著者は、DESCの文化と初期支援する秀のping王に感謝します。著者は（ODKにK12-GM081266は、MGCに、K99-DE022059 AHJにK08-DE022377-02 OHに、そしてR01-DE021420）国立衛生研究所からの奨学金や助成金によって部分的に資金を供給している。

1。成体マウスから低ヘミ顎を分析<ol><li>このプロトコルを実行する前に、必要な制度の承認を得て、すべての動物保護ガイドラインに準拠するようにしてください。標準動物実験委員会の手続きを承認用いて動物を安楽死させる。この作業のために、CO 2窒息頸椎脱臼に続いて使用されました。 <ol><li>オプション：カミソリの刃を使用して、身体の残りの部分とは別のヘッド。 </li></ol></li><li>ネックラインに下唇から肌を削除します。 </li><li>メスを用いて、緩く接続されている2下の切歯の間の切開を行います。圧力を加える際に、下顎は、2つの半分に分割される。 </li><li>あごを切り離す顎の顆および時間的な下顎関節や顎の筋肉と一緒にカット間のメスをウェッジ。 </li><li>骨だけが残るまで、すべての筋肉、腱、靭帯を削除します。 </li></ol> 2。切歯を隔離<ol><li> ＃15メスを用いて、慎重に膜状領域の近位端に遠位端から45°の角度で骨を削り取る。エッジでの骨片を含む任意の残りの骨を離れて選ぶ、メスの先端を使用してください。ゆっくりとわずか3 番目の臼歯の下に始まり、下顎骨の舌側皮質板で離れピックアップし始める。 </li><li>切歯はClを含む領域に向けて慎重に作業し、きれいになるまで続ける。 </li><li>下歯槽神経束を取り外します。 </li><li>最初のCLに近位骨や組織を除去するクリーンなカットを作る。 </li><li>およそ2 番目の臼歯の下に、メスで遠位側に第二のカットを行います。近位切歯領域が、その後の骨と近位切歯の内側表面との間にメスを挿入して切開されています。組織を引き裂くしないように、これは慎重に行う必要があります。切開運動は、近位 - 遠位である。 ALTERNATIVEステップ： 私の上の領域に沿って可能な限り多くの骨を除去するncisor。エリアがクリアされたら、慎重にCLの領域に近いエナメル部分を処理するためにサイズを5ピンセットを用いて全体の門歯を削除します。 </li><li>低付着プレートに4℃で3〜4時間、1×PBS中の2％コラゲナーゼで解剖した組織（代替ステップ2.5のように、 図3Eのように孤立し、CL、または全体の切歯のどちらか）を配置します。 1月10日切歯のために、12ウェルプレートにウェルあたり100μlを使用しています。 10を超える切歯の場合は、6ウェルの低付着プレートにウェルあたり500μlを使用する。 </li></ol> 3。顕微解剖唇頚椎ループ<ol><li>冷たいDMEM/F12培地中のコラゲナーゼおよび場所からのCL領域を除去。 </li><li>サイズ5鉗子またはインスリン注射器（1 CC、28 G½）のいずれかを使用して、CLの下端部に慎重に引き抜いてください。上皮は無傷のまま間葉はバラバラになります。 CLと「翼状」構造として、横方向に拡張し、隣接する上皮が表示されます。 </li><li>削除する両側にV字型の切開を行って、すぐに氷上に冷たいDMEM/F12中に置くことで、隣接する上皮から頂芽。 </li><li> 1.5mlマイクロチューブに集め、すべてのCLのために繰り返します。 </li><li>標本をマイクロチューブをスピンダウンし、メディアを取り出し、100μlの細胞剥離溶液と交換してください。 </li><li> 37℃で30分間、細胞剥離溶液中で組織をインキュベートする</li><li>優しくピペッティング千μlの低接着性ピペットチップを使用して、単一細胞懸濁液を生成するために組織を解離する。細胞はまた、解離を達成するために、滅菌細胞濾過器に配置することができる。 </li><li>組織培養プラスチック又は他の所望の材料で血球計数器、プレートで細胞を数える。 </li></ol> DESCsの4。初代培養<ol><li> 6ウェルプレート中の細胞をプレート1-6でのn×10 20の濃度でマウスEGF組換えタンパク質を補充したDMEM/F12から構成さDESC培地中で4細胞/ mlのμg/ mlの、25 ng / mlの1×B27サプリメント、および1％抗生物質溶液（ペニシリン、100 U / mlのストレプトマイシン、50μg/ ml）をFGFの濃度で組換えタンパク質。 </li><li> 5％CO 2 DESC培地1ml中で37℃で6ウェルプレートに播種した細胞を成長させる。細胞接着およびコロニー形成を可能にするために播種後5日間、初期の文化を乱すことがありません。 </li><li>最初のメディアの変化のために、新しいメディアの半分の容量（50​​0μL）と古いメディアの容量（50​​0μL）の半分を交換してください。メディアを変更するときに、顕微鏡下でコロニーを確認してください。 </li><li>最初のメディアが変更された後、2日ごとに（1-2）中にメディアを変更し続けています。メディアを変更するときに、顕微鏡下でコロニーを確認してください。 </li></ol> 5。パッセージDESCs <ol><li>培地を吸引し、細胞を洗浄し、事前に温めたPBSで3回。 </li><li>食塩水に予め温め0.25％トリプシン-EDTAを添加し、デタッチDESCs 10〜15分間37℃でインキュベートする。 </li><li>細胞を収集するための組織培養プレートに沿ってピペット優しく、トリプシンを中和するためにDESCメディアを追加し、15ミリリットルコニカルチューブ内の場所。 </li><li> 2分間800×gで細胞をスピンダウン。 </li></ol>

