Assessing the Permeability of Engineered Capillary Networks in a 3D Culture

Many pathologies are characterized by poor blood vessel growth and reduced nutrient delivery to the surrounding tissue, introducing a need for tissue engineered blood vessels. Our lab has developed a 3D co-culture method to grow interconnected networks of pericyte-invested capillaries, which can anastamose with host vasculature following implantation to restore blood flow to ischemic tissues. However, if the engineered vessels contain endothelial cells (ECs) that are misaligned or contain wide junctional gaps, they may function improperly and behave more like the pathologic vessels that nourish tumors. The purpose of this study was to test the resistance to permeability of these networks in vitro, grown with different stromal cell types, as a metric of vessel functionality. A fluorescent dextran tracer was used to visualize transport across the endothelium and the pixel intensity was quantified using a customized MATLAB algorithm. In fibroblast-EC co-cultures, the dextran tracer easily penetrated through the vessel wall and permeability was high through the first 5 days of culture, indicative of vessel immaturity. Beyond day 5, dextran accumulated at the periphery of the vessel, with very little transported across the endothelium. Quantitatively, permeability dropped from initial levels of 61% to 39% after 7 days, and to 7% after 2 weeks. When ECs were co-cultured with bone marrow-derived mesenchymal stem cells (MSCs) or adipose-derived stem cells (AdSCs), much tighter control of permeability was achieved. Relative to the EC-fibroblast co-cultures, permeabilities were reduced 41% for the EC-MSC co-cultures and 50% for the EC-AdSC co-cultures after 3 days of culture. By day 14, these permeabilities decreased by 68% and 77% over the EC-fibroblast cultures. Co-cultures containing stem cells exhibit elevated VE-cadherin levels and more prominent EC-EC junctional complexes when compared to cultures containing fibroblasts. These data suggest the stromal cell identity influences the functionality and physiologic relevance of engineered capillary networks

3D сфероиды - клеточная модель для изучения воздействия гипоксии на эпикардиальное микроокружение

Fundamental research in recent years has allowed us to reassess the molecular and cellular mechanisms of cardiac ontogenesis and its repair after damage. The epicardium, the outer, tightly adjoining layer of the cardiac wall formed by epicardial mesothelial cells, collagen and elastic fibers, has gained special relevance as an important participant of reparative processes. Better insight into poorly understood epicardial function is challenged due to anatomical issues and lack of relevant cellular models.The aim of this study was to develop a spheroid 3D model of the epicardial microenvironment and determine responses of spheroids to hypoxia.Materials and methods. Spheroids were harvested in V-shaped culture dishes with a low adhesion coating. Immunofluorescent staining of cryosections, histological methods and real-time PCR were used for characterization of cultured spheroids.Results. We demonstrated that cultivation of cells under low adhesion conditions in V-shaped culture dishes resulted in the formation of spheroids with an average size of 136+21 µm and cell viability rates of over 98%. The cells in the spheroids cultured under normoxic conditions formed tight junctions and were characterized by a low level of proliferation and the ability to synthesize extracellular matrix proteins. Under hypoxia cells in the spheroids showed partial loss of intercellular contacts, acquired a spindle shape, started to express HIF1a, SNAIL, COL1Al and accumulate collagen. All these features demonstrated the activation of mesothelial(endothelial)-mesenchymal transition strongly resembling epicardial cellular responses to ischemia in vivo.Conclusion. An epicardial spheroid cell culture model suitable for study cellular responses to hypoxic environment was developed. This model can be used to clarify mechanisms regulating epicardial microenvironment and test new targeted candidate drugs.Фундаментальные исследования последних лет позволили переосмыслить молекулярные и клеточные механизмы онтогенеза сердца и его репарации после повреждения. Особую актуальность приобретает изучение эпикарда — наружного, плотно примыкающего к миокарду слоя сердечной стенки, образованного гетерогенной популяцией клеток эпикардиального мезотелия, коллагеновыми и эластическими волокнами, являющегося важным участником репаративных процессов. Изучение эпикарда затруднено в связи с анатомическими ограничениями и отсутствием релевантных клеточных моделей.Цель исследования. Разработка 3D модели эпикардиального микроокружения и оценка влияния гипоксии на ее характеристики.Материал и методы. Сборку сфероидов проводили в V-образных культуральных чашках с низкоадгезионным покрытием. Характеристику сфероидов выполняли с использованием иммунофлуоресцентного окрашивания криосрезов, гистологических методов, ПЦР в реальном времени.Результаты. Культивирование клеток в низкоадгезионных условиях в V-образных культуральных чашках ведет к формированию сфероидов, имеющих размер 136±21 мкм и показатели жизнеспособности клеток более 98%. Клетки в составе сфероидов, культивированных в условиях нормоксии, образовывали плотные межклеточные контакты, характеризовались низким уровнем пролиферации и способностью синтезировать белки внеклеточного матрикса. В условиях гипоксии клетки сфероидов частично утрачивали межклеточные контакты, приобретали веретенообразную форму, экспрессировали HIF1a, SNAI1, ACTA2, FN1, COL1A1 и накапливали коллаген, что указывает на признаки активации мезотелиально-мезенхимального перехода и сходные черты с клеточным ответом эпикарда на острое ишемическое повреждение in vivo.Заключение. На основе клеточного сфероида разработали и охарактеризовали модель эпикарда, которая может реализовать клеточный ответ на воздействие гипоксического стимула и быть использована для изучения механизмов регуляции эпикардиального микроокружения, тестирования лекарственных препаратов направленного действия

