Name: Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells (Video) | JoVE Journal
Uploaded: 2018-06-13T14:00:00.000+00:00
Duration: 8 min 52 s
Description: Learn to model osteosarcoma using Li-Fraumeni Syndrome patient-derived iPSCs differentiated into osteoblasts. This protocol enables mechanistic studies, biomarker identification, and drug screening for cancer research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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 not need any approval by UTHealth human research ethics committee.
1. Generation of LFS iPSCs (Figure 1A)
<ol>
	<li>On day -2, plate 2 x 104 LFS patient fibroblasts into one well of a 6-well plate, and culture the cells with fibroblast medium (Table 1) at 37 &#176;C in a humidified 5% CO2 incubator. Ensure that the fibroblasts reach 50 - 60% confluence on the day of transduction (Day 0).</li>
	<li>Perform Sendai virus transduction on Day 0. To do this, replace the spent medium with pre-warmed fibroblast medium. Thaw the commercial Sendai virus reprogramming kit (see Table of Materials) on ice. Infect LFS fibroblasts with mixed Sendai viruses according to the manufacturer&#39;s instructions.</li>
	<li>Maintenance of the transduced cells in fibroblast medium (Day 1 - 6):
	<ol>
		<li>On the day 1, 24 h after transduction, remove the fibroblast medium containing remaining Sendai virus and replace it with 2 mL fresh fibroblast medium. Culture the transduced fibroblasts at 37 &#176;C in a humidified 5% CO2 incubator.</li>
		<li>Change the medium with the fibroblast medium every other day from the Day 2 - 6.</li>
	</ol>
	</li>
	<li>Maintenance of the transduced cells in the feeder culture condition (Day 7 - 28):
	<ol>
		<li>Prepare irradiated CF1 mouse embryonic fibroblast (MEF) culture dishes on Day 6. To do this coat six 100 mm tissue culture dishes by adding 5 mL of 0.1% gelatin to each plate at room temperature for 30 min. Aspirate the 0.1% gelatin after coating.</li>
		<li>Remove MEFs from liquid nitrogen container. Thaw MEFs using the commercially available thawing system following the manufacturer&#39;s instructions. Plate the cells on the gelatin-coated plates with MEF/MSC culture medium (Table 1) at a seeding density of around 6.7 x 105 cells per 100 mm tissue culture dish.</li>
		<li>Inoculate the transduced cells on MEF plates (Day 7): Trypsinize the transduced cells using 1 mL of 0.25% Trypsin-EDTA per 100 mm dish for 2 - 4 min at room temperature. Neutralize trypsin with 9 mL of fibroblast medium. Transfer cells to conical tubes and centrifuge cells at 230 x g at 37 &#176;C for 4 min. Plate the transduced fibroblasts on the six MEF dishes prepared on Day 6 and culture them in 8 mL of fibroblast medium per dish. 
		NOTE: The confluency of MEFs is around 20% before seeding transduced cell on MEF plates.</li>
		<li>On the day 8, discard the fibroblast medium and replace with hESC medium (Table 1).</li>
		<li>Wait for the emergence of iPS clones from Days 9 - 28 (Figure 1C). Change the spent hESC medium every other day and monitor the emergence of iPS clones under the microscope. Wait for iPSCs clones big enough to be observed by the naked eye and then proceed to expand iPS clones in feeder-free culture condition.</li>
	</ol>
	</li>
	<li>Expansion of the emerged iPS clones in feeder-free culture condition (Day 29 - 79~99):
	<ol>
		<li>To prepare the matrix-coated plate, coat a 48-well plate with 120 &#181;L of the basement membrane matrix coating solution (Table 1) per well. Make sure the basement membrane matrix coating solution covers the well and coats the culture dish surface. Leave it at room temperature for 1 h. Aspirate the coating solution immediately before use and add 250 &#181;L of hESC medium per well with 2 &#181;M ROCK inhibitor Thiazovivin.</li>
