Abstract-We report that human embryonic stem cells contain a population of vascular progenitor cells that have the ability to differentiate into endothelial-like and smooth muscle (SM)-like cells. Vascular progenitor cells were isolated from EBs grown in suspension for 10 days and were characterized by expression of the endothelial/hematopoietic marker CD34 (CD34 ϩ cells). When these cells are subsequently cultured in EGM-2 (endothelial growth medium) supplemented with vascular endothelial growth factor-165 (50 ng/mL), they give rise to endothelial-like cells characterized by a cobblestone cell morphology, expression of endothelial markers (platelet endothelial cell-adhesion molecule-1, CD34, KDR/Flk-1, vascular endothelial cadherin, von Willebrand factor), incorporation of acetylated low-density lipoprotein, and formation of capillary-like structures when placed in Matrigel. In contrast, when CD34 ϩ cells are cultured in EGM-2 supplemented with platelet-derived growth factor-BB (50 ng/mL), they give rise to SM-like cells characterized by spindle-shape morphology, expression of SM cell markers (␣-SM actin, SM myosin heavy chain, calponin, caldesmon, SM ␣-22), and the ability to contract and relax in response to common pharmacological agents such as carbachol and atropine but rarely form capillary-like structures when placed in Matrigel. Implantation studies in nude mice show that both cell types contribute to the formation of human microvasculature. Some microvessels contained mouse blood cells, which indicates functional integration with host vasculature. Therefore, the vascular progenitors isolated from human embryonic stem cells using methods established in the present study could provide a means to examine the mechanisms of endothelial and SM cell development, and they could also provide a potential source of cells for vascular tissue engineering. [3][4][5] Isolating a population of human progenitor cells with potential for cell number expansion and differentiation into both ECs and SMCs with high efficiency could benefit the area of tissue engineering. 2,5,6 Embryonic stem cells (ESCs) are a potential cell source for induction of tissue vascularization. 7 Prior studies have derived ECs and SMCs cells from a common progenitor (Flk-1 ϩ cells) from mouse 6 and monkey ESCs, 8 but not from human ESCs (hESCs). We previously reported that hESCs can spontaneously generate ECs with definitive properties. 9 These cells were isolated based on the expression of platelet EC-adhesion molecule-1 (PECAM1) from embryoid bodies (EBs) grown in suspension for 13 to 15 days. Using the same endothelial 10 or other (eg, CD34 11,12 ) markers, others have isolated endothelial progenitor cells with the ability to differentiate into mature endothelium. In addition, it has been reported that hESCs can differentiate into mesodermal cells that can give rise to ECs and SMCs 13 ; however, it is not clear that these cells were derived from the same progenitor.Here we report that cells isolated from EBs at day 10 and expressing the hematop...

We propose a new methodology to enhance the vascular differentiation of human embryonic stem cells (hESCs) by encapsulation in a bioactive hydrogel. hESCs were encapsulated in a dextran-based hydrogel with or without immobilized regulatory factors: a tethered RGD peptide and microencapsulated VEGF 165 . The fraction of cells expressing vascular endothelial growth factor (VEGF) receptor KDR/Flk-1, a vascular marker, increased up to 20-fold, as compared to spontaneously differentiated embryoid bodies (EBs). The percentage of encapsulated cells in hydrogels with regulatory factors expressing ectodermal markers including nestin or endodermal markers including α-fetoprotein decreased 2 or 3 fold, respectively, as compared to EBs. When the cells were removed from these networks and cultured in media conditions conducive for further vascular differentiation, the number of vascular cells was higher than the number obtained through EBs, using the same media conditions. Functionalized dextran-based hydrogels could thus enable derivation of vascular cells in large quantities, particularly endothelial cells, for potential application in tissue engineering and regenerative medicine. 1-INTRODUCTIONDuring normal embryogenesis, human embryonic stem cells (hESCs) differentiate along different lineages in the context of complex three-dimensional tissue structures, where the extracellular matrix (ECM) and different growth factors play an important role in this process. The three-dimensional ECM provides structural support for higher level of tissue organization and remodelling [1]. Significant differences were found in the differentiation profile of ESCs when cultured in a three-dimensional (3D) versus two-dimensional (2D) system [2,3] or 3D scaffold system versus embryoid body (EB) system [4,5]. In this last case, mouse ESCs cultured within tantalum scaffolds differentiate at higher extent into hematopoietic cells than EBs [4], while hESCs cultured in alginate scaffolds express significantly more vascular markers than EBs [5]. Moreover, the culture of hESCs within 3D poly(α-hydroxy esters) scaffolds with media containing different growth factors induced their differentiation into 3D structures with || To whom reprint requests should be addressed at: Department of Chemical Engineering, E25-342 Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139. E-mail: rlanger@mit.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [1,6]. However, these growth factors were supplied from outside the scaffold, and thus their activity may be affected by diffusion lim...

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Purpose: We review outcomes of posterior tracheopexy for tracheomalacia in esophageal atresia (EA) patients, comparing primary treatment at the time of initial EA repair versus secondary treatment.Methods: All EA patients who underwent posterior tracheopexy from October 2012 to September 2016 were retrospectively reviewed. Clinical symptoms, tracheomalacia scores, and persistent airway intrusion were collected. Indication for posterior tracheopexy was the presence of clinical symptoms, in combination with severe tracheomalacia as identified on bronchoscopic evaluation, typically defined as coaptation in one or more regions of the trachea. Secondary cases were usually those with chronic respiratory symptoms who underwent bronchoscopic evaluation, whereas primary cases were those found to have severe tracheomalacia on routine preoperative dynamic tracheobronchoscopy at the time of initial EA repair.results: A total of 118 patients underwent posterior tracheopexy: 18 (15%) primary versus 100 (85%) secondary cases. Median (interquartile range) age was 2 months (1-4 months) for primary (22% type C) and 18 months (8-40 months) for secondary (87% type C) cases (p < 0.001). There were statistically significant improvements in most clinical symptoms postoperatively for primary and secondary cases, with no significant differences in any postoperative symptoms between the two groups (p > 0.1). Total tracheomalacia scores improved significantly in primary (p = 0.013) and secondary (p < 0.001) cases. Multivariable Cox regression analysis indicated no differences in persistent airway intrusion requiring reoperation between primary and secondary tracheopexy adjusting for imbalances in age and EA type (p = 0.67).conclusion: Posterior tracheopexy is effective in treating severe tracheomalacia with significant improvements in clinical symptoms and degree of airway collapse on bronchoscopy. With no significant differences in outcomes between primary and secondary treatment, posterior tracheopexy should be selectively considered at the time of initial EA repair.