A Novel Hypothesis and Characterization to Isolate Microvascular Endothelial Cells Simultaneously with Adipose-Derived Stem Cells from the Human Adipose-Derived Stromal Vascular Fraction

Ryu Yeon Hee,Moon Suk-Ho,Kim Ki Joo,Jun Young Joon,Oh Deuk-Young,Kim Sang‐Heon,Rhie Jong-Won 한국조직공학과 재생의학회 2021 조직공학과 재생의학 Vol.18 No.3

Background: Angiogenesis and vasculogenesis are essential processes for successful tissue regeneration in tissue engineering and regenerative medicine. The adipose-derived stromal vascular fraction (SVF) is not only a source of adipose stem cells (ASC) but also a suitable source of microvascular endothelial cells because it is a rich capillary network. So, we propose a new hypothesis for isolating adipose-derived human microvascular endothelial cells (HMVEC-A) from the SVF and developed a dual isolation system that isolates two cell types from one tissue. Method: To isolate HMVEC-A, we analyzed the supernatant discarded when ASC is isolated from the adipose-derived SVF. Based on this analysis, we assumed that the SVF adherent to the bottom of the culture plate was divided into two fractions: the stromal fraction as the ASC-rich fraction, and the vascular fraction (VF) as the endothelial cells-rich fraction floating in the culture supernatant. VF isolation was optimized and the efficiency was compared, and the endothelial cells characteristics of HMVEC-A were confirmed by flow cytometric analysis, immunocytochemistry (ICC), a DiI-acetylated low-density lipoprotein (DiI-Ac-LDL) uptake, and in vitro tube formation assay. Results: Consistent with the hypothesis, we found a large population of HMVEC-A in the VF and isolated these HMVEC-A by our isolation method. Additionally, this method had higher yields and shorter doubling times than other endothelial cells isolation methods and showed typical morphological and phenotypic characteristics of endothelial cells. Conclusion: Cells obtained by the method according to our hypothesis can be applied as a useful source for studies such as tissue-to-tissue networks, angiogenesis and tissue regeneration, patient-specific cell therapy, and organoid chips. Background: Angiogenesis and vasculogenesis are essential processes for successful tissue regeneration in tissue engineering and regenerative medicine. The adipose-derived stromal vascular fraction (SVF) is not only a source of adipose stem cells (ASC) but also a suitable source of microvascular endothelial cells because it is a rich capillary network. So, we propose a new hypothesis for isolating adipose-derived human microvascular endothelial cells (HMVEC-A) from the SVF and developed a dual isolation system that isolates two cell types from one tissue. Method: To isolate HMVEC-A, we analyzed the supernatant discarded when ASC is isolated from the adipose-derived SVF. Based on this analysis, we assumed that the SVF adherent to the bottom of the culture plate was divided into two fractions: the stromal fraction as the ASC-rich fraction, and the vascular fraction (VF) as the endothelial cells-rich fraction floating in the culture supernatant. VF isolation was optimized and the efficiency was compared, and the endothelial cells characteristics of HMVEC-A were confirmed by flow cytometric analysis, immunocytochemistry (ICC), a DiI-acetylated low-density lipoprotein (DiI-Ac-LDL) uptake, and in vitro tube formation assay. Results: Consistent with the hypothesis, we found a large population of HMVEC-A in the VF and isolated these HMVEC-A by our isolation method. Additionally, this method had higher yields and shorter doubling times than other endothelial cells isolation methods and showed typical morphological and phenotypic characteristics of endothelial cells. Conclusion: Cells obtained by the method according to our hypothesis can be applied as a useful source for studies such as tissue-to-tissue networks, angiogenesis and tissue regeneration, patient-specific cell therapy, and organoid chips.

