著者が明らかに何もありません。

<ol>
	<li>Maruyama, T., Yoshimura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+mechanisms+for+differentiation+and+regeneration+of+the+uterine+endometrium.">Molecular and cellular mechanisms for differentiation and regeneration of the uterine endometrium.</a> Endocrine Journal. 55, 795-810 (2008).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+Stromal+Decidualization+Responds+Reversibly+to+Hormone+Stimulation+and+Withdrawal.">Endometrial Stromal Decidualization Responds Reversibly to Hormone Stimulation and Withdrawal.</a> Endocrinology. 157, 2432-2446 (2016).</li><li>Ahn, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cAMP-Response+Element-Binding+3-Like+Protein+1+(CREB3L1)+is+Required+for+Decidualization+and+its+Expression+is+Decreased+in+Women+with+Endometriosis.">cAMP-Response Element-Binding 3-Like Protein 1 (CREB3L1) is Required for Decidualization and its Expression is Decreased in Women with Endometriosis.</a> Current Molecular Medicine. 16, 276-287 (2016).</li><li>Gellersen, B., Brosens, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+decidualization+of+the+human+endometrium+in+reproductive+health+and+failure.">Cyclic decidualization of the human endometrium in reproductive health and failure.</a> Endocrine Reviews. 35, 851-905 (2014).</li><li>Telgmann, R., Gellersen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marker+genes+of+decidualization:+activation+of+the+decidual+prolactin+gene.">Marker genes of decidualization: activation of the decidual prolactin gene.</a> Human Reproduction Update. 4, 472-479 (1998).</li><li>Camden, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+regulation+by+estrogen+in+breast+cancer+1+(GREB1)+is+a+novel+progesterone-responsive+gene+required+for+human+endometrial+stromal+decidualization.">Growth regulation by estrogen in breast cancer 1 (GREB1) is a novel progesterone-responsive gene required for human endometrial stromal decidualization.</a> Molecular Human Reproduction. 23, 646-653 (2017).</li><li>Kommagani, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Promyelocytic+Leukemia+Zinc+Finger+Transcription+Factor+Is+Critical+for+Human+Endometrial+Stromal+Cell+Decidualization.">The Promyelocytic Leukemia Zinc Finger Transcription Factor Is Critical for Human Endometrial Stromal Cell Decidualization.</a> PLoS Genetics. 12, e1005937 (2016).</li><li>Large, M. J., DeMayo, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+embryo+implantation+and+endometrial+decidualization+by+progesterone+receptor+signaling.">The regulation of embryo implantation and endometrial decidualization by progesterone receptor signaling.</a> Molecular and Cellular Endocrinology. 358, 155-165 (2012).</li><li>Zhang, X. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mesenchymal-epithelial+transition+during+in+vitro+decidualization.">The mesenchymal-epithelial transition during in vitro decidualization.</a> Reproductive Sciences. 20, 354-360 (2013).</li><li>Kommagani, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+the+glycolytic+flux+by+steroid+receptor+coactivator-2+is+essential+for+endometrial+decidualization.">Acceleration of the glycolytic flux by steroid receptor coactivator-2 is essential for endometrial decidualization.</a> PLoS Genetics. 9, e1003900 (2013).</li><li>Ramathal, C. Y., Bagchi, I. C., Taylor, R. N., Bagchi, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+decidualization:+of+mice+and+men.">Endometrial decidualization: of mice and men.</a> Seminars in Reproductive Medicine. 28, 17-26 (2010).</li><li>Tamura, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Function+of+a+Transcription+Factor+WT1+in+Regulating+Decidualization+in+Human+Endometrial+Stromal+Cells+and+Its+Molecular+Mechanism.">Novel Function of a Transcription Factor WT1 in Regulating Decidualization in Human Endometrial Stromal Cells and Its Molecular Mechanism.</a> Endocrinology. 158, 3696-3707 (2017).</li><li>Vinketova, K., Mourdjeva, M., Oreshkova, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Decidual+Stromal+Cells+as+a+Component+of+the+Implantation+Niche+and+a+Modulator+of+Maternal+Immunity.">Human Decidual Stromal Cells as a Component of the Implantation Niche and a Modulator of Maternal Immunity.</a> Journal of Pregnancy. , 8689436 (2016).</li><li>Wu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+endometritis+modifies+decidualization+in+human+endometrial+stromal+cells.">Chronic endometritis modifies decidualization in human endometrial stromal cells.</a> Reproductive Biology and Endocrinology. 15, 16 (2017).</li><li>Garcia-Elias, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+quantification+methods+and+optimizing+protocols+for+microarray+hybridization+of+circulating+microRNAs.">Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs.</a> Scientific Reports. 7, 7725 (2017).</li><li>Brosens, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+endometrial+fibroblasts+immortalized+by+simian+virus+40+large+T+antigen+differentiate+in+response+to+a+decidualization+stimulus.">Human endometrial fibroblasts immortalized by simian virus 40 large T antigen differentiate in response to a decidualization stimulus.</a> Endocrinology. 137, 2225-2231 (1996).</li><li>Koukouritaki, S. B., Margioris, A. N., Gravanis, A., Hartig, R., Stournaras, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dexamethasone+induces+rapid+actin+assembly+in+human+endometrial+cells+without+affecting+its+synthesis.">Dexamethasone induces rapid actin assembly in human endometrial cells without affecting its synthesis.</a> Journal of Cellular Biochemistry. 65, 492-500 (1997).</li></ol>

