Name: modeling osteosarcoma using li-fraumeni syndrome patient-derived induced pluripotent stem cells
Uploaded: 2018-06-13T14:00:00
Description: Watch this Scientific Journal Video about Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells at JoVE.com

R. Z. UTHealth yenilik tarafından kanser önleme araştırma eğitim programı Pre-Doctoral bursu için (kanser önleme ve RP160015 vermek Texas Araştırma Enstitüsü) desteklenir. J.T. ilk bağlı hastane, Sun Yat-sen üniversite Ke Lin Program tarafından desteklenir. Ö-Tayback kanser araştırma'CPRIT uzman ve NIH için yol bağımsızlık Ödülü R00 CA181496 ve CPRIT Ödülü RR160019 tarafından desteklenir.

Bu eser Texas Üniversitesi Sağlık Bilimleri Merkezi Houston (UTHealth) hayvan refahı Komitesi tarafından kabul edildi. Deneyler ile laboratuvar hayvan Tıp & bakım (hangi Amerikan Derneği tarafından akredite olmuştur laboratuvar (AAALAC International) için hayvan bakım CLAMC) için UTHealth Merkezi tarafından belirlenmiş standartlara sıkı uygun olarak gerçekleştirilir. Bu çalışmanın insan denekler bir ("Hayır insan konularda araştırma") senaryosunda NIH SF424 belgeleri tarafından tanımlandığı...

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Takahashi, K., 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131 (5), 861-872 (2007).</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=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318 (5858), 1917-1920 (2007).</li><li>Yagi, T., 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=Modeling+familial+Alzheimer's+disease+with+induced+pluripotent+stem+cells.">Modeling familial Alzheimer's disease with induced pluripotent stem cells.</a> Hum Mol Genet. 20 (23), 4530-4539 (2011).</li><li>Dimos, J. T., 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=Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321 (5893), 1218-1221 (2008).</li><li>Moretti, 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=Patient-specific+induced+pluripotent+stem-cell+models+for+long-QT+syndrome.">Patient-specific induced pluripotent stem-cell models for long-QT syndrome.</a> N Engl J Med. 363 (15), 1397-1409 (2010).</li><li>Itzhaki, 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=Modelling+the+long+QT+syndrome+with+induced+pluripotent+stem+cells.">Modelling the long QT syndrome with induced pluripotent stem cells.</a> Nature. 471 (7337), 225-229 (2011).</li><li>Carvajal-Vergara, X., 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=Patient-specific+induced+pluripotent+stem-cell-derived+models+of+LEOPARD+syndrome.">Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome.</a> Nature. 465 (7299), 808-812 (2010).</li><li>Lee, D. 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=Modeling+familial+cancer+with+induced+pluripotent+stem+cells.">Modeling familial cancer with induced pluripotent stem cells.</a> Cell. 161 (2), 240-254 (2015).</li><li>Mulero-Navarro, 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=Myeloid+Dysregulation+in+a+Human+Induced+Pluripotent+Stem+Cell+Model+of+PTPN11-Associated+Juvenile+Myelomonocytic+Leukemia.">Myeloid Dysregulation in a Human Induced Pluripotent Stem Cell Model of PTPN11-Associated Juvenile Myelomonocytic Leukemia.</a> Cell Rep. 13 (3), 504-515 (2015).</li><li>Kotini, A. 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=Functional+analysis+of+a+chromosomal+deletion+associated+with+myelodysplastic+syndromes+using+isogenic+human+induced+pluripotent+stem+cells.">Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells.</a> Nat Biotechnol. 33 (6), 646-655 (2015).</li><li>Kotini, A. 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=Stage-Specific+Human+Induced+Pluripotent+Stem+Cells+Map+the+Progression+of+Myeloid+Transformation+to+Transplantable+Leukemia.">Stage-Specific Human Induced Pluripotent Stem Cells Map the Progression of Myeloid Transformation to Transplantable Leukemia.</a> Cell Stem Cell. 20 (3), 315-328 (2017).</li><li>Crespo, 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=Colonic+organoids+derived+from+human+induced+pluripotent+stem+cells+for+modeling+colorectal+cancer+and+drug+testing.">Colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing.</a> Nat Med. 23 (7), 878-884 (2017).</li><li>Gingold, J., Zhou, R., Lemischka, I. R., Lee, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Cancer+with+Pluripotent+Stem+Cells.">Modeling Cancer with Pluripotent Stem Cells.</a> Trends Cancer. 2 (9), 485-494 (2016).</li><li>Lin, Y. 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=Osteosarcoma:+Molecular+Pathogenesis+and+iPSC+Modeling.">Osteosarcoma: Molecular Pathogenesis and iPSC Modeling.</a> Trends Mol Med. 23 (8), 737-755 (2017).</li><li>Zhou, 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=Li-Fraumeni+Syndrome+Disease+Model:+A+Platform+to+Develop+Precision+Cancer+Therapy+Targeting+Oncogenic+p53.">Li-Fraumeni Syndrome Disease Model: A Platform to Develop Precision Cancer Therapy Targeting Oncogenic p53.</a> Trends Pharmacol Sci. 38 (10), 908-927 (2017).</li><li>Lian, Q., 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=Derivation+of+clinically+compliant+MSCs+from+CD105+,+CD24-+differentiated+human+ESCs.">Derivation of clinically compliant MSCs from CD105+, CD24- differentiated human ESCs.</a> Stem Cells. 25 (2), 425-436 (2007).</li><li>Zhou, 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=A+homozygous+p53+R282W+mutant+human+embryonic+stem+cell+line+generated+using+TALEN-mediated+precise+gene+editing.">A homozygous p53 R282W mutant human embryonic stem cell line generated using TALEN-mediated precise gene editing.</a> Stem Cell Res. 27, 131-135 (2018).</li></ol>

