この作品は、NIH / NCIの助成金R01CA - 118916によって資金を供給される

1。ヒト皮膚から上皮幹細胞を抽出する<ol><li>上皮幹細胞を単離する手順を開始する前に、1つは、それぞれのメディア、試薬を（表1参照）を準備する必要があります。 </li><li>フェイスリフトの手順またはパンチ生検から新鮮な大人の人間の頭皮の皮膚が収集され、その後4℃DMEM / 10％FBS /一晩ディスパーゼ（4 mg / mL）を℃でインキュベートする37℃で2〜4時間のインキュベーションでは、° Cにも効果的です。皮膚片は、浸透する酵素を可能にするために1cmの最大幅になります。 </li><li>滅菌シャーレに皮膚を転送する、皮膚の表面近くに毛幹を把握し、しっかりとスムーズに引っ張って皮膚からそれぞれの髪をやってのける。 、解剖顕微鏡下でその形態に基づいて、休止期の段階で卵胞を選択し（図1A）転送包は、15 mLの滅菌チューブにバルジ領域を切り取る。カットなら成長期の毛包は、毛包&quot;膨らみ&quot;の領域（図1B）を取得するために卵胞の上側1 / 3に使用することができます。 </li><li>定期的に振盪しながら室温で15-20分のための0.05％トリプシン- EDTA（GIBCO）およびヴェルセン（0.53 mMのEDTA 4NA、GIBCO）（1:1）の混合物に分離された毛包の断片をインキュベート、追加、4 mLのDMEM + 10 ％FBSは、反応を停止し、800rpmで5分間スピンダウンする。また、卵胞の組織片を外植片は、単一の卵胞から培養細胞をトリプシン消化することなく文化に配置することができます。 </li><li> EGFことなく、1 mLのKCM（ケラチノサイト培地）で（セルを失うのを避けるために〜0.2から0.5 mLを保存）注意深く上清を捨て、再びサスペンド。 </li></ol> 2。文化プライマリ上皮幹細胞<ol><li>培養前に、上皮幹細胞は、マイトマイシンC処理した3T3 - J2細胞（フィーダー細胞）は、次のように準備する必要があります。 3T3 - J2細胞は治療前に10％FBSを含むDMEM中で培養されています。培養メディアを取り出して、PBSで2回細胞を洗浄し、37℃で2時間無血清DMEMで15μg/ mLのマイトマイシンC ° C、5％CO 2でセルを扱う。マイトマイシンC含有DMEMを除去し、PBSで2回洗い、細胞を使用できるようになります。 （マイトマイシンC処理した3T3 - J2細胞はまた、事前に準備し、一日で37インキュベート培養一次上皮幹細胞、° C、5％CO 2の前に細胞を-80℃で融解し、シードで保存。15万することができます6ウェルプレートのウェル当たり3T3 - J2細胞を推奨します。 </li><li> 37℃で一晩EGFなしKCMにおけるマイトマイシンC処理した3T3 - J2細胞上でステップ1.5からシード孤立した毛包幹細胞は、° C、5％CO 2、翌日、EGF含有KCMに変更。細胞を37で成長している· CO 2 5％を含む湿った大気中のC。すべてのケラチノサイトは、KCMは2日毎にEGFを含有する供給および14-20日のために栽培されている。顕微鏡下で毎日のセルを確認してください。毛包幹細胞は、コロニー（図2A）を形成します。毛包の外植片は、10〜14日間（図2B）の後にoutgrowthsを形成します。 </li></ol> 3。パッセージ上皮​​幹細胞<ol><li>一度PBSで細胞を洗浄する。ヴェルセン（RTに予め温めておいた）処理により3T3 - J2フィーダー細胞を取り外します。インキュベートの文化が5分間ヴェルセンにくぼんだ、その後、振とうし、フィーダー細胞をオフに吸引除去する。 </li><li>ヴェルセンまたはPBSに一度のいずれかで毛包幹細胞を洗う。 </li><li>予め温めておいた追加（37 ° C、クリティカル）トリプシン- EDTA（2X）および37℃7分以上デタッチする毛包幹細胞のための必要に応じてのためのC。もはや15分以内が最適です。 </li><li> 、トリプシンを停止するには、KCM W / O EGFを追加軽く、200グラムでスピンダウン、細胞を分散させるために5分を上下にピペッティングし。 </li><li>マイトマイシンC処理した3T3 - J2細胞層上にEGF、カウントおよび再プレートなしKCMと再サスペンド細胞。 </li></ol> 4。上皮幹細胞を不死化初代細胞、さらに成体幹細胞は、培養中の連続継代し、数ヶ月後に老化に達する。