Endothelial Cell Products as a Key Player in Hypoxia-Induced Nerve Cell Injury after Stroke

Cho, Chul-Min,Ha, Se-Un,Bae, Hae-Rahn,Huh, Jae-Taeck The Korean Neurosurgical Society 2006 Journal of Korean neurosurgical society Vol.40 No.2

Objective : Activated endothelial cells mediate the cascade of reactions in response to hypoxia for adaptation to the stress. It has been suggested that hypoxia, by itself, without reperfusion, can activate the endothelial cells and initiate complex responses. In this study, we investigated whether hypoxia-induced endothelial products alter the endothelial permeability and have a direct cytotoxic effect on nerve cells. Methods : Hypoxic condition of primary human umbilical vein endothelial cells[HUVEC] was induced by $CoCl_2$ treatment in culture medium. Cell growth was evaluated by 3,4,5-dimethyl thiazole-3,5-diphenyl tetrazolium bromide [MTT] assay Hypoxia-induced products [$IL-1{\beta},\;TGF-{\beta}1,\;IFN-{\gamma},\;TNF-{\alpha}$, IL-10, IL-6, IL-8, MCP-l and VEGF] were assessed by enzyme-linked immunosorbent assay. Endothelial permeability was evaluated by Western blotting. Results : Prolonged hypoxia caused endothelial cells to secrete IL -6, IL -8, MCP-1 and VEGF. However, the levels of IL -1, IL -10, $TNF-{\alpha},\;TGF-{\beta},\;IFN-{\gamma}$ and nitric oxide remained unchanged over 48 h hypoxia. Hypoxic exposure to endothelial cells induced the time-dependent down regulation of the expression of cadherin and catenin protein. The conditioned medium taken from hypoxic HUVECs had the cytotoxic effect selectively on neuroblastoma cells, but not on astroglioma cells. Conclusion : These results suggest the possibility that endothelial cell derived cytokines or other secreted products with the increased endothelial permeability might directly contribute to nerve cell injury followed by hypoxia.

혈관내피세포 채취의 원천으로 인간 지방조직의 활용

박봉욱,하영술,김진현,조희영,정명희,김덕룡,김욱규,김종렬,장중희,변준호,Park, Bong-Wook,Hah, Young-Sool,Kim, Jin-Hyun,Cho, Hee-Young,Jung, Myeong-Hee,Kim, Deok-Ryong,Kim, Uk-Kyu,Kim, Jong-Ryoul,Jang, Jung-Hui,Byun, June-Ho 대한악안면성형재건외과학회 2010 Maxillofacial Plastic Reconstructive Surgery Vol.32 No.4

Purpose: Adipose tissue is located beneath the skin, around internal organs, and in the bone marrow in humans. Its main role is to store energy in the form of fat, although it also cushions and insulates the body. Adipose tissue also has the ability to dynamically expand and shrink throughout the life of an adult. Recently, it has been shown that adipose tissue contains a population of adult multipotent mesenchymal stem cells and endothelial progenitor cells that, in cell culture conditions, have extensive proliferative capacity and are able to differentiate into several lineages, including, osteogenic, chondrogenic, endothelial cells, and myogenic lineages. Materials and Methods: This study focused on endothelial cell culture from the adipose tissue. Adipose tissues were harvested from buccal fat pad during bilateral sagittal split ramus osteotomy for surgical correction of mandibular prognathism. The tissues were treated with 0.075% type I collagenase. The samples were neutralized with DMEM/and centrifuged for 10 min at 2,400 rpm. The pellet was treated with 3 volume of RBC lysis buffer and filtered through a 100 ${\mu}m$ nylon cell strainer. The filtered cells were centrifuged for 10 min at 2,400 rpm. The cells were further cultured in the endothelial cell culture medium (EGM-2, Cambrex, Walkersville, Md., USA) supplemented with 10% fetal bovine serum, human EGF, human VEGF, human insulin-like growth factor-1, human FGF-$\beta$, heparin, ascorbic acid and hydrocortisone at a density of $1{\times}10^5$ cells/well in a 24-well plate. Low positivity of endothelial cell markers, such as CD31 and CD146, was observed during early passage of cells. Results: Increase of CD146 positivity was observed in passage 5 to 7 adipose tissue-derived cells. However, CD44, representative mesenchymal stem cell marker, was also strongly expressed. CD146 sorted adipose tissue-derived cells was cultured using immuno-magnetic beads. Magnetic labeling with 100 ${\mu}l$ microbeads per 108 cells was performed for 30 minutes at $4^{\circ}C$ a using CD146 direct cell isolation kit. Magnetic separation was carried out and a separator under a biological hood. Aliquous of CD146+ sorted cells were evaluated for purity by flow cytometry. Sorted cells were 96.04% positivity for CD146. And then tube formation was examined. These CD146 sorted adipose tissue-derived cells formed tube-like structures on Matrigel. Conclusion: These results suggest that adipose tissue-derived cells are endothelial cells. With the fabrication of the vascularized scaffold construct, novel approaches could be developed to enhance the engineered scaffold by the addition of adipose tissue-derived endothelial cells and periosteal-derived osteoblastic cells to promote bone growth.