		<li>On the Day 28, aspirate the spent hESC medium and wash iPS clones with 1x DPBS. Pick up visible iPS clones with a 1 mL pipette tip and transfer into a well of a treated 48-well plate (step 1.4.1). Seed one colony per well of 48-well plate.</li>
		<li>Centrifuge the plate at 230 x g for 4 min to facilitate the cell attachment. Culture iPS clones at 37 &#176;C in a humidified 5% CO2 incubator.</li>
		<li>The following day (Day 29), remove the spent hESC medium and add 250 &#181;L of the commercially available iPSC medium in each well.</li>
		<li>Maintain iPS clones at 37 &#176;C in a humidified 5% CO2 incubator, and change the iPSC medium every other day.</li>
		<li>When iPS clones reach 85% confluence, subculture cells at a 1:10 ratio by culture area. Add 60 &#181;L of the commercial passaging solution to detach the cells and incubate at 37 &#176;C for 3 min.</li>
		<li>Seed half of the detached iPSCs on a basement membrane matrix-coated 12-well plate in 1 mL of hESC medium with 2 &#181;M ROCK inhibitor Thiazovivin. Culture iPS clones in a 12-well plate with 1 mL of the commercially available iPSC medium per well and subculture iPSCs at a 1:10 ratio until they reach passage 10. 
		NOTE: iPSCs are sub cultured every 5 - 7 days at a 1:10 ratio. It takes up to 10 weeks for iPS clones to reach passage 10. iPSC characterizations including cell morphology, alkaline phosphatase (AP) activity, and the expression of pluripotency factors and hESC markers, can be examined after confirming the removal of Sendai virus in iPSCs (usually demonstrated after around 10 passages) (Figure 1B-E). Antibody and primer information for iPSC characterization are included in Table 2 and 4.</li>
	</ol>
	</li>
</ol>
2. Differentiation of LFS iPSCs to Mesenchymal Stem Cells (MSCs) (Figure 2A)
<ol>
	<li>Culture iPSC clones on MEF plates for at least 2 weeks (Day ~ -14 - 0): Prepare the MEF dishes (100 mm tissue culture dishes) as previously described (1.3.1 - 1.3.2). Maintain iPSCs in hESC medium and change medium every other day. Whenever iPSCs reach 90% confluence, subculture iPSCs by 1:10 dilution.</li>
	<li>Removal of MEFs from iPSCs (Day 0):
	<ol>
		<li>After maintaining iPSCs on MEFs for 2 weeks, culture iPSCs in 100 mm tissue culture dishes until cells reach 80 - 90% confluence. Remove the spent hESC medium and wash iPSCs with 5 mL of 1x DPBS. Add 1 mL of the cell detachment solution to detach the cells and incubate at room temperature for 3 min.</li>
		<li>Add 9 mL of MEF/MSC culture medium to the plate to neutralize the cell detachment solution activity and transfer the detached cells to a new 100 mm tissue culture dish without any coating.</li>
		<li>Incubate the cells at room temperature for 30 min to allow the MEFs to attach to the plate.</li>
		<li>Carefully collect the supernatant which contains unattached iPSCs, and centrifuge at 230 x g at 4 &#176;C for 4 min. 
		NOTE: Carelessly flushing the plate bottom leads to detachment of MEFs.</li>
	</ol>
	</li>
	<li>Differentiation of iPSCs toward MSCs (Day 0 - 28):
	<ol>
		<li>Coat three wells of a 6-well plate with 1 mL of 0.1% gelatin per well at room temperature for 30 min.</li>
		<li>Remove the supernatant of the collected iPSCs from 2.2.4.</li>
		<li>Resuspend iPSCs in 6 mL of MSC differentiation medium (Table 1) and seed cells in the three coated wells with 2 mL per well (Day 0).</li>
		<li>Change the MSC differentiation medium every two days to remove unattached and/or dead cells (Day 1 - 28).</li>
	</ol>
	</li>
	<li>Maturation of iPSC-derived MSCs (Day 28 - 45):
	<ol>
		<li>Remove the spent MSC differentiation medium and wash cells with 1 mL of 1x DPBS.</li>
