実りある議論のため管理者にラボと Thijmen ヴァン Vliet と紫外線による老化のプロトコルのデータを共有するためのメンバーに感謝いたします。

<ol>
	<li>Hayflick, L., Moorhead, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serial+cultivation+of+human+diploid+cell+strains.">The serial cultivation of human diploid cell strains.</a> Experimental Cell Research. 25, 585-621 (1961).</li><li>Mu&#241;oz-Esp&#237;n, D., Serrano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence:+from+physiology+to+pathology.+Nature+reviews.">Cellular senescence: from physiology to pathology. Nature reviews.</a> Molecular cell biology. 15, 482-496 (2014).</li><li>Sharpless, N. E., Sherr, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forging+a+signature+of+in+vivo+senescence.">Forging a signature of in vivo senescence.</a> Nature Reviews Cancer. 15 (7), 397-408 (2015).</li><li>Correia-Melo, C., 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=Mitochondria+are+required+for+pro-ageing+features+of+the+senescent+phenotype.">Mitochondria are required for pro-ageing features of the senescent phenotype.</a> The EMBO Journal. 10, e201592862 (2016).</li><li>Loaiza, N., Demaria, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence+and+tumor+promotion:+Is+aging+the+key?.">Cellular senescence and tumor promotion: Is aging the key?.</a> Biochimica et Biophysica Acta (BBA) - Reviews on Cancer. , (2016).</li><li>Coppe, J. P., 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=Senescence-associated+secretory+phenotypes+reveal+cell-nonautonomous+functions+of+oncogenic+RAS+and+the+p53+tumor+suppressor.">Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.</a> PLoS Biology. 6 (12), 2853-2868 (2008).</li><li>Baker, D. 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=Naturally+occurring+p16(Ink4a)-positive+cells+shorten+healthy+lifespan.">Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan.</a> Nature. 530 (7589), 184-189 (2016).</li><li>Xu, M., 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=Targeting+senescent+cells+enhances+adipogenesis+and+metabolic+function+in+old+age.">Targeting senescent cells enhances adipogenesis and metabolic function in old age.</a> Elife. 4, e12997 (2015).</li><li>Baker, D. 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=Clearance+of+p16Ink4a-positive+senescent+cells+delays+ageing-associated+disorders.">Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders.</a> Nature. 479 (7372), 232-236 (2011).</li><li>Jeon, O. 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=Local+clearance+of+senescent+cells+attenuates+the+development+of+post-traumatic+osteoarthritis+and+creates+a+pro-regenerative+environment.">Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment.</a> Nature Medicine. 23 (6), 775-781 (2017).</li><li>Demaria, M., 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=Cellular+Senescence+Promotes+Adverse+Effects+of+Chemotherapy+and+Cancer+Relapse.">Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse.</a> Cancer Discovery. 7 (2), 165-176 (2017).</li><li>Chang, 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=Clearance+of+senescent+cells+by+ABT263+rejuvenates+aged+hematopoietic+stem+cells+in+mice.">Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice.</a> Nature Medicine. 22 (1), 78-83 (2016).</li><li>Soto-Gamez, A., Demaria, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+interventions+for+aging:+the+case+of+cellular+senescence.">Therapeutic interventions for aging: the case of cellular senescence.</a> Drug Discov Today. 22 (5), 786-795 (2017).</li><li>Childs, B. G., 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=Senescent+cells:+an+emerging+target+for+diseases+of+ageing.">Senescent cells: an emerging target for diseases of ageing.</a> Nature Reviews Drug Discovery. 16 (10), 718-735 (2017).</li><li>Marthandan, S., 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=Conserved+genes+and+pathways+in+primary+human+fibroblast+strains+undergoing+replicative+and+radiation+induced+senescence.">Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence.</a> Biological Research. 49, 34 (2016).</li><li>Marthandan, S., 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=Conserved+Senescence+Associated+Genes+and+Pathways+in+Primary+Human+Fibroblasts+Detected+by+RNA-Seq.">Conserved Senescence Associated Genes and Pathways in Primary Human Fibroblasts Detected by RNA-Seq.</a> PLoS One. 11 (5), e0154531 (2016).</li><li>Hernandez-Segura, 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=Unmasking+Transcriptional+Heterogeneity+in+Senescent+Cells.">Unmasking Transcriptional Heterogeneity in Senescent Cells.</a> Current Biology. 27 (17), 2652-2660 (2017).</li><li>Le, O. N., 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=Ionizing+radiation-induced+long-term+expression+of+senescence+markers+in+mice+is+independent+of+p53+and+immune+status.">Ionizing radiation-induced long-term expression of senescence markers in mice is independent of p53 and immune status.</a> Aging Cell. 9 (3), 398-409 (2010).