Origins of the Tumor Microenvironment: Quantitative Assessment of Adipose-Derived and Bone Marrow–Derived Stroma

To meet the requirements for rapid tumor growth, a complex array of non-neoplastic cells are recruited to the tumor microenvironment. These cells facilitate tumor development by providing matrices, cytokines, growth factors, as well as vascular networks for nutrient and waste exchange, however their precise origins remain unclear. Through multicolored tissue transplant procedures; we have quantitatively determined the contribution of bone marrow-derived and adipose-derived cells to stromal populations within syngeneic ovarian and breast murine tumors. Our results indicate that subpopulations of tumor-associated fibroblasts (TAFs) are recruited from two distinct sources. The majority of fibroblast specific protein (FSP) positive and fibroblast activation protein (FAP) positive TAFs originate from mesenchymal stem/stromal cells (MSC) located in bone marrow sources, whereas most vascular and fibrovascular stroma (pericytes, α-SMA+ myofibroblasts, and endothelial cells) originates from neighboring adipose tissue. These results highlight the capacity for tumors to utilize multiple sources of structural cells in a systematic and discriminative manner

Native human adipose stromal cells: localization, morphology and phenotype

International audienceObjectives:Beside having roles in energy homeostasis and endocrine modulation, adipose tissue (AT) is now considered a promising source of mesenchymal stromal cells (adipose-derived stromal cells or ASCs) for regenerative medicine. Despite numerous studies on cultured ASCs, native human ASCs are rarely investigated. Indeed, the phenotype of ASCs in their native state, their localization within AT and comparison with bone marrow-derived mesenchymal stromal cells (BM-MSCs) has been poorly investigated.Design:To address these issues, the stroma vascular fraction (SVF) of human AT was extracted and native cell subtypes were isolated by immunoselection to study their clonogenic potential in culture. Immunohistology on samples of human AT in combination with reconstruction of confocal sections were performed in order to localize ASCs.Results:Compared with BM-MNCs, all native ASCs were found in the CD34(+) cell fraction of the AT-SVF. Native ASCs expressed classical mesenchymal markers described for BM-MSCs. Interestingly, CD34 expression decreased during ASC cell culture and was negatively correlated with cell proliferation rate. Immunohistological analysis revealed that native ASCs exhibited specific morphological features with protrusions. They were found scattered in AT stroma and did not express in vivo pericytic markers such as NG2, CD140b or alpha-smooth muscle actin, which appeared during the culture process. Finally, ASCs spontaneous commitment to adipocytic lineage was enhanced in AT from obese humans.Conclusions:The use of complementary methodological approaches to study native human ASCs revealed their immunophenotype, their specific morphology, their location within AT and their stemness. Furthermore, our data strongly suggest that human ASCs participate in adipogenesis during AT development.International Journal of Obesity advance online publication, 25 January 2011; doi:10.1038/ijo.2010.269

SPARC functions as an inhibitor of adipogenesis

Adipogenesis, a key step in the pathogenesis of obesity, involves extensive ECM remodeling, changes in cell-ECM interactions, and cytoskeletal rearrangement. Matricellular proteins regulate cell-cell and cell-ECM interactions. Evidence in vivo and in vitro indicates that the prototypic matricellular protein, SPARC, inhibits adipogenesis and promotes osteoblastogenesis. Herein we discuss mechanisms underlying the inhibitory effect of SPARC on adipogenesis. SPARC enhances the Wnt/β-catenin signaling pathway and regulates the expression and posttranslational modification of collagen. SPARC might drive preadipocytes away from the status of growth arrest and therefore prevent terminal differentiation. SPARC could also decrease WAT deposition through its negative effects on angiogenesis. Therefore, several stages of white adipose tissue accumulation are sensitive to the inhibitory effects of SPARC

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Intramyocardial Injection of Plasmid Encoding Platelet Growth Factor Increases Epicardial-Mediated Post Infarction Myocardial Vascularization (Experimental Study)

Increasing incidence of ischemic diseases and limited resources for their treatment stimulate increased interest in studying the mechanisms of vascularization and finding new approaches for its promotion. One of these approaches is gene therapy aimed at activating the epicardium to produce the vascular precursor cells and microenvironment for the «assembly» of de novo vessels.The aim is to investigate the possibility of activating epicardial cells and post infarction cardiac vascularization by injecting a genetic construct encoding PDGFBB.Material and methods. A model of experimental myocardial infarction in a rat with subsequent intramyocardial injection of normal saline solution, control plasmid and plasmid encoding PDGFBB was used. The study of PDGFBB effect on epicardial cell activity was performed on the ex vivo model, as well as in vitro mesothelial cell culture.Results. Post infarction injection of plasmid encoding PDGFBB increases the density of the vascular network in the peri-infarct area as well as migration of pericytes to the injured zone. PDGFBB promotes activation of epicardial cell pool and expression of smooth muscle cell markers in them (shown on the ex vivo model), as well as stimulates activation of epithelial-mesenchymal transition (in vitro).Conclusion. Intramyocardial injection of a genetic construct encoding PDGFBB after an experimental myocardial infarction stimulated vascularization of the peri-infarction zone, which may have been partially due to the activation of the epicardial cell pool