この研究は、MTLに健康研究助成R01-DE021683-01と健康研究助成金R01-DE019434の国立衛生研究所の国立研究所によってサポートされていました。 MTCDCWにハワード·ヒューズ医学研究所研究員は、ACSフランクリン·マーティン学部研究フェローシップ、小児再生医療のためのHagey研究所、スタンフォード大学小児保健研究所学部奨学生賞によってサポートされていました。

注：すべての患者の試料はインフォームドコンセントを得て、実験的なプロトコルは、スタンフォード大学の治験審査委員会（プロトコル＃2188と＃9999）によって審査され、承認された。 1.細胞の単離および培養： <ol><li>一般/局所麻酔下で腹部、脇腹、および/または大腿部領域の選択科目lipoaspirationを受けた健康女性患者からのヒト皮下脂肪組織を入手します。治験審査委員会（IRB）の承認は、人間の組織からのASCを単離するプロトコルのために得られていることを確認し、そのような材料での作業中に制度的安全上の注意に従ってください。 </li><li> lipoaspirated脂肪組織からSVFを得るために、最初の1倍の滅菌リン酸緩衝生理食塩水（PBS）等容量の脂肪吸引物を3回洗浄する。慎重に吸引し、底の水層を捨てる。 </li><li>私はハンクのコラゲナーゼ0.075％の種類：コラゲナーゼ消化緩衝液を準備平衡塩類溶液（HBSS）。 FACS緩衝液を準備します。PBS中の2％FBS、1％のP188と1％のペニシリン - ストレプトマイシンを。市販の0.22μmの細孔サイズ速い流れのポリエーテルスルホンフィルターを使用して両方のソリューションをフィルタリングします。 </li><li>洗浄した脂肪組織を消化するために、37°C​​（約180シェイク/分）で60分間、振盪水浴中でしっかりと消化槽をコラゲナーゼ消化緩衝液の等量を追加して配置する。 注：これは、必要とされるよりも、これは（ すなわち 、コラゲナーゼ消化緩衝液の250ミリリットル、1リットルの滅菌フラスコ内の洗浄した脂肪組織の250ミリリットル）しながら中に最大の消化を可能にするので、より大きな容積消化容器を使用するのが最適です。 </li><li> FACS緩衝液の等量を添加し、室温で5分間静置させることによって酵素活性を中和する。次に、4℃で20分間233×gで遠心。 </li><li>慎重に吸引し、高密度SVFペレットを乱さないように注意しながら、上清を捨てる。 </li><li> RT赤5〜10mlの中でペレットを再懸濁ペレットサイズに基づいて、細胞溶解緩衝液。 RTで5分間233×gで5分間遠心分離を室温で座って解決のままにしておきます。 </li><li>上清を吸引し、ペレットの大きさに基づいて、伝統的な増殖培地5〜10mlの添加することによりペレットを再懸濁（ダルベッコ改変イーグル培地[DMEM] / 10％FBS / 1％ペニシリン - ストレプトマイシン溶液[ペンストレプトマイシン]）。 </li><li>細胞破片を除去するためには100μmナイロン細胞ストレーナーを通してサスペンションをフィルタリングします。 </li><li> 4℃で5分間233×gで遠心分離し、ペレットを中断することなく、上清を捨てる。 </li><li>ペレットサイズに基づいて、伝統的な増殖培地5-10中にペレットを再懸濁。 </li><li> 15センチメートル標準培養皿に2×10 6個の細胞を配置し、O / N 37°C / 21％O 2、5％CO 2で初代培養を確立する。増殖培地中で37℃/ 21％O 2、5％CO 2での自発的な分化を防止するために、サブコンフルエントなレベルに維持する。 注：完全に中の細胞密度コンフルエント15cmのプレートは、約4-6×10 6細胞である。 </li></ol> 2.染色<ol><li>ソート/染色前36時間、37℃/ 21％O 2、5％CO 2、伝統的な増殖培地中での培養のASC。 </li><li> （製造業者のプロトコルに従って）のAccutaseを用いて培養プレートからリフトのASC。 </li><li>一度（2％FBSおよび1％のペン - ストレプトマイシンを含むPBS）をFACS緩衝液で細胞を洗浄する。 </li><li> 4℃で5分間233×gで遠心し。 </li><li>ペレットを中断することなく、上清を捨てる。 