Faculty Bibliography

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and often incurable disease. To uncover therapeutic vulnerabilities, we first developed T-ALL patient-derived tumor-xenografts (PDX) and exposed PDX cells to a library of 433 clinical-stage compounds in vitro. We identified 39 broadly active compounds with anti-leukemia activity. Since endothelial cells (ECs) can alter drug responses in T-ALL, we developed an endothelial cells (ECs) / T-ALL co-culture system. We found that ECs provide pro-tumorigenic signals and mitigate drug responses to individual T-ALL PDX. ECs broadly rescued several compounds in most of the models, while other drugs were rescued only in individual PDXs suggesting unique crosstalk interactions and/or intrinsic tumor features. Mechanistically, co-cultured T-ALL and ECs underwent bi-directional transcriptomic changes at the single-cell level, highlighting distinct "education signatures". These changes were linked to a bi-directional regulation of multiple pathways in T-ALL and ECs. Remarkably, in-vitro EC-educated T-ALL cells mirrored ex-vivo splenic T-ALL at the single-cell resolution. Lastly, five effective drugs from the two drug screenings were tested in vivo and shown to effectively delay tumor growth/dissemination and prolonging the overall survival (OS). We anticipate that this T-ALL-EC platform can contribute to elucidating leukemia-microenvironment interactions and identify effective compounds and therapeutic vulnerabilities.

Histone variants and the associated post-translational modifications that govern the stemness of haematopoietic stem cells (HSCs) and differentiation thereof into progenitors (HSPCs) have not been well defined. H3.3 is a replication-independent H3 histone variant in mammalian systems that is enriched at both H3K4me3- and H3K27me3-marked bivalent genes as well as H3K9me3-marked endogenous retroviral repeats. Here we show that H3.3, but not its chaperone Hira, prevents premature HSC exhaustion and differentiation into granulocyte-macrophage progenitors. H3.3-null HSPCs display reduced expression of stemness and lineage-specific genes with a predominant gain of H3K27me3 marks at their promoter regions. Concomitantly, loss of H3.3 leads to a reduction of H3K9me3 marks at endogenous retroviral repeats, opening up binding sites for the interferon regulatory factor family of transcription factors, allowing the survival of rare, persisting H3.3-null HSCs. We propose a model whereby H3.3 maintains adult HSC stemness by safeguarding the delicate interplay between H3K27me3 and H3K9me3 marks, enforcing chromatin adaptability.

Jak3 is the only non-promiscuous member of the Jak family of secondary messengers. Studies to date have focused on understanding and targeting the cell-autonomous role of Jak3 in immunity, while functional Jak3 expression outside the hematopoietic system remains largely unreported. We show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow. The bone marrow niche is understood as a network of different cell types that regulate hematopoietic function. We show that the Jak3-/- bone marrow niche is deleterious for the maintenance of long-term repopulating hematopoietic stem cells (LT-HSCs) and that JAK3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. This work may serve to identify a novel function for a highly specific tyrosine kinase in the bone marrow vascular niche and to further characterize the LT-HSC function of sinusoidal endothelium.

Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood-brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.

Brain microvascular endothelial cells (BMECs) possess unique properties that are crucial for many functions of the blood-brain-barrier (BBB) including maintenance of brain homeostasis and regulation of interactions between the brain and immune system. The generation of a pure population of putative brain microvascular endothelial cells from human pluripotent stem cell sources (iBMECs) has been described to meet the need for reliable and reproducible brain endothelial cells in vitro. Human pluripotent stem cells (hPSCs), embryonic or induced, can be differentiated into large quantities of specialized cells in order to study development and model disease. These hPSC-derived iBMECs display endothelial-like properties, such as tube formation and low-density lipoprotein uptake, high transendothelial electrical resistance (TEER), and barrier-like efflux transporter activities. Over time, the de novo generation of an organotypic endothelial cell from hPSCs has aroused controversies. This perspective article highlights the developments made in the field of hPSC derived brain endothelial cells as well as where experimental data are lacking, and what concerns have emerged since their initial description.

Haematopoietic stem cells (HSCs) are the only adult stem cells with a demonstrated clinical use, even though a tractable method to maintain and expand human HSCs in vitro has not yet been found. Owing to the introduction of transplantation strategies for the treatment of haematological malignancies and, more recently, the promise of gene therapy, the need to improve the generation, manipulation and scalability of autologous or allogeneic HSCs has risen steeply over the past decade. In that context, reprogramming strategies based on the expression of exogenous transcription factors have emerged as a means to produce functional HSCs in vitro. These approaches largely stem from the assumption that key master transcription factors direct the expression of downstream target genes thereby triggering haematopoiesis. Both somatic and pluripotent cells have been used to this end, yielding variable results in terms of haematopoietic phenotype and functionality. Here, we present an overview of the haematopoietic reprogramming methods reported to date, provide the appropriate historical context and offer some critical insight about where the field stands at present.

The ability to generate hematopoietic stem cells (HSCs) in vitro would have an immeasurable impact on many areas of clinical practice, including trauma, cancer, and congenital disease. In this protocol, we describe a stepwise approach that converts adult murine endothelial cells (ECs) to HSCs, termed 'reprogrammed ECs into hematopoietic stem and progenitor cells' (rEC-HSPCs). The conversion, which is achieved without cells transitioning through a pluripotent state, comprises three phases: induction, specification, and expansion. Adult ECs are first isolated from Runx1-IRES-GFP; Rosa26-rtTa mice and maintained in culture under EC growth factor stimulation and Tgfβ inhibition. In the first (induction) phase of conversion (days 0-8), four transcription factors (TFs)-FosB, Gfi1, Runx1, and Spi1 (FGRS)-are expressed transiently, which results in endogenous Runx1 expression. During the second (specification) phase (days 8-20), endogenous Runx1+ FGRS-transduced ECs commit to a hematopoietic fate and no longer require exogenous FGRS expression. Finally, the vascular niche drives robust proliferation of rEC-HSPCs during the expansion phase (days 20-28). The resulting converted cells possess a transcriptomic signature and long-term self-renewal capacity indistinguishable from those of adult HSCs. In this protocol, we also describe functional in vitro and in vivo assays that can be used to demonstrate that rEC-HSPCs are competent for clonal engraftment and possess multi-lineage reconstitution potential, including antigen-dependent adaptive immune function. This approach thus provides a tractable strategy for interrogating the generation of engraftable hematopoietic cells, advancing the mechanistic understanding of hematopoietic development and HSC self-renewal.