この仕事はから国民衛生の健康 (NIH) の資金でサポートされている/児童保健および人間の開発 (NICHD) 助成金 (R00 HD080742) ワシントン州大学医科大学院スタートアップ研究資金 R.K.子宮内膜生検サンプルを提供するため、ワシントン大学の不妊クリニックに感謝したいと思います。

女性の月経周期は、それによって分泌細胞が上皮のようにラウンド、3、8に ESC のエストロージェンを誘導中黄体期のプロゲステロンのレベルの上昇によって特徴付けられます。エストロージェンの開始は、依存する生物です。ヒトでは、エストロージェンは、自発的にプロゲステロン濃度の上昇マウス胚盤胞の存在10,<sup ...

生殖年齢の女性がホルモンによって調節される子宮内膜の増殖・分化・月経1 として知られているプロセスで妊娠の準備のためにその後放出の毎月のサイクルを受ける初潮の段階と月経閉止期の間 ,2。ひと子宮内膜のような物理的な変更が必要1子宮壁に適切な胚移植です。形態学的および生化学的適応を含む子宮内膜の変化は卵巣性ステロイド ホルモンのエストロゲンとプロゲステロン (P4) を介して月経サイクルを通して仲介される3,4,5。増殖 (または卵胞) フェーズの中で排卵エストロゲン レベルが高く、子宮内膜肥厚を開始します。次の排卵、分泌 (または黄体) 相は丸め、上皮のような脱落膜細胞線維芽細胞のような外観から子宮内膜間質細胞 (ESC) の形態学的変化を誘導する P4 濃度の大幅な上昇を促進します。エストロージェン4,6として知られているプロセス。注入の失敗とその後初期流産胚4,7,8根本原因として不適切なエストロージェンを設置します。したがって、エストロージェン分子機構の解明、診断と妊娠初期の損失の治療に有利であります。
現在、子宮内膜間質細胞のエストロージェンの根本的な影響を探検するいくつかの手法が利用されています。生体内で、マウス子宮機械的刺激 (すなわち、, 傷) を介してエストロージェンの人工的に誘導することができますまたは油ホルモン プライミング子宮9で注入。人間とは異なる、この合成の刺激は、胚盤胞の存在、齧歯動物の10、11エストロージェンの開始のために必要なステップの外観を提供することで子宮内腔の分化を促進します。したがって、並進合併症の動物モデルと人間の生体内で調査を囲む倫理的なジレンマに関連するのためエストロージェン ベース モデルはほとんどが正常に研究体外。
本研究では被験者は両方のローカル英語とスペイン語の新聞広告の配置によって募集しています。この研究に適した候補として識別される科目は、研究コーディネーター、潜在的なリスクの完全な情報開示の説明に会うため持って来られます。潜在的なリスクの完全な理解の確認後、被験者の同意は書面と口頭の両方の形態で得られます。被写体の同意には、(1) 今後の研究目的のための組織の瀉血 (2) 長期的なストレージを受けるし、(3) 収集した組織標本から初代培養の作成に同意する権限が含まれています。同意、次科目は、人種/民族および/または機密保持のための権利を許可された自己で完了するフォームを与えられています。その後の訪問は、サブジェクトの月経周期に基づく子宮内膜生検を達成する予定です。この研究に募ったボランティア 2012年国勢調査によって文書化されたセントルイス都市圏の民族、人種的な人口を反映し、妊娠中の女性、胎児、胚を含む任意の脆弱な人口の参加を伴わない18 歳未満の年齢、または他の弱者の子供。生検サンプル コレクションに参加するための資格要件は、(1) 歳の 18-45 年 (2) (3) を持たない現在の妊娠や避妊薬ホルモン/子宮内のデバイスの使用規則的な月経周期 (25-32 日) を持つが現在膣感染症や性感染症 (5) ない現在の抗生物質治療と (6) を持たない登録 (4) 前 30 日間ないこと現在の異常な Pap 汚れ。
この研究では、ひと子宮内膜間質細胞 (HESC) を培養し、人工的に誘導した体外ホルモン (エストラジ オール (E2)、酢酸メドロキシプロゲステロン (MPA)、および環状アデノシンのサプリメントからエストロージェンを受けることに一リン酸塩 (キャンプ)) 媒体に。このメソッドは、エストロージェンの程度は、ホルモン治療日数の合計数に基づいて変更されます。細胞骨格再配置に伴い、ホルモン補充は脱落膜細胞が分泌のような資質2,4を発生生化学的適応を誘発します。プロラクチン (PRL) とインスリン様成長因子結合蛋白質 1 (IGFBP1) などの認刻極印の遺伝子の表現を活用して、確認、HESC エストロージェン5,12,の程度を定量化すること13,14します。 重要なことは、遺伝子の特定の打撃を行うこのプロトコルの生存性も実証されています。

