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 Video at JoVE.com

R. Z. is supported by UTHealth Innovation for Cancer Prevention Research Training Program Pre-Doctoral Fellowship (Cancer Prevention and Research Institute of Texas grant RP160015). J.T. is supported by the Ke Lin Program of the First Affiliated Hospital of Sun Yat-sen University. D.-F.L. is the CPRIT scholar in Cancer Research and supported by NIH Pathway to Independence Award R00 CA181496 and CPRIT Award RR160019.

This work was approved by The University of Texas Health Science Center at Houston (UTHealth) Animal Welfare Committee. The experiments are performed in strict accordance with the standards established by the UTHealth Center for Laboratory Animal Medicine &#38; Care (CLAMC) which is accredited by American Association for Laboratory Animal Care (AAALAC International).The human subjects in this study fall under Scenario A (&#34;No Human Subjects Research&#34;) as defined by the NIH SF424 documentation. Therefore, it does n...

<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>

To achieve higher MSC differentiation efficiency, several aspects are critical. One is the culture condition of iPSCs before initiating MSC differentiation. The protocol presented in the manuscript is based on previous studies 9,17. iPSCs need to be cultured on MEFs for at least 2 weeks. Maintaining iPSCs in good conditions on MEFs are critical for cells to attach on the gelatin-coated plate for MSC differentiation. Another important aspect is the density of iPSC...

Between 2006 and 2007, several breakthrough findings from the laboratories of Drs. Shinya Yamanaka and James A. Thomson led to the development of induced pluripotent stem cells (iPSCs)1,2,3. By reprogramming somatic cells with defined transcriptional factors to form iPSCs, researchers were able to generate cells with key characteristics namely, pluripotency, and self-renewal, which was previously thought to only exist in human embryonic stem cells (hESCs). iPSCs could be generated from any individual or patient and did not have to be derived from embryos, vastly expanding the repertoire of available diseases and backgrounds for the study. Since then, patient-derived iPSCs have been used to recapitulate the phenotype of various human diseases, from Alzheimer&#39;s disease4 and amyotrophic lateral sclerosis5 to long QT syndrome6,7,8.
These advances in iPSC research also have opened new avenues for the cancer research. Several groups recently have used patient iPSCs to model cancer development under a susceptible genetic background9,10,11, with successful application demonstrated to date in osteosarcoma9, leukemia10,11,12, and colorectal cancer13. Although iPSC-derived cancer models are still in their infancy, they have demonstrated great potential in phenocopying disease-associated malignancies, elucidating pathological mechanisms, and identifying therapeutic compounds14.
Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder caused by TP53 germline mutation15. Patients with LFS are predisposed to a various type of malignancies including osteosarcoma, making LFS iPSCs and their derived cells particularly well-suited to studying this malignancy16. An iPSC-based osteosarcoma model was first established in 2015 using LFS patient-derived iPSCs9 subsequently differentiated into mesenchymal stem cells (MSCs) and then to osteoblasts, the originating cells of osteosarcoma. These LFS osteoblasts recapitulate osteosarcoma-associated osteogenic differentiation defects and oncogenic properties, demonstrating the model potential as a &#34;bone tumor in a dish&#34; platform. Interestingly, genome-wide transcriptome analyses reveal aspects of an osteosarcoma gene signature in LFS osteoblasts and that features of this LFS gene expression profile are correlated with poor prognosis in osteosarcoma9, indicating the potential of LFS iPSCs disease models to reveal features of clinical relevance.
This manuscript provides a detailed description of how to use LFS patient-derived iPSCs to model osteosarcoma. It details the generation of LFS iPSCs, differentiation of iPSCs to MSCs and then to osteoblasts, and use of an in vivo xenograft model using LFS osteoblasts. The LFS disease model comprises several advantages, most notably the ability to generate unlimited cells at all stages of osteosarcoma development for mechanistic studies, biomarker identification, and drug screening9,14,16.
In summary, the LFS iPSC-based osteosarcoma model offers an attractive complementary system for advancing osteosarcoma research. This platform also provides a proof-of-concept for cancer modeling using patient-derived iPSCs. This strategy described below can be readily extended to model malignancies associated with other genetic disorders with cancer predispositions.

