Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Xiao, Lin; McCann, James V.; Dudley, Andrew C.

doi:10.3791/53072

Cited by 15 publications

(11 citation statements)

References 40 publications

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“…The membrane‐associated factors ( PLPP3, SDC2, MXRA8 ) identified in our study are of special interest, since they could be used to solve a common problem observed in tissue cultures. Purified cell types of enzymatically digested organs are often contaminated with FBs . The newly identified membrane‐associated FB markers might represent a novel tool to specifically label and remove unwanted FBs from isolated primary cell suspensions.…”

Section: Discussionmentioning

confidence: 99%

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Deciphering the functional heterogeneity of skin fibroblasts using single‐cell RNA sequencing

et al. 2020

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Though skin fibroblasts (FB) are the main cell population within the dermis, the different skin FB subsets are not well characterized and the traditional classification into reticular and papillary FBs has little functional relevance. To fill the gap of knowledge on FB diversity in human skin, we performed single‐cell RNA sequencing. Investigation of marker genes for the different skin cell subtypes revealed a heterogeneous picture of FBs. When mapping reticular and papillary FB markers, we could not detect cluster specificity, suggesting that these two populations show a higher transcriptional heterogeneity than expected. This finding was further confirmed by in situ hybridization, showing that DPP4 was expressed in both dermal layers. Our analysis identified six FB clusters with distinct transcriptional signatures. Importantly, we could demonstrate that in human skin DPP4+ FBs are the main producers of factors involved in extracellular matrix (ECM) assembly. In conclusion, we provide evidence that hitherto considered FB markers are not ideal to characterize skin FB subpopulations in single‐cell sequencing analyses. The identification of DPP4+ FBs as the main ECM‐producing cells in human skin will foster the development of anti‐fibrotic treatments for the skin and other organs.

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Section: Discussionmentioning

confidence: 99%

“…Purified cell types of enzymatically digested organs are often contaminated with FBs. 50,51 The newly identified membrane-associated FB markers might represent a novel tool to specifically label and remove unwanted FBs from isolated primary cell suspensions. The feasibility of such an approach merits further investigations.…”

Section: Discussionmentioning

confidence: 99%

Deciphering the functional heterogeneity of skin fibroblasts using single‐cell RNA sequencing

et al. 2020

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“…A major challenge for the universal use of these isolation techniques is the heterogeneity of the endothelial cell population in different vascular beds and the high risk of contamination with fibroblasts and vascular mural cells. However, refinements of the isolation techniques to avoid such contaminations have been reported 24 . Using these techniques, mostly mature endothelial cells are isolated from tissues.…”

Section: Discussionmentioning

confidence: 99%

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Haynes

Huyck

James

et al. 2018

JoVE

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Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease. This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) >35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2-3 g of fat. These cells can be expanded in culture over 10-14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5-6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPARγ agonist-rosiglitazone. This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

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“…To study the dynamic functions of TECs, it is necessary to use TECs freshly isolated from tumors. Several isolating techniques of TECs from tumors, similar to the methods for isolating the endothelial cells from organs other than tumors (Marelli-Berg et al, 2000;Onoe et al, 2005a;Kajimoto et al, 2010), have been used to elucidate the characteristics of TECs (Amin et al, 2006;Dudley et al, 2008;Matsuda et al, 2010;Xiao et al, 2015). Often, however, we cannot obtain sufficient TECs using these techniques: TECs are indeed a small population within the tumor constituent cells, and their low number does not allow the purification of a sufficient number of cells of high purity.…”

Section: Introductionmentioning

confidence: 99%

Isolation of tumor endothelial cells from murine cancer

Taguchi

Onoe

Yoshida

et al. 2019

Journal of Immunological Methods

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Tumor endothelial cells (TECs), which constitute the lining of the tumor blood vessels, have various characteristics as tumor constituent cells. In this study, we describe a novel method for the isolation of highly pure, fresh TECs, which form a small population within the tumor. Tumors were first dissected from tumor-bearing mice and digested to a single cell suspension with Collagenase Type II; the single cells were then separated by density gradient centrifugation. TECs were enriched by CD31-positive selection using magnetic activated cell sorting and subsequently purified by fluorescence activated cell sorting. The high purity of the obtained cells was verified by flow cytometry. Upon cell culture, the isolated cells showed a polygonal shape and a cobblestone appearance, which are features of the endothelial cells. Furthermore, a functional assay revealed that the TECs suppressed the proliferation of CD8+ T cells in vitro. We believe that the isolation method described in this study will enable the further elucidation of the characteristics of TECs.

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Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Cited by 15 publications

References 40 publications

Deciphering the functional heterogeneity of skin fibroblasts using single‐cell RNA sequencing

Deciphering the functional heterogeneity of skin fibroblasts using single‐cell RNA sequencing

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Isolation of tumor endothelial cells from murine cancer

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