공동배양한 암세포와 혈관내피세포의 미세구조에 미치는 Nocodazole의 영향

김주영,송인환,이종용,성언기,이융창 대한해부학회 1999 Anatomy & Cell Biology Vol.32 No.4

태아피부 혈관 및 배양한 태아진피 미세혈관 내피세포에서 E-selectin 발현에 관한 연구

나동균,유원민,박병윤,이광훈 大韓成形外科學會 2000 Archives of Plastic Surgery Vol.27 No.2

Inflammation is the characteristics of scar formation which is abscent in fetal wound healing. The adhesion molecules such as selection groups are believed to have key roles for migration of inflammatory cells through the microvascular endothelial cells to the wound. The purpose of this study was to evaluate the expression of E-selection on the cultured human fetal and neonate dermal microvascular endothelial cells. The back skin of spontaneously delivered dead fetus (IUP 18-22wks) and circumcised prepuce skin of neonate were used. Human fetal dermal microvascular endothelial cells (HFDMEC) were isolated by extracting microvascular segment from trypsin treated fetal and neonate skin tissue and isolated by sieving with nylon mesh and then by 35% Perocoll gradient centrifugation. Further purification was done with the Ulex europaeus I coated magnetic dynabead. To confirm the fetal and neonatal endothelial cells, expression of factor Ⅷ antigen on cell surface and uptake of acetylated low-density lipoprotein were checked. Expression of E-selection on cultured fetal and neonatal endothelial cells in response to IL-1αTNF-αINF-rwas examined by ELISA. And the expression of E-selection on fetal and neonatal dermal microvascular endothelial cells was examined by immunohistochemical study using monoclonal 3B7 anti E-selection antibody in cultured fetal and neonatal skin. The expression of E-selection on endothelial cells was not significantly different between fetal and neonatal endothelial cells. This expression was augmented 10 times more by IL-1αTNF-αINF-r. Augmented endothelial E-selection expression by IL-1αTNF-αINF-rshowed peak level 4 hours after stimulation and return to baseline level after 48 hrs. This time course was similar in both fetal and neonatal endothelial cells. Immunohistochemically, the expression of E-selection molecule of unstimulated fetal and neonatal tissue was not observed. However, on both fetal and neonatal tissue cultured for 4 hours after stimulation by 100u/ml of IL-1 and 100u/ml of TNF, expression of E-selection molecule in microvasculature of upper dermis was observed and this expression persisted for up to 16 hours of culture. Also after culturing for 48hrs with 500u/ml of IFN, expression of E-selection was observed in the microvessels of upper dermis. In conclusion, we could not find any differences between the fetal and neonate skin in the expression of E-selection on the endothelial cell spontaneously or stimulated by IL-1αTNF-αINF-rin vivo and vitro which means the expression of E-selection may not be an important mechanism of scarless wound healing in fetus.

