Casein Kinase 1 Phosphomimetic Mutations Negatively Impact Connexin-43 Gap Junctions in Human Pluripotent Stem Cell-Derived Cardiomyocytes.

The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has shown promise in preclinical models of myocardial infarction, but graft myocardium exhibits incomplete host-graft electromechanical integration and a propensity for pro-arrhythmic behavior. Perhaps contributing to this situation, hPSC-CM grafts show low expression of connexin 43 (Cx43), the major gap junction (GJ) protein, in ventricular myocardia. We hypothesized that Cx43 expression and function could be rescued by engineering Cx43 in hPSC-CMs with a series of phosphatase-resistant mutations at three casein kinase 1 phosphorylation sites (Cx43-S3E) that have been previously reported to stabilize Cx43 GJs and reduce arrhythmias in transgenic mice. However, contrary to our predictions, transgenic Cx43-S3E hPSC-CMs exhibited reduced Cx43 expression relative to wild-type cells, both at baseline and following ischemic challenge. Cx43-S3E hPSC-CMs showed correspondingly slower conduction velocities, increased automaticity, and differential expression of other connexin isoforms and various genes involved in cardiac excitation-contraction coupling. Cx43-S3E hPSC-CMs also had phosphorylation marks associated with Cx43 GJ internalization, a finding that may account for their impaired GJ localization. Taken collectively, our data indicate that the Cx43-S3E mutation behaves differently in hPSC-CMs than in adult mouse ventricular myocytes and that multiple biological factors likely need to be addressed synchronously to ensure proper Cx43 expression, localization, and function.

Sphingosine-1-phosphate receptor 3 potentiates inflammatory programs in normal and leukemia stem cells to promote differentiation.

Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis, driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature myeloid cells and influences stemness programs and lineage determination in HSC by activating stress myelopoiesis, such roles in LSC are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator which drives myeloid differentiation and activates inflammatory programs in both HSC and LSC. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across AML patient cohorts, each with distinct phenotypic and clinical properties. S1PR3 was high in LSC and blasts of mature myeloid samples with linkages to chemosensitivity, while S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset.

Sphingolipid Modulation Activates Proteostasis Programs to Govern Human Hematopoietic Stem Cell Self-Renewal.

Cellular stress responses serve as crucial decision points balancing persistence or culling of hematopoietic stem cells (HSCs) for lifelong blood production. Although strong stressors cull HSCs, the linkage between stress programs and self-renewal properties that underlie human HSC maintenance remains unknown, particularly at quiescence exit when HSCs must also dynamically shift metabolic state. Here, we demonstrate distinct wiring of the sphingolipidome across the human hematopoietic hierarchy and find that genetic or pharmacologic modulation of the sphingolipid enzyme DEGS1 regulates lineage differentiation. Inhibition of DEGS1 in hematopoietic stem and progenitor cells during the transition from quiescence to cellular activation with N-(4-hydroxyphenyl) retinamide activates coordinated stress pathways that coalesce on endoplasmic reticulum stress and autophagy programs to maintain immunophenotypic and functional HSCs. Thus, our work identifies a linkage between sphingolipid metabolism, proteostatic quality control systems, and HSC self-renewal and provides therapeutic targets for improving HSC-based cellular therapeutics.

SIRT1-dependent anti-senescence effects of cell-deposited matrix on human umbilical cord mesenchymal stem cells.

Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are considered an attractive cell source for tissue regeneration. However, environmental oxidative stress can trigger premature senescence in MSCs and thus compromises their regenerative potential. Extracellular matrix (ECM) derived from MSCs has been shown to facilitate cell proliferation and multi-lineage differentiation. This investigation evaluated the effect of cell-deposited decellularized ECM (DECM) on oxidative stress-induced premature senescence in UC-MSCs. Sublethal dosages of H2 O2 , ranging from 50 µm to 200 µm, were used to induce senescence in MSCs. We found that DECM protected UC-MSCs from oxidative stress-induced premature senescence. When treated with H2 O2 at the same concentration, cell proliferation of DECM-cultured UC-MSCs was twofold higher than those on standard tissue culture polystyrene (TCPS). After exposure to 100 µm H2 O2 , fewer senescence-associated ß-galactosidase-positive cells were observed on DECM than those on TCPS (17.6  ± â€…4.0% vs. 60.4  ± â€…6.2%). UC-MSCs cultured on DECM also showed significantly lower levels of senescence-related regulators, such as p16INK4α and p21. Most importantly, DECM preserved the osteogenic differentiation potential of UC-MSCs with premature senescence. The underlying molecular mechanisms involved the silent information regulator type 1 (SIRT1)-dependent signalling pathway, confirmed by the fact that the SIRT1 inhibitor nicotinamide counteracted the DECM-mediated anti-senescent effect. Collagen type I, rather than fibronectin, partially contributed to the protective effect of decellularized matrix. These findings provide a new strategy of using stem cell-deposited matrix to overcome the challenge of cellular senescence and to facilitate the clinical application of MSCs in regenerative medicine. Copyright © 2017 John Wiley & Sons, Ltd.