<ol>
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著者らは、開示することは、特別の利害関係はありません。

上皮細胞は、40年以上前に21〜24第一に成功培養し、さらに最近では、上皮幹細胞の正常な分離25〜27は、上皮生物学の知識を進んでいる。我々は、成体マウスの切歯のDESCs、歯の生物学とエナメル質の形成に重要な洞察をもたらす可能性を秘めている幹細胞の比較的代役集団を単離するためのプロトコルを報告している。このプロトコルは、最初は毛包28から上皮幹細?...

脊椎動物のユニークな特徴の一つは、歯の進化である。この器官に関連する分子経路及び形態学の専門分野が発達し、進化生物学者5によるものを含む、いくつかの観点から検討されているように歯は、研究の多くの分野のための重要なモデルシステムとなっています。より最近では、再生医療の分野は、モデルとしての歯を使用して貴重な洞察を得るために始めている。特に、歯科用上皮幹細胞の発見は、重要な進歩であった6-13。 すべてのげっ歯類は、その成長のこれらの歯の成体幹細胞制御を研究するためのアクセス可能なモデルシステムを作り、幹細胞によって支えられて継続的に成長している切歯を有する。遺伝系統トレース実験8,9,12,14が続き、1970年代10,11における標識実験は、DESCsは切歯の近位領域に存在することを明らかにした。ステム細胞唇子宮頸ループ（CL）として知られている推定のニッチの外唇側に移動、上の上皮の区画に子孫、およびトランジット増幅（TA）細胞（ 図1）と呼ばれる細胞の集団に貢献しています。具体的には、DESCsは、外エナメル上皮（OEE）に存在し、星状胞体（SR）8,9,14、および内エナメル上皮（IEE）は、細胞周期の限られた数を経て進行してから移動し、TA細胞を生じさせる遠位側切歯の長さ（ 図1）に沿って。マウス切歯で分化エナメル芽細胞は、1日15,16で約350ミクロンの驚くべき割合で切歯に沿って遠位方向に移動し続けています。彼らが移動すると、細胞が成熟したエナメル芽細胞と角質アラト（SI）の細胞に分化する。エナメルマトリックスの全層を堆積した後、エナメル芽細胞の多くは、アポトーシスを受け、残りのセルのサイズが縮小し、エナメル質の成熟を制御する&lt;&gt; 17 SUP。このようなSr等の唇のCLでの他の細胞型の系統は、それほど明確であり、間充織18内および舌のCLでの幹細胞に関するデータのみ出現し始めている。 マウス切歯モデルを使用して、グループの数は、遺伝的経路と自然の幹細胞に基づいた臓器再生に関与する細胞生物学的プロセスを解明するために取り組んできました。しかし、唇CLが困難な初代細胞での作業になり約5000のマウス切歯あたりと推定された細胞の数が比較的少なく、含まれています。そのため、努力が文化に作られ、 生体内で達成可能ではない実験的なアプローチに新たな扉を開くために体外 6,16,19,20 におけるこれらの細胞を拡大してきた。ほとんどの最近の研究は、これらの細胞が両方の自己再生および培養13発現細胞アメロゲニンに分化できることを実証した。ここでは、目のための方法を説明し、実証するマウス唇CLからの細胞のE信頼性と一貫性の文化。

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isolation and culture of dental epithelial stem cells from the adult mouse incisor

歯の再生と刷新の根底にある細胞および分子メカニズムを理解することは、大きな関心1-4の話題になると、マウスの切歯は、これらのプロセスのためのモデルを提供しています。この驚くべき器官は、動物の生涯を通じて継続的に成長し、（舌側）（唇側）唇と舌頚椎ループ（CL）領域内に収容された成体幹細胞のアクティブなプールから必要なすべての細胞型を生成します。唇CLからのみの歯科幹細胞は、唇表面のエナメル質、歯の外側カバーを、生成エナメル芽細胞を生じさせる。この非対称エナメル形成は切歯先端に擦り傷を可能にし、近位の切歯における前駆細胞や幹細胞は歯の組織が常に補充されていることを確認。 試験管内で 、これらの前駆細胞もしくは幹細胞を単離および増殖する能力は、その拡張を可能にし、例えばHなどの生体内では達成できない多くの実験、への扉を開きます潜在的な幹細胞調節因子のIGHスループット·テスト。ここでは記述し、マウスの切歯の唇CLからの培養細胞への信頼性と一貫性のある方法を示す。

マウスヘミ-下顎骨の1つを連続的に成長切歯三大臼歯根付いた（ 図1A）を含む。すべての歯は、象牙質とエナメル質、歯冠の2鉱化コンポーネント（ 図1Aおよび1A &#39;）で構成されています。切歯を収容2つのステム細胞ニッチは、唇および舌CL CLと呼ばれ、エナメル質は、唇側（ 図1A &#39;）のみに形成されている。 DESCsは切歯エナメル質の形成...

Watch this Scientific Journal Video about Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor at JoVE.com

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

継続的に成長しているマウスの門歯は、歯科上皮幹細胞（DESCs）から歯の組織の更新を研究するためのモデルを提供します。一貫して確実切歯からこれらの細胞を取得し、in vitroでそれらを拡張するための堅牢なシステムをここで報告されている。

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-culture-dental-epithelial-stem-cells-from-adult-mouse

Research

JoVE Journal

Biology

15.0K ビュー.  University of California, San Francisco. 継続的に成長しているマウスの門歯は、歯科上皮幹細胞（DESCs）から歯の組織の更新を研究するためのモデルを提供します。一貫して確実切歯からこれらの細胞を取得し、in vitroでそれらを拡張するための堅牢なシステムをここで報告されている。 

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

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

マウスの眼から網膜幹細胞の単離

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

生物学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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 ...

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, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

分離と性質、大人マウス心臓から心臓の幹細胞の分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

心房筋細胞、心室筋細胞、および非筋細胞の同時分離と培養

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

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 ...