지방조직내 혈관내피세포와 전구세포의 효율적인 분리와 분화

김정남,이중호,오득영,유결,전영준,문석호,서제원,안상태,김상헌,이종원 한국조직공학과 재생의학회 2011 조직공학과 재생의학 Vol.8 No.1s

Recently, it has been documented that human adipose tissue (AT) has mesenchymal stem cells which can be differentiated into multiple cell lineage including endothelia cells, and there are many attempts to differentiate these cells into endothelial cells for the experimental and clinical study about angiogenesis. However, it seems to be that endothelial cells and their precursor cells in AT could be discarded in the process of extracting adipose derived stromal cells (ADSCs). In this study, we evaluated that endothelial differentiation of discardable cells. After the isolation of ADSCs, 6 hour and 24 hour non adherent floating cells were collected and characterized by the flow cytometry analysis using CD29, CD31, CD34 and CD90 cell surface markers. Also, we cultured these cells in the endothelial medium and RT-PCR for endothelial-specific mRNA was performed. Lastly, CD31 staining and Dillabeled ac-LDL uptake study were performed to confirm endothelial differentiation. In vitro experiment indicated that ADSCs showed a spindle like shape, but 6 hour and 24 hour non adherent floating cells showed a cobblestone like shape. When 6 hour and 24 hour non adherent floating cells were incubated for up to 1 weeks in endothelial medium, they detected to express a variety of endothelial-specific mRNA. Differentiated cells were also found to be able to uptake ac-LDL and stain CD 31. These results suggest that a subset of 6 hour and 24 hour non adherent floating cells derived from AT can give rise to cells with an endothelial cell-like phenotype, in vitro, at high percentages,which could be applied to in vivo vasculogenesis.

수입 각막에서 보존 기간 동안의 내피세포 감소율이 수술 후 내피세포 생존에 미치는 영향

이국,황규연,김만수.Kook Lee. MD. Kyu Yeon Hwang. MD. Man Soo Kim. MD 대한안과학회 2013 대한안과학회지 Vol.54 No.6

Purpose: To investigate the influence of preoperative endothelial cell loss on the outcome of keratoplasty for keratoconus in imported donor corneas. Methods: Eighteen imported corneas used in keratoplasty for keratoconus patients were evaluated. Corneal endothelial cell density at the moment of preservation was obtained from the medical records and was measured immediately before the keratoplasty. Correlation of the endothelial cell loss count before and after keratoplasty was analyzed and postoperative endothelial cell loss count according to the range of preoperative endothelial cell loss was evaluated. Results: Mean endothelial cell loss before and after keratoplasty was 258.94 ± 128.58 cells/mm2 and 355.44 ± 371.83 cells/mm2, respectively. There was a positive correlation between preoperative and postoperative endothelial cell loss count (r = 0.431, p = 0.074). The results showed statistically significant higher endothelial cell loss count after keratoplasty in the range above 250 cells/mm2 rather than below 250 cells/mm2 of preoperative endothelial cell loss count (p = 0.033). Conclusions: The preoperative decrease in endothelial cell density affected the endothelial cell loss after keratoplasty for keratoconus in imported donor corneas.

In Vitro Culture of Endothelial Cell and Smooth Muscle Cell for Studying Vascular Diseases

Kim, Joo-Young Yeungnam University College of Medicine 2010 Yeungnam University Journal of Medicine Vol.27 No.2

암세포의 전이, 죽상경화증, 당뇨성 망막병증과 같은 병적인 과정에서 혈관내피세포는 핵심적인 역할을 담당한다. 죽상경화증의 죽종 형성에 혈관민무늬근육세포가 직접적으로 관여한다. 배꼽정맥, 혈관내벽, 그리고 망막에 있는 이들 내피세포들은 다양한 효소용액들을 이용하여 얻는다. 순수하게 분리된 이들 세포는 내피세포와 관련된 질병의 시험관 내 연구에 있어 중요한 모델이다. 이러한 관점에서 볼 때 대동맥 벽의 중간막에서 분리한 후 배양한 민무늬근육세포도 죽상경화증의 발병을 설명할 수 있다. 이 종설에서는 사람배꼽정맥내피세포(HUVEC),대동맥의 내피세포 및 민무늬근육세포, 그리고 망막미세혈관내피세포(RMEC)의 분리 뿐 만 아니라 이들 세포를 이용한 질병연구에 관한 논문들을 소개하고자 한다. Endothelial cells play a key role in pathological processes such as cancer cell metastasis, atherosclerosis, and diabetic retinopathy. Vascular smooth muscle cells directly involve in the formation of atheroma in atherosclerosis. Some kinds of the endothelial cells are simply harvested from the umbilical veins, the tunica intima of aortic walls, the retina using various enzymes solutions. Those purely isolated cells provide a powerful tool in vitro studies of the endothelial cell related diseases. In this context, the cultured smooth muscle cells after the isolation from the tunica media of aortic walls are also used for elucidating the pathogenesis of atherosclerosis. Here, I briefly introduce articles that include the isolation of human umbilical vein endothelial cells(HUVEC), aortic endothelial and smooth muscle cells, retinal microvascular endothelial cells(RMEC), as well as the diseases' applications of these cells.

