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...

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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.

<table border="0" cellpadding="0" cellspacing="0" style="width:828px;" width="828">
	<tbody>
		<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>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">200 &#181;L Pipet Tip</td>
			<td style="width:157px;">USA Scientific</td>
			<td style="width:136px;">1111-0706</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">5 mL Serological Pipette</td>
			<td style="width:157px;">SARSTEDT</td>
			<td style="width:136px;">86.1253.001</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<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>
			<td></td>
		</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>
			<td></td>
		</tr>
		<tr height="42">
			<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>
			<td></td>
		</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>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;MEM</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">10-022-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">KnockOut DMEM/F-12</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">12660012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">FBS Opti-Gold</td>
			<td style="width:157px;">GenDEPOT</td>
			<td style="width:136px;">F0900-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">KnockOut Serum Replacement</td>
			<td style="width:157px;">ThermoFisher</td>
			<td style="width:136px;">A3181502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Penicillin-Streptomycin</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:136px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">MEM Nonessential Amino Acids</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">25-025-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">L-Glutamine Solution</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:136px;">G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">2-Mercaptoethanol</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:136px;">M3148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Human FGF-basic (bFGF)</td>
			<td style="width:157px;">PEPROTECH</td>
			<td style="width:136px;">100-18B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Recombinant Human PDGF-AB</td>
			<td style="width:157px;">PEPROTECH</td>
			<td style="width:136px;">100-00AB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">&#946;-Glycerophosphate</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:136px;">G9422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Dexamethasone</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:136px;">A4902</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:327px;">Ascorbic Acid</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:136px;">A5960</td>
			<td></td>
		</tr>
		<tr height="23">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Accutatse Cell Detachment Solution</td>
			<td style="width:157px;">Corning</td>
			<td style="width:136px;">25-058-CI</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Thiazovivin (ROCK Inhibitor)</td>
			<td style="width:157px;">Calbiochem</td>
			<td style="width:136px;">420220</td>
			<td></td>
		</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 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 at JoVE.com

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

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.

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.

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Cancer Research

8.8K visualizações.  The University of Texas Health Science Center at Houston. 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 mo...