Name: isolation, expansion, and adipogenic induction of cd34+cd31+ endothelial cells from human omental and subcutaneous adipose tissue
Uploaded: 2018-07-17T14:00:00
Description: Watch this Scientific Journal Video about Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue at JoVE.com

Författarna vill uppmärksamma Becky Marquez, klinisk samordnare på Sentara bariatrisk Center, för hennes hjälp med processen att patienten screening och samtycker. Denna forskning stöddes av R15HL114062 till Anca D. Dobrian.

Den institutionella styrelsen granskningskommittén vid östra Virginia Medical School godkände forskning och mänsklig fettvävnad provtagning används i studien. Informerade samtycke inhämtades från patienterna.
1. beredning av buffertar, Media och instrument
<ol><li>Förbereda en Krebs Ringer Bicarbonate-Buffered lösning (KRBBS): 135 mM natriumklorid, kaliumklorid 5 mM, 1 mM magnesiumsulfat, 0,4 mM kalium fosfatbuffert dibasiskt, 5,5 mM glukos, 1 mM adenosin, 0,01% antibiotika/antimyc...

<ol>
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Fokus för denna uppsats är att tillhandahålla metoder för isolering, expansion och adipogena induktion av CD34 + CD31 + endotelceller från visceral och subkutant depåer av mänsklig fettvävnad.
Metoder har rapporterats för isolering av endotelceller från olika kärlbäddar av gnagare eller människa som involverar främst metoder med CD31 antikroppar antingen fluorescently märkt eller kopplat till magnetiska pärlor18, 19 <su...

Senaste rönen visar att både möss och människor, kan en delmängd av celler som är bosatta i den fettvävnad vaskulatur göras åtskillnad mellan in i antingen vit eller beige/bruna adipocyter1,2,3. Fenotypen av sådana celler är föremål för kontroverser, med underlagen endotelceller, glatt muskulatur/pericyt eller ett spektrum av mellanliggande fenotyper4,5,6,7. Tillämpningsområdet för utveckla denna metod var att testa den adipogena potentialen av CD34 + CD31 + endothelial celler isolerade från olika fett depåer från överviktiga människor. Andra studier i litteraturen fokuserar på adipogena som är potentiell av den totala stromal vaskulära fraktionen eller kända fettceller föräldraparets2,8,9. Eftersom nuvarande teknik kan rikta särskilt fettvävnad endotelceller för drogen leverans10, är förstå potentialen hos sådana celler att genomgå adipogena induktion mot vit eller beige adipocyter viktigt framtida riktade terapier.
Olika grupper rapporterade kombinationen av CD31 och CD34 markörer som surrogat att isolera endotelceller från mänsklig fettvävnad11,12,13. Typiskt, isolering utförs med två sekventiella steg och ett positivt urval använder magnetiska pärlor. I denna rapport utnyttjades immunoseparation med hjälp av CD34 + magnetiska pärlor kombinerat med CD31 plast pärlor. Vi hittade denna teknik överlägsen den sekventiella magnetiska immunoseparation när det gäller bevarandet av typiska kullersten endothelial morfologi. Dessutom var vi kunna generera tillräckligt med celler krävs för expansion och adipogena induktion från så lite som 1 – 2 g fett. Ett litet urval biopsi av underhudsfett är tillräckligt för att producera den erforderliga mängden av celler för nedströms tillämpningar. Denna aspekt är potentiellt viktigt, särskilt om denna metod kommer att utnyttjas för screening för en lyhördhet för adipogena induktion hos försökspersoner.
Till skillnad från andra system som rapporterats i litteraturen, den här metoden utnyttjar endast två ingredienser för adipogena induktion av CD34 + CD31 + celler: en PPARγ-agonist – rosiglitazon — och humant insulin. Ännu viktigare, faller mängden insulin används inom intervallet normal/hög cirkulerande post-absorptive insulin i människor14. Graden av lyhördhet för insulin celler in vitro-, mätt med Akt fosforylering, korrelerar inte med deras förmåga att reagera på induktion cocktail. Intressant nog använder denna induktion cocktail och experimentella förhållanden, en blandning av vitt och beige/brun celler erhölls som bestäms av storlek och antal intracellulära lipid droppar och uttrycket av molekylära markörer. Detta enkla och kostnadseffektiva induktion protokoll tillsammans med den kvantitativa utvärderingen av fenotypen av responder cellerna (vit vs beige) möjliggör en screening av agenter som potentiellt kan förändra balansen i differentierade beige : vit adipocyter.
Denna metod erbjuder också en translationell strategi för att förstå de underliggande mekanismerna bakom adipogenesis av vaskulär endotelial stamfäder i mänsklig fettvävnad. Med denna specifika isolering/differentiering teknik, kan utredare förhöra olika vägar ansvarar för adipogenesis i en delmängd av vaskulära endotelceller från olika fett depåer i lean och överviktiga människor.