<table><tbody><tr><td>Opti-MEM I (1X) Reduced Serum Medium</td><td>Gibco</td><td>31985-070</td><td>For decidualization media</td></tr><tr><td>DMEM / F12 (1:1) (1X)</td><td>Gibco</td><td>11330-032</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Gibco</td><td>15250-061</td><td>For cell count</td></tr><tr><td>PureLink RNA Mini Kit</td><td>Invitrogen</td><td>12183018A</td><td>For RNA Isolation/Purification</td></tr><tr><td>TaqMan 2X Universal PCR Master Mix</td><td>Applied Biosystems</td><td>4304437</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit</td><td>Applied Biosystems</td><td>4374967</td><td></td></tr><tr><td>Eukaryotic 18S rRNA Endogenous Control</td><td>Life Technologies</td><td>4319413E</td><td>For qPCR internal control</td></tr><tr><td>TaqMan Gene Expression Assay Prolactin Probe</td><td>Applied Biosystems</td><td>4331182</td><td>For qPCR internal control</td></tr><tr><td>TaqMan Gene Expression Assay IGFBP1 Probe</td><td>Applied Biosystems</td><td>4331182</td><td>For qPCR internal control</td></tr><tr><td>Phalloidin-iFluor 488 Reagent - CytoPainter</td><td>Abcam</td><td>ab176753</td><td></td></tr><tr><td>Human Prolactin ELISA Kit</td><td>Invitrogen</td><td>EHIAPRL</td><td></td></tr><tr><td>16% Paraformaldehyde</td><td>Alfa Aesar</td><td>30525-89-4</td><td>Fixative</td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen</td><td>13778150</td><td>Transfection Reagent</td></tr><tr><td>ProLong Gold antifade reagent with DAPI</td><td>Invitrogen</td><td>P36935</td><td>For mounting</td></tr><tr><td>0.25% Trypsin-EDTA (1X)</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Charcoal Stripped Fetal Bovine Serum</td><td>Sigma</td><td>F6765-500ML</td><td></td></tr><tr><td>Sodium Bicarbonate (7.5%)</td><td>Gibco</td><td>25080-094</td><td></td></tr><tr><td>Ficoll-Paque PLUS Reagent</td><td>Fisher Scientific</td><td>45001749</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum</td><td>Sigma</td><td>C0130-1G</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma</td><td>DN25-100MG</td><td></td></tr><tr><td>Medroxyprogesterone 17-acetate</td><td>Sigma</td><td>M1629-1G</td><td>For EPC Media</td></tr><tr><td>Estradiol</td><td>Sigma</td><td>E1024-1G</td><td>For EPC Media</td></tr><tr><td>cAMP</td><td>Sigma</td><td>A6885-100MG</td><td>For EPC Media</td></tr><tr><td>Fine straight stitch scissors</td><td>Fine Science Tools</td><td>15396-00</td><td></td></tr><tr><td>TaqMan Gene Expression Assay NCOA2 Probe</td><td>Applied Biosystems</td><td>4351372</td><td></td></tr><tr><td>Fetal Bovine Serum, heat inactivated</td><td>Gibco</td><td>10-082-147</td><td></td></tr><tr><td>Antibiotic-antimycotic</td><td>Thermo Scientific</td><td>15240062</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution&amp;nbsp;</td><td>Corning</td><td>21-021-cv</td><td></td></tr><tr><td>50 