人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导
Summary
脂肪组织血管祖细胞中白、米色脂肪体的分化具有改善肥胖代谢的潜力。我们描述了 CD34+CD31+ 内皮细胞与人的脂肪分离的协议, 并为随后的体外扩增和分化为白色和米色脂肪。讨论了几种下游应用。
Abstract
肥胖伴随着脂肪组织的广泛重塑, 主要是通过脂肪细胞肥大。极端脂肪细胞的生长导致胰岛素、局部缺氧和炎症反应不良。通过刺激祖细胞功能性白细胞的分化, 可以预防脂肪细胞的自由基肥大, 从而可以改善脂肪组织的代谢健康, 同时减少炎症。另外, 通过刺激米色/褐色脂肪细胞的分化, 总能量消耗可以增加, 导致体重减轻。这种方法可以防止肥胖症的发病率, 如2型糖尿病和心血管疾病的发展。
本文描述了 CD31 和 CD34 标记的人脂肪组织内皮细胞的一个子集对白色和米色脂肪体的分离、扩增和分化。该方法相对便宜, 不劳动密集型。它需要进入人体脂肪组织和皮下仓库是适合取样。对于这个协议, 肥胖症患者的新鲜脂肪组织样本 [身体质量指数 (BMI) > 35] 是在减肥手术过程中收集的。使用序贯 immunoseparation 从基质血管的分数, 足够的细胞产生的小到 2–3 g 的脂肪。这些细胞可以在10–14天内进行培养, 可以冷冻保存, 并保留其脂肪的性质, 传代直至通过5–6。细胞被治疗14天与脂肪鸡尾酒使用人胰岛素和 PPARγ激动剂-罗格列酮的组合。
这种方法可用于获取分子机制的概念实验的证明, 推动脂肪内皮细胞的脂肪反应, 或筛选新的药物, 可以提高脂肪反应, 无论是对白色或米色/棕色脂肪细胞分化。使用小皮下活检, 这种方法可用于筛选非应答者的临床试验目的是刺激米色/棕色和白色脂肪细胞的治疗肥胖和共同疾病。
Introduction
最近的证据显示, 在小鼠和人类中, 在脂肪组织血管中的细胞子集可以被区分成白色或米色/褐色脂肪体1,2,3。这种细胞的表型是一个争议的主题, 有证据支持内皮细胞, 平滑肌/周细胞, 或一光谱的中间表型4,5,6,7。这种方法的发展范围是测试从肥胖人群中分离出不同脂肪库的 CD34+CD31+ 内皮细胞的脂肪电位。文献中的其他研究集中在脂肪电位的总基质血管分数或已知脂肪细胞祖细胞2,8,9。由于目前现有的技术可以专门针对脂肪组织内皮细胞的药物传递10, 了解这种细胞的潜力接受脂肪诱导向白色或米色脂肪体是重要的未来靶向治疗。
不同的组报告, CD31 和 CD34 标记作为代理人, 以隔离内皮细胞从人脂肪组织11,12,13。通常情况下, 隔离是使用两个连续步骤和一个使用磁性珠子的正选择来执行的。在本报告中, 利用 CD34+ 磁珠结合 CD31 塑料珠 immunoseparation。我们发现这种技术优于顺序磁性 immunoseparation 的保存典型的鹅卵石内皮形态学。此外, 我们能够产生足够的细胞, 以扩大和脂肪诱导开始从尽可能少的脂肪 g。小样本皮下脂肪活检足以为下游应用生产所需的细胞数量。这方面是潜在的重要, 特别是如果这一方法将被用于筛选对脂肪诱导的人的对象的反应。
与文献报道的其他系统不同, 这种方法仅利用两种成分脂肪诱导 CD34+CD31+ 细胞: PPARγ激动剂-罗格列酮-和人胰岛素。重要的是, 使用胰岛素的数量在正常/高范围内循环后吸收胰岛素的人14。Akt 磷酸化测定的细胞体外对胰岛素的反应程度与它们对诱导性鸡尾酒反应的能力没有关联。有趣的是, 使用这种诱导鸡尾酒和实验条件, 由白色和米色/褐色细胞的混合, 由细胞内脂质滴的大小和数量和分子标记的表达决定。这种简单和经济高效的诱导协议以及对应答器细胞表型的定量评估 (白色与米色) 允许筛选可能改变差异性米色平衡的药剂。: 白色脂肪细胞。
该方法还为了解人体脂肪组织中血管内皮祖细胞成脂的基本机制提供了一种平移方法。使用这种特殊的隔离/分化技术, 调查人员可以审问各种途径, 负责成脂在血管内皮细胞的子集, 从不同脂肪库的瘦身和肥胖的人。
Protocol
Representative Results
Discussion
本文的重点是为 CD34+CD31+ 内皮细胞从内脏和皮下的脂肪组织的分离、扩增和脂肪诱导提供一种方法。
已报告的方法, 以隔离从不同的啮齿动物或人类的血管床内皮细胞, 主要涉及使用 CD31 抗体荧光标记或耦合磁性珠18,19,20,21,22,23….
