EZH1 repression generates mature iPSC-derived CAR T cells with enhanced antitumor activity.

Human induced pluripotent stem cells (iPSCs) provide a potentially unlimited resource for cell therapies, but the derivation of mature cell types remains challenging. The histone methyltransferase EZH1 is a negative regulator of lymphoid potential during embryonic hematopoiesis. Here, we demonstrate that EZH1 repression facilitates in vitro differentiation and maturation of T cells from iPSCs. Coupling a stroma-free T cell differentiation system with EZH1-knockdown-mediated epigenetic reprogramming, we generated iPSC-derived T cells, termed EZ-T cells, which display a highly diverse T cell receptor (TCR) repertoire and mature molecular signatures similar to those of TCRαß T cells from peripheral blood. Upon activation, EZ-T cells give rise to effector and memory T cell subsets. When transduced with chimeric antigen receptors (CARs), EZ-T cells exhibit potent antitumor activities in vitro and in xenograft models. Epigenetic remodeling via EZH1 repression allows efficient production of developmentally mature T cells from iPSCs for applications in adoptive cell therapy.

CellComm infers cellular crosstalk that drives haematopoietic stem and progenitor cell development.

Intercellular communication orchestrates a multitude of physiologic and pathologic conditions. Algorithms to infer cell-cell communication and predict downstream signalling and regulatory networks are needed to illuminate mechanisms of stem cell differentiation and tissue development. Here, to fill this gap, we developed and applied CellComm to investigate how the aorta-gonad-mesonephros microenvironment dictates haematopoietic stem and progenitor cell emergence. We identified key microenvironmental signals and transcriptional networks that regulate haematopoietic development, including Stat3, Nr0b2, Ybx1 and App, and confirmed their roles using zebrafish, mouse and human models. Notably, CellComm revealed extensive crosstalk among signalling pathways and convergence on common transcriptional regulators, indicating a resilient developmental programme that ensures dynamic adaptation to changes in the embryonic environment. Our work provides an algorithm and data resource for the scientific community.

Sequential regulation of hemogenic fate and hematopoietic stem and progenitor cell formation from arterial endothelium by Ezh1/2.

Across species, hematopoietic stem and progenitor cells (HSPCs) arise during embryogenesis from a specialized arterial population, termed hemogenic endothelium. Here, we describe a mechanistic role for the epigenetic regulator, Enhancer of zeste homolog-1 (Ezh1), in vertebrate HSPC production via regulation of hemogenic commitment. Loss of ezh1 in zebrafish embryos favored acquisition of hemogenic (gata2b) and HSPC (runx1) fate at the expense of the arterial program (ephrinb2a, dll4). In contrast, ezh1 overexpression blocked hematopoietic progression via maintenance of arterial gene expression. The related Polycomb group subunit, Ezh2, functioned in a non-redundant, sequential manner, whereby inhibition had no impact on arterial identity, but was capable of blocking ezh1-knockdown-associated HSPC expansion. Single-cell RNA sequencing across ezh1 genotypes revealed a dropout of ezh1+/- cells among arterial endothelium associated with positive regulation of gene transcription. Exploitation of Ezh1/2 modulation has potential functional relevance for improving in vitro HSPC differentiation from induced pluripotent stem cell sources.

Noninvasive Assessment of Jugular Venous Pressure via Force-Coupled Single Crystal Ultrasound.

OBJECTIVE: we have developed a handheld device for noninvasive quantitative assessment of jugular venous pressure (JVP). METHODS: we used a single crystal ultrasound coupled to a force-sensing load cell to measure JVP based on the force necessary to collapse the internal jugular vein (IJV) walls. We used a gelatin-based model system of the IJV to test the ability of single crystal ultrasound to identify the IJV and verified the cross-sectional position and diameter of the vessels with conventional imaging ultrasound. We also tested our prototype device on healthy human volunteers. RESULTS: experiments on model system demonstrated that vessel diameters determined with single crystal ultrasound were in close agreement with the diameters derived from conventional 2-D ultrasound. Proof-of-concept human experiments demonstrate that single crystal ultrasound can detect the IJV in basal and collapsed states, as compared to gold-standard sonography (insert stats). Assessment of JVP in human volunteers was physiologically consistent with and sensitive to postural changes (supine JVP 6.6 ± 2.4 mmHg; standing JVP 4.2 ± 1.9 mmHg (p < 0.0001). CONCLUSION: noninvasive assessment of JVP could prove valuable in informing rapid clinical decision-making across various pathologies and conditions leading to derangements in intravascular volume status.

Alginate encapsulation parameters influence the differentiation of microencapsulated embryonic stem cell aggregates.

Pluripotent embryonic stem cells (ESCs) have tremendous potential as tools for regenerative medicine and drug discovery, yet the lack of processes to manufacture viable and homogenous cell populations of sufficient numbers limits the clinical translation of current and future cell therapies. Microencapsulation of ESCs within microbeads can shield cells from hydrodynamic shear forces found in bioreactor environments while allowing for sufficient diffusion of nutrients and oxygen through the encapsulation material. Despite initial studies examining alginate microbeads as a platform for stem cell expansion and directed differentiation, the impact of alginate encapsulation parameters on stem cell phenotype has not been thoroughly investigated. Therefore, the objective of this study was to systematically examine the effects of varying alginate compositions on microencapsulated ESC expansion and phenotype. Pre-formed aggregates of murine ESCs were encapsulated in alginate microbeads composed of a high or low ratio of guluronic to mannuronic acid residues (High G and High M, respectively), with and without a poly-L-lysine (PLL) coating, thereby providing four distinct alginate bead compositions for analysis. Encapsulation in all alginate compositions was found to delay differentiation, with encapsulation within High G alginate yielding the least differentiated cell population. The addition of a PLL coating to the High G alginate prevented cell escape from beads for up to 14 days. Furthermore, encapsulation within High M alginate promoted differentiation toward a primitive endoderm phenotype. Taken together, the findings of this study suggest that distinct ESC expansion capacities and differentiation trajectories emerge depending on the alginate composition employed, indicating that encapsulation material physical properties can be used to control stem cell fate.