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

Os autores desejam reconhecer Becky Marquez, o coordenador clínico no centro de bariátrica Sentara, pela sua assistência com o processo do paciente, triagem e concordando. Esta pesquisa foi apoiada pelo R15HL114062 para Anca D. Dobrian.

O Comitê de placa de revisão institucional na Eastern Virginia Medical School aprovado a pesquisa e a coleta de amostras de tecido adiposo humano utilizados no estudo. Termo de consentimento informado foram coletado dos pacientes.
1. preparação de Buffers, meios e instrumentos
<ol><li>Preparar uma solução de Krebs Ringer Bicarbonate-Buffered (KRBBS): 135 milímetros de cloreto de sódio, 5 mM de cloreto de potássio, sulfato de magnésio 1 mM, 0,4 milímetros de fosfato de potássio di...

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O foco deste artigo é fornecer uma metodologia para o isolamento, a expansão e a indução de adipogenic de CD34 + CD31 + células endoteliais de depósitos viscerais e subcutâneas de tecido adiposo humano.
Metodologias foram relatadas para o isolamento de células endoteliais de vários leitos vasculares de roedores ou humanos que envolvem principalmente técnicas usando anticorpos CD31 fluorescente etiquetado ou acoplado a grânulos magnético18, <sup cl...

Evidência recente mostra que, tanto nos ratos e nos seres humanos, um subconjunto de células que residem na vasculatura do tecido adiposo pode ser diferenciado em adipócitos branco ou bege/marrom1,2,3. O fenótipo de tais células é um assunto controverso, com evidências que sustentam as células endoteliais, musculares lisas/Pericito ou um espectro de fenótipos intermediários4,5,6,7. O escopo de desenvolver esta metodologia foi testar o potencial de adipogenic de CD34 + CD31 + células endoteliais isoladas de diferentes depósitos de gordura dos humanos obesos. Outros estudos na literatura estão focando o adipogenic potencial de fração total do estroma vascular ou do adipócito conhecido progenitores2,8,9. Desde tecnologias actualmente existentes podem alvejar especificamente tecido adiposo células endoteliais por drogas entrega10, compreendendo o potencial de tais células adipogenic submeter-se a indução no sentido de adipócitos brancos ou bege é importante para terapias alvo futuras.
Diferentes grupos relataram a combinação de marcadores CD31 e CD34 como substitutos para isolar as células endoteliais de tecido adiposo humano11,12,13. Normalmente, o isolamento é realizado usando duas etapas sequenciais e uma seleção positiva usando esferas magnéticas. Neste relatório, foi utilizada a immunoseparation usando CD34 + grânulos magnéticos combinados com grânulos plásticos CD31. Encontramos essa técnica superior para o sequencial immunoseparation magnético com relação a preservação da morfologia endotelial típico de paralelepípedos. Além disso, fomos capazes de gerar células suficientes, necessárias para a expansão e adipogenic indução a partir de tão pouco quanto 1-2 g de gordura. A biópsia de pequena amostra de gordura subcutânea é suficiente para produzir a quantidade necessária de células para aplicações a jusante. Este aspecto é potencialmente importante, particularmente se esse método será utilizado para a seleção para uma capacidade de resposta à indução de adipogenic em cobaias humanas.
Ao contrário de outros sistemas relatados na literatura, este método utiliza apenas dois ingredientes para a indução de adipogenic do CD34 + CD31 + células: um agonista do PPARγ — rosiglitazone — e da insulina humana. Importante, a quantidade de insulina usada cai dentro do intervalo normal/alta de circulação post-absorptive insulina em seres humanos14. O grau de sensibilidade à insulina de células em vitro, medido por fosforilação da Akt, não se correlaciona com a capacidade de responder para a indução de cocktail. Curiosamente, usando este condições de cocktail e experimental de indução, uma mistura de brancas e bege/marrom células foram obtidos conforme determinado pelo tamanho e número de gotículas lipídicas intracelulares e a expressão de marcadores moleculares. Este protocolo de indução simples e econômica, juntamente com a avaliação quantitativa do fenótipo das células Respondente (branco vs bege) permite um rastreio dos agentes que potencialmente podem alterar o equilíbrio de bege diferenciada : branco adipócitos.
Este método também fornece uma abordagem translacional para compreender os mecanismos subjacentes da adipogenesis de progenitoras endoteliais vasculares no tecido adiposo humano. Usando esta técnica de isolamento específico/diferenciação, investigadores podem interrogar várias vias responsáveis para adipogenesis em um subconjunto de pilhas endothelial vasculares de vários depósitos de gordura em humanos obesos e magros.