Daha yüksek MSC farklılaşma verim elde etmek için çeşitli yönleri önemlidir. Bir kültür iPSCs MSC ayırt etme işlemini başlatmadan önce bir durumdur. El yazması sunulan Protokolü önceki çalışmalar 9,17at dayanır. iPSCs en az 2 hafta MEFs üzerinde kültürlü olmanız gerekiyor. İPSCs iyi bakımı MEFs koşullara hücreler MSC farklılaşma için jelatin kaplı tabakta iliştirmek için kritik öneme sahiptir. Bir başka önemli yönü farkl?...

2006 ve 2007 arasında laboratuvarları Drs. Shinya Yamanaka ve James A. Thomson birkaç atılım bulgular indüklenmiş pluripotent kök hücreler (iPSCs)1,2,3gelişmesine yol açtı. Somatik hücre formu iPSCs için tanımlanmış transkripsiyon faktörleri ile yeniden programlama tarafından araştırmacılar yani anahtar özelliklere sahip hücreleri elde edebilecektir, Nanog ve daha önce sadece için düşünüldü kendini yenileme, insan embriyonik kök hücre var (hESCs). iPSCs herhangi bir birey ya da hasta oluşturulabilir ve embriyo, büyük ölçüde elde edilebilir hastalıklar ve arka planlar çalışma için repertuar genişleyen türetilen gerek yoktu. O zamandan beri iPSCs hasta kaynaklı çeşitli insan hastalıkları, Alzheimer hastalığı4 ve amyotrofik lateral skleroz5 uzun QT sendromu6,7, fenotip özetlemek için kullanılmıştır 8.
Bu gelişmeler IPSC araştırma Ayrıca kanser araştırmaları için yeni yollar açtı. Çeşitli gruplar son zamanlarda bugüne osteosarkom9gösterdiği başarılı uygulama modeli kanser geliştirme altında bir duyarlı genetik arka plan9,10,11, hasta iPSCs kullanmış, Lösemi10,11,12ve kolorektal kanser13. IPSC kaynaklı kanser modelleri hala onların bebeklik olmakla birlikte, patolojik mekanizmalar elucidating ve terapötik bileşiklerin14tanımlayan phenocopying maligniteler, hastalık ilişkili büyük potansiyel göstermiştir.
Li-Fraumeni sendromu (LFS) TP53 germline mutasyon15tarafından neden olduğu bir otozomal dominant kalitsal kanser hastalıktır. LFS hastalarda osteosarkom, dahil olmak üzere maligniteler çeşitli bir tür LFS iPSCs ve türetilmiş hücrelerine özellikle iyi bu malignite16eğitimi için uygun hale yatkındır. LFS hasta kaynaklı iPSCs9 daha sonra kullanarak Ayrıştırılan mezenkimal kök hücre (MSCs) ve sonra kaynaklanan dokusunu için hücreleri bir IPSC tabanlı osteosarkom modeli ilk 2015 yılında kuruldu osteosarkom. Bu LFS dokusunu Osteojenik farklılaşma osteosarkom ilişkili kusurlar ve oncogenic özellikleri, bir "bir tabak içinde kemik tümörü" platform olarak potansiyel modeli gösteren özetlemek. İlginçtir, genom-wide transcriptome analizleri bir osteosarkom gen İmzada LFS dokusunu yönlerini açığa vurmak ve bu şekil-in bu LFS gen ifade profil osteosarkom9, kötü prognoz ile ilişkili gösteren LFS iPSCs hastalık modelleri potansiyelini klinik özellikleri ortaya çıkarmak için.
Bu el yazması LFS hasta kaynaklı iPSCs modeli osteosarkom için kullanmak hakkında ayrıntılı bir açıklama sağlar. LFS iPSCs, iPSCs MSCs ve dokusunu için sonra farklılaşma ve LFS dokusunu kullanarak bir in vivo xenograft modeli kullanımı nesil ayrıntıları. LFS hastalık modeli birçok avantajı, özellikle mekanik çalışmaları, biyomarker teşhis ve ilaç9,14eleme için her aşamasında osteosarkom gelişme sınırsız hücreler oluşturma yeteneği oluşur, 16.
Özetle, LFS osteosarkom IPSC tabanlı modeli osteosarkom araştırma ilerlemek için çekici bir tamamlayıcı sistem sunar. Bu platform da bir kanıtı-of-concept hasta kaynaklı iPSCs kullanarak kanser modelleme için sağlar. Aşağıda açıklanan bu strateji modeli maligniteler kanser eğilimlerin ile diğer genetik bozuklukları ile ilişkili için kolayca genişletilebilir.

