Name: isolation and culture of adult epithelial stem cells from human skin
Uploaded: 2011-03-31T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Adult Epithelial Stem Cells from Human Skin at JoVE.com

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?...

<ol>
	<li>Jones, P. H., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+human+epidermal+stem+cells+from+transit+amplifying+cells+on+the+basis+of+differences+in+integrin+function+and+expression.">Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.</a> Cell. 73, 713-724 (1993).</li><li>Lyle, S., Christofidou-Solomidou, M., Liu, Y., Elder, D. E., Albelda, S., Cotsarelis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C8/144B+monoclonal+antibody+recognizes+cytokeratin+15+and+defines+the+location+of+human+hair+follicle+stem+cells.">The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells.</a> J. Cell. Sci. 111, 3179-3188 (1998).</li><li>Bac, S. P., Reneha, A. G., Potte, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells:+the+intestinal+stem+cell+as+a+paradigm.">Stem cells: the intestinal stem cell as a paradigm.</a> Carcinogenesis. 21, 469-476 (2000).</li><li>Slac, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+epithelial+tissues.">Stem cells in epithelial tissues.</a> Science. 287, 1431-1433 (2000).</li><li>It, M., Li, Y., Yan, Z., Nguye, J., Lian, F., Morri, R. J., Cotsarelis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+the+hair+follicle+bulge+contribute+to+wound+repair+but+not+to+homeostasis+of+the+epidermis.">Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis.</a> Nat Med. 11, 1351-134 (2005).</li><li>Spradlin, A., Drummond-Barbos, D., Kai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+find+their+niche.">Stem cells find their niche.</a> Nature. 414, 98-104 (2001).</li><li>Taylo, G., Lehre, M. S., Jense, P. J., Su, T. T., Lavke, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+follicular+stem+cells+in+forming+not+only+the+follicle+but+also+the+epidermis.">Involvement of follicular stem cells in forming not only the follicle but also the epidermis.</a> Cell. 102, 451-461 (2000).</li><li>Oshim, H., Rocha, A., Kedzi, C., Kobayash, K., Barrandon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+renewal+of+hair+follicles+from+adult+multipotent+stem+cells.">Morphogenesis and renewal of hair follicles from adult multipotent stem cells.</a> Cell. 104, 233-245 (2001).</li><li>Morri, R. J., Li, Y., Marle, L., Yan, Z., Trempu, C., L, S., Li, J. S., Sawick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotsarelis+G+Capturing+and+profiling+adult+hair+follicle+stem+cells.">Cotsarelis G Capturing and profiling adult hair follicle stem cells.</a> Nat. Biotechnol. 22, 411-417 (2004).</li><li>Ro, C., Roch, M., Gu, Z., Photopoulo, C., Ta, Q., Lyle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-potentiality+of+a+new+immortalized+epithelial+stem+cell+line+derived+from+human+hair+follicles.">Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles.</a> In vitro Cell. &#38; Dev. Biol. 44, 236-244 (2008).</li><li>Ohyama, M., Vogel, J. C. G. e. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=delivery+to+the+hair+follicle.">delivery to the hair follicle.</a> J Investig Dermatol Symp Proc. 8, 204-206 (2003).</li><li>Sugiyama-Nakagiri, Y., Akiyama, M., Shimizu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+follicle+stem+cell-targeted+gene+transfer+and+reconstitution+system.">Hair follicle stem cell-targeted gene transfer and reconstitution system.</a> Gene Ther. 13, 732-737 (2006).</li><li>Stenn, K. S., Cotsarelis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineering+the+hair+follicle:+fringe+benefits+of+stem+cell+technology.">Bioengineering the hair follicle: fringe benefits of stem cell technology.</a> Curr Opin Biotechnol. 16, 493-497 (2005).</li><li>Hoeller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+and+rapid+method+to+construct+skin+equivalents+from+human+hair+follicles+and+fibroblasts.+Exp+Dermatol+10.">An improved and rapid method to construct skin equivalents from human hair follicles and fibroblasts. Exp Dermatol 10.</a> , 264-271 (2001).</li><li>Navsaria, H. A., Ojeh, N. O., Moiemen, N., Griffiths, M. A., Frame, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reepithelialization+of+a+full-thickness+burn+from+stem+cells+of+hair+follicles+micrografted+into+a+tissue-engineered+dermal+template+(Integra).">Reepithelialization of a full-thickness burn from stem cells of hair follicles micrografted into a tissue-engineered dermal template (Integra).</a> Plast Reconstr Surg. 113, 978-981 (2004).</li><li>Werni, M., Meissne, A., Forema, R., Brambrin, T., K, M., Hochedlinge, K., Bernstei, B. E., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li><li>Par, I. H., Zha, R., Wes, J. A., Yabuuch, A., Hu, H., Inc, T. A., Lero, P. H., Lensc, M. W., Dale, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, 141-146 (2008).</li><li>Kazantsev, A., Goltso, A., Zinchenk, R., Grigorenk, A. P., Abrukov, A. V., Moliak, Y. K., Kirillo, A. G., Gu, Z., Lyl, S., Ginte, E. K., Rogae, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hair+growth+deficiency+is+linked+to+a+genetic+defect+in+the+phospholipase+gene+LIPH.">Human hair growth deficiency is linked to a genetic defect in the phospholipase gene LIPH.</a> Science. 314, 982-985 (2006).</li><li>Tola, J., Ishida-Yamamot, A., Riddl, M., McElmurr, R. T., Osbor, M., Xi, L., Lun, T., Slatter, C., Uitt, J., Christian, A. M., Wagne, J. E., Blaza, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+epidermolysis+bullosa+by+transfer+of+wild-type+bone+marrow+cells.">Amelioration of epidermolysis bullosa by transfer of wild-type bone marrow cells.</a> Blood. 113, 1167-1174 (2009).</li><li>Wagne, J. E., Ishida-Yamamot, A., McGrat, J. A., Hordinsk, M., Keen, D. R., Riddl, M. J., Osbor, M. J., Lun, T., Dola, M., Blaza, B. R., Tolar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+transplantation+for+recessive+dystrophic+epidermolysis+bullosa.">Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.</a> N Engl J Med. 363, 629-639 (2010).</li><li>Muraue, E. M., Gach, Y., Grat, I. K., Klausegge, A., Mus, W., Grube, C., Meneguzz, G., Hintne, H., Baue, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Correction+of+Type+VII+Collagen+Expression+in+Dystrophic+Epidermolysis+Bullosa.">Functional Correction of Type VII Collagen Expression in Dystrophic Epidermolysis Bullosa.</a> J Invest Dermatol. , (2010).</li><li>Y, H., Kuma, S. M., Kossenko, A. V., Show, L., X, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+with+neural+crest+characteristics+derived+from+the+bulge+region+of+cultured+human+hair+follicles.">Stem cells with neural crest characteristics derived from the bulge region of cultured human hair follicles.</a> J Invest Dermatol. 130, 1227-1236 (2010).</li><li>Nishimur, E. K., Grante, S. R., Fishe, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+hair+graying:+incomplete+melanocyte+stem+cell+maintenance+in+the+niche.">Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche.</a> Science. 307, 720-724 (2005).</li></ol>

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 ...

Research

JoVE Journal

Biology

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

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 ...

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

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

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 of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

三次元混合細胞スフェロイド共培養から乳腺上皮細胞の分離

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

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

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

NaÃƒÆ’Ã‚Â¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

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

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

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

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

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 ...