Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea–induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage–dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.

BackgroundThe cell-material interaction is a complex bi-directional and dynamic process that mimics to a certain extent the natural interactions of cells with the extracellular matrix. Cells tend to adhere and rearrange adsorbed extracellular matrix (ECM) proteins on the material surface in a fibril-like pattern. Afterwards, the ECM undergoes proteolytic degradation, which is a mechanism for the removal of the excess ECM usually approximated with remodeling. ECM remodeling is a dynamic process that consists of two opposite events: assembly and degradation.Methodology/Principal FindingsThis work investigates matrix protein dynamics on mixed self-assembled monolayers (SAMs) of –OH and –CH3 terminated alkanethiols. SAMs assembled on gold are highly ordered organic surfaces able to provide different chemical functionalities and well-controlled surface properties. Fibronectin (FN) was adsorbed on the different surfaces and quantified in terms of the adsorbed surface density, distribution and conformation. Initial cell adhesion and signaling on FN-coated SAMs were characterized via the formation of focal adhesions, integrin expression and phosphorylation of FAKs. Afterwards, the reorganization and secretion of FN was assessed. Finally, matrix degradation was followed via the expression of matrix metalloproteinases MMP2 and MMP9 and correlated with Runx2 levels. We show that matrix degradation at the cell material interface depends on surface chemistry in MMP-dependent way.Conclusions/SignificanceThis work provides a broad overview of matrix remodeling at the cell-material interface, establishing correlations between surface chemistry, FN adsorption, cell adhesion and signaling, matrix reorganization and degradation. The reported findings improve our understanding of the role of surface chemistry as a key parameter in the design of new biomaterials. It demonstrates the ability of surface chemistry to direct proteolytic routes at the cell-material interface, which gains a distinct bioengineering interest as a new tool to trigger matrix degradation in different biomedical applications.

Protein adsorption and cellular behavior depend strongly on the wettability of substrates. Such studies are scarce for surfaces exhibiting extreme values of contact angles. Fibronectin (FN) adsorption and adhesion of MC3T3-E1 cells were investigated on superhydrophobic polystyrene (SH-PS) surfaces and compared with the corresponding smooth polystyrene (PS) substrate and the control glass. The FN surface density was lower on the SH-PS than on PS, and the adsorbed protein showed altered conformation of cell adhesion domains, as obtained by ELISA with monoclonal antibodies. Cell adhesion occurred on the SH-PS without the formation of mature focal adhesions, as assessed by immunofluorescence for vinculin, talin and paxillin. Correspondingly, the development of the actin cytoskeleton was delayed and without the presence of defined F-actin fibers. FAK phosphorylation was reduced on SH-PS, as compared with PS and the control glass. Also, cell contractility was diminished on the SH-PS as revealed by phosphorylation of myosin light chain (pMLC). Likewise, FN reorganization and secretion were impaired on the superhydrophobic surfaces. Cell proliferation was significantly lower in SH-PS as compared with PS up to 21 days of culture.

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WileyBallester Beltrán, J.; Lebourg, MM.; Salmerón Sánchez, M. (2013). Dorsal and ventral stimuli in sandwich-like microenvironments. Effect on cell differentiation. Dorsal stimulation of C2C12 myoblasts using sandwich-like microenvironments enhances cell differentiation. Dorsal integrin-mediated adhesion within sandwich culture triggers intracellular signal cascades and increase myoblast differentiation. Sandwich-like models are proposed as a versatile tool to study cell behavior in a quasi-3D environment under well-controlled conditions. Keywords: 3D matrix adhesion, fibronectin, integrins, multilayers, myoblasts, sandwich 2 ABSTRACT: While most of the in vivo extracellular matrices are 3D, most of the in vitro cultures are 2D -where only ventral adhesion is permitted-thus modifying cell behavior as a way to self-adaptation to this unnatural environment. We hypothesize that the excitation of dorsal receptors in cells already attached on a 2D surface (sandwich culture) could cover the gap between 2D and 3D cell-material interactions and result in a more physiological cell behavior.In this study we investigate the role of dorsal stimulation on myoblast differentiation within different poly(l-lactic acid) (PLLA) sandwich-like microenvironments, including plain material and aligned fibers. Enhanced cell differentiation levels were found for cells cultured with dorsal fibronectin-coated films. Seeking to understand the underlying mechanisms, experiments were carried out with (i) different types of dorsal stimuli (FN, albumin, FN after blocking the RGD integrin-binding site and activating dorsal cell integrin receptors), (ii) in the presence of an inhibitor of cell contractility and (iii) increasing the frequency of culture medium changes to assess the effect of paracrine factors. Furthermore, FAK and integrin expressions, determined by western blotting, revealed differences between cell sandwiches and 2D controls. Results show a stimuli-dependent response to dorsal excitation, proving that integrin outside-in signaling is involved in the enhanced cell differentiation. Due to their easiness and versatility, these sandwich-like systems are excellent candidates to get deeper insights into the study of 3D cell behavior and to direct cell fate within multilayer constructs.

From tissue morphogenesis to homeostasis, cells continuously experience and respond to physical, chemical, and biological cues commonly presented in gradients. In this article, we focus our discussion on the importance of nano/micro topographic cues on cell activity, and the role of anisotropic milieus play on cell behavior, mostly adhesion and migration. We present the need to study physiological gradients in vitro. To do this, we review different cell migration mechanisms and how adherent cells react to the presence of complex tissue-like environments and cell-surface stimulation in 2D and 3D (e.g., ventral/dorsal anisotropy).

SummaryThe production of blood cells and their precursors from human pluripotent stem cells (hPSCs) in vitro has the potential to make a significant impact upon healthcare provision. We demonstrate that the forward programming of hPSCs through overexpression of GATA1, FLI1, and TAL1 leads to the production of a population of progenitors that can differentiate into megakaryocyte or erythroblasts. Using “rainbow” lentiviral vectors to quantify individual transgene expression in single cells, we demonstrate that the cell fate decision toward an erythroblast or megakaryocyte is dictated by the level of FLI1 expression and is independent of culture conditions. Early FLI1 expression is critical to confer proliferative potential to programmed cells while its subsequent silencing or maintenance dictates an erythroid or megakaryocytic fate, respectively. These committed progenitors subsequently expand and mature into megakaryocytes or erythroblasts in response to thrombopoietin or erythropoietin. Our results reveal molecular mechanisms underlying hPSC forward programming and novel opportunities for application to transfusion medicine.

The identification of parameters influencing cell response is of paramount importance for the design of biomaterials that will act as synthetic scaffolds for cells to anchor, grow and, eventually, become specialised tissues. Cells interact with materials through an intermediate layer of proteins adsorbed on the material surface. It is known that the density and conformation of these proteins determine cell behaviour. Here we show that the strength of protein/material interactions, which has received very limited attention so far, is key to understand the cellular response to biomaterials. Very strong protein/material interactions reduce the ability of cells to mechanically reorganize proteins at the material interface which results in enhanced matrix degradation, leading ultimately to compromised cell viability.