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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">0.25% Trypsin-EDTA Solution</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:136px;">T4049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Collagenase, Type II&#160;&#160;</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">17101015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Human NANOG Antibody</td>
			<td style="width:157px;">R&#38;D System</td>
			<td style="width:136px;">AF1997</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">OCT4 Antibody (H-134)</td>
			<td style="width:157px;">Santa Cruz</td>
			<td style="width:136px;">sc-9081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Human/Mouse SSEA-4 PE-conjugated Antibody</td>
			<td style="width:157px;">R&#38;D System</td>
			<td style="width:136px;">FAB1435P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Alexa Fluor 555 Mouse Anti-Human TRA-1-81 Antigen</td>
			<td style="width:157px;">DB Biosciences</td>
			<td style="width:136px;">560123</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Alexa Fluor 488 Donkey Anti-Goat IgG (H+L)</td>
			<td style="width:157px;">Jackson ImmunoResearch</td>
			<td style="width:136px;">705-545-003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L)</td>
			<td style="width:157px;">Jackson ImmunoResearch</td>
			<td style="width:136px;">111-545-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">PE Mouse Anti-Human CD105</td>
			<td style="width:157px;">eBioscience</td>
			<td style="width:136px;">12-1057-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">FITC Mouse Anti-Human CD44</td>
			<td style="width:157px;">DB Biosciences</td>
			<td style="width:136px;">555478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">PE Mouse Anti-Human CD73</td>
			<td style="width:157px;">DB Biosciences</td>
			<td style="width:136px;">550257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">PE Mouse Anti-Human CD166</td>
			<td style="width:157px;">DB Biosciences</td>
			<td style="width:136px;">560903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">FITC Mouse Anti-Human CD24</td>
			<td style="width:157px;">DB Biosciences</td>
			<td style="width:136px;">555427</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Donkey Serum</td>
			<td style="width:157px;">Jackson ImmunoResearch</td>
			<td style="width:136px;">017-000-121</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Goat Serum</td>
			<td style="width:157px;">Jackson ImmunoResearch</td>
			<td style="width:136px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Alkaline Phosphatase Staining Kit II</td>
			<td style="width:157px;">Stemgent</td>
			<td style="width:136px;">00-0055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Alizarin Red S</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:136px;">A5533</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">TRIzol Reagent</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">15596018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Chloroform</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">C298-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">2-Propanol</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">A416-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Ethanol, Absolute, Molecular Biology Grade</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">BP28184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">DNase I, RNase-free (1 U/&#181;L)</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">EN0521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">iScript cDNA Synthesis Kit</td>
			<td style="width:157px;">BioRad</td>
			<td style="width:136px;">1708891BUN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">iQ SYBR Green Supermix</td>
			<td style="width:157px;">BioRad</td>
			<td style="width:136px;">1708884</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Matrigel Matrix High Concentration (HC), Phenol-Red Free</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">354262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">1 mL Slip Tip Syringe, 26 Gauge x 5/8 Inch</td>
			<td style="width:157px;">DB Biosciences</td>
			<td style="width:136px;">309597</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling osteosarcoma using li-fraumeni syndrome patient-derived induced pluripotent stem cells

Li-Fraumeni sendromu (LFS) bir otozomal dominant kalitsal kanser hastalıktır. LFS hastalarda tümör, osteosarkom--en sık birincil olmayan hematolojik maligniteler çocukluk ve ergenlik de dahil olmak üzere çeşitli tür yatkındır. Bu nedenle, LFS bu malignite eğitim için ideal bir model sağlar. IPSC yöntemlerden yararlanarak osteosarkom LFS ilişkili başarılı bir şekilde örnek alınarak LFS hasta iPSCs mezenkimal kök hücre (MSCs) ve ardından dokusunu--menşei osteosarkom hücreleri ayırt tarafından. Bu LFS dokusunu osteosarkom, osteosarkom patogenezinde operasyona için bir çekici model sistemi sağlayan oncogenic özelliklerini özetlemek. Bu el yazması bir protokol LFS hasta fibroblastlar, farklılaşma MSCs, MSCs farklılaşma dokusunu ve LFS dokusunu kullanarak vivo içinde tumorigenesis için iPSCs dan iPSCs üretimi için gösterir. Bu IPSC hastalık modeli potansiyel biyolojik veya LFS ilişkili osteosarkom için tedavi hedefleri tanımlamak için genişletilebilir.

Bu iletişim kuralı LFS IPSC üretimi, MSC farklılaşma, osteoblast farklılaşma ve in vivo tumorigenesis tahlil LFS MSC kaynaklı dokusunu kullanarak da dahil olmak üzere yordamları sunar.
LFS iPSCs piyasada bulunan Sendai virüs seti yeniden programlama kullanarak fibroblastlar gelen nesil için şeması Şekil 1Aile gösterilir. Sendai virüs alan teslim Yamanaka dört faktörlerin ...

Watch this Scientific Journal Video about Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells at JoVE.com

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Burada, bir protokol indüklenmiş pluripotent kök hücreler (iPSCs) üretimi için Li-Fraumeni sendromu (LFS) hasta türetilmiş fibroblastlar, iPSCs mezenkimal kök hücre (MSCs) yoluyla farklılaşma üzerinden dokusunu ve modelleme içinde vivo mevcut LFS hasta kaynaklı dokusunu kullanarak tumorigenesis.