isolation, expansion, and adipogenic induction of cd34+cd31+ endothelial cells from human omental and subcutaneous adipose tissue

Övervikt åtföljs av en omfattande ombyggnad av fettvävnad primärt via fettceller hypertrofi. Extrema fettceller tillväxt resulterar i en dålig reaktion på insulin, lokal syrebrist och inflammation. Genom att stimulera differentiering av funktionella vita fettceller från stamfäder, radikala hypertrofi av fettceller befolkningen kan förhindras och, följaktligen, fettvävnad metabol hälsa kan förbättras tillsammans med en minskning av inflammation. Också, genom att stimulera en differentiering av beige/bruna adipocyter, kroppens totala energiförbrukningen kan ökas, vilket resulterar i viktminskning. Denna strategi kan förhindra utvecklingen av fetma komorbiditet såsom typ 2-diabetes och hjärt-kärlsjukdom.
Detta dokument beskriver isolering, expansion och differentiering av vitt och beige adipocyter från en delmängd av mänsklig fettvävnad endotelceller som samtidigt uttrycker de CD31 och CD34 markörerna. Metoden är relativt billigt och är inte arbetsintensiva. Det kräver tillgång till mänsklig fettvävnad och subkutan depån är lämplig för provtagning. För detta protokoll, färska fettvävnad prover från sjukligt överviktiga patienter [BMI (Kroppsmasseindex) > 35] samlas under fetmakirurgi förfaranden. Använder en sekventiell immunoseparation från stromal vaskulär fraktionen, produceras tillräckligt med celler från så lite som 2 – 3 g fett. Dessa celler kan utökas i kultur under 10 – 14 dagar, kan förvaras i fryst tillstånd och behåller sina adipogena egenskaper med passaging upp till passage 5 – 6. Cellerna behandlas i 14 dagar med en adipogena cocktail med en kombination av humaninsulin och en PPARγ-agonist-rosiglitazon.
Denna metod kan användas för att erhålla bevis på konceptet experiment på molekylära mekanismer som driver adipogena svaren i fett endotelceller eller för screening nya läkemedel som kan förbättra adipogena svar riktas antingen mot vit eller beige/bruna fettceller differentiering. Använder små subkutana biopsier, kan denna metod användas för att sålla bort non-responder ämnen för kliniska prövningar som syftade till att stimulera beige/brun och vit adipocyter för behandling av fetma och komorbiditet.

Våra protokollet syftar till att ge en in vitro -Metod för att bestämma den adipogena potentialen av CD34 + CD31 + vaskulära celler från olika depåer av mänsklig fettvävnad. Ett förenklat flödesschema visas i figur 1A. Det första steget använder ett positivt urval av CD34 uttrycker cellerna resulterar i > 95% CD34 + celler i befolkningen av de färska isolerade cellerna (figur 1A). Viktigast av allt, går den...

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

Differentiering av vitt och beige adipocyter från fettvävnad vaskulär föräldraparets björnar metabola förbättringspotentialen i fetma. Vi beskriver protokoll för en CD34 + CD31 + endotelceller isolering från mänskliga fett och en efterföljande in vitro- expansion och differentiering i vitt och beige adipocyter. Flera efterföljande program diskuteras.

