Name: Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients (Video) | JoVE Journal
Uploaded: 2016-10-16T14:00:00.000+00:00
Duration: 9 min 31 s
Description: Learn to generate induced-pluripotent stem cells from fibroblast-like synoviocytes using a lentiviral system without feeder cells. This method enables rheumatoid arthritis pathology research and regenerative medicine applications.

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5 minutes

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

Ethics Statement: This study protocol was approved by the institutional review board of The Catholic University of Korea (KC12TISI0861).
1. Synoviocyte Isolation and Expansion
<ol>
	<li>Synoviocyte Isolation
	<ol>
		<li>Sterilize two pairs of surgical scissors and one pair of forceps.</li>
		<li>Transfer the synovial tissue to a 100 mm dish and wash with 5 ml of phosphate-buffered saline (PBS) containing 1% penicillin/streptomycin.</li>
		<li>Cut off the yellowish fat tissue and bone residues. Transfer the trimmed tissue to a well of a 6-well plate and add 5 ml of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 20% Fetal bovine serum (FBS).</li>
		<li>Chop the tissues with the scissors until the pieces are small enough to penetrate a disposable pipette.</li>
		<li>Transfer the tissue-containing media to a 50 ml conical tube. Harvest the remaining material by adding 5 ml of DMEM with 20% FBS to the 6-well plate and then transfer to the tube.</li>
		<li>Thaw collagenase on ice. Add collagenase to a final concentration of 0.01% and seal the tube with parafilm. Incubate in a water bath at 37 &#176;C with shaking for 4 hr.</li>
		<li>After incubation, fill the tube with DMEM with 20% FBS, until the total volume is 50 ml and centrifuge at 300 x g, RT for 10 min.</li>
		<li>Remove the supernatant without disturbing the pellet and add 40 ml of media to resuspend the pellet.</li>
		<li>Repeat steps 1.1.10-1.1.11.</li>
		<li>Resuspend the pellet in 25 ml of DMEM with 20% FBS and wait for the large clumps of tissue to sink to the bottom.</li>
		<li>Transfer the supernatant to a 100 mm dish and incubate at 37 &#176;C in 5% CO2 for 14 day.</li>
	</ol>
	</li>
	<li>Synoviocyte Maintenance and Expansion
	<ol>
		<li>Discard the used media from the plate and wash the cells with 5 ml of PBS.</li>
		<li>Add 1 ml PBS/1 mM EDTA and incubate at 37 &#176;C in 5% CO2 for 2 min.</li>
		<li>Tap the dish gently and transfer the cells to a 15 ml conical tube. Centrifuge the cells at 250 x g, RT for 2 min.</li>
		<li>Remove the supernatant without disturbing the pellet and resuspend the pellet in 30 ml of DMEM with 20% FBS.</li>
		<li>Transfer the cells to 3 x 100 mm dishes, without leaving any visible leftover material.</li>
		<li>Replace the media with fresh media every 3 d. Split the cells at 80% confluency using 1 ml PBS/1 mM EDTA. Maintain until passage 3 before use. Divide each dish of cells into 3 dishes in every split. 
		NOTE: After reaching passage 3, cells that are not going to be used immediately can be frozen.</li>
	</ol>
	</li>
</ol>
2. Reprogramming FLSs Using Lentiviruses-encoding Yamanaka Factors
<ol>
	<li>Transduction (D0)
	<ol>
		<li>Seed 3 &#215; 104 cells per well of a 6-well plate in growth media (500 ml of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin). Incubate the cells O/N at 37 &#176;C in 5% CO2.</li>
		<li>The following day, remove one vial of lentivirus containing 4 Yamanaka factors: Oct4, Klf4, Sox2 and c-Myc from the freezer and thaw at 4 &#176;C. Note: Lentivirus was produced by the procedure described in our previous study11.</li>
		<li>While thawing the virus, change the media to FLS growth media (20% FBS plus antibiotics in DMEM) containing 10 &#181;g/ml hexadimethrine bromide and 50 &#181;g/ml ascorbic acid.</li>
		<li>After changing the media, add 30 &#181;l of lentivirus to the cells and mix gently. To improve infection, centrifuge the plate at 680 x g, 35 &#176;C for 30 min.</li>
		<li>After centrifugation, incubate the cells at 37 &#176;C in 5% CO2.</li>
	</ol>
	</li>
	<li>Maintenance Until Reprogramming is Visible
	<ol>
		<li>For 3 day, replace the media daily with FLS growth media containing 0.1 mM sodium butyrate and 50 &#181;g/ml ascorbic acid.</li>
		<li>The next day, replace the media with a mixture of FLS growth media and iPSC media (1:1 ratio) containing 0.1 mM sodium butyrate and 50 &#181;g/ml ascorbic acid. 
		Note: The components of the iPSC media is given in the materials/equipment list.</li>