		<li>Trypsinize MSCs with 0.5 mL of 0.25% Trypsin-EDTA per well at room temperature for 3 min.</li>
		<li>Neutralize trypsin by 1.5 mL of the MEF/MSC culture medium and collect MSCs from three wells into one 15 mL conical tube. Centrifuge at 230 x g at 4 &#176;C for 4 min.</li>
		<li>Remove the supernatant and resuspend MSCs in 3 mL of MEF/MSC culture medium and plate on one 60 mm 0.1% gelatin-coated plate (Day 28).</li>
		<li>Culture MSCs in MEF/MSC culture medium and change medium every two days. When the cells reach 100% confluence, subculture MSCs at a 1:3 ratio using 0.25% Trypsin-EDTA. 
		NOTE: The MSCs display a fibroblast-like morphology (Figure 2B). When MSCs grow in a high density, elongated MSCs can form a swirl-like pattern (Figure 2B).</li>
		<li>Perform the immunofluorescent staining to evaluate iPSC-derived MSCs by examining MSC surface markers CD44, CD73, CD105, and CD166 (Figure 2C). 
		NOTE: The dilution of antibodies are shown in Table 2. Immunofluorescent staining of live cells is performed according to manufacturer&#39;s instructions.</li>
		<li>After confirmation, expand MSCs at a 1:3 ratio in 100 mm tissue culture dishes. Freeze down vials (3 vials per 100 mm tissue culture dish) using the freezing medium containing 10% DMSO and 90% FBS. 
		NOTE: To enrich the MSC population, MSCs can be sorted using CD105+, CD24- criteria by flow cytometry9,17. iPSC-derived MSCs differentiated using the PDGF-AB-based method commonly can be maintained for 2 - 3&#160;months in culture without loss of MSC identity9. Freeze MSCs from an early passage number after confirming MSC characteristics.</li>
	</ol>
	</li>
</ol>
3. Differentiation of LFS MSCs to Osteoblasts (Figure 3A)
<ol>
	<li>Preparation of MSCs for the osteogenic differentiation (Day -1)
	<ol>
		<li>To ensure an adequate quantity of cells to complete the osteogenic differentiation protocol, begin with MSCs cultured in 100 mm tissue culture dishes to 95% confluence. Remove the spent culture medium and wash MSCs with 5 mL of 1x DPBS.</li>
		<li>Trypsinize MSCs with 1 mL of 0.25% trypsin-EDTA per 100 mm dish at room temperature for 3 min. To ensure the cells are not over trypsinized, stop trypsinization when 50% of the cell detachment is observed under the microscope.</li>
		<li>Neutralize trypsin by 9 mL of MEF/MSC culture medium and collect MSCs from 3 wells into one 15 mL conical tube. Centrifuge at 230 x g at 4 &#176;C for 4 min.</li>
		<li>Aspirate the supernatant, resuspend MSCs in 5 mL of MSC medium, and count the number of cells by a hemocytometer.</li>
		<li>Plate proper number of MSCs on a culture plate. 
		NOTE: The detail of cell numbers of 12-well plate, 6 well-plate, and 100 mm tissue culture dishes are summarized in Table 3.</li>
	</ol>
	</li>
	<li>Induction of osteogenic differentiation by osteoblast differentiation medium (ODM) (Table 1) (Day 0 - 24):
	<ol>
		<li>Aspirate MEF/MSC culture medium and replace with the proper volume of ODM (1 mL, 2 mL, and 8 mL for each well of 12-well plate, each well of 6-well plate, and 100 mm tissue culture dish, respectively) to differentiate MSCs to osteoblasts (Day 0).</li>
		<li>Aspirate the spent ODM and replace with the proper volume of ODM every two days during the osteogenic differentiation process.</li>
		<li>Perform the alkaline phosphatase (AP) and the Alizarin Red S (ARS) staining to detect bone-associated alkaline phosphatase produced by preosteoblasts and mineral deposition produced by mature osteoblasts, respectively, on Day 3, 6, 9, 12, 15, 18, 21, and 24 (Figure 3B). 
		NOTE: AP staining and ARS staining are performed using commercially available kit following manufacturer protocol.</li>
		<li>Examine the expression levels of preosteoblast and mature osteoblast markers (ALPL and COL1A1 for preosteoblasts; PTH1R and BGLAP for mature osteoblasts) by qRT-PCR (Figure 3C). 