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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Plastic ware</td>
			<td style="width:157px;"></td>
			<td style="width:136px;"></td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">100 mm Dish</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">430107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">60 mm Dish</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">430166</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">6-well Plate</td>
			<td style="width:157px;">Falcon</td>
			<td style="width:136px;">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">12-well Plate</td>
			<td style="width:157px;">Falcon</td>
			<td style="width:136px;">353043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">48-well Plate</td>
			<td style="width:157px;">Falcon</td>
			<td style="width:136px;">353078</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">1 mL Pipet Tip</td>
			<td style="width:157px;">USA Scientific</td>
			<td style="width:136px;">1111-2721</td>
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			<td height="21" style="height:21px;width:327px;">200 &#181;L Pipet Tip</td>
			<td style="width:157px;">USA Scientific</td>
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			<td height="21" style="height:21px;width:327px;">10 &#181;L Pipet Tip</td>
			<td style="width:157px;">USA Scientific</td>
			<td style="width:136px;">1111-3700</td>
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		</tr>
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			<td height="21" style="height:21px;width:327px;">5 mL Serological Pipette</td>
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			<td height="21" style="height:21px;width:327px;">10 mL Serological Pipette</td>
			<td style="width:157px;">SARSTEDT</td>
			<td style="width:136px;">86.1254.001</td>
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			<td height="21" style="height:21px;width:327px;">25 mL Serological Pipette</td>
			<td style="width:157px;">SARSTEDT</td>
			<td style="width:136px;">86.1685.001</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">50 mL Tube, PP</td>
			<td style="width:157px;">SARSTEDT</td>
			<td style="width:136px;">62.547.100</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">15 mL Tube, PP</td>
			<td style="width:157px;">SARSTEDT</td>
			<td style="width:136px;">62.554.100</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Culture materials and Reagents</td>
			<td style="width:157px;"></td>
			<td style="width:136px;"></td>
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		</tr>
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			<td height="42" style="height:42px;width:327px;">CytoTune- iPS 2.0 Sendai Reprogramming Kit</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:136px;">A16517</td>
			<td style="width:208px;">Commercial Sendai virus reprogramming kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Corning hESC-Qualified Matrix</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">354277</td>
			<td style="width:208px;">Basement membrane matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">CF1 MEFs, irradiated</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">A34180</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">DMEM</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:136px;">D5671</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">DMEM/F12</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">10-090-CV</td>
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			<td height="21" style="height:21px;">&#945;MEM</td>
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			<td height="21" style="height:21px;width:327px;">StemMACS iPS-Brew XF</td>
			<td style="width:157px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-104-368</td>
			<td>Commercial iPSC medium</td>
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			<td height="21" style="height:21px;width:327px;">KnockOut DMEM/F-12</td>
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			<td style="width:136px;">12660012</td>
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			<td height="21" style="height:21px;width:327px;">FBS Opti-Gold</td>
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			<td style="width:136px;">A3181502</td>
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			<td style="width:136px;">100-00AB</td>
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			<td style="width:136px;">G9422</td>
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			<td height="21" style="height:21px;width:327px;">Dexamethasone</td>
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			<td height="23" style="height:23px;width:327px;">Ascorbic Acid</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:136px;">A5960</td>
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			<td height="23" style="height:23px;width:327px;">Dulbecco&#39;s Phosphate-Buffered Saline, 1x (DPBS)</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">21-031-CV</td>
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			<td height="21" style="height:21px;width:327px;">StemMACS Passaging Solution XF</td>
			<td style="width:157px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-104-688</td>
			<td>Commercial passaging solution</td>
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			<td style="width:136px;">T4049</td>
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			<td height="21" style="height:21px;width:327px;">Collagenase, Type II&#160;&#160;</td>
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			<td style="width:136px;">17101015</td>
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			<td height="21" style="height:21px;width:327px;">Human NANOG Antibody</td>
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			<td height="21" style="height:21px;width:327px;">OCT4 Antibody (H-134)</td>
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			<td height="42" style="height:42px;width:327px;">Alexa Fluor 555 Mouse Anti-Human TRA-1-81 Antigen</td>
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			<td style="width:136px;">560123</td>
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			<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 syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

This protocol presents the procedures including LFS iPSC generation, MSC differentiation, osteoblast differentiation, and in vivo tumorigenesis assay using LFS MSC-derived osteoblasts.
Scheme for the generation of LFS iPSCs from fibroblasts by using a commercially available Sendai virus reprogramming kit is shown in Figure 1A. Sendai virus-based delivery of the Yamanaka four factors is a no...

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

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells Video (Video) | JoVE

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

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

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

Research

JoVE Journal

Cancer Research

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma Video (Video) | JoVE

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

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

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

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

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

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

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts Video (Video) | JoVE

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

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants Video (Video) | JoVE

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment Video (Video) | JoVE

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

A Melanoma Patient-Derived Xenograft Model

A Melanoma Patient-Derived Xenograft Model Video (Video) | JoVE

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

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells Video (Video) | JoVE

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

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells Video (Video) | JoVE

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

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts Video (Video) | JoVE

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