This study was supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant (371697-2011, AT). The authors want to acknowledge the help of bariatric surgeons Drs S. Biron, F-S. Hould, S. Lebel, O. Lescelleur, P. Marceau as well as Christine Racine and Caroline Gagnon from the IUCPQ Tissue Bank. We thank Mr Jacques Cadorette from the IUCPQ&#8217;s audiovisual services for video shooting and editing.

倫理声明：プロジェクトが前に患者の募集にIUCPQの研究倫理委員会によって承認されています。この記事/ビデオの目的のために、私たちは2人の患者から組織を得た：1）57キロのBMIが50.7キロ/ m 2であり、2）35歳の女性患者のBMIが62歳の男性患者/ m 2である 。実験は、両方の脂肪のコンパートメントで行うことができるが、このビデオの目的のために一種の脂肪区画に限られていた。ビデオの技術的側面は、患者1で行ったとFABP4免疫蛍光は、患者2からの脱分化細胞を用いて行った。 1.サンプル処理<ol><li>腹腔鏡肥満手術の時に大網および皮下脂肪区画から脂肪組織を収集するために外科医に依頼。 </li><li>すぐに室温で実験室に脂肪のサンプルを持ってきて、すぐに処理します。 </li><li>非無菌雰囲気の中で、研究室での消化を実行します。ザ·細胞は、最終的には培養室に移し、無菌条件下で培養される。汚染を避けるために、消化の前に蒸留し、濾過水をKRH緩衝液を調製し、濾過（0.22μmのフィルター）をたどる。フラスコとプレートの準備のための細胞培養フード中のエタノールの前に転送で十分にきれいなチューブ。 </li><li>事前加重ディッシュとレコード重量に脂肪組織を置きます。パラフィン包埋の前に少なくとも24時間、室温で10％ホルマリン緩衝液中の各組織サンプル（1cm未満2）の小片を固定する。免疫組織化学実験（技術図示せず）のために、この埋め込まれたサンプルを使用してください。 </li><li>全体の脂肪組織（ -技術図示していない例えば 、遺伝子発現）上のさらなる研究のために-80℃で保存する前に、液体窒素中で50 mlチューブとフラッシュ凍結に別の作品を配置します。 </li></ol> 2.コラゲナーゼ消化<ol><li> 50ミリリットルtuの中の残りの脂肪組織片を置き消化のためにも。 </li><li>消化管中の試料のグラム当たりのコラゲナーゼ（350 U / ml）を補充したKRH-WBを4mlを加える。 </li><li>はさみで脂肪組織をみじん切りに。 </li><li> 45分のインキュベーション（最大1時間）のために、シェーカーで37℃、90回転数の最大値を、みじん切り脂肪組織懸濁液を置きます。 </li></ol>脂肪細胞および前脂肪細胞の精製<ol><li>プラスチック製のビーカーにメッシュ400のナイロンを通して脂肪の少ないチャンクと半透明のソリューションを注ぐ。 </li><li>ピンセットで、ナイロンメッシュ上の細胞調製をこするとKRH-WBの5ミリリットルで洗浄する。 </li><li>微妙にその中にプラスチック管および管端に取り付けられた60ccのシリンジで50ミリリットルチューブに濾過し、細胞懸濁液を転送します。 </li><li>成熟脂肪細胞との懸濁液を、細胞が浮遊することによって、バッファの先頭に到達することを可能にする、約10分間放置する。 </li><li>ゆっくりと60ccのsyrinを使用して、チューブの底にバッファを吸引GE吸引。 </li><li>洗ってKRH-WBの20ミリリットルを追加します。 2追加の洗浄のためのステップ3.4から繰り返します。 </li><li>細胞の量に応じて、5または10 mlの最終容積に脂肪細胞の懸濁液をもたらすためにバッファを集める。セクション5のステップで追求しています。 </li><li>所望であれば、さらに、初代細胞培養の遠心分離によって60ccのシリンジ（3000回転、RT、5分）で集めバッファから間質 - 血管画分を回収し（技術が示されていない）。 </li></ol> 4.成熟脂肪細胞細胞数<ol><li>負荷の計数室（血球計）で穏やかに振盪脂肪細胞懸濁液10μl。四重に細胞数を実行します。 </li><li>孤立した成熟細胞の数を計算します。 </li></ol> T-25フラスコに5.成熟脂肪細胞脱分化<ol><li> DMEM / F12-20％ウシ血清でボリュームの3/4に25cm 2の組織培養フラスコを埋める。 </li><li>細胞数に応じて、フラスコに50万成熟細胞を注ぐ。 </li&gt; <li>培地で50ミリリットルチューブを使用して完全にフラスコを記入し、できるだけ多くの気泡を除去。 </li><li>フラスコ上の未換気キャップをねじ込みます。 </li><li>汚染を避けるためにインキュベーションの前にエタノールでフラスコを清掃してください。 </li><li>細胞接着が中断される場合があり文化の動きを避けるために、それに触れることなく、週に逆さまにフラスコをインキュベートする。 </li><li>反転した培養7日目に、フラスコを逆にする前に、静かにフラスコを操作し、突然の動きを避け、吸引によりフラスコ内のすべてのメディアを削除します。 </li><li> DMEM-F12-20％ウシ血清の12ミリリットルを追加し、標準的な技術を用いて細胞を養う。ろ過、ベントキャップフラスコに添加してもよい。 </li></ol> 6ウェルプレートに6成熟脂肪細胞脱分化<ol><li> 6ウェルプレートの各ウェルの底にカバースリップを置き</li><li>各カバースリップの上に½ &quot;プラスチックブッシングを追加します。 </li><li> 20％のウシ血清DMEM培地の8ミリリットルで井戸を埋める。 </li><li>各プラスチックブッシングにカバースリップを置く。 </li><li>スライドとスライドの下で細胞を注入するチューブ（ウェル当たり50,000細胞）との間にピペットチップを挿入します。 </li><li>週5％CO 2、37℃で標準的な細胞培養インキュベーター中でプレートをインキュベートする。 </li><li> 20％のウシ血清を補充した培地の各ウェルを含む2ミリリットル中に付着した細胞を用いた逆カバースリップと文化を追求する。 </li><li>免疫蛍光（技術が示されていない）を含むいくつかの目的のために脱分化を受けている細胞でカバーガラスを使用してください。 </li></ol>

