This research was supported in part by Program for Creating Start-ups from Advanced Research and Technology (START Program) from the Japan Society for the Promotion of Science (ST261006IP, TM) and by Program for the Strategic Research Foundation at Private Universities (2014-2019) (S1411018, TM) from the Ministry of Education, Sports, Science and Technology.

人間の皮下脂肪のサンプルは、形成外科、泌尿器科、小児外科と日本大学板橋病院（東京、日本）の整形外科の部門で手術を受ける患者から得ました。患者は書面によるインフォームドコンセントを与えた、と医学の倫理委員会日本大学の学校は研究を承認しました。 1.組織標本<ol><li>研究室に手術室からの組織サンプルを持参してください。 </li><li>一旦室温（RT）で、50mlチューブ中で、次いで、10cmのプラスチック皿に試料を置く（ - ）洗浄1-2 PBS 5 mlの脂肪組織のG。 </li></ol> 2.コラゲナーゼ消化<ol><li>組織サンプルを含む皿に（w / v）のコラゲナーゼ溶液（コラゲナーゼII型）の無菌の0.1％の10ミリリットルを追加し、 - （）PBSからサンプルを削除します。 </li><li>それは構成さピューレに到達するまで、約15分間、外科用ハサミで組織をミンチency。 </li><li>続いて、シェーカー上で穏やかに撹拌しながら50mlのチューブ中で30分間（60 rpm）で37℃で0.1％（w / v）のコラゲナーゼ溶液中で細分化した組織を消化します。 </li><li> 50ミリリットルチューブに100μmのフィルターを通して消化されたサンプルをフィルタリングします。次に、フィルターを介して、2％FBSを補充した（ 材料の表を参照）、DCMの10ミリリットルを渡します。 </li></ol> 3.細胞の単離<ol><li>室温で1分間、100×gでろ過し、細胞を遠心。 </li><li>この時点で、DCMの調製5mlを50mlチューブ中の2％FBSを補充しました。 </li><li>次に、千μlのピペットを用いて、浮遊脂肪細胞を策定し、培地を含む50 mlチューブに細胞を追加します。 </li><li>その後、細胞をDCM媒体を混合するために穏やかに50ミリリットルチューブを振ります。 </li><li>その後、室温で1分間、100×gでチューブを遠心します。 </li><li> 18 G針と20ミリリットルの注射器を使用して、チューブの底に培地を捨てます。 </li><li>次に、DCM秒の10ミリリットルを追加します。採取した細胞を、2％FBSをupplemented。 </li><li>穏やかに50ミリリットルチューブを振とうすることにより細胞を用いて、DCMを混ぜます。 </li><li>室温で1分間、100×gでチューブを遠心。 </li></ol> 4.めっきセル<ol><li> 12.5センチメートルフラスコに20％FBSを補充DCMまたはDMEMの41ミリリットルを追加します。 </li><li>次に、200μlのピペットを用いて、フラスコに脂肪細胞の40μlを添加します。 </li><li>その後、DCMまたはDMEMでフラスコを埋めるには、20％FBSを補充しました。重要なステップ：この時点で、細胞を培地全体に広がっていることを確認してください。視覚的に剥離した細胞を確認し、その後フラットそれを維持するラウンドペトリ皿の蓋の上にフラスコを保ちます。 </li><li>最後に、インキュベータの内側にフラスコを置き、7日間37℃、5％CO 2インキュベーター内で邪魔されずにそのままにしておきます。 </li><li>インキュベーションの週に続いて、フラスコを反転し、DFAT細胞を確認してください。この点fibroblで以来、10を説明するよう次に、DFAT細胞の生成能力を評価しますAST様細胞（DFAT細胞）は、脂肪細胞とは異なる領域に配置されています。 </li><li> DCMまたはDMEMを5mlを加える前に細胞培養培地を除去した後、フラスコに、20％FBSを補充しました。 DFAT細胞は、培地を交換した後、別の週のために成長することを可能にします。 </li><li>細胞カウンターを用いて、種々の細胞培養条件（DMEM対DCM）で生成DFAT細胞の数を数えます。 </li></ol>

<ol>
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The authors have nothing to disclose.