Declarações
The authors have nothing to disclose.
Acknowledgements
作者希望在森塔拉减肥中心的临床协调员贝基. 马奎斯的帮助下, 她的病人筛查和同意的过程。这项研究得到了 R15HL114062 对 Anca d Dobrian 的支持。
Materials
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Large Equipment |
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Biosafety Cabinet |
Nuaire |
nu-425-400 |
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Cell Culture Incubator |
Thermo-Fisher Scientific |
800 DH |
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Water Bath |
Forma Scientific |
2568 |
Reciprocal Shaker |
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RT-PCR Machine |
BIO-RAD |
CFX96-C1000 |
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Electrophoresis Box |
BIO-RAD |
Mini PROTEAN 3 Cell |
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Transblot Box |
BIO-RAD |
Mini Trans-Blot Cell |
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Electrophoresis Power Supply |
BIO-RAD |
PowerPac Basic |
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ELISA Reader |
Molecular Devices |
SpectraMax M5 |
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Blot Reader |
LI-COR |
Odyssey |
Near Infrared |
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Refrigerated Centrifuge |
Eppendorf |
5810 R |
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Tabletop Centrifuge |
Eppendorf |
MiniSpin Plus |
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Fluorescent Microscope |
Olympus |
BX50 |
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Inverted Microscope |
Nikon |
TMS |
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KRBSS Buffer |
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HEPES |
Research Products International |
H75030 |
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Sodium bicarbonate |
Sigma-Aldrich |
792519 |
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Calcium chloride dihydrate |
Sigma-Aldrich |
C7902 |
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Potassium phosphate monobasic |
Sigma-Aldrich |
P5655 |
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Magnesium sulfate |
Sigma-Aldrich |
M2643 |
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Sodium chloride |
Sigma-Aldrich |
746398 |
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Sodium phosphate monobasic monohydrate |
Sigma-Aldrich |
S9638 |
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Potassium chloride |
Sigma-Aldrich |
P9333 |
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Glucose |
Acros Organics |
410950010 |
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Adenosine |
Acros Organics |
164040250 |
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Bovine Serum Albumin |
GE Healthcare Bio-Sciences |
SH30574.02 |
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Penicillin/Streptomycin |
Thermo-Fisher Scientific |
15070063 |
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Tissue Digestion |
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20 mL Syringe |
Global Medical |
67-2020 |
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Nylon Mesh, 250 µm |
Sefar |
03-250/50 |
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Pipetting Needles |
Popper |
7934 |
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Fine Scissors |
Fine Science Tools |
14058-11 |
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Tissue Forceps |
George Tiemann & Co |
160-20 |
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Collagenase, Type I |
Worthington Biochemical |
LS004196 |
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Petri Dishes, 100 mm |
USA Scientific |
5666-4160 |
TC Treated |
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Eppendorf Tubes, 1.5 mL |
USA Scientific |
1615-5500 |
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Conical Tubes, 15 mL |
Nest Scientific |
601052 |
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Conical Tubes, 50 mL |
Nalgene |
3119-0050 |
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Scintillation Vials |
Kimble |
74505-20 |
Tissue Dicing |
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Cell Isolation |
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Cellometer |
Nexcelom |
Auto 2000 |
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Cellometer Slides |
Nexcelom |
CHT4-SD100-002 |
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Cellometer Viability Stain |
Nexcelom |
CS2-0106-5mL |
Acridine Orange/Propidium Iodine |
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Anti-CD34 Magnetic Beads |