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The authors declare no conflict of interest.

天井培養技術と成熟脂肪細胞の脱分化は、天然の脂肪組織の小サンプルから脂肪幹細胞を得るための新たなアプローチである。我々の経験など2のそれに基づいて、組織の1グラムを25cm 2のフラスコにプレートし、均質性がPoloni及び共同研究者3によって実証されているDFAT細胞の集団を得るために十分である。脂肪細胞の脱分化は、独立して自分の年齢、性別、その他の...

In vitro dedifferentiation of mature adipocytes is achieved through a technique called ceiling culture1. Because of their natural tendency to float in aqueous solutions, isolated mature adipocytes adhere to the surface of an inverted flask fully filled with culture medium. Over a few days, cells modify their spherical morphology and become fibroblast-like cells. The resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent2. Research articles on adipocyte dedifferentiation, especially on human cells, are limited. However, they have already provided interesting information regarding multipotency, cell phenotype and replicative capacity of DFAT cells2. Mature adipocytes originating from various fat compartments have been successfully dedifferentiated including those originating from human visceral and subcutaneous adipose tissues2-4. In addition to these depots, Kishimoto and collaborators sampled adipose tissue from the buccal fat pads and dedifferentiated adipocytes into DFAT cells5. Matsumoto and collaborators successfully generated subcutaneous DFAT cells from patients covering a wide range of ages, and the majority of cells had a high proliferative rate and less than 6% of senescence even after 10 passages in culture2.
DFAT cells have been successfully re-differentiated into several lineages, including adipogenic, osteogenic, chondrogenic and neurogenic lineages2,3,6. These cells express several embryonic stem cell markers such as Nanog and the four identified pluripotent factors Oct4, c-myc, Klf4 and Sox23. They also express markers specific to each of the three germ layers7. In addition, DFAT cells are similar to Bone Marrow-derived Mesenchymal Stem Cells (BM-derived MSC) based on their epigenetic signature3. Exploiting the stem cell capacity of DFAT cells, many groups have investigated their potential to treat or improve various diseases8,9. Improvements of pathologic conditions, such as infracted cardiac tissue, spinal cord injury and urethral sphincter dysfunction, have been observed with DFAT cell injections in rat models of disease10-12. 
In addition to the stem cell properties of DFAT cells, they may represent a new cellular model for adipocyte physiology studies. The 3T3-L1 cell line is often used for this purpose as these cells differentiate into adherent, lipid-storing adipocytes under adipogenic stimulation13. However, these cells originate from mouse embryo tissue13. Also, depot-specificity cannot be investigated with this model and it may not fully reflect human adipocyte physiology14. Other laboratories work with isolated adipose cells from murine fat depots, but fat distribution is not dimorphic in mice and anatomical configuration of the rodent's abdominal cavity prevents from extrapolating directly to humans15. In order to study adipocytes in the context of the physiopathology of human obesity, consideration of body fat distribution and fat depot-specific differences has become essential16. Some limitations of primary preadipocyte cultures, including cell quantities obtained from adipose tissue biopsy samples and their senescence after a few passages in culture, created the need for alternate models. Perrini and collaborators investigated depot-specificity in gene expression of DFAT cells originating from visceral and subcutaneous fat and compared them to adipose-derived stem cells (ASC) from the same fat depot. They demonstrated that differences in gene expression and function where mainly found between depots than between cell types, suggesting that DFAT cells are physiologically close to ASC from the same depot. DFAT cells may represent an interesting alternative to available models for studies on fat distribution in the pathophysiology of human obesity. Moreover, ceiling culture is a promising method to obtain adult stem cells for tissue engineering purposes. 
Here, we describe collagenase digestion, a widely-used technique to isolate mature adipocytes from the subcutaneous and/or visceral fat samples17, and the subsequent steps to perform ceiling culture and dedifferentiate these cells into multipotent, fibroblast-like cells.