</li><li>Hall, J. 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=C/EBPalpha+regulates+CRL4(Cdt2)-mediated+degradation+of+p21+in+response+to+UVB-induced+DNA+damage+to+control+the+G1/S+checkpoint.">C/EBPalpha regulates CRL4(Cdt2)-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint.</a> Cell Cycle. 13 (22), 3602-3610 (2014).</li><li>Nitiss, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+DNA+topoisomerase+II+in+cancer+chemotherapy.">Targeting DNA topoisomerase II in cancer chemotherapy.</a> Nature Reviews Cancer. 9 (5), 338-350 (2009).</li><li>Pazolli, E., 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=Chromatin+remodeling+underlies+the+senescence-+associated+secretory+phenotype+of+tumor+stromal+fibroblasts+that+supports+cancer+progression.">Chromatin remodeling underlies the senescence- associated secretory phenotype of tumor stromal fibroblasts that supports cancer progression.</a> Cancer Research. 72, 2251-2261 (2012).</li><li>Venturelli, S., 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=Differential+induction+of+apoptosis+and+senescence+by+the+DNA+methyltransferase+inhibitors+5-azacytidine+and+5-aza-2'-deoxycytidine+in+solid+tumor+cells.">Differential induction of apoptosis and senescence by the DNA methyltransferase inhibitors 5-azacytidine and 5-aza-2'-deoxycytidine in solid tumor cells.</a> Molecular Cancer Therapeutics. 12, 2226-2236 (2013).</li><li>Tennant, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Trypan+Blue+Technique+for+Determination+of+Cell+Viability.">Evaluation of the Trypan Blue Technique for Determination of Cell Viability.</a> Transplantation. 2, 685-694 (1964).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Lee, B. Y., 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=Senescence-associated+&#946;-galactosidase+is+lysosomal+&#946;-galactosidase.">Senescence-associated &#946;-galactosidase is lysosomal &#946;-galactosidase.</a> Aging Cell. 5, 187-195 (2006).</li><li>Kopp, H. G., Hooper, A. T., Shmelkov, S. V., Rafii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-galactosidase+staining+on+bone+marrow.+The+osteoclast+pitfall.">Beta-galactosidase staining on bone marrow. The osteoclast pitfall.</a> Histology and Histopathology. 22 (9), 971-976 (2007).</li><li>Dimri, G. P., 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=A+biomarker+that+identifies+senescent+human+cells+in+culture+and+in+aging+skin+in+vivo.">A biomarker that identifies senescent human cells in culture and in aging skin in vivo.</a> Proceedings of the National Academy of Sciences. 92 (20), 9363-9367 (1995).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proceedings of the National Academy of Sciences. 105 (7), 2415-2420 (2008).</li><li>Sherr, C. J., McCormick, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RB+and+p53+pathways+in+cancer.">The RB and p53 pathways in cancer.</a> Cancer Cell. 2 (2), 103-112 (2002).</li><li>Bunz, F., 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=Requirement+for+p53+and+p21+to+sustain+G2+arrest+after+DNA+damage.">Requirement for p53 and p21 to sustain G2 arrest after DNA damage.</a> Science. 282 (5393), 1497-1501 (1998).</li><li>Severino, J., Allen, R. G., Balin, S., Balin, A., Cristofalo, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+beta-galactosidase+staining+a+marker+of+senescence+in+vitro+and+in+vivo.">Is beta-galactosidase staining a marker of senescence in vitro and in vivo.</a> Experimental Cell Research. 257 (1), 162-171 (2000).</li><li>Stolzing, A., Coleman, N., Scutt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-induced+replicative+senescence+in+mesenchymal+stem+cells.">Glucose-induced replicative senescence in mesenchymal stem cells.</a> Rejuvenation Research. 9 (1), 31-35 (2006).</li><li>Blazer, S., 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=High+glucose-induced+replicative+senescence:+point+of+no+return+and+effect+of+telomerase.">High glucose-induced replicative senescence: point of no return and effect of telomerase.</a> Biochemical and Biophysical Research Communications. 296 (1), 93-101 (2002).</li><li>Wiley, C. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MTOR+and+autophagy:+converging+on+senescence+or+quiescence.">MTOR and autophagy: converging on senescence or quiescence.</a> Autophagy. 9 (2), 260-262 (2013).</li><li>Meuter, 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=Markers+of+cellular+senescence+are+elevated+in+murine+blastocysts+cultured+in+vitro:+molecular+consequences+of+culture+in+atmospheric+oxygen.">Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen.</a> Journal of Assisted Reproduction and Genetics. 31 (10), 1259-1267 (2014).</li><li>Coppe, J. 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ここで説明したプロトコル最適化した一次線維芽細胞、特に BJ と WI-38 細胞。複製老化、電離放射線、ドキソルビシンのためのプロトコルは (HCA2 および IMR90) 線維芽細胞と他の細胞型 (すなわち新生児メラノサイトとケラチノ サイトまたは iPSC 由来心筋細胞) の他のタイプに正常に適用されています。本研究室では。ただし、その他の細胞のタイプのための適応は、シード細胞、メソッドおよ...