StemCell Technologies |
18056 |
Kit |
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EasySep Magnet |
StemCell Technologies |
18000 |
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Anti-CD31 Plastic Beads |
pluriSelect USA |
19-03100-10 |
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pluriSelect 10x Wash Buffer |
pluriSelect USA |
60-00080-10 |
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pluriSelect Connector Ring |
pluriSelect USA |
41-50000-03 |
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pluriSelect Detachment Buffer |
pluriSelect USA |
60-00046-12 |
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pluriSelect Incubation Buffer |
pluriSelect USA |
60-00060-12 |
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pluriSelect S Cell Strainer |
pluriSelect USA |
43-50030-03 |
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Cell Culture |
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6-well Plates |
USA Scientific |
CC7682-7506 |
TC Treated |
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4-well chambered slides |
Corning Life Sciences |
354559 |
Fibronectin coated |
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4-well chambered slides |
Thermo-Fisher Scientific |
154526PK |
Uncoated glass |
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Human Adipose Microvascular Endothelial Cells (HAMVEC) |
Sciencell Research Laboratories |
7200 |
Primary cell line |
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Endothelial Cell Media (ECM) |
ScienCell Research Laboratories |
1001 |
Complete Kit |
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DMEM/F12 Basal Media |
Thermo-Fisher Scientific |
11320082 |
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Fetal Bovine Serum (FBS) |
Rocky Mountain Biologicals |
FBS-BBT |
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Insulin |
Lilly |
U-100 |
Humalog |
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Rosiglitazone |
Sigma-Aldrich |
R2408 |
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Cell Analysis |
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Oil Red O Dye |
Sigma-Aldrich |
O0625 |
Prepared in isopropanol |
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96 well plates |
USA Scientific |
1837-9600 |
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96 well PCR plates |
Genesee Scientific |
24-300 |
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RNA Extraction |
Zymo Research |
R2072 |
Kit |
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cDNA Synthesis |
BIO-RAD |
1708841 |
Supermix |
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JumpStart PCR Polymerase |
Sigma-Aldrich |
D9307-250UN |
Hot start, with PCR Buffer N |
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Magnesium Chloride Solution |
Sigma-Aldrich |
M8787-5ML |
3 mM final in PCR reaction |
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dNTPs |
Promega |
U1515 |
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TaqMan AdipoQ |
Thermo-Fisher Scientific |
Hs00605917_m1 |
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TaqMan CIDEA |
Thermo-Fisher Scientific |
Hs00154455_m1 |
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TaqMan RPL27 |
Thermo-Fisher Scientific |
Hs03044961_g1 |
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TaqMan UCP1 |
Thermo-Fisher Scientific |
Hs00222453_m1 |
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BCA Assay |
Sigma-Aldrich |
QPBCA-1KT |
Kit |
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Bis-acrylamide |
BIO-RAD |
1610146 |
40% stock solution |
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Ammonium Persulfate |
BIO-RAD |
1610700 |
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TEMED |
BIO-RAD |
1610800 |
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Tris |
Sigma-Aldrich |
T1503 |
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Glycine |
BIO-RAD |
1610718 |
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Sodium Dodecal Sulfate |
Sigma-Aldrich |
L3771 |
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EDTA |
Fisher Scientific |
S311-100 |
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Bromophenol Blue |
Sigma-Aldrich |
B8026 |
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Blot Membrane |
EMD Millipore |
IPFL00010 |
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Methanol |
Fisher Scientific |
A452-SK4 |
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Odyssey Blocking Buffer, Tris |
LI-COR |
927-50000 |
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Anti-AKT antibody |
Cell Signaling Technology |
2920S |
Mouse monoclonal |
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Anti-pAKT antibody |
Cell Signaling Technology |
9271S |
Rabbit polyclonal |
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Anti-UCP1 antibody |
Abcam |
ab10983 |
Rabbit polyclonal |
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Anti-Mouse IgG antibody |
LI-COR |
926-68070 |
Goat Polyclonal, IRDye 680RD |
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Anti-Rabbit IgG antibody |
LI-COR |
926-32211 |
Goat Polyclonal, IRDye 800CW |
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Anti-Rabbit IgG antibody |
Jackson ImmunoResearch |
111-025-003 |
Goat Polyclonal, TRITC |
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Phosphate Buffer Saline |
Thermo-Fisher Scientific |
10010049 |
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37% Formaldehyde Solution |
Electron Microscopy Sciences |
15686 |
4% solution for cell fixation |
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Normal Goat Serum |
Vector Laboratories |
S-1000 |
10% blocking solution |
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Triton X-100 |
Sigma-Aldrich |
X-100 |
0.1% permeabilization solution |
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DAPI |
Thermo-Fisher Scientific |
D1306 |
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Calcein AM |
Thermo-Fisher Scientific |
65-0853-39 |
Cell fluorescent visualization |
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Matrigel Basement Membrane Matrix |
Corning Life Sciences |
356231 |
Growth factor reduced |
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DiI labeled Acetylated LDL |
Thermo-Fisher Scientific |
L3484 |
Referências
- Tang, W., et al. White fat progenitor cells reside in the adipose vasculature. Science. 322 (5901), 583-586 (2008).
- Lee, Y. H., Petkova, A. P., Granneman, J. G. Identification of an adipogenic niche for adipose tissue remodeling and restoration. Cell Metabolism. 18 (3), 355-367 (2013).
- Min, S. Y., et al. Human ‘brite/beige’ adipocytes develop from capillary networks, and their implantation improves metabolic homeostasis in mice. Nature Medicine. 22 (3), 312-318 (2016).
- Gupta, R. K., et al. Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells. Cell Metabolism. 15 (2), 230-239 (2012).
- Tran, K. V., et al. The vascular endothelium of the adipose tissue gives rise to both white and brown fat cells. Cell Metabolism. 15 (2), 222-229 (2012).
- Rodeheffer, M. S., Birsoy, K., Friedman, J. M. Identification of white adipocyte progenitor cells in vivo. Cell. 135 (2), 240-249 (2008).
- Scott, M. A., Nguyen, V. T., Levi, B., James, A. W. Current methods of adipogenic differentiation of mesenchymal stem cells. Stem Cells and Development. 20 (10), 1793-1804 (2011).
- Macotela, Y., et al. Intrinsic differences in adipocyte precursor cells from different white fat depots. Diabetes. 61 (7), 1691-1699 (2012).
- Lee, Y. H., Petkova, A. P., Mottillo, E. P., Granneman, J. G. In vivo identification of bipotential adipocyte progenitors recruited by beta3-adrenoceptor activation and high-fat feeding. Cell Metabolism. 15 (4), 480-491 (2012).
- Xue, Y., Xu, X., Zhang, X. Q., Farokhzad, O. C., Langer, R. Preventing diet-induced obesity in mice by adipose tissue transformation and angiogenesis using targeted nanoparticles. Proceedings of the National Academy of Sciences of the United States of America. 113 (20), 5552-5557 (2016).
- Villaret, A., et al. Adipose tissue endothelial cells from obese human subjects: differences among depots in angiogenic, metabolic, and inflammatory gene expression and cellular senescence. Diabetes. 59 (11), 2755-2763 (2010).
- Miranville, A., et al. Improvement of postnatal neovascularization by human adipose tissue-derived stem cells. Circulation. 110 (3), 349-355 (2004).
- Sengenes, C., Lolmede, K., Zakaroff-Girard, A., Busse, R., Bouloumie, A. Preadipocytes in the human subcutaneous adipose tissue display distinct features from the adult mesenchymal and hematopoietic stem cells. Journal of Cellular Physiology. 205 (1), 114-122 (2005).
- Moller, D. E., Flier, J. S. Insulin resistance–mechanisms, syndromes, and implications. The New England Journal of Medicine. 325 (13), 938-948 (1991).
- Pittenger, M. F., et al. Multilineage potential of adult human mesenchymal stem cells. Science. 284 (5411), 143-147 (1999).