Li-Fraumeni sendromu hasta kaynaklı indüklenmiş Pluripotent kök hücreler kullanılarak osteosarkom modelleme

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

This Li-Fraumeni syndrome iPSC-based osteosarcoma model can help answer key questions in the osteosarcoma research field about using patient iP cells in cancer modeling. The main advantage of this model is that it allows the generation unlimited cells at all stages of osteosarcoma development for mechanistic studies, biomarker identification, and drug screening. Ruoji will be demonstrating the cell culture procedure and I will be demonstrating the animal procedure.After two weeks of mouse embryonic fibroblast, or MEF, co-culture wash the 80 to 90%confluent iPS cell culture with five milliliters of DPBS followed by the addition of one milliliter of cell detachment solution to the cells for a three minute incubation at room temperature. It is critical to maintain iPS cells in good conditions on MEFs for at least 14 days for the cells to be able to attach to a gelatin-coated plate for their differentiation into mesenchymal stem cells. When the cells have detached from the plate bottom add nine milliliters of MEF/MSC culture medium to the cells to neutralize the cell detachment solution and transfer the cell suspension into an uncoated 100 millimeter tissue culture plate.Incubate the cells at room temperature for 30 minutes to allow the MEFs to attach to the plate and carefully collect the unattached iPS cell-containing supernatant. Centrifuge the iPS cell solution and resuspend the pellet in six milliliters of MSE differentiation medium. Then seed two milliliters of cells into each of three 0.1%gelatin-coated wells of a six-well culture plate and place the plate in the cell culture incubator for 28 days feeding the cells with fresh medium every two days to remove any unattached or dead cells.On day 28 large masses of differentiated cells will be visible with fibroblast-like MSCs observed at the edges of the clusters. Wash the cell cultures with one milliliter of DPBS per well followed by their detachment with 0.5 milliliters of 0.25%Trypsin-EDTA per well at room temperature. After three minutes neutralize the Tyrypsin with 1.5 milliliters of MEF/MSE culture medium per well and pool the detached MSEs into a single 15 milliliter conical tube for their centrifugation.Resuspend the pellet in three milliliters of MEF/MSE culture medium and seed the cells onto a 0.1%gelatin-coated 60 millimeter plate, changing the medium every two days until the cells reach 100%confluence. After splitting the 100%confluent culture at a one to three ratio the MSCs will proliferate quickly demonstrating a fibroblast-like morphology and formi&ng a swirl-like culture pattern when the culture reaches a high density. The iPS cell-derived MSCs can also be examined by a immunofluorescent staining for mesenchymal stem cell markers at any point during their differentiation.To differentiate the MSE into osteoblasts plate the appropriate number of MSE from a 95%confluent cell culture onto a cell culture plate in the appropriate volume of MEF/MSC culture medium for 24 hours. Then begin replacing the supernatant with the appropriate volume of osteoblast differentiation medium the next day and every two days after until the end of the culture period. To detect the bone-associated alkaline phosphatase