		NOTE: Positive AP staining is expected after Day 9 of osteogenic differentiation and positive ARS staining can be expected after Day 21 of osteogenic differentiation. The MSCs growing in MEF/MSC culture medium can be served as a negative control of AP staining and ARS staining.</li>
	</ol>
	</li>
</ol>
4. The Xenograft Model to Study In Vivo Tumorigenesis of LFS MSC-derived Osteoblasts (Figure 4A)
<ol>
	<li>Differentiation of LFS MSCs to osteoblasts:
	<ol>
		<li>Seed 3 - 4.5 x 105 MSCs in 100 mm tissue culture dish. Prepare five dishes for one subcutaneous injection.</li>
		<li>Culture MSCs in ODM to differentiate MSCs to osteoblasts as described in 3.2 until Day 14.</li>
	</ol>
	</li>
	<li>Preparation of differentiated osteoblasts for subcutaneous injection:
	<ol>
		<li>To prepare osteoblast detachment solution (Table 1), dissolve 1 g type II collagenase in 1,000 mL of &#945;MEM medium to make the collagenase II solution (1 mg/mL). Keep the collagenase II solution at -20 &#176;C. Mix 0.25% Trypsin-EDTA and the collagenase II solution at a 1:1 ratio to make the osteoblasts detachment solution.</li>
		<li>Remove the spent ODM and wash differentiated osteoblasts with 5 mL of 1x DPBS per 100 mm dish.</li>
		<li>Add 1.5 mL of osteoblast detachment solution per 100 mm dish and incubate dishes at 37 &#176;C for 30 min. Examine and ensure the osteoblast detachment by microscopy.</li>
		<li>Neutralize the osteoblasts detachment solution by ODM and collect the detached osteoblasts in a 50 mL conical tube. Centrifuge at 230 x g at 4 &#176;C for 5 min.</li>
		<li>Remove the supernatant and resuspend cells in 25 mL of ODM medium. Pass the cells through a 70 &#181;m cell strainer to remove aggregated cells. Count the cell numbers using a hemocytometer.</li>
		<li>Prepare 1 x 107 osteoblasts per subcutaneous injection, and centrifuge cells at 230 x g at 4 &#176;C for 4 min.</li>
		<li>Resuspend the 1 x 107 osteoblasts in 50 &#181;L ice-cold 1x DPBS. Mix differentiated osteoblasts with 50 &#181;L of phenol-red free basement membrane matrix (osteoblasts suspension and basement membrane matrix were mixed at a 1:1 ratio) and maintain osteoblasts on ice before injection.</li>
	</ol>
	</li>
	<li>In vivo xenograft tumor model of LFS MSC-derived osteoblasts:
	<ol>
		<li>Anesthetize 8-week-old female immunocompromised NU/NU mice in a chamber supplying 5% (v/v) inhaled isoflurane in 1 L/min of oxygen (provided by CLAMC). After the animal becomes recumbent, switch the anesthetic delivery system to the nose cone, supplying 2% (v/v) inhaled isoflurane in 200mL/min of oxygen (provided by CLAMC). Monitor the depth of anesthesia by checking the corneal and pedal reflexes to make the mice are sufficiently anesthetized and are not suffering from discomfort during the procedure.</li>
		<li>Disinfect the area to be injected with alternating swabs of chlorhexidine and 70% isopropanol. Use 26 G needle to perform subcutaneous injection of basement membrane matrix-mixed LFS osteoblasts into one side of hind legs of 8-week-old immunocompromised NU/NU mice (Figure 4A).</li>
		<li>Monitor mice by performing weekly palpation at injection sites for growth of subcutaneous nodular densities. Tumor formation can be observed around 6-10 weeks after injection (Figure 4B).</li>
		<li>Euthanize the mice by carbon dioxide asphyxiation method followed by cervical dislocation. Dissect the developed tumors. Determine the tumor grade and the differentiation index, preosteoblasts, and mature osteoblasts by various staining methods (e.g. Hematoxylin and Eosin (H&#38;E) staining, Picro Sirius Red staining, and von Kossa staining respectively).</li>
	</ol>
	</li>
</ol>

