.

Canonical Wnt signaling regulates the self-renewal of most if not all stem cell systems. In the blood system, the role of Wnt signaling has been the subject of much debate but there is consensus that high Wnt signals lead to loss of reconstituting capacity. To better understand this phenomenon, we have taken advantage of a series of hypomorphic mutant Apc alleles resulting in a broad range of Wnt dosages in hematopoietic stem cells (HSCs) and performed whole-genome gene expression analyses. Gene expression profiling and functional studies show that HSCs with APC mutations lead to high Wnt levels, enhanced differentiation, and diminished proliferation but have no effect on apoptosis, collectively leading to loss of stemness. Thus, we provide mechanistic insight into the role of APC mutations and Wnt signaling in HSC biology. As Wnt signals are explored in various in vivo and ex vivo expansion protocols for HSCs, our findings also have clinical ramifications.

The nuclear effector of Wnt-signaling, Tcf1, functions as a T-cell-specific tumor suppressor for development of lymphomas.

The HMG-box factor Tcf1 is required during T-cell development in the thymus and mediates the nuclear response to Wnt signals. Tcf1(-/-) mice have previously been characterized and show developmental blocks at the CD4-CD8- double negative (DN) to CD4+CD8+ double positive transition. Due to the blocks in T-cell development, Tcf1(-/-) mice normally have a very small thymus. Unexpectedly, a large proportion of Tcf1(-/-) mice spontaneously develop thymic lymphomas with 50% of mice developing a thymic lymphoma/leukemia at the age of 16 wk. These lymphomas are clonal, highly metastatic, and paradoxically show high Wnt signaling when crossed with Wnt reporter mice and have high expression of Wnt target genes Lef1 and Axin2. In wild-type thymocytes, Tcf1 is higher expressed than Lef1, with a predominance of Wnt inhibitory isoforms. Loss of Tcf1 as repressor of Lef1 leads to high Wnt activity and is the initiating event in lymphoma development, which is exacerbated by activating Notch1 mutations. Thus, Notch1 and loss of Tcf1 functionally act as collaborating oncogenic events. Tcf1 deficiency predisposes to the development of thymic lymphomas by ectopic up-regulation of Lef1 due to lack of Tcf1 repressive isoforms and frequently by cooperating activating mutations in Notch1. Tcf1 therefore functions as a T-cell-specific tumor suppressor gene, besides its established role as a Wnt responsive transcription factor. Thus, Tcf1 acts as a molecular switch between proliferative and repressive signals during T-lymphocyte development in the thymus.

NK cells can generate from precursors in the adult human liver.

Hepatic NK cells constitute ≈ 40% of hepatic lymphocytes and are phenotypically and functionally distinct from blood NK cells. Whether hepatic NK cells derive from precursors in the BM or develop locally from hepatic progenitors is still unknown. Here, we identify all five known sequential stages of NK-cell development in the adult human liver and demonstrate that CD34(+) hepatic progenitors can generate functional NK cells. While early NK-cell precursors (NKPs) were similar in liver and blood, hepatic stage 3 NKPs displayed immunophenotypical differences, suggesting the onset of a liver-specific NK-cell development. Hepatic stage 3 NKPs were RORC(neg) and did not produce IL-17 or IL-22, excluding them from the lymphoid tissue-inducer (LTi) subset. In vitro culture of hepatic NKPs gave rise to functional NK cells exhibiting strong cytotoxicity against K562 targets. To determine whether hepatic NKPs are stably residing in the liver, we analyzed donor and recipient-derived cells in transplanted livers. Shortly after liver transplantation all donor NKPs in liver grafts were replaced by recipient-derived ones, indicating that hepatic NKPs are recruited from the bloodstream. Together, our results show that NKPs are continuously recruited from peripheral blood into the liver and can potentially differentiate into liver-specific NK cells.