produced by preosteoblasts and the mineral deposition produced by mature osteoblasts perform alkaline phosphatase and Alizarin Red S staining, respectively, according to the manufacturer's protocol at the appropriate experimental time points.To set up an in vivo tumorigenesis model with the differentiated osteoblasts seed the appropriate number of MSC into five 100 millimeter dishes to start osteoblastic differentiation. On day 14 of osteoblastic differentiation wash five culture plates of osteoblasts with five milliliters of DPBS and treat the cells with 1.5 milliliters of osteoblast detachment solution per dish. After a 30 minute incubation at 37 degrees celsius confirm detachment of the cells by light microscopy and neutralize the detachment solution with fresh osteoblast differentiation medium.Pool the cells in a 50 milliliter conical tube for their centrifugation and resuspend the pellet in 25 milliliters of fresh osteoblast differentiation medium. Filter the cell solution through 70 micron strainer to remove any aggregated cells prior to counting and set aside 1 X 10 to the 7 cells per subcutaneous injection. Pellet the cells by centrifugation and reconstitute the cells at a 1 X 10 to the 7 osteoblasts per 50 microliters of ice-cold DPBS concentration.Mix the osteoblasts with an equal volume of phenol-red free basement membrane matrix and place the cells on ice until their injection. After confirming a lack of response to toe pinch in an eight-week old female immuno-compromised nude mouse load the cells into a one milliliter syringe equipped with the 27 gauge needle and subcutaneously inject 100 microliters of the cells into the disinfected flank of the animal. Then monitor the mice with weekly palpitations at the injection site for the growth of subcutaneous nodular densities.Tumor formation should be observed between six to 10 weeks after injection. Established LFS iPS cell clones should exhibit a typical human embryonic stem cell morphology and demonstrate a positive alkaline phosphatase activity. LFS iPS cells also highly express pluripotency factor mRNAs comparable to human embryonic stem cell lines and much higher than levels observed in parental fibroblasts.Successfully differentiated LFS MSCs also express typical MSC markers including CD44, CD73, CD105 and CD166 as assessed by immunofluoresent staining. When the MSCs are subjected to osteogenic differentiation signals the differentiated cells begin to exhibit positive alkaline phosphatase activity by day nine and mineral deposition by day 21. The differentiating osteoblasts also demonstrate an upregulated expression of pre-osteoblast and mature osteoblast genes throughout their differentiation process.Six to 10 weeks after osteoblast injection LFS osteoblast derived tumors demonstrate immature osteoblast characteristics including positive alkaline phosphatase activity, positive collagen matrix deposition, and negative mineralization. After watching this video you should have a good understanding of how to differentiate Li-Fraumeni Syndrome iPS Cells into osteoblasts and to generate an in vivo osteosarcoma model using patient-derived osteoblasts. In addition to osteosarcoma this Li-Fraumeni Syndrome iPS cell-based disease model can be also applied to other Li-Fraumeni Syndrome related malignancies such as soft tissue sarcoma, breast cancer, and brain tumors.