FACS緩衝液0.5〜1 mlの細胞を再懸濁し（細胞数および所望の抗体の量に依存する）。 注：細胞懸濁液の体積は、FACS抗体製造業者の推奨に従って、抗体濃度を決定する。しかし、1の開始濃度を用いて抗体濃度の滴定、：それは抗体を使用する最初の時間であれば100は、通常は推奨されている。 </li><li>の合計数血球計数器を用いて細胞。 </li><li>未染色試料および単色のコントロールのために使用されて1.5 ml遠心管中で予備100μlのアリコート（使用する蛍光抗体の数に応じて）。 </li><li> （製造者の指示に従って、所定の濃度で）適切な蛍光標識されたモノクローナル抗体を追加し、遮光し氷上で20〜30分、インキュベートする。 <ol><li>骨形成亜集団にラベルを付けるには：1：50の濃度になるようにFACS緩衝液中の抗CD90（APC抗ヒトCD90（はThy1））または抗CD105（FITC抗ヒトCD105（エンドグリン））を希釈します。 </li><li>他の細胞集団（ 例えば 、造血および内皮細胞）を標識する：希釈した抗CD45（抗ヒトCD45パシフィックブルー）、抗CD34（抗ヒトCD34 APC）、および/ ​​または抗CD31（抗ヒトCD31 PE ）FACSで1時50分の濃度にバッファー。 </li><li>直接結合した抗体を使用していない場合は、適切なストレプトアビジン結合第二に、ビオチン化一次抗体を使用する100：そのような1の希釈でストレプトアビジンPE-Cy7は、などの進の抗体。 </li></ol></li><li>インキュベーション後、細胞を2回洗浄する。上清を吸引し、4℃で5分間、233×gでFACS緩衝液および遠心機でチューブを埋める。 </li><li>細胞塊を除去するために70μmのナイロン細胞ストレーナーを通して400〜500μlのFACSバッファおよびフィルタサンプル中の細胞を再懸濁。 </li><li>フィルタートップでFACSチューブに標識細胞を移し、分析の間、サンプルを氷上で維持する。 </li></ol> 3.蛍光活性化細胞ソーティング注：以下の手順では、蛍光活性化細胞選別（FACS）または熟練した技術者の支援に予備知識を義務付ける。 <ol><li>前方散乱（FSC）、側方散乱（SSC）を調べるために、設計解析プロットを適切なFACSソフトウェアを使用して、フィコエリトリン（PE）-Texasレッド、パシフィックブルー、フルオレセインイソチオシアネート（FITC）、アロフィコシアニン（APC）および任意の他のフルオロフォアの使用dは細胞を標識する。 </li><li>セルソーター、簡単に渦に細胞を再懸濁するために、各サンプルをロードする前に。 </li><li>細胞破片を排除するために、ベースライン自己蛍光を決定するために、全細胞集団のためにゲートを設定するために、未染色試料の分析を開始する。 </li><li>未染色試料にヨウ化プロピジウム（PI）を追加し、全細胞集団内の生細胞の集団を可視化するためにそれを分析する。この集団の周りにゲートを構築します。 </li><li>蛍光補正を行うために、各蛍光色素のための単色対照試料を分析する。 </li><li>ソートにゲートの位置を設定するための染色サンプルを分析します。 </li><li>死細胞を染色するために、染色されたサンプルにPIを追加します。 </li><li>標識された細胞集団をソートします。ソート培地を含む1.5 mlの遠心チューブまたは円錐管（15ml）中のどちらかに変換する。 </li><li>手動での細胞の必要量が得られた後に選別止める。 </li><li>私達取得された細胞集団のFACS、ごく一部分析することによって、高純度チェックを行う（ 例えば 、500細胞）る。 注：この手順はソートされた細胞集団の純度を確認した。理想的には、純度が90〜95％より大きくなければならない。 </li><li> 10％FBSを含む新鮮な培地1mlにソートし、再懸濁の4°Cの直後の終了で5分間233×gで遠心分離細胞。 注：使用される媒体の量は、細胞ペレットのサイズに基づいて増加させることができる。 </li><li>ゼラチン被覆プレート上のプレートは、細胞生存率を改善する。最終的な細胞収量に応じて、6ウェルプレート中の12ウェルプレートでウェル当たり100,000細胞を、ウェル当たり300,000細胞をプレーティング。 </li></ol>