	</ol>
	</li>
	<li>Splitting Cells for Colony Formation
	<ol>
		<li>Prepare a vitronectin-coated 6-well plate.
		<ol>
			<li>Add 60 &#181;l vitronectin to 6 ml PBS without Ca2+ and Mg2+. Put 2 ml of mixture into each wells and incubate in RT for at least 1 hr. Note: The working concentration of vitronectin is 5 &#181;g/mL.</li>
		</ol>
		</li>
		<li>On D5, wash the cells with PBS.</li>
		<li>Add 1 ml PBS/1 mM EDTA to detach the cells and incubate at 37 &#176;C, 5% CO2 for 2 min.</li>
		<li>Harvest the cells and centrifuge at 250 x g, RT for 2 min.</li>
		<li>Split the cells at 3 different ratios (1:3, 1:6, and 1:9) to achieve different confluencies. Add 900 &#181;l of media to the cell pellet and resuspend. Add 300, 150, and 100 &#181;l of the cell mixture per well of a 6-well plate to achieve a ratio of 1:3, 1:6, and 1:9, respectively.</li>
		<li>Replace the media daily with iPSC media until colonies appear. Colonies will appear after about D18. Note: At this stage, iPSC colonies co-exist with the non-reprogrammed FLSs.</li>
	</ol>
	</li>
	<li>Colony picking
	<ol>
		<li>Prepare a 48-well vitronectin-coated plate by adding 500 &#181;l vitronectin to the wells, and incubate at RT for at least 1 hr.</li>
		<li>Place the microscope on a clean bench, and remove the 6-well plate from the incubator.</li>
		<li>Remove the vitronectin solution from the 48-well plate and add 500 &#181;l iPSC media supplemented with 10mM Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor.</li>
		<li>Using a 10p pipette tip, cut around the colony. Transfer the picked colony to one well of the 48-well plate.</li>
		<li>After picking several colonies, incubate the cells at 37 &#176;C, 5% CO2.</li>
		<li>Maintain the cells until the colonies are big enough for transfer. Note: We usually spilt the cells when the colony gets out of the visible field of the microscope, when viewed at 100X magnification.</li>
	</ol>
	</li>
</ol>
3. Immunofluorescence Staining
<ol>
	<li>Cell Preparation
	<ol>
		<li>Place a sterile 18 mm cover glass into a 12-well plate.</li>
		<li>Add 1 ml of PBS to cool and rinse the cover glass.</li>
		<li>Replace with 1 ml of a 10 &#181;g/ml vitronectin solution.</li>
		<li>Incubate the plate at RT for at least 1 hr.</li>
		<li>Discard the vitronectin solution and plate iPSCs in the vitronectin-coated 12-well plate and culture for 7 days at 37 &#176;C, 5% CO2, changing the media daily.</li>
	</ol>
	</li>
	<li>Cell Staining
	<ol>
		<li>Discard the culture media and wash the cells with PBS once.</li>
		<li>Fix the cells in 0.4% paraformaldehyde (PFA) for 30 min at RT.</li>
		<li>Permeabilize the cells with 0.1% Triton X-100 for 5 min at RT.</li>
		<li>Remove the permeabilization solution and block with PBS containing 2% bovine serum albumin (BSA) for 30 min at RT.</li>
		<li>Dilute the antibodies in PBS containing 2% BSA according to Table 1. Incubate the cells with the primary antibodies for 2 hr at RT.</li>
		<li>Add the secondary antibody (diluted 1:200) and incubate the cells for 1 hr at RT, avoiding light.</li>
		<li>Treat the cells with 1 &#181;l/mL DAPI for 10 min.</li>
		<li>Place the cover glass on top of the slide glass with antifade reagent and incubate at RT for 24 hr, avoiding light.</li>
		<li>Verify expression with a fluorescence microscope.</li>
	</ol>
	</li>
</ol>
4. Real-time Polymerase Chain Reaction (RT-PCR)
<ol>
	<li>Extract mRNA from the cell pellet using the guanidinium thiocyanate-phenol-chloroform extraction method11.</li>
	<li>Amplify cDNA from 2 &#181;g of total mRNA using reverse transcription11.</li>
	<li>Mix the components required for PCR using 2 &#181;l of cDNA template11.</li>
	<li>Perform RT-PCR and verify the results by gel electrophoresis11.</li>
</ol>
5. Alkaline Phosphatase (AP) Staining
<ol>
	<li>Culture iPSCs for 5-7 days at 37 &#176;C, 5% CO2 prior to staining.</li>
	<li>Aspirate the media and fix the cells with 4% PFA for 1 min.</li>
	<li>Discard the fixative and rinse the cells with 1X rinse buffer.</li>
	<li>Prepare the reagents for AP staining. Mix the reagents in the following ratio: Fast Red Violet : Naphthol AS-BI phosphate solution : water = 2:1:1.</li>
	<li>Incubate the cells with the staining solution at RT for 15 min, avoiding light.</li>
	<li>Discard the staining solution and rinse the cells with rinse buffer.</li>
	<li>Cover the cells with PBS to prevent drying and verify expression using a bright-field microscope.</li>
</ol>

