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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 & Care (CLAMC) which is accredited by American Association for Laboratory Animal Care (AAALAC International).The human subjects in this study fall under Scenario A ("No Human Subjects Research") 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....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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