<ol><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Modeling+familial+Alzheimer%27s+disease+with+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Hum+Mol+Genet%22%5BJournal%5D)">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/pubmed?term=((Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Patient-specific+induced+pluripotent+stem-cell+models+for+long-QT+syndrome%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">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/pubmed?term=((Modelling+the+long+QT+syndrome+with+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Patient-specific+induced+pluripotent+stem-cell-derived+models+of+LEOPARD+syndrome%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Modeling+familial+cancer+with+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Myeloid+Dysregulation+in+a+Human+Induced+Pluripotent+Stem+Cell+Model+of+PTPN11-Associated+Juvenile+Myelomonocytic+Leukemia%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">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/pubmed?term=((Functional+analysis+of+a+chromosomal+deletion+associated+with+myelodysplastic+syndromes+using+isogenic+human+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Stage-Specific+Human+Induced+Pluripotent+Stem+Cells+Map+the+Progression+of+Myeloid+Transformation+to+Transplantable+Leukemia%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">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/pubmed?term=((Colonic+organoids+derived+from+human+induced+pluripotent+stem+cells+for+modeling+colorectal+cancer+and+drug+testing%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">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/pubmed?term=((Modeling+Cancer+with+Pluripotent+Stem+Cells%5BTitle%5D)+AND+%22Trends+Cancer%22%5BJournal%5D)">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/pubmed?term=((Osteosarcoma%3A+Molecular+Pathogenesis+and+iPSC+Modeling%5BTitle%5D)+AND+%22Trends+Mol+Med%22%5BJournal%5D)">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/pubmed?term=((Li-Fraumeni+Syndrome+Disease+Model%3A+A+Platform+to+Develop+Precision+Cancer+Therapy+Targeting+Oncogenic+p53%5BTitle%5D)+AND+%22Trends+Pharmacol+Sci%22%5BJournal%5D)">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/pubmed?term=((Derivation+of+clinically+compliant+MSCs+from+CD105%2B%2C+CD24-+differentiated+human+ESCs%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">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/pubmed?term=((A+homozygous+p53+R282W+mutant+human+embryonic+stem+cell+line+generated+using+TALEN-mediated+precise+gene+editing%5BTitle%5D)+AND+%22Stem+Cell+Res%22%5BJournal%5D)">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>