<ol><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>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>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>Zhou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+human+induced+pluripotent+stem+cells+from+urine+samples%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Generation of human induced pluripotent stem cells from urine samples.</a> Nat Protoc. 7 (12), 2080-2089 (2012).</li>
<li>Haase, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+induced+pluripotent+stem+cells+from+human+cord+blood%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Generation of induced pluripotent stem cells from human cord blood.</a> Cell Stem Cell. 5 (4), 434-441 (2009).</li>
<li>Loh, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+induced+pluripotent+stem+cells+from+human+blood%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Generation of induced pluripotent stem cells from human blood.</a> Blood. 113 (22), 5476-5479 (2009).</li>
<li>Aasen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnol. 26 (11), 1276-1284 (2008).</li>
<li>Scott, D. L., Wolfe, F., Huizinga, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rheumatoid+arthritis%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Rheumatoid arthritis.</a> Lancet. 376 (9746), 1094-1108 (2010).</li>
<li>Chang, S. K., Gu, Z., Brenner, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblast-like+synoviocytes+in+inflammatory+arthritis+pathology%3A+the+emerging+role+of+cadherin-11%5BTitle%5D)+AND+%22Immunol+Rev%22%5BJournal%5D)">Fibroblast-like synoviocytes in inflammatory arthritis pathology: the emerging role of cadherin-11.</a> Immunol Rev. 233 (1), 256-266 (2010).</li>
<li>Bartok, B., Firestein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblast-like+synoviocytes%3A+key+effector+cells+in+rheumatoid+arthritis%5BTitle%5D)+AND+%22Immunol+Rev%22%5BJournal%5D)">Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis.</a> Immunol Rev. 233 (1), 233-255 (2010).</li>
<li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+disease-specific+induced+pluripotent+stem+cells+from+patients+with+rheumatoid+arthritis+and+osteoarthritis%5BTitle%5D)+AND+%22Arthritis+Res+Ther%22%5BJournal%5D)">Generation of disease-specific induced pluripotent stem cells from patients with rheumatoid arthritis and osteoarthritis.</a> Arthritis Res Ther. 16 (1), R41(2014).</li>
</ol>

The authors have nothing to disclose.

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...