Research

JoVE Journal

Cancer Research

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

Kanser kurulması İnsan Konvansiyonel Osteosarkom Hücre Kültürleri Kök

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

Kanser Araştırmaları

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Tümör ve Mezenkimal Kök hücreler arasında ekstrasellüler vezikül aracılı iletişim tanımlamak için osteosarkom preklinik fare modeli

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Akış Sitometresi lösemi kök hücre birincil Akut Miyeloid Lösemi ve hasta-türetilen-xenografts, tanı ve takip yukarı tahmin etmek için

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

Sarkom hasta türevi xenografts tümör-başlatma hücrelerinin yalıtım ve karakterizasyonu

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

A Melanoma Patient-Derived Xenograft Model

Bir melanom hasta-türetilen xenograft model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

Fizyolojik hasta, kanser kök hücrelerini hedef alan anti-neoplastik Ilaç taraması için 3B kürler türetilmiştir

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

Tümör Antijene Özgü T Hücrelerinin Üretimi için İnsan Kaynaklı Pluripotent Kök Hücrelerin Kullanılması

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

Yapısal DNA Alterasyon Analizi ve Hasta Kaynaklı Ksenogreftler Kullanılarak Kanserde Hedefli Tedavilerin Test Edilmesi

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Kombine Koşullu Knockdown ve Uyarlanmış Küre Oluşumu Çalışma Hasta Kaynaklı Mide Kanseri Kök Hücrelerin bir Stemness-Associated Gene Çalışma

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

Hasta Kaynaklı Glioma Kök Hücreleri Kullanarak GBM için Potansiyel Kombinasyon Tedavisini Belirlemek için Hızlı Tarama İş Akışı

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...