Two-Cell Spheroid Angiogenesis Assay System Using Both Endothelial Colony Forming Cells and Mesenchymal Stem Cells

( Sajita Shah ),( Kyu-tae Kang ) 한국응용약물학회 2018 Biomolecules & Therapeutics(구 응용약물학회지) Vol.26 No.5

Most angiogenesis assays are performed using endothelial cells. However, blood vessels are composed of two cell types: endothelial cells and pericytes. Thus, co-culture of two vascular cells should be employed to evaluate angiogenic properties. Here, we developed an in vitro 3-dimensional angiogenesis assay system using spheroids formed by two human vascular precursors: endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs). ECFCs, MSCs, or ECFCs+MSCs were cultured to form spheroids. Sprout formation from each spheroid was observed for 24 h by real-time cell recorder. Sprout number and length were higher in ECFC+MSC spheroids than ECFC-only spheroids. No sprouts were observed in MSC-only spheroids. Sprout formation by ECFC spheroids was increased by treatment with vascular endothelial growth factor (VEGF) or combination of VEGF and fibroblast growth factor-2 (FGF-2). Interestingly, there was no further increase in sprout formation by ECFC+MSC spheroids in response to VEGF or VEGF+FGF-2, suggesting that MSCs stimulate sprout formation by ECFCs. Immuno-fluorescent labeling technique revealed that MSCs surrounded ECFC-mediated sprout structures. We tested vatalanib, VEGF inhibitor, using ECFC and ECFC+MSC spheroids. Vatalanib significantly inhibited sprout formation in both spheroids. Of note, the IC₅₀ of vatalanib in ECFC+MSC spheroids at 24 h was 4.0 ± 0.40 µM, which are more correlated with the data of previous animal studies when compared with ECFC spheroids (0.2 ± 0.03 µM). These results suggest that ECFC+MSC spheroids generate physiologically relevant sprout structures composed of two types of vascular cells, and will be an effective pre-clinical in vitro assay model to evaluate pro- or anti-angiogenic property.

폴리우레탄 인공혈관에 대한 혈관내피세포의 친화성: 예비동물실험

안승현,전영민,장학,박정희,민경원 대한성형외과학회 2009 Archives of Plastic Surgery Vol.36 No.4

Purpose: Autologous vessels remain the gold standard for vascular grafts in microanastomoses. However, they are sometimes unavailable and have a limited long-term patency. Synthetic vessels have high success rates in large-diameter reconstructions but failed when used as small-diameter grafts due to graft occlusion. It has been proven that endothelial cell seeding improves prosthesis performance and long-term patency. Among polyurethane, PET and ePTFE, polyurethane has the best affinity to endothelial cells and mechanical properties closest to human vessels. We examined the ability of endothelial cells to attach to a polyurethane graft manufactured by the electrospinning method. Methods: Endothelial cells, which were cultured from porcine internal jugular veins, were attached to polyurethane grafts with an internal diameter of 3mm. The same cells were attached to allogeneic decellularized porcine internal carotid artery grafts as controls. Both of the 10mm-long grafts were exposed to endothelial cells in a well for 1 hour. Each well contained 2×105 endothelial cells. The graft materials were rotated through 90 degrees every 15 minutes in order to minimize the effect of gravity. The extent of cell attachment was examined with the MTT assay. Results: The MTT assay showed good incorporation of endothelial cells into both grafts. For the evaluation of affinity, the number of attached cells was counted at 10 fields of microscopic examination with×40 magnification. Endothelial cells adhered more to polyurethane grafts (mean, 127.4±6.2 cells) compared to porcine artery grafts (mean 45.8±5.1 cells)(p<0.05,Mann-Whitney test). Conclusion: In this study, we attached porcine endothelial cells to polyurethane grafts, manufactured by electrospinning. The grafts exhibited a better affinity to endothelial cells than allogeneic decellularized porcine internal carotid artery grafts. It is suggested that the time required for endothelial cells to attach to decellulized artery grafts may be longer than that which is required for attachment to polyurethane grafts.