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 cells, avoiding the ethical hurdles.
Usually, iPSCs are generated by the delivery of four exogenous genes: Oct4, Sox2, Klf4, and c-Myc. These Yamanaka factors are originally delivered using lentiviral and retroviral systems. The first iPSCs were derived from mouse somatic cells3. Afterwards, the technique was applied to human dermal fibroblasts1,2. Subsequent studies successfully generated iPSCs from various sources, such as urine4, blood5,6, keratinocytes7, and several other cell types. However, there are some somatic cells that have not been used in reprogramming, and screening of the reprogramming capabilities of various cell types from specific tissues in disease state, is still required.
Rheumatoid arthritis (RA) is a disease that can strike all joints and lead to autoimmune conditions in other organs. RA affects about 1% of adults in the developed world. It is a rather common disease and its incidence increases each year8. However, RA is hard to identify in the early stages and oncebone destruction occurs there is no treatment that can recover the damage. Moreover, drug efficacy differs from patient to patient, and it is hard to predict the medicine that is required. Therefore, the development of a drug-screening method is needed, and a cell material that can reflect the conditions of RA is required.
Fibroblast-like Synoviocytes (FLSs) are an active cellular participant in the pathogenesis of RA9,10. FLSs exist in the synovial intimal lining between the joint capsule and cavity, which is also referred to as the synovium. By supporting the joint structure and providing nutrients to the surrounding cartilage, FLSs usually play a crucial role in joint function and maintenance. However, FLSs in RA have an invasive phenotype. RA FLSs have a cancer-like phenotype, eventually destroying the surrounding bone by infinite proliferation10. With this unique characteristic, FLSs can be used as a promising material that can reflect the pathobiology of RA. Yet, these cells are rarely produced, and the cell phenotypes alter as the cells go through several passages in in vitro conditions. Therefore, it can be complicated to use RA FLSs as a tool that can represent the patient&#39;s condition.
Theoretically, RA patient-derived iPSCs (RA-iPSCs) can become an ideal tool for drug screening and further research. Generated iPSCs have self-renewal ability and can be maintained and expanded in vitro. With pluripotency, these cells can be differentiated into mature chondrocyte and osteocyte lineages, which can contribute cell material for specific research in RA and other bone-related diseases11.
In this study, we demonstrate how to isolate and expand FLSs from a surgically removed synovium, and how to generate RA-iPSCs from FLSs using lentiviruses containing Yamanaka factors.

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

generation of induced-pluripotent stem cells using fibroblast-like synoviocytes isolated from joints of rheumatoid arthritis patients

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

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.

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 ...

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 ...

