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Developmental Biology

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Published: October 16th, 2016

DOI:

10.3791/54072

1CiSTEM Laboratory, Convergent Research Consortium for Immunologic Disease, Division of Rheumatology, Seoul St. Mary's Hospital, Republic of Korea, 2Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, Institute of Medical Science, Republic of Korea, 3College of Medicine, The Catholic University of Korea, Republic of Korea

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Pluripotent stem cells are the next-generation platform in various clinical and biological fields. They are a promising tool that can be used in disease modeling, drug screening, and regenerative medical therapy. Human Embryonic Stem Cells (hESCs) were mainly used to study and understand pluripotent cells. However, isolated by the destruction of the human blastocyst, hESCs are associated with several ethical concerns. In 2007, Dr. Shinya Yamanaka and his team reversed the cell programming process and developed stem cells from human adult somatic cells1,2. Therefore, unlike hESCs, induced-Pluripotent Stem Cells (iPSCs) can be generated from mature somatic ce....

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Ethics Statement: This study protocol was approved by the institutional review board of The Catholic University of Korea (KC12TISI0861).

1. Synoviocyte Isolation and Expansion

  1. Synoviocyte Isolation
    1. Sterilize two pairs of surgical scissors and one pair of forceps.
    2. Transfer the synovial tissue to a 100 mm dish and wash with 5 ml of phosphate-buffered saline (PBS) containing 1% penicillin/streptomycin.
    3. Cut off the yellowish fat tissue and bone residues. Transfer the trimm.......

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In this study, we describe a protocol to generate iPSCs from FLSs using a lentiviral system. Figure 1A shows a simple scheme of the FLS isolation protocol. Following surgical removal of the synovium, the tissue was chopped into small pieces using surgical scissors. Collagenase was added to isolate the cells from the clumps of tissue. Cells were incubated for 14 days before further processing. Figure1B shows the morphology of the isolated .......

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Before the discovery of iPSCs, scientists mainly used ESCs to study stem cell biology and other cell lineages through differentiation. However, ESCs originate from the inner mass of a blastocyst, which is an early-stage embryo. To isolate ESCs, destruction of the blastocyst is inevitable, raising ethical issues that are impossible to overcome. Moreover, although ESCs have stemness characteristics and pluripotency, they cannot be obtained from individuals and are sometimes not an ideal tool for personalized analysis and d.......

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This work was supported by the Research Program funded by the Korea Centers for Disease Control and Prevention (HI13D2188).

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Name Company Catalog Number Comments
100mm Dish TPP 93100
6-well Plate TPP 92006
50 mL Cornical Tube SPL 50050
15 mL Cornical Tube SPL 50015
10 mL Disposable Pipette Falcon 7551
5 mL Disposable Pipette Falcon 7543
12-well Plate TPP 92012
FLS Isolation Materials
Surgical Scissors
Surgical Forcep
DPBS Life Technologies 14190-144
DMEM Life Technologies 11995-073
Penicilin Streptomycin Sigma Aldrich P4333
Fetal Bovine Serum Life Technologies 16000-044
Collagenase Sigma Aldrich C6885-100MG
Parafilm Sigma Aldrich 54956
PBS/1 mM EDTA Life Technologies 12604-039
iPSC Generation Materials
DMEM Life Technologies 11885
MEM Non-Essential Amino Acids Solution (100X) Life Technologies 11140-050
β-Mercaptoethanol Sigma Aldrich M3148
Polybrene Chemicon TR-1003-G
Penicilin Streptomycin Life Technologies P4333
Fetal Bovine Serum Life Technologies 16000-044
DPBS Life Technologies 14190-144
Lentivirus
DMEM/F12, HEPES Life Technologies 11330-057 iPSC media ingredient (500 mL)
Sodium Bicarbonate Life Technologies 25080-094 iPSC media ingredient (Conc.: 543 μg/mL)
Sodium Selenite Sigma Aldrich S5261 iPSC media ingredient  (Conc.: 14 ng/mL)
Human Transfferin Sigma Aldrich T3705 iPSC media ingredient (Conc.: 10.7 μg/mL)
Basic FGF2 Peprotech 100-18B iPSC media ingredient  (Conc.: 100 ng/mL)
Human Insulin Life Technologies 12585-014 iPSC media ingredient (Conc.: 20 μg/mL)
Human TGFβ1 Peprotech 100-21 iPSC media ingredient (Conc.: 2 ng/mL)
Ascorbic Acid Sigma Aldrich A8960 iPSC media ingredient  (Conc.: 64 μg/mL)
Polybrene Chemicon TR-1003
Sodium Butyrate Sigma Aldrich B5887
Vitronectin Life Technologies A14700
ROCK Inhibitor Sigma Aldrich Y0503
Guality Control Materials
18 mm Cover Glass Superior HSU-0111580
4% Paraformaldyhyde Tech & Innovation BPP-9004
Triton X-100 BIOSESANG 9002-93-1
Bovine Serum Albumin Vector Lab SP-5050
Anti-SSEA4 Antibody Millipore MAB4304
Anti-Oct4 Antibody Santa Cruz SC9081
Anti-TRA-1-60 Antibody Millipore MAB4360
Anti-Sox2 Antibody Biolegend 630801
Anti-TRA-1-81 Antibody Millipore MAB4381
Anti-Klf4 Antibody Abcam ab151733
Alexa Fluor 488 goat anti-mouse IgG (H+L) antibody Molecular Probe A11029
Alexa Fluor 594 goat anti-rabbit IgG (H+L) antibody Molecular Probe A11037
DAPI Molecular Probe D1306
Prolong gold antifade reagent Invitrogen P36934
Slide Glass, Coated Hyun Il Lab-Mate HMA-S9914
Trizol Invitrogen 15596-018
Chloroform Sigma Aldrich 366919
Isoprypylalcohol Millipore 109634
Ethanol Duksan 64-17-5
RevertAid First Strand cDNA Synthesis kit Thermo Scientfic K1622
i-Taq DNA Polymerase iNtRON BIOTECH 25021
UltraPure 10X TBE Buffer Life Technologies 15581-044
loading star Dyne Bio A750
Agarose Sigma-Aldrich 9012-36-6
1kb (+) DNA ladder marker Enzynomics DM003
Alkaline Phosphatase Millipore SCR004

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  8. Scott, D. L., Wolfe, F., Huizinga, T. W. Rheumatoid arthritis. Lancet. 376 (9746), 1094-1108 (2010).
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  10. Bartok, B., Firestein, G. S. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev. 233 (1), 233-255 (2010).
  11. Lee, J., et al. Generation of disease-specific induced pluripotent stem cells from patients with rheumatoid arthritis and osteoarthritis. Arthritis Res Ther. 16 (1), R41 (2014).

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