The authors have nothing to disclose.

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><tbody><tr><td>&lt;strong&gt;Plastic ware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>100 mm Dish</td><td>Corning</td><td>430107</td><td></td></tr><tr><td>60 mm Dish</td><td>Corning</td><td>430166</td><td></td></tr><tr><td>6-well Plate</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>12-well Plate</td><td>Falcon</td><td>353043</td><td></td></tr><tr><td>48-well Plate</td><td>Falcon</td><td>353078</td><td></td></tr><tr><td>1 mL Pipet Tip</td><td>USA Scientific</td><td>1111-2721</td><td></td></tr><tr><td>200 &amp;micro;L Pipet Tip</td><td>USA Scientific</td><td>1111-0706</td><td></td></tr><tr><td>10 &amp;micro;L Pipet Tip</td><td>USA Scientific</td><td>1111-3700</td><td></td></tr><tr><td>5 mL Serological Pipette</td><td>SARSTEDT</td><td>86.1253.001</td><td></td></tr><tr><td>10 mL Serological Pipette</td><td>SARSTEDT</td><td>86.1254.001</td><td></td></tr><tr><td>25 mL Serological Pipette</td><td>SARSTEDT</td><td>86.1685.001</td><td></td></tr><tr><td>50 mL Tube, PP</td><td>SARSTEDT</td><td>62.547.100</td><td></td></tr><tr><td>15 mL Tube, PP</td><td>SARSTEDT</td><td>62.554.100</td><td></td></tr><tr><td>&lt;strong&gt;Culture materials and Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CytoTune- iPS 2.0 Sendai Reprogramming Kit</td><td>Invitrogen</td><td>A16517</td><td>Commercial Sendai virus reprogramming kit</td></tr><tr><td>Corning hESC-Qualified Matrix</td><td>Corning</td><td>354277</td><td>Basement membrane matrix</td></tr><tr><td>CF1 MEFs, irradiated</td><td>ThermoFisher</td><td>A34180</td><td></td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D5671</td><td></td></tr><tr><td>DMEM/F12</td><td>Corning</td><td>10-090-CV</td><td></td></tr><tr><td>&amp;alpha;MEM</td><td>Corning</td><td>10-022-CV</td><td></td></tr><tr><td>StemMACS iPS-Brew XF</td><td>Miltenyi Biotec</td><td>130-104-368</td><td>Commercial iPSC medium</td></tr><tr><td>KnockOut DMEM/F-12</td><td>ThermoFisher</td><td>12660012</td><td></td></tr><tr><td>FBS Opti-Gold</td><td>GenDEPOT</td><td>F0900-050</td><td></td></tr><tr><td>KnockOut Serum Replacement</td><td>ThermoFisher</td><td>A3181502</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>MEM Nonessential Amino Acids</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>L-Glutamine Solution</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>Human FGF-basic (bFGF)</td><td>PEPROTECH</td><td>100-18B</td><td></td></tr><tr><td>Recombinant Human PDGF-AB</td><td>PEPROTECH</td><td>100-00AB</td><td></td></tr><tr><td>&amp;beta;-Glycerophosphate</td><td>Sigma-Aldrich</td><td>G9422</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>A4902</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A5960</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline, 1x (DPBS)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>StemMACS Passaging Solution XF</td><td>Miltenyi Biotec</td><td>130-104-688</td><td>Commercial passaging solution</td></tr><tr><td>Accutatse Cell Detachment Solution</td><td>Corning</td><td>25-058-CI</td><td>Cell detachment solution</td></tr><tr><td>Thiazovivin (ROCK Inhibitor)</td><td>Calbiochem</td><td>420220</td><td></td></tr><tr><td>0.25% Trypsin-EDTA Solution</td><td>Sigma-Aldrich</td><td>T4049</td><td></td></tr><tr><td>Collagenase, Type II&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher</td><td>17101015</td><td></td></tr><tr><td>Human NANOG Antibody</td><td>R&amp;amp;D System</td><td>AF1997</td><td></td></tr><tr><td>OCT4 Antibody (H-134)</td><td>Santa Cruz</td><td>sc-9081</td><td></td></tr><tr><td>Human/Mouse SSEA-4 PE-conjugated Antibody</td><td>R&amp;amp;D System</td><td>FAB1435P</td><td></td></tr><tr><td>Alexa Fluor 555 Mouse Anti-Human TRA-1-81 Antigen</td><td>DB Biosciences</td><td>560123</td><td></td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Goat IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>705-545-003</td><td></td></tr><tr><td>Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>111-545-144</td><td></td></tr><tr><td>PE Mouse Anti-Human CD105</td><td>eBioscience</td><td>12-1057-42</td><td></td></tr><tr><td>FITC Mouse Anti-Human CD44</td><td>DB Biosciences</td><td>555478</td><td></td></tr><tr><td>PE Mouse Anti-Human CD73</td><td>DB Biosciences</td><td>550257</td><td></td></tr><tr><td>PE Mouse Anti-Human CD166</td><td>DB Biosciences</td><td>560903</td><td></td></tr><tr><td>FITC Mouse Anti-Human CD24</td><td>DB Biosciences</td><td>555427</td><td></td></tr><tr><td>Donkey Serum</td><td>Jackson ImmunoResearch</td><td>017-000-121</td><td></td></tr><tr><td>Goat Serum</td><td>Jackson ImmunoResearch</td><td>005-000-121</td><td></td></tr><tr><td>Alkaline Phosphatase Staining Kit II</td><td>Stemgent</td><td>00-0055</td><td></td></tr><tr><td>Alizarin Red S</td><td>Sigma-Aldrich</td><td>A5533</td><td></td></tr><tr><td>TRIzol Reagent</td><td>ThermoFisher</td><td>15596018</td><td></td></tr><tr><td>Chloroform</td><td>ThermoFisher</td><td>C298-500</td><td></td></tr><tr><td>2-Propanol</td><td>ThermoFisher</td><td>A416-4</td><td></td></tr><tr><td>Ethanol, Absolute, Molecular Biology Grade</td><td>ThermoFisher</td><td>BP28184</td><td></td></tr><tr><td>DNase I, RNase-free (1 U/&amp;micro;L)</td><td>ThermoFisher</td><td>EN0521</td><td></td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>BioRad</td><td>1708891BUN</td><td></td></tr><tr><td>iQ SYBR Green Supermix</td><td>BioRad</td><td>1708884</td><td></td></tr><tr><td>Matrigel Matrix High Concentration (HC), Phenol-Red Free</td><td>Corning</td><td>354262</td><td></td></tr><tr><td>1 mL Slip Tip Syringe, 26 Gauge x 5/8 Inch</td><td>DB Biosciences</td><td>309597</td><td></td></tr></tbody></table>

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

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...

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 ...

modeling-osteosarcoma-using-li-fraumeni-syndrome-patient-derived

Research

JoVE Journal

Cancer Research

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

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

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

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

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

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

Learn to model osteosarcoma using Li-Fraumeni Syndrome patient-derived iPSCs differentiated into osteoblasts. This protocol enables mechanistic studies, biomarker identification, and drug screening for cancer research.

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

Explore More Videos