mL conical polypropylene Falcon tube</td><td>Corning</td><td>352098</td><td></td></tr><tr><td>Dumont #5/45 Forceps</td><td>Fine Science Tools</td><td>11251-35</td><td></td></tr><tr><td>100 x 15 mm Glass Petri dish</td><td>VWR</td><td>75845-546</td><td></td></tr><tr><td>40 micron cell strainer</td><td>Midsci</td><td>229481</td><td></td></tr><tr><td>Isotemp 215 Water bath</td><td>Fisher Scientific</td><td>FS-215</td><td></td></tr><tr><td>LSE Centrifuge&amp;nbsp;</td><td>Corning</td><td>6755</td><td></td></tr><tr><td>Human NCOA2 siRNA&amp;nbsp;</td><td>Dharmacon</td><td>L-020159-00</td><td>Gene specific qPCR probe</td></tr><tr><td>Steri-cycle i160 CO2 Incubator&amp;nbsp;</td><td>Thermo Scientific</td><td>51030301</td><td></td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td>ND-2000</td><td>For RNA quantification</td></tr><tr><td>Phosphate Buffered Saline&amp;nbsp;</td><td>Fisher Scientific</td><td>50146771</td><td></td></tr><tr><td>75 cm Canted Neck Cell Culture flask</td><td>Corning</td><td>430641U</td><td></td></tr><tr><td>6 well Non-Pyrogenic Cell Culture Plate</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Pipet Controller Ultra</td><td>Corning</td><td>4099</td><td></td></tr><tr><td>Photoshop</td><td>Adobe</td><td>19.0.1.334</td><td></td></tr><tr><td>25 cm Canted Neck Cell Culture flask</td><td>Corning</td><td>7200876</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100-1L</td><td></td></tr><tr><td>15 mL conical polypropylene Falcon tube</td><td>Corning</td><td>352099</td><td></td></tr><tr><td>10 mL Syringe</td><td>BD</td><td>302995</td><td></td></tr><tr><td>1300 Series A2 Biological Safety Hood</td><td>Thermo Scientific</td><td>1377</td><td></td></tr><tr><td>accuspin Micro17 centrifuge</td><td>Fisher Scientific</td><td>13100675</td><td></td></tr><tr><td>7500 Fast real time PCR system</td><td>Applied Biosystems</td><td>3052632</td><td></td></tr><tr><td>EVOS FL Immunofluorescence Microscope</td><td>Life Technologies</td><td>01414-155G-291</td><td></td></tr><tr><td>Leica Inverted Light Microscope</td><td>Leica</td><td>DMi1</td><td></td></tr><tr><td>Illrustrator</td><td>Adobe</td><td>22.0.1.253</td><td></td></tr><tr><td>Excel Spreadsheets</td><td>Microsoft</td><td>2016</td><td></td></tr><tr><td>Deckglaser 18 mm cover glasses</td><td>NeuVitro</td><td>GG-18</td><td></td></tr><tr><td>Reichert Bright-Line Hemacytometer</td><td>Sigma</td><td>Z359629-1EA</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Fisher Bioreagents</td><td>BP176-100</td><td>For RNA lysis buffer</td></tr><tr><td>1.2mL External Threaded Polypropylene Cryogenic Vial</td><td>Corning</td><td>430658</td><td>For freezing HESC cells&amp;nbsp;</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma</td><td>D2650-100mL</td><td>For freezing media</td></tr></tbody></table>