Isolering, Expansion och adipogena induktion av CD34 + CD31 + Endothelial celler från mänskliga Omental och subkutan fettvävnad

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

This method can help answer key questions in the obesity field, such as identifying the key factors and mechanisms that prevent a robust adipogenic response during adipose tissue remodeling. The main advantage of this technique is that it allows isolation of CD34+CD31+endothelial cells from human adipose tissue starting with small amounts of tissue. We first had the idea for this method when we wanted to identify cells in human adipose vasculature that respond to adipogenic stimulation.Visual demonstration of this method is critical, as the cell isolation steps are difficult to practice due to the oftentimes limited access to fresh human adipose tissue. Collect human omental and subcutaneous adipose tissue from human subjects undergoing bariatric surgery. Immediately after tissue extraction, keep the human omental and subcutaneous adipose tissue in separate vials containing Hank's buffered salt solution with 50 micrograms per milliliter of penicillin streptomycin at room temperature.Once the sample arrives at the lab after the tissue extraction, carefully clean and remove fibrotic and cauterized sections of the tissue by using 70%ethanol-swabbed scissors and tweezers. Weigh the adipose tissue and partition it into five gram aliquots for the collagenase digestion. Use two pairs of scissors to finely mince five grams of the adipose tissue in five milliliters of a collagenase solution in a scintillation vial at room temperature.Add an additional 10 milliliters of collagenase solution to the minced tissue for 15 milliliters total. Digest the samples for one hour at 37 degrees Celsius in a water bath with continuous shaking. Following digestion, pour the samples into the 20 milliliter blunt end syringe.Filter the sample through 250 micron nylon mesh into the 50 milliliter conical tube to separate adipocytes and stromal vascular cells from undigested tissue. Wash out the scintillation vial with 10 milliliters of KRBBS, and pour the wash through the filter to collect residual cells before incubating the filtered samples for five minutes at room temperature. Then, utilize a 20 milliliter syringe and a 20 gauge by six inch pipetting needle to remove stromal vascular fraction layer and transfer it to a clean 50 milliliter conical tube.Add 10 milliliters of KRBBS and allow the samples to sit on the bench for five minutes at room temperature. Keep the stromal vascular fractions from both depots on ice for further processing. Spin the stromal vascular fractions at 500 times G for five minutes at 4 degrees Celsius.Remove the samples from the centrifuge and carefully pour off the supernatant, keeping the pellet containing the stromal vascular fraction cells. Gently re-suspend the cell pellets in five milliliters of PBS. After spinning the samples again, remove the supernatant, re-suspend the cells in one milliliter of PBS, and count the cells.Pellet the samples for a third time, and re-suspend the pellet in 100 microliters of CD34 isolation buffer. Add 10 microliters of CD34 cocktail and incubate the sample for 15 minutes at room temperature. Then add five microliters of magnetic beads and incubate the sample for 10 minutes at room temperature.Next, add 2.5 milliliters of CD34 isolation buffer to each sample, and place the samples into the magnet for five minutes at room temperature. Invert the magnet for five seconds to pour off the isolation buffer. After repeating this process four times, remove the tube from the magnet and add two milliliters of CD34 isolation buffer.Following another centrifugation, re-suspend the CD34+cell pellets in one milliliter of CD34 isolation buffer and count the cells. Pellet the cells again, and re-suspend the pellet in 250 microliters of Buffer B and 250 microliters of wash buffer. Next, thoroughly re-suspend the CD31 beads by vortexing the tube, then add 20 microliters of CD31 beads to the re-suspended cell pellet and incubate the sample with rocking for 30 minutes at room temperature.Prior to the cell separation, add one milliliter of wash buffer to equilabrate the strainer. Then, apply the prepared mixture of re-suspended cells in CD31 beads to the strainer. Wash the strainer with five milliliters of wash buffer in a circular motion for a total volume of 20 milliliters.Attach the connector to a sterile 50 milliliter conical tube and close the leur lock. After attaching the strainer to the connector, add one milliliter of wash buffer along the wall of the strainer, then add one milliliter of activated Buffer D along the wall of the strainer. Swirl the sample gently, and incubate it for 10 minutes at room temperature.Next, add one milliliter of wash buffer and separate the cells from the beads by pipetting up and down 10 times. Open the leur lock and allow the detached cells to flow into the 50 milliliter