The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodeling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, Hand2, MEF2C and Tbx5 can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodeling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.

Rationale In early heart development, platelet derived growth factor (PDGF) receptor expression in the heart ventricles is restricted to the epicardium. Previously, we showed that PDGFRβ is required for coronary vascular smooth muscle cell (cVSMC) development, but a role for PDGFRα, has not been identified. Therefore, we investigated the combined and independent roles of these receptors in epicardial development. Objective To understand the contribution of PDGF receptors in epicardial development and epicardial derived cell fate determination. Methods and results By generating mice with epicardial specific deletion of the PDGF receptors, we found that epicardial EMT was defective. Sox9, an SRY-related transcription factor, was reduced in PDGF receptor-deficient epicardial cells, and overexpression of Sox9 restored epicardial migration, actin reorganization, and EMT gene expression profiles. The failure of epicardial EMT resulted in hearts that lacked epicardial-derived cardiac fibroblasts and cVSMC. Loss of PDGFRα, resulted in a specific disruption of cardiac fibroblast development, while cVSMC development was unperturbed. Conclusions Signaling through both PDGF receptors is necessary for epicardial EMT and formation of epicardial mesenchymal derivatives. PDGF receptors also have independent functions in the development of specific epicardial derived cell fates.

Abstract-The epicardium plays an essential role in coronary artery formation and myocardial development, but signals controlling the development and differentiation of this tissue are not well understood. To investigate the role of platelet-derived growth factor receptor (PDGFR)␤ in development of epicardial-derived vascular smooth muscle cells (VSMCs), we examined PDGFR␤ Ϫ/Ϫ and PDGFR␤ epicardial mutant hearts. We found that PDGFR␤ Ϫ/Ϫ hearts failed to form dominant coronary vessels on the ventral heart surface, had a thinned myocardium, and completely lacked coronary VSMCs (cVSMCs). This constellation of defects was consistent with a primary defect in the epicardium. To verify that these defects were specific to epicardial derivatives, we generated mice with an epicardial deletion of PDGFR␤ that resulted in reduced cVSMCs distal to the aorta. The regional absence of cVSMCs suggested that cVSMCs could arise from 2 sources, epicardial and nonepicardial, and that both were dependent on PDGFR␤. In the absence of PDGFR␤ signaling, epicardial cells adopted an irregular actin cytoskeleton, leading to aberrant migration of epicardial cells into the myocardium in vivo. In addition, PDGF receptor stimulation promoted epicardial cell migration, and PDGFR␤-driven phosphoinositide 3Ј-kinase signaling was critical for this process. Our data demonstrate that PDGFR␤ is required for the formation of 2 distinct cVSMC populations and that loss of PDGFR␤-PI3K signaling disrupts epicardial cell migration. , and past data have demonstrated that many coronary VSMCs (cVSMCs) are derived from the embryonic epicardium. 2,3 Whereas several genes have been identified that are essential for the formation, attachment, and spreading of the epicardium, few genes have been identified that are essential during epithelial-to-mesenchymal transition (EMT) and subsequent differentiation into cVSMCs and cardiac fibroblasts.Platelet-derived growth factor receptor (PDGFR) tyrosine kinases are 1 family of signaling proteins that are potentially involved in epicardial cell function. Analyses in the mouse have shown that PDGFR␤ signaling promotes proliferation and migration of VSMCs in multiple vascular beds including the heart. 4 -8 Therefore, we investigated the function of PDGFR␤ signaling during epicardial development. We have examined PDGFR␤ Ϫ/Ϫ , epicardial-specific PDGFR␤ mutant, and PDGFR␤ signaling-deficient embryos. We discovered that epicardial deletion resulted in the absence of cVSMCs distal to the aorta and that PDGFR␤ signaling through phosphoinositide 3Ј-kinase (PI3K) was required for proper cytoskeletal organization in epicardial cells. Our results designate PDGF receptor signaling as another growth factor system involved in epicardial development.

Epidermal growth factor receptor (EGFR) signaling is a potent driver of glioblastoma, a malignant and lethal form of brain cancer. Disappointingly, inhibitors targeting receptor tyrosine kinase activity are not clinically effective, and EGFR persists on the plasma membrane to maintain tumor growth and invasiveness. Here we show that endolysosomal pH is critical for receptor sorting and turnover. By functioning as a leak pathway for protons, the Na+/H+ exchanger NHE9 limits luminal acidification to circumvent EGFR turnover and prolong downstream signaling pathways that drive tumor growth and migration. In glioblastoma, NHE9 expression is associated with stem/progenitor characteristics, radiochemoresistance, poor prognosis and invasive growth in vitro and in vivo. Silencing or inhibition of NHE9 in brain tumor initiating cells attenuates tumorsphere formation and improves efficacy of EGFR inhibitor. Thus, NHE9 mediates inside-out control of oncogenic signaling and is a highly druggable target for pan-specific receptor clearance in cancer therapy.

