Alhaji Osman Smith1,2, Seyram Yao Adzraku1,2, Wen Ju1,2,3, Jianlin Qiao4,5,6, Kailin Xu7,8,9, Lingyu Zeng10,11,12. 1. Blood Diseases Institute, Xuzhou Medical University, Xuzhou, 221002, China. 2. Key Laboratory of Bone Marrow Stem Cell, Xuzhou, 221002, Jiangsu Province, China. 3. Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221002, China. 4. Blood Diseases Institute, Xuzhou Medical University, Xuzhou, 221002, China. jianlin.qiao@gmail.com. 5. Key Laboratory of Bone Marrow Stem Cell, Xuzhou, 221002, Jiangsu Province, China. jianlin.qiao@gmail.com. 6. Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221002, China. jianlin.qiao@gmail.com. 7. Blood Diseases Institute, Xuzhou Medical University, Xuzhou, 221002, China. lihmd@163.com. 8. Key Laboratory of Bone Marrow Stem Cell, Xuzhou, 221002, Jiangsu Province, China. lihmd@163.com. 9. Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221002, China. lihmd@163.com. 10. Blood Diseases Institute, Xuzhou Medical University, Xuzhou, 221002, China. zengly2000@163.com. 11. Key Laboratory of Bone Marrow Stem Cell, Xuzhou, 221002, Jiangsu Province, China. zengly2000@163.com. 12. Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221002, China. zengly2000@163.com.
Abstract
BACKGROUND: In the bone marrow microenvironment (BM), endothelial cells are individual cells that form part of the sinusoidal blood vessels called the "bone marrow endothelial-vascular niche." They account for less than 2% of the bone marrow cells. They play essential functions by generating growth and inhibitory factors that promote the hematopoietic stem cells (HSCs) regulation. In response to inflammatory stimuli, the BMECs increase in proliferation to maintain the blood vessels' integrity within the BM. The inflammatory response releases cytokines such as tumor necrosis factor-alpha (TNF-α) that promote vascular endothelial cells' expansion and upregulation of adhesion molecules (ICAM-1 and VCAM-1, respectively) in the BM. However, the evaluation of mouse BMECs in the bone marrow microenvironment is scared by a lack of mouse bone marrow endothelial cell primary culture METHODS: Two steps approach for isolation of bone marrow endothelial cells (BMECs) from mice. In brief, the bone marrow cells extracted from the mice long bones were cultured overnight with Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS) and antibiotics to separate between marrow-derived adherent and non-adherent cells. The floating cells were discarded, and the adhered section detached with accutase and BMECs selected using CD31 microbeads. The isolated BMECs were cultured in a dish pre-coated with rat-tail collagen type 1 with endothelial cells medium supplement with growth factors. The cells were verified by confocal microscopy for morphology and tube formation by matrigel assay. We validate the cells' purity by flow cytometry, RT-qPCR, immunofluorescence staining, and immunoblotting by established BMEC markers, PECAM-1, VE-cadherin, vascular endothelial cell growth factor receptor-2 (VEGFR2), CD45, E-selectin, and endothelial selectin adhesion molecule (ESAM). Lastly, we characterize BMEC activation with recombinant TNF-α. RESULTS: Our method clearly defined the cells isolated have the characteristics of BMECs with the expression of CD31, VE-cadherin, E-selectin, VEGFR-2, and ESAM. The cells' response to TNF-α indicates its inflammatory function by increasing proliferation and upregulation of adhesion molecules. CONCLUSIONS: This study outline a simple new technique of isolating mouse BMEC primary culture and a suitable method to evaluate the function and dysregulation of BMEC in in vitro studies using mouse models.
BACKGROUND: In the bone marrow microenvironment (BM), endothelial cells are individual cells that form part of the sinusoidal blood vessels called the "bone marrow endothelial-vascular niche." They account for less than 2% of the bone marrow cells. They play essential functions by generating growth and inhibitory factors that promote the hematopoietic stem cells (HSCs) regulation. In response to inflammatory stimuli, the BMECs increase in proliferation to maintain the blood vessels' integrity within the BM. The inflammatory response releases cytokines such as tumor necrosis factor-alpha (TNF-α) that promote vascular endothelial cells' expansion and upregulation of adhesion molecules (ICAM-1 and VCAM-1, respectively) in the BM. However, the evaluation of mouse BMECs in the bone marrow microenvironment is scared by a lack of mouse bone marrow endothelial cell primary culture METHODS: Two steps approach for isolation of bone marrow endothelial cells (BMECs) from mice. In brief, the bone marrow cells extracted from the mice long bones were cultured overnight with Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS) and antibiotics to separate between marrow-derived adherent and non-adherent cells. The floating cells were discarded, and the adhered section detached with accutase and BMECs selected using CD31 microbeads. The isolated BMECs were cultured in a dish pre-coated with rat-tail collagen type 1 with endothelial cells medium supplement with growth factors. The cells were verified by confocal microscopy for morphology and tube formation by matrigel assay. We validate the cells' purity by flow cytometry, RT-qPCR, immunofluorescence staining, and immunoblotting by established BMEC markers, PECAM-1, VE-cadherin, vascular endothelial cell growth factor receptor-2 (VEGFR2), CD45, E-selectin, and endothelial selectin adhesion molecule (ESAM). Lastly, we characterize BMEC activation with recombinant TNF-α. RESULTS: Our method clearly defined the cells isolated have the characteristics of BMECs with the expression of CD31, VE-cadherin, E-selectin, VEGFR-2, and ESAM. The cells' response to TNF-α indicates its inflammatory function by increasing proliferation and upregulation of adhesion molecules. CONCLUSIONS: This study outline a simple new technique of isolating mouse BMEC primary culture and a suitable method to evaluate the function and dysregulation of BMEC in in vitro studies using mouse models.