1961 年、ヘイフリック、ムーアヘッドは、文化の主要な線維芽細胞が連続通路1の後の増殖の可能性を失うことを報告します。このプロセスは、各細胞分裂後シーケンシャル テロメア短縮が原因です。テロメア批判的に短い長さに達する、増殖の不可逆的な停止を活性化する DNA 損傷応答 (DDR) によって認識されます-複製老化としても定義されています。複製老化細胞のマイトジェンとアポトーシス シグナル2,3の両方に依存しないレンダリングする永久的な細胞周期の停止の状態を誘導するために知られている多くの刺激の 1 つされて。老化プログラムは通常高リソソーム活動、ミトコンドリア、核変化、クロマチン再編成、小胞体ストレス、DNA の損傷と老化関連を含む追加の機能によって特徴付けられる分泌型表現型 (SASP)3,4。老化細胞体に複数の機能がある: 開発、創傷治癒およびがん抑制2。同様に、彼らは高齢化と、逆説的に、腫瘍の進行5で重要な役割を再生する知られています。老化の負の値、および部分的に矛盾した効果は、SASP6頻繁に原因です。
最近では、マウスから老化細胞の除去が寿命延長し老化機能7,8,9,10、11,の多くの除去につながることを示した12します。 同じ方法で複数の薬を老化細胞 (senolytics) を排除するか、または SASP13,14を対象に開発されています。アンチエイジング治療の可能性は、フィールドに多くの注目を集めている最近。
細胞の老化に関連したメカニズムの研究と薬理学的介入の上映前のヴィヴォモデルでは、特に主線維芽細胞上に依存します。老化の表現型の変化は観察と細胞の種類、刺激および時間ポイント3,15を含む様々 な要因に依存して多様な老化誘導因子によって活性化されるいくつかの一般的な機能がありますが、 16,17。勉強し、老化細胞をターゲットの不均一性を考慮することが不可欠です。したがって、このプロトコルは一連のさまざまな治療法を使用して、主に線維芽細胞老化を誘導するために使用されるメソッドを提供するために目指しています。それで説明しますが、メソッドは他の細胞型に適応できます。
別に複製老化老化を誘発する他の 5 つの治療について述べる: 電離放射線、紫外線 (UV)、ドキソルビシン、酸化ストレスおよびエピジェネティックな変化 (すなわち DNA 脱メチル化やヒストンのアセチル化の推進).電離放射線と紫外線の両方、直接 DNA 損傷を引き起こすし、適切な用量でトリガー老化18,19。ドキソルビシンまた間を DNA にインターカ レートによって DNA の損傷によって主に老化を引き起こすし、トポイソメラーゼ II の機能を中断、中止 DNA 修復機構20。老化に不可欠な遺伝子の発現は、DNA メチル化とヒストンのアセチル化によって制御されます通常。結果として、ヒストン脱アセチル化酵素阻害剤 (例えば、酪酸ナトリウム、サハ) と DNA demethylating (例えば5 字) エージェントは、正常細胞21,22老化をトリガーします。
最後に、4 つの老化細胞に関連付けられている最も一般的なマーカーの説明される: 老化関連-β-ガラクトシダーゼ (SA β gal) EdU 取り込みアッセイ、細胞周期調節因子の過剰発現による DNA 合成率の活動とサイクリン依存性キナーゼ阻害剤 p16 と p21 と、SASP のメンバーの分泌の過剰発現