conical tube. Wash the strainer 10 times with one milliliter of wash buffer each time.Discard the connector and strainer, and centrifuge the samples at 300 times G for 10 minutes at 4 degrees Celsius. Finally, re-suspend the cell pellet in two milliliters of complete EGM2 media for culture. Grow the cells in six-well tissue culture treated plates with complete EGM2 media in a 37 degrees Celsius, 5%carbon dioxide incubator.Replace the media every three to four days until the cells reach 80 to 90%confluence. Then, seed approximately 100 to 200 thousand cells into six-well plates, ensuring duplicate wells for each depot, and culture them in complete EGM2 media for two days. After two days, aspirate off any growth media.Replace the media with two milliliters of DMEM/F-12 media with 5%FBS and 50 micrograms per milliliter of penicillin streptomycin for the control cells. For the treatment cells, replace the media with two milliliters of adipogenesis media. Incubate the cells at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for 13 days replacing the respective media every three to four days.Representative results showing the typical endothelial cobblestone-like morphology of the CD34+CD31+cells after two passages in culture is shown. Also demonstrated is the ability of cells to form tubules when cultured in Matrigel as expected. Uniform uptake of fluorescently labeled acetylated LDL shows the presence of a homogenous cell population with endothelial identity.Shown here is Oil Red O staining of neutral lipid droplets in CD34+CD31+cells following 13 days of adipogenic induction. Representative responses of cells from omental and subcutaneous depots of three different human subjects are shown to illustrate the large variability in response to adipogenic induction between fat depots of the same donor and between donors. This image displays fluorescent intracellular lipid staining in CD34+C31+cells after adipogenic induction for 13 days.The image shows cells with unilocular lipids, multilocular lipids, and no lipid accumulation. Real time PCR shows the expression of the pan adipocyte marker adiponectin and the brown beige markers CIDEA and UCP-1. These results confirm that cells following adipogenic induction express molecular markers consistent with a mixed white beige adipocyte phenotype.Once mastered, this technique can be done in five hours if it is performed properly. While attempting this procedure, it is important to remember to use fresh human adipose tissue that was collected and not stored longer than two hours. Following this procedure, other methods like flow cytometry, immunohistochemistry, and RT-PCR can be performed to identify specific subsets of responding cells, and expression of mature adipocyte markers that can be utilized to identify a beige brown adipocyte phenotype.After it's development, this technique paved the way for researchers in the filed of obesity to explore roles of adipose tissue, vascular cells, and adipogenesis in humans. After watching this video, you should have a good understanding of how to isolate, culture, and expand CD34+31+cells. Don't forget that working with human tissues can be extremely hazardous, and precautions, such as bio-safety regulations, should always be taken while performing this procedure.The implications of this technique extend toward therapy of obesity associated comorbidities, such as insulin resistance, because it identifies adipogenic response of cells to a potential therapeutic intervention.

Research

JoVE Journal

Immunology and Infection

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Expansion, rening och Functional Assesment of Human Peripheral Blood NK-celler

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

Utbyggnad av Human Peripheral Blood γδ T-celler med hjälp zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Isolering av humana navelvenendotelceller och deras användning i studier av neutrofilinhibitorfaktor Transmigration Under flödesförhållanden

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Isolation of Adipose Tissue Immune Cells

Isolering av immun fettvävnad celler

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Isolering av myeloida dendritiska celler och epitelceller från Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Isolering av leukocyter från Human Maternal-foster Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

Isolering av Viral Replication Fack-berikade underkärn Fraktioner från adenovirus-infekterade Normal humana celler

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

Bedömning av Synaptic gränssnittet för primära humant T-celler från perifert blod och lymfatisk vävnad

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Isolering av makrofager subsets och stromaceller från Human-och mus-Myokarprover

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Pneumokocker infektion av primära humana endotelceller i konstant flöde

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...