The overall goal of this experiment is to generate induced-pluripotent stem cells, or IPCs, from fibroblast-like synoviocytes isolated from the synovial tissue of a rheumatoid arthritis inflamed joint. Currently, iPSC's can be generated from various human primary cells. We successfully generated iPSC's from human fibroblast-like synoviocytes, which are the key pathological cells of rheumatoid joints.This may help to investigate the pathology of rheumatoid arthritis. This periodical can help answer key questions about degeneration of IPSC's, using RA-FLSs. Begin by placing the synovial tissue in a 100 millimeter dish and washing the tissue with 5 milliliters of PBS supplemented with antibiotics.Remove the yellowish fat tissue and bone residue. Then transfer the trimmed tissue into one well of a six well plate containing 5 milliliters of DMEM supplemented with FBS and use scissors to mince the tissue until the pieces are small enough to penetrate a disposable pipet tip. Transfer the medium to a 50 milliliter conical tube, then add 5 milliliters of fresh DMEM plus FBS to the well to collect any remaining tissue and transfer the entire well contents to the tube.Next, add ice-thawed collagenase to a final concentration of 0.01%to the tube and seal the tube with parafilm for incubation in a 37 degree Celsius water bath with shaking. After four hours, fill the tube with fresh DMEM plus FBS to a final volume of 50 milliliters and collect the tissue fragments by centrifugation. Remove the supernatant without disturbing the pellet then re-suspend the cells in 40 milliliters of fresh medium for another centrifugation.Re-suspend the second pellet in 25 miilliters of fresh DMEM plus FBS and allow the large clumps of tissue to settle. Then transfer the supernatant into a new 100 millimeter dish and incubate the synoviocytes at 37 degrees Celsius and 5%CO2 for 14 days. To expand the synoviocyte culture discard the used medium and wash the cells in 5 milliliters of PBS.Remove the PBS and dissociate the cells with 1 milliliter of EDTA in the cell culture incubator for 2 minutes. At the end of the incubation, tap the dish gently to detach any remaining adherent cells and transfer the resulting cell suspension into a 15 milliliter conical tube for centrifugation. Re-suspend the pellet in 30 milliliters of DMEM and FBS and split the cells into 3 new 100 millimeter dishes.Feed the synoviocytes with fresh medium every 3 days, splitting the cells into 3 new cultures at 80%confluency as just demonstrated. On transduction Day zero, see 3 x 10 to the 4th FLS's per well onto a 6 well plate in fresh growth medium for an overnight incubation at 37 degrees Celsius in 5%CO2. The next day, thaw 1 vial of Lenti-virus containing 4 Yamanaka factors at 4 degrees Celsius and replace the FLS culture medium with FLS growth medium supplemented with hexadimethrine bromide and ascorbic acid.When the vial contents have thawed add 30 microliters of lenti-virus to the cells and gently mix to homogeneously distribute the virus. To improve the infection, centrifuge the plate and return the cells to the incubator replacing the medium every day with fresh FLS growth medium containing sodium butyrate and ascorbic acid for 3 days. On the 4th day, replace the medium with a mixture of FLS growth and iPSC medium containing sodium butyrate and ascorbic acid.On day 5 add 60 microliters of vitronectin to 6 milliliter of PBS without calcium and magnesium and coat 3 wells of a 6 well plate with 2 milliliters of the solution each for at least an hour at room temperature. While the vitronectin is setting, wash the transduced cells with PBS and detach them with EDTA as demonstrated. After collecting the dissociated cells by centrifuguation, re-suspend the pellet in 900 microliters of fresh medium.Remove the excess vitronectin from the wells and add 300, 150, and 100 microliters of cells into individual wells. Then return the cells to the incubator, replacing the medium daily with fresh iPSC medium until colonies appear. To pick the colonies, first add 500 microliters of iPCS medium, supplemented with ROCK inhibitor to each well of a vitronectin-coated 48 well plate.Then, place the FLS cultures under a dissecting microscope on a clean bench, and use a 10 microliter pipet tip to cut around a colony of interest. Using a 200 microliter pipet tip, transfer the picked colony to 1 well of the 48 well plate. When all of the colonies have been picked, place the plate in the cell culture incubator until the colonies are big enough for transfer.Splitting the cells when the colonies are out of the visible field of the microscope, as viewed at 100X magnification. In this image, the morphology of the isolated FLS after an initial 14 day culture can be observed. The cells exhibit the characteristics of fibroblasts in general with a similar expression of the fibrotic markers vimentin and fibronectin as well as a low expression of the macrophage-like synoviocyte marker CD-68.8 to 11 days after the Yamanaka factor lenti-viral transduction, small colonies begin to appear that can be picked and amplified for 5 to 10 passages.These rheumatoid arthritis iPSC's express alkaline phosphatase indicating that they remain undifferentiated. The cells also express pluripotent markers as confirmed by RT-PCR and immunofluorescence analysis. Rheumatoid arthritis iPCS's exhibit a normal chromosomal pattern of 44+XY as determined by karyotyping.Furthur, 12 weeks after their injection into SCID mice, the rheumatoid arthritis iPSC formed teratoma, that display diverse tissue phenotypes. With their germ layer differentiation confirmed by immunofluorescent staining. Once mastered, iPSC's can be generated from FLSs with synovium.After watching the video, you should have a better understanding of how to isolate from synovial tissue and how to induce reprogramming using them. These RAF-FLS-derived iPSC's can be useful for studying regeneration or drug screening in rheumatology. Once these iPSC's are established, various kinds of target cells or organs can be generated.Which may be useful for studying the disease or other future applications, such as gene editing and so forth.

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Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients (Video) | JoVE Journal

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

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