The ion transporter NKCC1 determines brain tumor cell migration by regulating the interplay between cell adhesion and growth factor signaling, and is a potential therapeutic target to treat brain cancer.

The advent of stem cell based therapies has brought regenerative medicine into an increased focus as a part of the modern medicine practice, with a potential to treat a myriad of intractable diseases in the future. Stem cells reside in a complex microenvironment presenting them with a multitude of potential cues that are chemical, physical, and mechanical in nature. Conventional techniques used for experiments involving stem cells can only poorly mimic the physiological context, and suffer from imprecise spatial and temporal control, low throughput, lack of scalability and reproducibility, and poor representation of the mechanical and physical cell microenvironment. Novel lab-on-a-chip platforms, on the other hand, can much better mimic the complexity of in vivo tissue milieu and provide a greater control of the parameter variation in a high throughput and scalable manner. This capability may be especially important for understanding the biology and cementing the clinical potential of stem cell based therapies. Here we review microfabrication- and microfluidics-based approaches to investigating the complex biology of stem cell responses to changes in the local microenvironment. In particular, we categorize each method based on the types of controlled inputs it can have on stem cells, including soluble biochemical factors, extracellular matrix interactions, homotypic and heterotypic cell-cell signaling, physical cues (e.g. oxygen tension, pH, temperature), and mechanical forces (e.g. shear, topography, rigidity). Finally, we outline the methods to perform large scale observations of stem cell phenotypes and high-throughput screening of cellular responses to a combination of stimuli, and many new emerging technologies that are becoming available specifically for stem cell applications.

Purpose Glioblastoma (GBM) is the most common adult primary malignant intracranial cancer. It is associated with poor outcomes due to its invasiveness and resistance to multimodal therapies. Human adipose-derived mesenchymal stem cells (hAMSCs) are a potential treatment because of their tumor tropism, ease of isolation, and ability to be engineered. In addition, bone morphogenetic protein 4 (BMP4) has tumor-suppressive effects on GBM and GBM brain tumor initiating cells (BTICs), but is difficult to deliver to brain tumors. We sought to engineer BMP4-secreting hAMSCs (hAMSCs-BMP4) and evaluate their therapeutic potential on GBM. Experimental Design The reciprocal effects of hAMSCs on primary human BTIC proliferation, differentiation, and migration were evaluated in vitro. The safety of hAMSC use was evaluated in vivo by intracranial co-injections of hAMSCs and BTICs in nude mice. The therapeutic effects of hAMSCs and hAMSCs-BMP4 on the proliferation and migration of GBM cells as well as the differentiation of BTICs, and survival of GBM-bearing mice were evaluated by intracardiac injection of these cells into an in vivo intracranial GBM murine model. Results hAMSCs-BMP4 targeted both the GBM tumor bulk and migratory GBM cells, as well as induced differentiation of BTICs, decreased proliferation, and reduced the migratory capacity of GBMs in vitro and in vivo. In addition, hAMSCs-BMP4 significantly prolonged survival in a murine model of GBM. We also demonstrate that the use of hAMSCs in vivo is safe. Conclusions Both unmodified and engineered hAMSCs are non-oncogenic and effective against GBM, and hAMSCs-BMP4 are a promising cell-based treatment option for GBM.

Summary During heart morphogenesis, epicardial cells undergo an epithelial to mesenchymal transition (EMT) and migrate into the subepicardium. The cellular signals controlling this process are poorly understood. Here, we show that epicardial cells exhibit two distinct mitotic spindle orientations, directed either parallel or perpendicular to the basement membrane. Cells undergoing perpendicular cell division subsequently enter the myocardium. We found that loss of β-catenin led to a disruption in adherens junctions and a randomization of mitotic spindle orientation. Loss of adherens junctions also disrupted Numb localization within epicardial cells and disruption of Numb and Numblike expression in the epicardium led to randomized mitotic spindle orientations. Taken together, these data suggest that directed mitotic spindle orientation contributes to epicardial EMT and implicate a junctional complex of β-catenin and Numb in the regulation of spindle orientation.