インビトロ脱分化に天井文化として知られているプロセスを経る成熟脂肪細胞は、より原始的な表現型に復帰し、増殖能力を得てもよいです。これらの細胞は、以下のように脱分化脂肪（DFAT）細胞と呼ばれます。 DFAT細胞の多分化能を評価しました。分析および遺伝子発現のフローサイトメトリー分析DFAT細胞のASC 6と比較して非常に均質であったことを明らかにしました。実際には、DFAT細胞の細胞表面抗原プロファイルは、ASCを6に見られるものと非常に類似しています。従って、DFAT細胞は、MSCと同様に機能することができます。 正常の20％ウシ血清7-8を添加したDMEM F12細胞培養培地を利用DFAT細胞を生成した他の基。我々は、10％FBSを補充した細胞培養培地を用いてDFAT細胞を産生することを試みているが、このプロトコルを用いて製造DFAT細胞の数は、Wよりも有意に低かったです鶏は、20％FBS（データは示していない）を補充しました。従って、FBS濃度および/またはその内容はDFAT細胞の脱分化及び/又はDFAT細胞の増殖のいずれかのために重要です。今後の研究の一環として、我々はさらに、血清コンテンツはDFAT細胞の生成および増殖を支配するメカニズムを解明しようとします。一方、DCMは、私たちの研究で使用したDMEM未満グルコース、脂肪酸および他の化学物質が含まれています。細胞培養におけるこの変動は、培地成分はDFAT細胞の発生と増殖の両方の効率を調節することができます。実際には、低グルコース濃度がわずかにDMEMを用いDFAT細胞の生成効率を向上させる（データは示さず）。 DFAT細胞の生成と成長にDCMの効果の基礎となる正確なメカニズムは不明であるが、我々は特定の培地成分の濃度は、これらの細胞事象を駆動することをここに仮定する。さらなる研究は、この点を明確にするために必要とされています。さらに、我々は限られをテストしているので、DMEM製剤の次元数は、さらに実験をDFAT細胞を産生する他のDMEM変動（ 例えば 、DMEMを含有するL-グルタミンまたはピリドキシン）の効率を比較するために必要とされます。 我々はDFAT細胞を産生することができるが、DFAT細胞の生成を支配する分子メカニズムはまだ不明です。同様の質問がiPS細胞11の産生を囲みます。これらの治療法のような臨床使用は、細胞の状態調節機構のさらなる調査が必要になります。したがって、我々は強くDFAT細胞が外部遺伝子形質導入の自由、細胞は幹細胞様の状態に戻す方法を解明するためのモデルを提供すると信じています。 DFAT細胞産生プロトコールは、従って、将来の臨床試験での使用のために現在に最適化されています。 コラゲナーゼ処理し、脂肪細胞の単離は、両方適正に行われることを確保することが正常にこのプロトコルを使用してDFAT細胞を生成する際に重要な要因です。 APPR培養中の微細なハサミや気泡の除去opriate使用もDFAT細胞増殖を増強するのに役立ちます。

細胞治療および組織工学、再生医療1-5の分野におけるホットなトピックです。これらの治療法は偉大な約束を保持している間、いくつかの技術的な問題は、現在、その臨床使用を妨げます。この点において、iPS細胞の生成のように、すべての組織工学的療法は、患者の安全性を維持するために、外部遺伝子形質導入を含まない細胞を産生しなければなりません。したがって、我々が正常に人間DFAT細胞6を生成するために、最初のグループでした。いくつかの他の研究グループがいるので、さらに我々のモデルの有用性を強調し、哺乳動物起源7-9のDFAT細胞を生成するために私たちの方法を採用しています。 いくつかの研究の過程で、我々は、細胞培養環境の質は、細胞培地の含有量を調整することによって修正することができることを見出しました。この知見は、DFAT細胞の産生および改善された細胞質の成功率の増加につながっています。両方の重要な要素で効率的に将来の臨床試験のための細胞を生成します。この点に関して、改善されたDFAT培地中で（DCM、組換えヒトインスリンを含有する細胞培地、血清アルブミン、L-グルタミン酸、いくつかの脂肪酸、およびコレステロール幹間葉する媒体と同様）とDFAT細胞の生成のための方法および増殖は、（DCMの内容の詳細は、リクエストに応じて入手可能です）を開発しました。使用して、この方法の高品質DFAT細胞が脂肪生成、骨形成、および軟骨細胞を含むいくつかの細胞型に分化する能力を生成しました。要するに、この検証済みの細胞培養プロトコルはDFAT細胞の質を高め、細胞治療および組織工学の臨床応用性を高めるために非常に有用であり得ます。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">CSTI303-MSC medium&#160;</td>
			<td style="width:143px;">CSTI</td>
			<td style="width:155px;">87-671</td>
			<td style="width:167px;">This medium is defined as DCM in the text</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:243px;">PBS(-)</td>
			<td style="width:143px;">Wako</td>
			<td style="width:155px;">166-23555</td>
			<td style="width:167px;">It does not contain Mg2+ and Ca2+</td>
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			<td height="21" style="height:21px;width:243px;">DMEM medium</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:155px;">11965-092</td>
			<td></td>
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			<td style="width:143px;">Sigma</td>
			<td style="width:155px;">172012</td>
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			<td height="42" style="height:42px;width:243px;">Falcon Cell Strainer 100um Yellow</td>
			<td style="width:143px;">CORNING LIFE SCIENCES&#160;</td>
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			<td height="63" style="height:63px;width:243px;">Falcon 12.5cm&#178; Rectangular Canted Neck Cell Culture Flask with Blue Vented Screw Cap</td>
			<td style="width:143px;">CORNING LIFE SCIENCES&#160;</td>
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			<td height="21" style="height:21px;width:243px;">18G needle</td>
			<td style="width:143px;">NIPRO</td>
			<td style="width:155px;">02-002</td>
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an efficient method to obtain dedifferentiated fat cells

