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

28.3K 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

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

背マウス皮膚から毛包幹細胞と表皮角化細胞の単離

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

発生生物学

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

人間の毛包から神経堤幹細胞の単離および培養

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

神経科学

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

世代、大人の肌からプライマリ ヒト角化培養

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

大人の皮膚組織から人間ケラチノ サイト主を特定する簡易で効率的な方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

ケラチノサイト幹細胞の可塑性をモデル化する高スループット表皮球面培養システムの確立

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

腹部皮膚からの成人ヒト皮膚線維芽細胞の分離と非積分法を用いた人工多能性幹細胞の生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

生物工学

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&#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 ...

生物学

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

ヒト包皮からの初代シュワン細胞、ケラチノサイト、および線維芽細胞の単離、培養、および特性評価

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

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 ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

成人マウス口蓋からの一次口腔角化細胞の分離と培養

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

ヒト皮膚から大人上皮幹細胞の単離と培養

要約

さらに動画を探す

背マウス皮膚から毛包幹細胞と表皮角化細胞の単離

人間の毛包から神経堤幹細胞の単離および培養

世代、大人の肌からプライマリヒト角化培養

大人の皮膚組織から人間ケラチノサイト主を特定する簡易で効率的な方法

ケラチノサイト幹細胞の可塑性をモデル化する高スループット表皮球面培養システムの確立

腹部皮膚からの成人ヒト皮膚線維芽細胞の分離と非積分法を用いた人工多能性幹細胞の生成

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

ヒト包皮からの初代シュワン細胞、ケラチノサイト、および線維芽細胞の単離、培養、および特性評価

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

成人マウス口蓋からの一次口腔角化細胞の分離と培養

ヒト皮膚から大人上皮幹細胞の単離と培養

要約

さらに動画を探す

背マウス皮膚から毛包幹細胞と表皮角化細胞の単離

人間の毛包から神経堤幹細胞の単離および培養

世代、大人の肌からプライマリ ヒト角化培養

大人の皮膚組織から人間ケラチノ サイト主を特定する簡易で効率的な方法

ケラチノサイト幹細胞の可塑性をモデル化する高スループット表皮球面培養システムの確立

腹部皮膚からの成人ヒト皮膚線維芽細胞の分離と非積分法を用いた人工多能性幹細胞の生成

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

ヒト包皮からの初代シュワン細胞、ケラチノサイト、および線維芽細胞の単離、培養、および特性評価

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

成人マウス口蓋からの一次口腔角化細胞の分離と培養

世代、大人の肌からプライマリヒト角化培養

大人の皮膚組織から人間ケラチノサイト主を特定する簡易で効率的な方法