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

A obesidade é acompanhada por uma extensa remodelação do tecido adiposo, principalmente através de hipertrofia dos adipócitos. Crescimento extremo dos adipócitos resulta em uma má resposta à insulina, hipóxia local e inflamação. Estimulando a diferenciação dos adipócitos brancos funcionais de progenitores, hipertrofia radical da população adipócito pode ser prevenida e, consequentemente, melhorar a saúde metabólica do tecido adiposo juntamente com uma redução de inflamação. Também, estimulando uma diferenciação dos adipócitos bege/marrom, o gasto de energia total do corpo pode ser aumentado, resultando em perda de peso. Esta abordagem poderia impedir o desenvolvimento de co-morbidades como diabetes tipo 2 e doença cardiovascular obesidade.
Este artigo descreve o isolamento, expansão e diferenciação dos adipócitos brancos e bege de um subconjunto de células endoteliais de tecido adiposo humano que expressam co os marcadores CD31 e CD34. O método é relativamente barato e não é trabalhoso. Ele requer acesso ao tecido adiposo humano e é adequado para amostragem o depósito subcutâneo. Para este protocolo, amostras de tecido adiposo fresco de indivíduos obesos mórbidos [índice de massa corporal (IMC) > 35] são recolhidos durante os procedimentos de cirurgia bariátrica. Usando um immunoseparation sequencial da fracção vascular do estroma, células suficientes são produzidas a partir de tão pouco como 2 – 3 g de gordura. Estas células podem ser expandidas em cultura durante 10 a 14 dias, podem ser criopreservadas e mantém suas propriedades de adipogenic com passagem até passagem 5 – 6. As células são tratadas por 14 dias com um coquetel, usando uma combinação de insulina humana e o agonista do PPARγ-rosiglitazone de adipogenic.
Esta metodologia pode ser usada para a obtenção de prova dos experimentos de conceito sobre os mecanismos moleculares que conduzem a respostas adipogenic nas células endoteliais adiposas, ou para a seleção de novos medicamentos que podem melhorar o adipogenic resposta dirigido também para o branco ou diferenciação dos adipócitos bege/marrom. Usando pequenas biópsias subcutâneas, esta metodologia pode ser usada para filtrar os assuntos não-Respondente para ensaios clínicos vistos estimular adipócitos bege/marrom e brancos para o tratamento da obesidade e co-morbidades.

Nosso protocolo visa proporcionar uma abordagem in vitro para determinar o potencial de adipogenic de CD34 + CD31 + vascular em células de diferentes depósitos de tecido adiposo humano. Um diagrama de fluxograma simplificado é mostrado na figura 1A. O primeiro passo usando uma seleção positiva de CD34 expressando células resulta em > 95% células CD34 + na população das células recém isoladas (figura 1A). Impor...

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

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

A diferenciação dos adipócitos brancos e bege de tecido adiposo vascular progenitores tem potencial para melhoria metabólica em obesidade. Descrevemos os protocolos para um CD34 + CD31 + isolamento de células endoteliais de gordura humana e uma subsequente em vitro expansão e diferenciação em adipócitos brancos e bege. Diversas aplicações a jusante são discutidas.

Isolamento, expansão e indução de Adipogenic de CD34 + CD31 + células endoteliais de tecido adiposo humano de Omental e subcutâneo

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

Expansão, Purificação e Avaliação Funcional de Células NK do sangue periférico

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

Imunologia e Infecção

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

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

Expansão do sangue periférico humano γδ células T usando zoledronato

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

Isolamento de Células endoteliais da veia umbilical humana e sua utilização no estudo de transmigração de Neutrófilos sob condições de fluxo

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

Isolamento de tecido adiposo de células imunes

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

O isolamento de células dendríticas mielóides e células epiteliais de timo humano

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

Isolamento de Leucócitos do Human Interface materno-fetal

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

Isolamento de Frações Sub-nuclear Viral Replication Compartimento enriquecidas de Adenovirus-infectados células normais Humanos

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

Avaliação da Interface sináptica de células T humano primário do sangue periférico e tecido linfoide

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

Isolamento de subconjuntos de macrófagos e células estromais de espécimes miocárdios humanos e de camundongos

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

Infecção pneumocócica de células endotélias humanas primárias em fluxo constante

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