主要な幹細胞の不死化には、この問題を緩和する効果的な方法です。 <ol><li> 2日間のフィーダー層（3T3 - J2）と文化の道1のプレート一次上皮幹細胞（〜35万細胞/ウェル、6ウェルプレートの）。 </li><li> DMEM 10％FBSを含む、プライマリ上皮幹細胞を播種後の培養PA317 LXSN 16E6E7細胞株は、一日。 （この行は、HPV16 E6とE7のオープンリーディングフレームをコードアンフォトロピックレトロウイルスLXSN16E6E7を生成し、安定的に多くの細胞型に感染して不死化するために使用することができる）。 </li><li> 2時間マイトマイシンCとPA317 LXSN 16E6E7細胞株（3T3 - J2細胞と同じプロトコル）を扱います。マイトマイシンCは、EGFとKCMの6日間一次上皮幹細胞、共培養にPA317 LXSN 16E6E7細胞株を扱うに追加します。 2日おきEGFメディアで新鮮なKCMを更新。 </li><li>ヴェルセンと3T3 - J2とPA317 LXSN 16E6E7セルを削除します。追加マイトマイシンC処理した3T3 - J2 NHP細胞（ネオマイシン、ハイグロマイシン、ピューロマイシン耐性、ウェルあたり200,000細胞）上皮幹細胞へ。細胞は、追加のためにG418（Gibco）を0.2 mg / mLの下で選択されていますitional 6日。 EGFメディア2日ごとに新鮮なG418を含むKCMを更新する。 存続上皮幹細胞は、幹細胞を不死化されます。 PA317 LXSN 16E6E7細胞と共培養されていない主な上皮幹細胞のウェルG418選択のためのコントロールとして設定する必要があります、このほかのすべてのセルが選択の6日後にG418によって殺されるべき。 </li></ol> 5。代表的な結果皮膚上皮幹細胞と不死化上皮幹細胞の早期成立には、フィーダー細胞に囲まれた小さなケラチノサイト（図2A）で構成されたタイトなコロニーを形成する。フィーダー層なしで、定義されているサプリメントと無血清培地で培養した場合、幹細胞が分散し、（それらが単一細胞や小さなクラスターとしての成長）タイトなコロニーを形成しません。不死化上皮幹細胞は、連続継代の&gt; 12ヶ月間の安定した表現型を維持するが、一次電池よりも厳重なコロニーを形成する傾向があった。彼らはこれらの不死化細胞はがん細胞の特性を有していないことを示す、軟寒天アッセイにおける足場非依存性コロニーを形成しません。プライマリおよび不死化の両方上皮幹細胞は毛包幹細胞マーカーサイトケラチン15（図3A）を発現し、表皮、毛包や皮脂腺の系統（図3B - D）に分化することができます。 <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="図1" /> 図1は、。髪の毛を羽をむしられた。ディスパーゼ処理皮膚から摘採した後、毛包には毛の長さを周囲の上皮のスリーブで髪の底部を周囲の細胞のボールを持って休止期クラブの毛（）のいずれかとして表示され、または成長期の毛（B）。成長期の膨らみが少なく明瞭である間休止期のバルジ領域は、ミクロ分析する明確な形態​​学的領域を形成する。成長期の電球はまた、単離され、培養することができる中継増幅のセルを表す行列のケラチノサイトを、含まれています。 <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="図2" /> 図2。幹細胞の培養。 （A）細胞（Fで指定された）のフィーダー層とKCMで培養皮膚のフォームタイト上皮コロニー（Eによって指定される）から上皮幹細胞。 （B）毛包の外植片は上皮outgrowthsを生じさせる。休止期のバルジ領域は、（Tが指定する）皿に取り付けられ、フィーダー細胞とともに上皮細胞のコロニーを囲まれています。 <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="図3" /> 図3：細胞分化の幹。 （A）幹細胞のコロニーは、サイトケラチン15の上皮マーカーの発現を維持する。 （B）皮膚上皮幹細胞は、K6Hf式によって決定された毛包系列に沿って分化させることができる。 （C）細胞は、空気 - 液体界面でのデ - epidermalized真皮または他のマトリックス上で培養することができ、角質層、顆粒層および有棘層で層別表皮を形成することになる。 （D）幹細胞をオイルレッド、正球と皮脂系統に沿って誘導することができる。