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著者のいずれも、この原稿に記載されている製品、デバイス、または薬物のいずれかに経済的利害関係を有していない。著者のいずれも、報告するためのあらゆる競合する経済的利害関係を有していない。

現在、ヒト脂肪組織のSVFからASCの均質な亜集団の単離は、望ましい目標ものの依然として厳しい。このような細胞は、骨格組織の形成および恒常性を研究するために使用することができるようにプロ骨形成ASC亜集団の単離は、特に望ましい。しかし、脂肪組織のSVFは、セル容量と分化能を食い止めるに関して有意な異質性を保有する。11この異質性の分子基盤は、細胞のプールされた...

The heterogeneous nature of stem cell populations is not yet fully understood and remains a major impediment to the development of clinically effective stem cell-based therapeutic applications. One of the most common ways to characterize a heterogeneous population of stem cells is to employ a cell sorting method, such as fluorescence-activated cell sorting (FACS), to separate cells based on their surface marker expression profiles. As sorting methods become more complex, it becomes possible to identify more distinct functional subpopulations of cells. Microfluidic-based technologies are becoming more and more frequently utilized in analysis of gene expression at the single cell level. Multiplexed quantitative polymerase chain reaction (qPCR) within a microfluidic chip allows for effective and reliable high-resolution, single cell transcriptional analysis.1-5
In a previous study using single cell transcriptional profiling of 48 genes, considerable transcriptional heterogeneity was observed among ASCs.6 However, the distribution of genes MSX2, BMP-5, BMP-7, ALP, OCN, RUNX2 exhibited a strong association with a cluster of cells possessing highly osteogenic transcriptional profiles. To isolate cells according to this osteogenic gene expression profile, surface antigen expression patterns were correlated with transcription patterns and surface marker expression of endoglin (CD105) was subsequently discovered to closely correlate with enhanced osteogenic differentiation potential of ASCs. Independent of CD105 expression, expression of surface receptor Thy-1 (CD90), a glycosyl-phosphatidylinositol-linked membrane protein previously shown by Chen et al. to be associated with osteoprogenitor cells, was also correlated with osteogenic gene expression.6,7 These findings provide the opportunity to prospectively isolate subpopulations within the larger heterogeneous pool of ASCs with increased osteogenic capacity for cell-based bone tissue engineering applications.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Name of Reagent/Material</td>
			<td style="width:152px;">Company</td>
			<td style="width:112px;">Catalog Number</td>
			<td style="width:79px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Disposable 250 mL Conical Tubes</td>
			<td style="width:152px;">Corning (Thomas Scientific)</td>
			<td style="width:112px;">2602A43</td>
			<td style="width:79px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:152px;">Gibco</td>
			<td style="width:112px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">DMEM, high glucose, GlutaMAX Supplement</td>
			<td style="width:152px;">Gibco</td>
			<td style="width:112px;">10566-016</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS, pH 7.4</td>
			<td style="width:152px;">Gibco</td>
			<td>10010-023</td>
			<td style="width:79px;"></td>
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			<td height="21" style="height:21px;">Betadine - Antiseptic Povidone/Iodine Solution</td>
			<td style="width:152px;">Purdue&#160;</td>
			<td>PFC-67618015017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#39;s Balanced Salt Solution, 1X</td>
			<td style="width:152px;">Cellgro</td>
			<td>21-023-CV</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum, Certified, US Origin</td>
			<td style="width:152px;">Gibco</td>
			<td>16000-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase from&#160;Clostridium histolyticum</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td>C0130-5G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ACCUTASE Cell Detachment Solution</td>
			<td style="width:152px;">Stem Cell Technologies</td>
			<td>7920</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">APC Mouse Anti-Human CD90</td>
			<td style="width:152px;">BD Pharmingen</td>
			<td style="width:112px;">559869</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">FITC Mouse anti-Human CD105 (Endoglin)</td>
			<td style="width:152px;">BD Pharmingen</td>
			<td style="width:112px;">561443</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Anti-Human CD45 eFluor 450 (Pacific Blue replacement)&#160;</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">48-9459-41</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Anti-Human CD34 APC</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">17-0349-41</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Anti-Human CD31 (PECAM-1) PE</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">12-0319-41</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Streptavidin PE-Cyanine7</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">25-4317-82</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">BD FACS Aria II instrument</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:112px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">BD FACSDiva Software</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:112px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

ヒトASC（ 図1A、1B）の高濃縮集団の単離に強化された骨形成の結果と細胞のマーカーとしてCD90を使用した。 ASCははパシフィックブルー結合抗ヒトCD45、FITC結合抗ヒトCD105、およびAPC結合抗ヒトCD90で染色した。ポストソート分析によって定量しソートした後、純度のレベルは、98％以上であった。 2つの新規な亜集団の将来の単離のために許可された転写プロ?...

Watch this Scientific Journal Video about Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis at JoVE.com

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

強化された骨形成のためのヒト脂肪由来間質細胞の単離および濃縮

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

isolation-enrichment-human-adipose-derived-stromal-cells-for-enhanced

Research

JoVE Journal

Developmental Biology

11.4K ビュー.  Stanford University School of Medicine. The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

ビデオ: Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

ブタ皮下脂肪組織から分離し、脂肪由来幹細胞の分化

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

発生生物学

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 ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

単離およびヒト胎盤組織由来の間葉系幹/間質細胞の増殖

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

遠心重力によって脂肪由来幹細胞の軟骨形成分化誘導

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

頭蓋冠欠損治癒モデルで使用するためのBMPR-IB +脂肪由来間質細胞の迅速な単離

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

マウス子宮内膜上皮と間質細胞の単離のために<em&gt;インビトロ</em&gt;脱落膜化

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

ヒト臍帯及び胎児胎盤由来間葉系間質細胞の単離および特徴付け

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

生体内でひと脂肪由来間葉系幹細胞が脂溶性膜の蛍光色素をラット膝関節変形性関節症モデルでの追跡

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

分離、カルチャ、および骨髄間質細胞とマウスから破骨細胞前駆細胞の分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

催不整脈性心筋症患者の心内膜心筋生検サンプルから心臓の間葉系間質細胞の分離と性状

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...