<table>
	<tbody>
		<tr>
			<td>DMEM Media - GlutaMAX</td>
			<td>Gibco</td>
			<td>31966-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S; 10,000 U/ml)</td>
			<td>Lonza</td>
			<td>DE17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>SC-202581</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water (not DEPC-Treated)</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Sarstedt</td>
			<td>833911002</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA Solution</td>
			<td>Lonza</td>
			<td>CC-5012</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Gibco</td>
			<td>18912-014</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>T9661-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;15 mL centrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430791</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Sarstedt</td>
			<td>83.3920</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Sarstedt</td>
			<td>83.3922</td>
			<td></td>
		</tr>
		<tr>
			<td>13mm round coverslips</td>
			<td>Sarstedt</td>
			<td>83.1840.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip</td>
			<td>Merck Chemicals</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Cesium137-source</td>
			<td></td>
			<td></td>
			<td>IBL 637 Cesium-137&#947;-ray machine</td>
		</tr>
		<tr>
			<td>UV radiation chamber</td>
			<td></td>
			<td></td>
			<td>Opsytec, Dr. G&#246;bel BS-02</td>
		</tr>
		<tr>
			<td>Doxorubicin dihydrochloride&#160;</td>
			<td>BioAustralis Fine Chemicals</td>
			<td>BIA-D1202-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma-Aldrich</td>
			<td>7722-84-1</td>
			<td></td>
		</tr>
		<tr>
			<td>5-aza-2&#8217;-deoxycytidine</td>
			<td>Sigma-Aldrich</td>
			<td>A3656</td>
			<td></td>
		</tr>
		<tr>
			<td>SAHA</td>
			<td>Sigma-Aldrich</td>
			<td>SML0061</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Butyrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B5887</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal (5-Bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside)</td>
			<td>Fisher Scientific</td>
			<td>7240-90-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>5949-29-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dibasic phosphate</td>
			<td>Acros organics</td>
			<td>7782-85-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferrocyanide&#160;</td>
			<td>Fisher Scientific</td>
			<td>14459-95-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferricyanide</td>
			<td>Fisher Scientific</td>
			<td>13746-66-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Merck Millipore</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Fisher Chemicals</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>25% glutaraldehyde</td>
			<td>Fisher Scientific</td>
			<td>111-30-8,7732-18-5</td>
			<td></td>
		</tr>
		<tr>
			<td>16% formaldehyde (w/v)</td>
			<td>Thermo-Fisher Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>EdU (5-ethynyl-2&#8217;-deoxyuridine)</td>
			<td>Lumiprobe</td>
			<td>10540</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-Cyanine3 azide (Sulfo-Cy3-Azide)</td>
			<td>Lumiprobe</td>
			<td>D1330</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) sulfate pentahydrate (Cu(II)SO4.5H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>209198</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Acros organics</td>
			<td>215682500</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS base</td>
			<td>Roche</td>
			<td>11814273001</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 Multiwell Plate 384, white&#160;</td>
			<td>Roche</td>
			<td>4729749001</td>
			<td></td>
		</tr>
		<tr>
			<td>Lightcycler 480 sealing foil&#160;</td>
			<td>Roche</td>
			<td>4729757001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sensifast Probe Lo-ROX kit&#160;</td>
			<td>Bioline</td>
			<td>BIO-84020</td>
			<td></td>
		</tr>
		<tr>
			<td>UPL Probe Library</td>
			<td>Sigma-Aldrich</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-6 DuoSet ELISA</td>
			<td>R&#38;D</td>
			<td>D6050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad TC20</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides</td>
			<td>Bio-Rad</td>
			<td>145-0017</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry incubator</td>
			<td>Thermo-Fisher Scientific</td>
			<td>Heratherm</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td>Merck Millipore</td>
			<td>1.10983</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm &#39;M&#39; laboratory film</td>
			<td>Bemis&#160;</td>
			<td>#PM992</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needles</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and validation of cellular senescence in primary human cells