組織工学や細胞治療は、臨床的に偉大な約束を保持します。この点に関して、このような間葉系幹細胞（MSC）のような多能性細胞は、損傷した器官に機能を回復するために、近い将来に、治療的に使用することができます。それにもかかわらず、MSCおよびそれらの増幅を囲む非効率を単離する高度に侵襲的な手順を含むいくつかの技術的な問題は、現在、これらの治療法の潜在的な臨床使用を妨げます。ここで、我々は、脱分化脂肪細胞（DFAT）、MSC様細胞の生成のための非常に効率的な方法をご紹介します。興味深いことに、DFAT細胞が脂肪生成、骨形成、および軟骨細胞を含むいくつかの細胞型に分化することができます。他のグループが以前に成熟した脂肪組織からDFAT細胞を生成するための様々な方法を提示しているが、我々の方法は、私たちはより効率的にDFAT細胞を産生することができます。この点において、我々は、20％FBSを補充したものDFAT培地（DCM）を実証します20％FBSを補充したDMEMよりDFAT細胞を、生成においてより効果的です。さらに、我々の細胞培養法で製造DFAT細胞は、いくつかの組織型に再分化することができます。このように、組織の脱分化の研究のために非常に興味深く、有用なモデルが提示されています。

<html>本研究では、DFAT細胞の生成のための方法およびツールキットは、（ 図1）に向上しました。我々の方法は、私たちは、DCMおよび20％FBS（ 図2A）を含むDMEM培地の両方を使用してDFAT細胞を生成することができます。このように、我々はDFAT細胞を生成するDCMとDMEMの効率を比較しました。この点で、DCMにかかわらず、脂肪細胞の数（ 図2AおよびB）を、DMEMに比べ3倍DFAT細胞増殖を増強しました。 10％FBS（データは示さず）を使用したプロトコルと比較した場合20％を用いてFBSは、細胞増殖を増強します。我々は、さらにDCMを用いて製造DFAT細胞が脂肪細胞への再分化および骨芽細胞（ 図3）へと分化する能力を有することが確認されました。アッセイのための方法は、我々の以前の研究6に記載されています。 7 / 54177fig1.jpg &quot;/&gt; 図1： 分離を示す概略、脱分化、および成熟脂肪細胞の培養物は、脂肪組織の小片を、0.1％コラゲナーゼで消化し ​​ました。遠心分離後、単房脂肪細胞浮遊層から単離しました。単離された細胞は、7日間、20％FBSを含む培地で満たされたフラスコ中で培養しました。 DFAT細胞に変換した後、培養の間、細胞が付着し、フラスコの上面に平坦化。次に、フラスコを反転させ、DFAT細胞は、従来の方法を用いて培養した。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54177/54177fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54177/54177fig2.jpg" /> 図2：DCM はDFAT細胞の生成を増強します。 （ <strong&gt; A）DMEMまたはDCM中での培養の14日後DFAT細胞コロニー。スケールバー= 200μmの（B）DMEMまたはDCM（平均±SD、n = 3）でのいずれかでDFAT細胞増殖の定量化。 DFAT細胞を生成する際にDCMとDMEMの効率を比較しました。増強細胞増殖をDCMを用いて観察されます。私たちは、製造業者の指示に従って、セルカウンタを使用して、DFAT細胞の増殖を定量化した。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54177/54177fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/54177/54177fig3.jpg" /> 図 3：DCMを使用して生成DFAT細胞の多電位ヒトDFAT細胞はDFAT細胞の再分化および分化能力を評価するための適切な誘導培地中で3週間培養しました。 C言語ellsは、骨形成（ALPおよびアリザリンレッドS）、脂肪生成（オイルレッドO）能力について分析した。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54177/54177fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about An Efficient Method to Obtain Dedifferentiated Fat Cells at JoVE.com

An Efficient Method to Obtain Dedifferentiated Fat Cells

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

脱分化脂肪細胞を得るために効率的な方法

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

an-efficient-method-to-obtain-dedifferentiated-fat-cells

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.9K ビュー.  Nihon University. We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

ビデオ: An Efficient Method to Obtain Dedifferentiated Fat Cells

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

幹細胞から網膜色素上皮細胞の効率的な導出

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

発生生物学

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

酵素法は、凝固した骨髄試料からの間葉系幹細胞を救出する

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

ヒト胚性幹細胞から生成された内胚葉細胞の精製のための迅速かつ効率的な方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

大人の皮膚線維芽細胞からのインスリン分泌細胞を得るために安全かつ簡便な方法として、エピジェネティック変換

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

脂肪デポ固有のSca1の免疫磁気分離<sup&gt;高</sup&gt;脂肪由来幹細胞（ASCに）

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

NKX6-1への多能性幹細胞の効率的分化<sup&gt; +</sup&gt;膵臓前駆細胞

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

血管内皮幹細胞の血管形成を視覚化するための新規乳腺脂肪移植技術

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

Immunodetections または PCR の試金に適したショウジョウバエの蛹の翼を解剖する迅速かつ効率的な方法

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

核の分布、蛋白質および細胞骨格を研究する線虫生殖細胞の解析

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

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 ...