isolation of human endometrial stromal cells for in vitro decidualization

分泌脱落膜に線維芽細胞のような外観からひと子宮内膜間質細胞 (HESC) の分化は、母親の子宮の子宮内に胚移植に必要な変換です。着床障害とそれに続く初期胚流産の根本的な原因として不適切なエストロージェンを設置します。したがって、エストロージェンの分子機構を理解することは成功した出生率向上に有利であります。人工エストロージェンの生体内研究は人間研究として動物モデル内での並進運動合併症に関連付けられている倫理的なジレンマのためしばしば制限しています。その結果、一次電池文化を通しての in vitroアッセイがホルモンを介してエストロージェンの変調を探るにしばしば用いられます。この研究は、悪名高くの分離と培養の培地にホルモンの補充によるその後の人工エストロージェンの詳しいプロトコルを提供します。さらに、この研究は、siRNA の脂質ベースの transfections を利用してノックダウンするうまく設計された方法興味の任意の遺伝子を提供します。このプロトコルにより、文化純度としてエストロージェンとの後の定量化の基礎となる分子機構を理解するための信頼性の高い方法としてこのモデルを活用する能力を最大化する製品の歩留まりの最適化decidualized 子宮内膜間質細胞因子を分泌します。

HESC 文化エストロージェン
次の分離、ひと子宮内膜間質細胞は 80-90% の confluency による単分子膜形成に培養し、体外エストロージェンの 10 nM E2、1 μ M MPA、50 μ M のキャンプと治療による (EPC)。エストロージェン体外に関連付けられている形態学的変化は、図 1 aに, 可視化さ?...

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Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

本稿ではエストロージェンの in vitroアッセイを行うひと子宮内膜間質細胞の分離文化の検証と最適化されたプロシージャ。さらに、この研究では、ひと子宮内膜間質細胞で Sirna を使用して特定の遺伝子の効率的にノックダウンする詳細な方法が提供します。

エストロージェン体外用ひと子宮内膜間質細胞の分離

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

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Developmental Biology

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

10.1K ビュー.  Washington University School of Medicine. 本稿ではエストロージェンの in vitroアッセイを行うひと子宮内膜間質細胞の分離文化の検証と最適化されたプロシージャ。さらに、この研究では、ひと子宮内膜間質細胞で Sirna を使用して特定の遺伝子の効率的にノックダウンする詳細な方法が提供します。

ビデオ: Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

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.

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 ...

発生生物学

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 ...

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. ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

子宮摘出標本から初代ヒト子宮内膜間質細胞の確立するための2つの方法

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

医学

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

多細胞ヒト初等子宮内膜オルガノイドの生成

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of decidualization leads to various endometrial disorders, such as infertility, recurrent miscarriage, and preterm birth. The development and use of the endometrial decidualization model in reproductive studies have been a highlight for reproductive researchers for a long time. The mouse has been extensively used in studying reproduction and decidualization. There are three well-established mouse models regarding decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). Among them, AD is considered a reliable model for mouse decidualization, which is easy to implement and close to NPD. This paper focuses on a modified method of the generation and application process of the mouse artificial decidualization model with ovariectomy to avoid ovarian effects, which can obtain highly reproducible results with small within group variances. This method provides a good and reliable animal model for the study of endometrial decidualization.

Here, we describe the method of generating an artificial decidualization model using the ovariectomized mouse, a classic endometrial decidualization experiment in the research field of endometrial decidualization.

子宮内膜脱落膜化研究のための卵巣摘出術を用いたマウス人工脱落膜モデルの生成

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

用語胎盤の胎児膜から主のひと脱落膜細胞の分離

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Establishing 3D Endometrial Organoids from the Mouse Uterus

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of luminal and glandular epithelium, a stromal compartment, a vascular network, and a complex immune cell population. Mouse models have been a powerful tool to study the endometrium, revealing critical mechanisms that control implantation, placentation, and cancer. The recent development of 3D endometrial organoid cultures presents a state-of-the-art model to dissect the signaling pathways that underlie endometrial biology. Establishing endometrial organoids from genetically engineered mouse models, analyzing their transcriptomes, and visualizing their morphology at a single-cell resolution are crucial tools for the study of endometrial diseases. This paper outlines methods to establish 3D cultures of endometrial epithelium from mice and describes techniques to quantify gene expression and analyze the histology of the organoids. The goal is to provide a resource that can be used to establish, culture, and study the gene expression and morphological characteristics of endometrial epithelial organoids.

This protocol describes methodologies to establish mouse endometrial epithelial organoids for gene expression and histological analyses.

マウス子宮からの3D子宮内膜オルガノイドの確立

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of ...

生物学

<meta charset="utf8"></head><body>胚の着床メカニズムを調査するための3Dプリントされた足場モデル

Co-culture Model Using Two Types of Adherent Cell Lines

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.

<meta charset="utf8"></head><body>著者スポットライト:上皮-間質および胚の相互作用を研究するためのin vitro子宮内膜モデル

The protocol shows a novel in vitro experimental model that can recapitulate the biology of two kinds of adherent cell lines with a three-dimensional (3D)-printed scaffold. The construction of this model and operating procedures, from cell preparation and cell culture to analysis and evaluation, are described.

2種類の接着細胞株を用いた共培養モデル

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications ...