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 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.
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'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 "bone tumor in a dish" 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.
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)
2. Differentiation of LFS iPSCs to Mesenchymal Stem Cells (MSCs) (Figure 2A)
3. Differentiation of LFS MSCs to Osteoblasts (Figure 3A)
4. The Xenograft Model to Study In Vivo Tumorigenesis of LFS MSC-derived Osteoblasts (Figure 4A)
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 non-integrating reprogramming method. Therefore, stable LFS iPSC clones should be free of Sendai virus genome and loss of exogenous OCT4, SOX2, KLF4, and c-MYC transgenes, which can be examined by RT-PCR using specific primers (Figure 1B). As it is suggested in the manufacturer's instructions, generation of Sendai virus-free iPSCs depends on both culture and passage conditions. Under the described culture conditions in this protocol, removal of Sendai virus in iPSCs usually can be detected after 10 passages (Figure 1B). The established LFS iPSC clones should exhibit the typical hESC morphology and show the positive AP activity (Figure 1C). LFS iPSCs highly express pluripotency factor mRNAs (NANOG, OCT4, SOX2, DPPA4, and REX1), comparable to hESC H1 line and much higher than parental fibroblasts (Figure 1D). The expression of pluripotency factors (NANOG, OCT4) and hESC markers (SSEA4 and TRA-1-81) can also be examined by immunofluorescent staining (Figure 1E).
iPSCs are maintained on MEFs for at least 14 days before initiating MSC differentiation (Figure 2A). Although a lot of cell death happen during MSC differentiation, masses of differentiated cells are visible on the cell culture plate and fibroblast-like MSCs at the edge of the cell masses can be observed at Day 28. (Figure 2B). After sub culturing differentiated cells in MEF/MSC culture medium, differentiated MSCs proliferate quickly and show fibroblast-like morphology around Day 35, and then gradually represent an elongated shape and form a swirl-like pattern at Day 40 (Figure 2B). Differentiated LFS MSCs express MSC markers, including CD44, CD73, CD105, and CD166 by immunofluorescence staining (Figure 2C).
Figure 3A outlines osteoblastic differentiation. When MSCs are subjected to osteogenic differentiation signals, the differentiated cells start to show positive alkaline phosphatase activity at Day 9 (Figure 3B). ARS staining can be used to detect mineral deposition produced by mature osteoblasts. The bright red color staining instead of brown color staining indicates the positive result of ARS staining, which can be observed after Day 21 (Figure 3B). The differentiating osteoblasts show increasing expression level of preosteoblast genes (ALPL and COL1A1) and mature osteoblast genes (BGLAP and PTH1R) during osteogenic differentiation.
In vivo xenograft model can be established by subcutaneously injecting LFS MSC-derived osteoblasts in NU/NU mice (Figure 4A). Tumors can be observed 6 – 10 weeks after subcutaneous injection (Figure 4B). The LFS osteoblast-derived tumors demonstrated immature osteoblast characteristics, positive AP activity (AP staining), positive collagen matrix deposition (picrosirius red staining) but negative mineralization (von Kossa staining) (Figure 4C).
Figure 1: iPSC generation from LFS patient fibroblasts. (A) Schematic diagram of iPSC generation. (B) RT-PCR detection of Sendai virus genome and transgenes (KOS (KLF4, OCT4, and SOX2), KLF4, and c-MYC) in the reprogrammed iPSC clone after 10 passages (left) and fibroblast post-infection Day 11 (right, positive control). LFS iPSC clone is Sendai virus free after 10 passages. GAPDH is shown as internal control. (C) Cell morphology of LFS iPSCs and AP staining. Scale bar, 50 µm (D) qRT-PCR of NANOG, SOX2, OCT4, DPPA4, and REX1 mRNA expression in LFS iPSCs. hESC H1 line and parental LFS fibroblasts are used as a positive and a negative control, respectively. The mRNA expression is normalized to GAPDH expression. The relative mRNA expression is adjusted to hESC H1 line as 1. (E) Immunostaining of hESC pluripotent transcription factors (NANOG and OCT4) and hESC surface markers (SSEA4 and TRA-1-81) in LFS iPSCs. Scale bar, 50 µm. The dilution of antibodies used in this study are shown in Table 2.The primer sequences are shown in Table 4. Please click here to view a larger version of this figure.
Figure 2: Differentiation of LFS iPSCs to MSCs. (A) Schematic diagram of PDGF-AB-induced MSC differentiation. (B) Cell morphology of LFS iPSC-derived MSCs at differentiation Day 28, Day 36, and Day 40+. Scale bar, 100 µm. (C) Immunofluorescence staining demonstrates that differentiated LFS MSCs exhibit CD44+, CD73+, CD105+, CD166+, and CD24– signature. Scale bar, 30 µm. The dilution of antibodies used in this study are shown in Table 2. Please click here to view a larger version of this figure.
Figure 3: Differentiation of LFS MSCs to osteoblasts. (A) Schematic diagram of osteogenic differentiation. (B) AP and ARS staining are performed at different time points (Day 3, 6, 9, 12, 15, 18, 21, and 24). Osteoblastic differentiation of LFS MSC-derived osteoblasts is expected to lead to positive AP staining around Day 9 and positive ARS staining at Day 21. (C) The qRT-PCR analysis demonstrates the increased expression of pre-osteoblast (ALPL and COL1A1) and mature osteoblast (PTH1R and BGLAP) genes during osteogenic differentiation. The mRNA expression is normalized to GAPDH expression. Expression levels were relative to cells at differentiation Day 0. The primer sequences are shown in Table 4. Please click here to view a larger version of this figure.
Figure 4: In vivo tumorigenesis of LFS MSC-derived osteoblasts. (A) Schematic diagram of tumor xenograft of LFS osteoblasts. (B) NU/NU mice bear LFS osteoblast-derived tumors after 10 weeks of subcutaneous injection. (C) LFS osteoblast-derived tumors were examined by H&E, AP, picrosirius red, and von Kossa staining for morphology, bone-associated AP, collagen, and mineral deposits, respectively. The LFS-derived tumors represent immature osteoblast characteristics showing positive AP activity, positive collagen, and negative mineral mineralization. Scale bar, 1 mm. Please click here to view a larger version of this figure.
Fibroblast Medium (500 mL) | |
DMEM | 440 mL |
Heat-inactivated FBS | 50 mL |
Antibiotics(Pen/Strep) (100x) | 5 mL |
Nonessential Amino Acid (100x) | 5 mL |
2-Mercaptoethanol | 3.5 µL |
MEF/MSC Culture Medium (500 mL) | |
DMEM | 440 mL |
Heat-inactivated FBS | 50 mL |
Antibiotics(Pen/Strep) (100x) | 5 mL |
L-Glutamine (100x) | 5 mL |
hESC medium (500 mL) | |
DMEM/F-12 | 384.5 mL |
KnockOut Serum Replacement | 100 mL (total: 20%) |
Nonessential Amino Acid (100x) | 5 mL |
Antibiotics (Pen/Strep) (100x) | 5 mL |
L-Glutamine (100x) | 5 mL |
bFGF (10 µg/mL) | 500 µL |
2-Mercaptoethanol | 3.5 µL |
MSC Differentiation Medium (500 mL) | |
KnockOut DMEM/F-12 or DMEM/F-12 | 445 mL |
KnockOut Serum Replacement | 50 mL |
bFGF (10 µg/mL) | 500 µL (10 ng/mL) |
PDGF-AB (25 µg/mL) | 200 µL (10 ng/mL) |
Nonessential Amino Acid (100x) | 5 mL |
2-Mercaptoethanol | 3.5 µL |
Osteoblast Differentiation Medium (ODM) (500 mL) | |
αMEM | 395 mL |
Heat-inactivated FBS | 50 mL |
10 mM β-Glycerophosphate | 50 mL (1.08 g in 50 mL αMEM) |
0.1 µM Dexamethasone (Light Sensitive) | 10 µL of 5 mM dexamethasone |
200 µM Ascorbic Acid (Light Sensitive) | 100 µL of 1 mM ascorbic acid |
Antibiotics (Pen/Strep) (100x) | 5 mL |
Matrigel coating solution (50 mL) | |
Basement Membrane Matrix | 2 mL |
DMEM/F-12 (pre-cold 4 °C) | 48 mL |
Osteoblast Detachment Solution (50 mL) | |
0.25% Trypsin-EDTA | 25 mL |
Collagenase II Solution (1 mg/mL) | 25 mL |
Note: Prepare osteoblast detachment solution fresh right before use. Collagenase II solution is stored at -20 °C for up to 6 months. |
Table 1: Compositions of fibroblast medium, MEF/MSC culture medium, hESC medium, MSC differentiation medium, ODM, and osteoblast detachment solution.
Antibody Name | Dilution |
NANOG | 1:500 |
OCT4 | 1:300 |
SSEA-4 PE-conjugated | 1:600 |
TRI-1-81 | 1:600 |
Donkey Anti-Goat IgG | 1:500 |
Goat Anti-Rabbit IgG | 1:500 |
CD105 | 1:500 |
CD44 | 1:500 |
CD73 | 1:500 |
CD166 | 1:500 |
CD24 | 1:500 |
Table 2: Antibody dilution.
Culture Plate | Seeding Density | Assay |
12-well Plate | 0.67×104 cells per well | Alkaline phosphatase staining (AP staining) |
Alizarin red S staining (ARS staining) | ||
6-well Plate | 2×104 cells per well | RT-PCR detection |
Preosteoblast markers: ALPL, COL1A1 | ||
Mature osteoblast markers: PTH1R, BGLAP | ||
100 mm Dish | 3 – 4.5×105 cells per plate | In vivo tumorigenesis |
Table 3: Seeding density of MSCs for osteoblastic differentiation.
Sendai Virus Primers | |
Target | Primer Sets |
SeV | Forward: GGATCACTAGGTGATATCGAGC |
Reverse: ACCAGACAAGAGTTTAAGAGATATGTATC | |
KOS (KLF4/OCT4/SOX2) | Forward: ATGCACCGCTACGACGTGAGCGC |
Reverse: ACCTTGACAATCCTGATGTGG | |
KLF4 | Forward: TTCCTGCATGCCAGAGGAGCCC |
Reverse: AATGTATCGAAGGTGCTCAA | |
c-MYC | Forward: TAACTGACTAGCAGGCTTGTCG |
Reverse: TCCACATACAGTCCTGGATGATGATG | |
RT-PCR Primers | |
Target | Primer Sets |
NANOG | Forward: TTTGTGGGCCTGAAGAAAACT |
Reverse: AGGGCTGTCCTGAATAAGCAG | |
SOX2 | Forward: AGAAGAGGAGAGAGAAAGAAAGGGAGAGA |
Reverse: GAGAGAGGCAAACTGGAATCAGGATCAAA | |
OCT4 | Forward: AACCTGGAGTTTGTGCCAGGGTTT |
Reverse: TGAACTTCACCTTCCCTCCAACCA | |
DPPA4 | Forward: GACCTCCACAGAGAAGTCGAG |
Reverse: TGCCTTTTTCTTAGGGCAGAG | |
REX1 | Forward: GCCTTATGTGATGGCTATGTGT |
Reverse: ACCCCTTATGACGCATTCTATGT | |
ALPL | Forward: GGGACTGGTACTCAGACAACG |
Reverse: GTAGGCGATGTCCTTACAGCC | |
COL1A1 | Forward: GTGCGATGACGTGATCTGTGA |
Reverse: CGGTGGTTTCTTGGTCGGT | |
PTH1R | Forward: AGTGCGAAAAACGGCTCAAG |
Reverse: GATGCCTTATCTTTCCTGGGC | |
BGLAP | Forward: GGCGCTACCTGTATCAATGG |
Reverse: GGCGCTACCTGTATCAATGG |
Table 4: Primer information.
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 iPSCs on MEFs before differentiation. 80 – 90% confluency of iPSCs is preferred to initiate MSC differentiation. The overgrowth of iPSCs will impair cell survival during the differentiation process. The last critical point is the seeding density when starting MSC differentiation. In our hands, high cell density promotes iPSC attachment to the gelatin-coated plate. One 100 mm dish iPSCs can be seeded into three wells of 6-well plate. Initiation of MSC differentiation by directly resuspending and plating iPSCs in MSC differentiation medium produces great stresses on iPSCs, therefore occasionally resulting in severe cell death after seeding. High density of iPSCs increases the success rate of MSC differentiation.
Unlike MSC differentiation, the osteoblastic differentiation process is more straightforward. To achieve reproducible differentiation results, ensure the initiating MSC numbers are consistent. Results of osteoblastic differentiation from the same cell line may vary from batch to batch, therefore, setting up differentiation for different lines at the same time will give more comparative results among groups. The increase of MSC numbers shorten the differentiation process indicated by positive AP and ARS staining at an earlier time point. Also, ensure the change of ODM medium is handled in a gentle way and powerful vacuum suction system is not recommended in the late differentiation stage (Day 18 – 24) due to the potential detachment of aggregated osteoblasts during culture process.
The differentiated osteoblasts used for in vivo xenograft experiment are osteoblasts at the Day 14 differentiation time point. The osteoblasts later than Day 14 may aggregate and be difficult to dissociate due to the huge accumulations of collagens and other bone matrix materials produced by osteoblasts. To prevent the difficulty of osteoblast dissociation, LFS MSCs can be seeded in 2 – 3-fold higher density in the initiation step of osteoblastic differentiation to facilitate osteoblast differentiation. LFS MSC-derived osteoblasts can be dissociated and collected at the Day 6 – 10 differentiation time point for in vivo tumorigenesis assay at the Day 6 – 7 differentiation time point. The LFS xenograft tumor model demonstrates LFS MSC-derived osteoblasts recapitulate in vivo tumorigenic ability, which provides an alternative platform to study LFS-associated osteosarcoma.
In summary, an LFS iPSC-based osteosarcoma model provides a valuable recourse for osteosarcoma research. In addition to osteosarcoma, LFS patients suffer from various other types of cancers such as soft tissue sarcoma, breast cancer, and brain tumor. Therefore, LFS iPSC-based disease models can be extended to model other LFS related malignancies. Combining LFS patient-derived iPSCs and engineered mutant p53 (mutp53) hESCs18, LFS disease model also has great value in delineating pathogenesis of mutp53 associated malignancies and developing novel therapeutic strategies targeting oncogenic p5316.
The authors have nothing to disclose.
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.
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 |