- Novikoff, A. B., Novikoff, P. M., Rosen, O. M., Rubin, C. S. Organelle relationships in cultured 3T3-L1 preadipocytes. The Journal of Cell Biology. 87 (1), 180-196 (1980).
- Satish, L., et al. Expression analysis of human adipose-derived stem cells during in vitro differentiation to an adipocyte lineage. BMC Medical Genomics. 8 (41), (2015).
- Gimbrone, M. A., Cotran, R. S., Folkman, J. Human vascular endothelial cells in culture. Growth and DNA synthesis. The Journal of Cell Biology. 60 (3), 673-684 (1974).
- Burridge, K. A., Friedman, M. H. Environment and vascular bed origin influence differences in endothelial transcriptional profiles of coronary and iliac arteries. American Journal of Physiology-Heart and Circulatory Physiology. 299 (3), H837-H846 (2010).
- Paruchuri, S., et al. Human pulmonary valve progenitor cells exhibit endothelial/mesenchymal plasticity in response to vascular endothelial growth factor-A and transforming growth factor-beta2. Circulation Research. 99 (8), 861-869 (2006).
- Hewett, P. W., Murray, J. C. Human microvessel endothelial cells: isolation, culture and characterization. In Vitro Cellular & Development Biology. 29 (11), 823-830 (1993).
- Hewett, P. W., Murray, J. C. Human lung microvessel endothelial cells: isolation, culture, and characterization. Microvascular Research. 46 (1), 89-102 (1993).
- Hewett, P. W., Murray, J. C., Price, E. A., Watts, M. E., Woodcock, M. Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue. In Vitro Cellular & Development Biology. 29 (4), 325-331 (1993).
- Xiao, L., McCann, J. V., Dudley, A. C. Isolation and culture expansion of tumor-specific endothelial cells. Journal of Visualized Experiments. (105), e53072 (2015).
- Berry, R., Rodeheffer, M. S., Rosen, C. J., Horowitz, M. C. Adipose tissue residing progenitors (adipocyte lineage progenitors and adipose derived stem cells (ADSC). Current Molecular Biology Reports. 1 (3), 101-109 (2015).
- Zhou, L., et al. In vitro evaluation of endothelial progenitor cells from adipose tissue as potential angiogenic cell sources for bladder angiogenesis. PLoS One. 10 (2), e0117644 (2015).
- Curat, C. A., et al. From blood monocytes to adipose tissue-resident macrophages: induction of diapedesis by human mature adipocytes. Diabetes. 53 (5), 1285-1292 (2004).
- Sidney, L. E., Branch, M. J., Dunphy, S. E., Dua, H. S., Hopkinson, A. Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem Cells. 32 (6), 1380-1389 (2014).
- Traktuev, D. O., et al. A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circulation Research. 102 (1), 77-85 (2008).
- Frontini, A., Giordano, A., Cinti, S. Endothelial cells of adipose tissues: a niche of adipogenesis. Cell Cycle. 11 (15), 2765-2766 (2012).
- Sengenes, C., Miranville, A., Lolmede, K., Curat, C. A., Bouloumie, A. The role of endothelial cells in inflamed adipose tissue. Journal of Internal Medicine. 262 (4), 415-421 (2007).
- Ong, W. K., et al. Identification of specific cell-surface markers of adipose-derived stem cells from subcutaneous and visceral fat depots. Stem Cell Reports. 2 (2), 171-179 (2014).
- Ong, W. K., Sugii, S. Adipose-derived stem cells: fatty potentials for therapy. The International Journal of Biochemistry & Cell Biology. 45 (6), 1083-1086 (2013).
- Tchkonia, T., et al. Abundance of two human preadipocyte subtypes with distinct capacities for replication, adipogenesis, and apoptosis varies among fat depots. American Journal of Physiology-Endocrinoloy and Metabolism. 288 (1), E267-E277 (2005).
- van de Vyver, M., Andrag, E., Cockburn, I. L., Ferris, W. F. Thiazolidinedione-induced lipid droplet formation during osteogenic differentiation. Journal of Endocrinology. 223 (2), 119-132 (2014).