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に記載の細胞抽出および培養方法は、驚くほど容易なと再現性です。我々は、継承された皮膚の欠陥18の患者を含む幅広い年齢範囲にわたって個人の数十から上皮幹細胞培養物を生成している。組織の収穫の日にプロセスを開始することをお勧めですが、細胞が必要な場合は翌日配達を促進する、数日間氷上メディアで実行可能なままになります。破棄されたフェイスリフトスキンは...

ケラチノサイトメディア（KCM） [DMEMとハムのF12（GIBCO、3:1）、アデニン（シグマ、180 mM）で、10％ウシ胎児血清（GIBCO）、コレラ毒素（ICN、0.1nm以下）、ペニシリン/ストレプトマイシン（GIBCO、100 U / mlのと、それぞれ100 mg / mlの、）、ヒドロコルチゾン（Sigma社、0.4 mg / mlの、1.1 mM）で、T/T3（トランスフェリン、GIBCO、5μg/ mlの、649 nMの、とトリヨード- L -チロニン、シグマ、2 nM）を、インスリン（Sigma社、5 mg / mlの、862 nm）、およびEGF（シグマ、10 ng / mlの、1.6 nM）を、pH 7.2の]

isolation and culture of adult epithelial stem cells from human skin

すべて自己再生組織の恒常性は、成体幹細胞に依存しています。未分化幹細胞は非対称分裂を受けるように、彼らは幹細胞の表現型と、幹細胞のニッチから移行急速な普及を受け、末期組織を再作成するには差別トランジット増幅細胞を（TA細胞）保持の娘細胞を生成する。 上皮幹細胞は 、in vivo 1-3 の試験管内増殖能の高いとのようなスローサイクリングのラベル保持細胞との細胞として表皮、毛包、および腸内で確認されている。成人、組織特異的幹細胞は、ストレス4-5の時間帯だけでなく、正常な生理学的代謝回転の間に置かれている組織の再生のための責任があります。また、幹細胞は、一般的に組織6内の複数の細胞型を生じさせる能力を有する、複数の強力であると考えられる。例えば、げっ歯類の毛包幹細胞は表皮、皮脂腺、および毛包7-9生成することができます。私たちは、人間の毛包のバルジ領域から幹細胞が多潜在力10を示すことが示されている。 幹細胞は発生生物学、分化、腫瘍形成とそれらの可能な治療的有用性のために研究するためのin vitroの系としてのユーティリティに起因する生物医学研究における貴重なツール、となっている。それは大人の上皮幹細胞はそのような外胚葉異形成、monilethrix、ネザートン症候群、メンケス病、遺伝性表皮水疱症と脱毛11月13日のような疾患の治療に有用となる可能性があります。さらに、このようなやけどの傷、慢性創傷や潰瘍のような他の皮膚の問題は、治療14,15に関連幹細胞の恩恵を受ける。多能性の状態（iPS細胞）16,17に成体細胞の再プログラミングのための可能性を考えると、人間の皮膚に容易にアクセス可能で拡張可能な成体幹細胞が誘導とを含む病気の広い範囲のダウンストリーム治療用細胞の貴重な源を提供することがあります糖尿病とパーキンソン病。

Watch this Scientific Journal Video about Isolation and Culture of Adult Epithelial Stem Cells from Human Skin at JoVE.com

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

人間の皮膚から実行可能な成人上皮幹細胞を単離の迅速、確実な方法が記載されている。方法は、毛包と細胞培養のための単一の細胞懸濁液または組織断片の分離の摘採が続く皮膚のコラーゲンマトリックスの酵素消化を、利用しています。

ヒト皮膚から大人上皮幹細胞の単離と培養

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

isolation-and-culture-of-adult-epithelial-stem-cells-from-human-skin

Research

JoVE Journal

Biology

28.2K ビュー.  University of Massachusetts Medical School. 人間の皮膚から実行可能な成人上皮幹細胞を単離の迅速、確実な方法が記載されている。方法は、毛包と細胞培養のための単一の細胞懸濁液または組織断片の分離の摘採が続く皮膚のコラーゲンマトリックスの酵素消化を、利用しています。

ビデオ: Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

分離と成人ヒト内皮コロニー形成前駆細胞の大規模拡張

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

生物学

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

大人と胚骨格筋のMicroexplantの文化と骨格筋幹細胞の単離

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

ヒト膵臓癌異種移植片由来の幹細胞の分離

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

初代ヒト線維芽細胞培養からナイーブ成人幹細胞の分離

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

人間の脂肪吸引物からの脂肪由来幹細胞を手動で隔離

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

筋衛星細胞の単離、培養、および移植

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

成体マウス切歯から歯科上皮幹細胞の単離および培養

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

新生児および成人マウス皮膚由来の初代マウスケラチノサイトの単離および培養

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...