Isolation, ekspansion og Adipogenic induktion af CD34 + CD31 + endotelceller fra menneskelige Omental og subkutane fedtvæv
Summary
Differentiering af hvide og beige adipocytter fra fedtvæv vaskulære stamfaderen bjørne potentiale for metaboliske forbedring i fedme. Vi beskriver protokoller for en CD34 + CD31 + endotel celle isolation fra menneskelige fedt og en efterfølgende in vitro- udvidelse og differentiering af hvide og beige adipocytter. Flere downstream ansøgninger behandles.
Abstract
Fedme er ledsaget af en omfattende ombygning af fedtvæv primært via adipocyt hypertrofi. Ekstreme adipocyt vækst resulterer i en dårlig reaktion på insulin, lokale hypoxi og betændelse. Ved at stimulere differentiering af funktionelle hvid adipocytter fra stamfaderen, radikale hypertrofi af befolkningens adipocyt kan forebygges, og derfor fedtvæv metabolisk sundhed kan forbedres med en reduktion af betændelse. Også, ved at stimulere en differentiering af beige/brun adipocytter, kroppens samlede energi udgifter kan øges, hvilket resulterer i vægttab. Denne tilgang kunne forhindre udviklingen af fedme Co-morbiditeter som type 2-diabetes og hjerte-kar-sygdom.
Dette papir beskriver den isolation, udvidelse og differentiering af hvide og beige adipocytter fra et undersæt af menneskers fedtvæv endotelceller, der samtidig udtrykker CD31 og CD34 markørerne. Metoden er relativt billige og ikke arbejdskrævende. Det kræver adgang til menneskers fedtvæv og subkutane depotet er velegnet til prøveudtagning. For denne protokol, friske fedtvæv prøver fra sygeligt overvægtige emner [body mass index (BMI) > 35] er indsamlet under bariatric kirurgi procedurer. Bruger en sekventiel immunoseparation fra de stromale vaskulære brøkdel, er nok celler produceret fra så lidt som 2-3 g fedt. Disse celler kan udvides i kultur over 10-14 dage, kan være cryopreserved og bevare deres adipogenic egenskaber med passaging op til passage 5 – 6. Cellerne er behandlet i 14 dage med en cocktail ved hjælp af en kombination af human insulin og PPARγ agonist-rosiglitazon adipogenic.
Denne metode kan anvendes til at indhente bevis for konceptet eksperimenter på molekylære mekanismer, der drev adipogenic svar i adipøst endotelceller, eller til screening af nye lægemidler, der kan forbedre adipogenic reaktion rettet enten mod hvid eller beige/brun adipocyt differentiering. Brug små subkutane biopsier, kan denne metode bruges til at frasortere ikke-responder emner for kliniske forsøg har til formål at stimulere beige/brun og hvid adipocytter til behandling af fedme og co-morbiditet.
Introduction
De seneste erfaringer viser, at både mus og mennesker et undersæt af celler bosat i fedtvæv Vaskulaturen kan opdeles i enten hvide eller beige/brun adipocytter1,2,3. Fænotypen af sådanne celler er genstand for kontroverser, med dokumentation for endotelceller, glat muskel/pericyte eller et spektrum af mellemliggende fænotyper4,5,6,7. Muligheder for at udvikle denne metode var at teste adipogenic potentialet af CD34 + CD31 + endotelceller isoleret fra forskellige fedt depoter fra fede mennesker. Andre undersøgelser i litteraturen fokuserer på den potentielle af den samlede stromale vaskulære fraktion eller kendte adipocyt stamfaderen2,8,9adipogenic. Da eksisterende teknologier kan målrette specielt fedtvæv endotelceller for drug delivery10, er forstå potentialet af sådanne celler til at gennemgå adipogenic induktion mod hvide eller beige adipocytter vigtigt for fremtidige målrettede behandlinger.
Forskellige grupper rapporterede kombination af CD31 og CD34 markører som surrogater at isolere endotelceller fra menneskers fedtvæv11,12,13. Typisk, isolering udføres, ved hjælp af to sekventielle trin og en positiv markering med magnetiske perler. I denne rapport, blev immunoseparation brug af CD34 + magnetiske perler kombineret med CD31 plastik perler udnyttet. Vi fandt denne teknik overlegen i forhold til den sekventielle magnetiske immunoseparation med hensyn til bevarelse af typiske brostensbelagte endotel morfologi. Også, vi var i stand til at generere nok celler kræves for ekspansion og adipogenic induktion starter fra så lidt som 1-2 g fedt. Et lille udsnit biopsi af subkutant fedt er nok til at producere den ønskede mængde af celler til downstream applikationer. Dette aspekt er potentielt vigtigt, især hvis denne metode vil blive udnyttet til screening for en lydhørhed over for adipogenic induktion i forsøgspersoner.
I modsætning til andre systemer, der er rapporteret i litteraturen, denne metode udnytter kun to ingredienser til adipogenic induktion af CD34 + CD31 + celler: en PPARγ agonist — rosiglitazon — og humant insulin. Vigtigere, falder mængden af insulin bruges inden for normal/høj vifte af cirkulerende post-absorptive insulin i mennesker14. Graden af lydhørhed over for insulin celler in vitro-, målt ved Akt fosforylering, korrelerer ikke med deres evne til at reagere på induktion cocktail. Interessant, bruger denne induktion cocktail og eksperimentelle betingelser, en blanding af hvid og beige/brun celler blev fremstillet som bestemmes af størrelsen og antallet af intracellulære lipid dråber og udtryk for molekylære markører. Denne enkle og omkostningseffektive induktion protokol sammen med den kvantitative evaluering af Fænotypen af responder celler (hvide vs beige) giver mulighed for en screening af agenter, der potentielt kan ændre balancen af differentierede beige : hvide adipocytter.
Denne metode giver også en translationel tilgang for at forstå de underliggende mekanismer for adipogenesis af vaskulære endotel ophav i menneskers fedtvæv. Med denne specifikke isolation/differentiering teknik, kan efterforskere afhøre forskellige veje ansvarlig for adipogenesis i en delmængde af vascular endothelial celler fra forskellige fedt depoter i magre og fede mennesker.
Protocol
Representative Results
Discussion
Fokus i dette papir er at fremlægge en metodologi til isolering, ekspansion og adipogenic induktion af CD34 + CD31 + endotelceller fra visceral og subkutane depoter af menneskers fedtvæv.
Metoder er blevet rapporteret for isolation i endothelial celler fra forskellige vaskulære senge af gnavere eller mennesker, der involverer primært teknikker ved hjælp af CD31 antistoffer enten fluorescently mærket eller koblet til magnetiske perler18, <sup class="xref…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne anerkende Becky Marquez, den kliniske koordinator på Sentara Bariatric Center, for hendes hjælp med processen med patienten screening og samtykkende. Denne forskning blev støttet af R15HL114062 til Anca D. Dobrian.
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 |
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