<table><tbody><tr><td>Bovine serum albumine</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Adenosine</td><td>Sigma</td><td>A4036</td><td></td></tr><tr><td>Ascorbic acid</td><td>Sigma</td><td>A0278</td><td></td></tr><tr><td>NaCl</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>KCl</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>HEPES</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>Glucose</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>Type I collagenase</td><td>Worthington Biochemical Corp</td><td>LS-004196</td><td></td></tr><tr><td>DMEM/F-12, HEPES, no phenol red</td><td>Gibco-Life Technologies</td><td>11039-021</td><td>Add to medium : 20% calf serum, gentamicin (50 &amp;micro;g/ml) and fungizone (2.5 &amp;micro;g/ml)</td></tr><tr><td>Calf Serum, iron supplemented, from formula-fed calves</td><td>Sigma</td><td>C8056-500 ml</td><td></td></tr><tr><td>1/2 In plastic bushing</td><td>Iberville</td><td>2704-CP</td><td>SKU:1000120918 (Home Depot)</td></tr><tr><td>Liquid nitrogen</td><td>Linde</td><td></td><td></td></tr><tr><td>Formalin soluton, neutral buffered, 10%</td><td>SIGMA</td><td>HT501128</td><td></td></tr><tr><td>Sterile tweezers</td><td></td><td></td><td></td></tr><tr><td>Sterile scissors</td><td></td><td></td><td></td></tr><tr><td>60 cc syringes</td><td>BD Syringe</td><td></td><td></td></tr><tr><td>Plastic tubing</td><td></td><td></td><td></td></tr><tr><td>Krebs-Ringer-Henseleit stock buffer (KRH)</td><td></td><td></td><td>Prepare stock buffer as following: 25 mM HEPES pH 7.6, 125 mM NaCl, 3.73 mM KCl, 5 mM CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O, 2.5 mM MgCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O, 1 mM K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;. Adjust pH to 7.4.</td></tr><tr><td>Krebs-Ringer-Henseleit-Working Buffer (KRH-WB)</td><td></td><td></td><td>Add the following components freshly to KRH buffer: 4% bovine serum albumin, 5 mM glucose, 0.1 &amp;micro;M adenosine, 560 &amp;micro;M ascorbic acid</td></tr><tr><td>KRH-WB supplemented with Type I collagenase</td><td></td><td></td><td>Add 350 U/ml of Type I collagenase</td></tr><tr><td>T25 unvented cap tissue culture flask</td><td>Sarsted or other brand</td><td></td><td></td></tr><tr><td>6-well tissue culture plate</td><td>BD Falcon or other brand</td><td></td><td></td></tr><tr><td>Microscope cover glass 22x22</td><td>Fisherbrand</td><td>12-542-B</td><td></td></tr><tr><td>Sterile beakers</td><td></td><td></td><td></td></tr></tbody></table>

generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

主な形態学的変化は、脱分化（ 図1）の間に脂肪細胞を成熟起こる。 図2に示すように、脱分化を受けている細胞は、蛍光分析のために抗FABP4抗体で染色した。線維芽細胞様細胞の大部分はしなかったのに対し、丸い形態を有する細胞は、FABP4タンパク質を発現した。脱分化した後、DFAT細胞は数回の継代のための標準的な手順で培養することができる。我々は、ヒト大...

Watch this Scientific Journal Video about Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures at JoVE.com

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

天井の培養における成熟脂肪細胞の脱分化を経てヒト脂肪幹細胞の生成

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

generation-human-adipose-stem-cells-through-dedifferentiation-mature

Research

JoVE Journal

Developmental Biology

11.7K ビュー.  IUCPQ Research Center. Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

ビデオ: Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

強化された骨形成のためのヒト脂肪由来間質細胞の単離および濃縮

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

発生生物学

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

ブタ皮下脂肪組織から分離し、脂肪由来幹細胞の分化

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

脱分化脂肪細胞を得るために効率的な方法

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

吸引脂肪から採取した脂肪由来間質細胞を使用したクリティカル·サイズの頭蓋冠欠損モデルの修復

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

医学

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

神経紋の分離、培養、脂肪形成誘導、周食脂肪組織由来の脂肪由来幹細胞

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

The protocol allows the generation of a pure adipocyte population from induced pluripotent stem cells (iPSCs). Retinoic acid is used to differentiate iPSCs into mesenchymal stem cells (MSCs) which are used for producing adipocytes. Then, a sorting approach based on Nile red staining is used to obtain pure adipocytes.

脂肪細胞関連疾患を研究するためのヒトiPS細胞を純粋な脂肪細胞集団に確実に分化させる(英語)

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

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

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

分離、拡張、および CD31 cd34 陽性 + + ひと大網と皮下脂肪組織から血管内皮細胞の脂肪分化誘導

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

免疫・感染学

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

マトリックス細胞代謝クロストークを研究するためのヒト3D細胞外マトリックス-脂肪細胞培養モデル

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

生物工学

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

ヒト大動脈血管周囲脂肪前駆細胞の分化能力

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

生物学

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

膜成熟性アディポサイト凝集培養(MAAC)を用いたヒト成熟性の単離と培養

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...