細胞の老化は、さまざまな有害な刺激への応答でアクティブに永久的な細胞周期の停止の状態です。細胞の老化の活性化は、腫瘍の抑制、組織改造と高齢化を含むさまざまな病態生理学的条件の特徴です。生体内で細胞老化の誘導はまだよく特徴付けられます。ただし、 ex vivo細胞老化を促進する刺激の数を使用できます。その中で、最も一般的な老化誘導が複製枯渇、電離放射線し非電離放射線、遺伝毒性薬、酸化ストレスと demethylating とエージェントをアセチル化します。ここでは、これらの刺激を使用して老化に線維芽細胞を誘導する方法で詳細な指示いたします。このプロトコルは、簡単に、初代培養細胞やがん細胞を含む細胞の種類ごとに適応できます。老化誘導の検証するための異なる方法についても述べる。特に、我々 はライソゾーム酵素老化関連 β ガラクトシダーゼ (SA β gal) 細胞周期の表現のレベル 5-エチニル-2'-デオキシウリジン (EdU) 取り込みアッセイを用いた DNA 合成率の活性の測定に焦点を当てるp16 の阻害剤と p21 と表情やメンバーの Senescence-Associated 分泌形質 (SASP) の分泌。最後に、私達は例の結果を提供し、これらのプロトコルのアプリケーションをさらに議論。

SA β gal 染色老化線維芽細胞の濃縮
Β-ガラクトシダーゼ (β gal) すべての細胞で発現していること、最適な pH 4.025,26のライソゾーム酵素であります。ただし、老化、中にリソソームのサイズが大きく、その結果、老化細胞蓄積 β gal。この酵素の増加量は、最適 pH 6.025</s...

Watch this Scientific Journal Video about Induction and Validation of Cellular Senescence in Primary Human Cells at JoVE.com

Induction and Validation of Cellular Senescence in Primary Human Cells

ひと初代細胞における細胞老化の誘導性

ここでは、一連の誘発のためのプロトコルおよび培養細胞の細胞老化の検証について述べる。私たちはさまざまな老化を誘発する刺激に焦点を当てるし、一般的な老化関連マーカーの定量化を記述します。私たちはモデルとして線維芽細胞を使用して技術的な詳細を提供するが、プロトコルは、さまざまな携帯電話モデルに合わせることができます。

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

induction-and-validation-of-cellular-senescence-in-primary-human-cells

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Cell Proliferation

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16.6K ビュー.  University of Groningen, University Medical Center Groningen. ここでは、一連の誘発のためのプロトコルおよび培養細胞の細胞老化の検証について述べる。私たちはさまざまな老化を誘発する刺激に焦点を当てるし、一般的な老化関連マーカーの定量化を記述します。私たちはモデルとして線維芽細胞を使用して技術的な詳細を提供するが、プロトコルは、さまざまな携帯電話モデルに合わせることができます。

ビデオ: Induction and Validation of Cellular Senescence in Primary Human Cells

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

タンパク質の削除の誘導を介して<em&gt;子宮内で</em&gt;エレクトロポレーションは、トランスジェニックモデルにおいて神経細胞の移動の欠損を定義する

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

発生生物学

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

ヒト骨格筋からの一次筋原細胞および線維芽細胞の単離および定量的免疫組織化学的特性評価

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

末梢血造血前駆細胞からヒト神経幹細胞の直接誘導

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

ヒト多能性幹細胞における転写因子の過剰発現によって、造血内皮と血液の直接誘導

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

ひと多能性幹細胞からプリミティブかつ決定的な造血前駆細胞の分化を監督

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

肉腫細胞における間葉上皮移行の誘導

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

傷害による老化と生体内での骨格筋におけるリプログラミングの評価

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

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 ...

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

文化、ゼブラフィッシュ初代培養細胞のトランスフェクション

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

癌遺伝子が誘導する老化の中に活性酸素種および分泌老化関連の表現型正常ひと線維芽細胞の定量測定

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...