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

摘要

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.

摘要

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.

引言

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 cells^1,2. 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 fibroblasts^1,2. Subsequent studies successfully generated iPSCs from various sources, such as urine⁴, blood^5,6, 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 RA^9,10. 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'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.

方案

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 trimmed tissue to a well of a 6-well plate and add 5 ml of Dulbecco's modified Eagle's medium (DMEM) with 20% Fetal bovine serum (FBS).
   4. Chop the tissues with the scissors until the pieces are small enough to penetrate a disposable pipette.
   5. 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.
   6. 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 °C with shaking for 4 hr.
   7. 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.
   8. Remove the supernatant without disturbing the pellet and add 40 ml of media to resuspend the pellet.
   9. Repeat steps 1.1.10-1.1.11.
   10. Resuspend the pellet in 25 ml of DMEM with 20% FBS and wait for the large clumps of tissue to sink to the bottom.
   11. Transfer the supernatant to a 100 mm dish and incubate at 37 °C in 5% CO₂ for 14 day.
2. Synoviocyte Maintenance and Expansion
   1. Discard the used media from the plate and wash the cells with 5 ml of PBS.
   2. Add 1 ml PBS/1 mM EDTA and incubate at 37 °C in 5% CO₂ for 2 min.
   3. 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.
   4. Remove the supernatant without disturbing the pellet and resuspend the pellet in 30 ml of DMEM with 20% FBS.
   5. Transfer the cells to 3 x 100 mm dishes, without leaving any visible leftover material.
   6. 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.

2. Reprogramming FLSs Using Lentiviruses-encoding Yamanaka Factors

1. Transduction (D0)
   1. Seed 3 × 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 °C in 5% CO₂.
   2. 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 °C. Note: Lentivirus was produced by the procedure described in our previous study¹¹.
   3. While thawing the virus, change the media to FLS growth media (20% FBS plus antibiotics in DMEM) containing 10 µg/ml hexadimethrine bromide and 50 µg/ml ascorbic acid.
   4. After changing the media, add 30 µl of lentivirus to the cells and mix gently. To improve infection, centrifuge the plate at 680 x g, 35 °C for 30 min.
   5. After centrifugation, incubate the cells at 37 °C in 5% CO₂.
2. Maintenance Until Reprogramming is Visible
   1. For 3 day, replace the media daily with FLS growth media containing 0.1 mM sodium butyrate and 50 µg/ml ascorbic acid.
   2. 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 µg/ml ascorbic acid.
      Note: The components of the iPSC media is given in the materials/equipment list.
3. Splitting Cells for Colony Formation
   1. Prepare a vitronectin-coated 6-well plate.
      1. Add 60 µl 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 µg/mL.
   2. On D5, wash the cells with PBS.
   3. Add 1 ml PBS/1 mM EDTA to detach the cells and incubate at 37 °C, 5% CO₂ for 2 min.
   4. Harvest the cells and centrifuge at 250 x g, RT for 2 min.
   5. Split the cells at 3 different ratios (1:3, 1:6, and 1:9) to achieve different confluencies. Add 900 µl of media to the cell pellet and resuspend. Add 300, 150, and 100 µl of the cell mixture per well of a 6-well plate to achieve a ratio of 1:3, 1:6, and 1:9, respectively.
   6. 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.
4. Colony picking
   1. Prepare a 48-well vitronectin-coated plate by adding 500 µl vitronectin to the wells, and incubate at RT for at least 1 hr.
   2. Place the microscope on a clean bench, and remove the 6-well plate from the incubator.
   3. Remove the vitronectin solution from the 48-well plate and add 500 µl iPSC media supplemented with 10mM Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor.
   4. Using a 10p pipette tip, cut around the colony. Transfer the picked colony to one well of the 48-well plate.
   5. After picking several colonies, incubate the cells at 37 °C, 5% CO₂.
   6. 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.

3. Immunofluorescence Staining

1. Cell Preparation
   1. Place a sterile 18 mm cover glass into a 12-well plate.
   2. Add 1 ml of PBS to cool and rinse the cover glass.
   3. Replace with 1 ml of a 10 µg/ml vitronectin solution.
   4. Incubate the plate at RT for at least 1 hr.
   5. Discard the vitronectin solution and plate iPSCs in the vitronectin-coated 12-well plate and culture for 7 days at 37 °C, 5% CO₂, changing the media daily.
2. Cell Staining
   1. Discard the culture media and wash the cells with PBS once.
   2. Fix the cells in 0.4% paraformaldehyde (PFA) for 30 min at RT.
   3. Permeabilize the cells with 0.1% Triton X-100 for 5 min at RT.
   4. Remove the permeabilization solution and block with PBS containing 2% bovine serum albumin (BSA) for 30 min at RT.
   5. Dilute the antibodies in PBS containing 2% BSA according to Table 1. Incubate the cells with the primary antibodies for 2 hr at RT.
   6. Add the secondary antibody (diluted 1:200) and incubate the cells for 1 hr at RT, avoiding light.
   7. Treat the cells with 1 µl/mL DAPI for 10 min.
   8. Place the cover glass on top of the slide glass with antifade reagent and incubate at RT for 24 hr, avoiding light.
   9. Verify expression with a fluorescence microscope.

4. Real-time Polymerase Chain Reaction (RT-PCR)

1. Extract mRNA from the cell pellet using the guanidinium thiocyanate-phenol-chloroform extraction method¹¹.
2. Amplify cDNA from 2 µg of total mRNA using reverse transcription¹¹.
3. Mix the components required for PCR using 2 µl of cDNA template¹¹.
4. Perform RT-PCR and verify the results by gel electrophoresis¹¹.

5. Alkaline Phosphatase (AP) Staining

1. Culture iPSCs for 5-7 days at 37 °C, 5% CO₂ prior to staining.
2. Aspirate the media and fix the cells with 4% PFA for 1 min.
3. Discard the fixative and rinse the cells with 1X rinse buffer.
4. 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.
5. Incubate the cells with the staining solution at RT for 15 min, avoiding light.
6. Discard the staining solution and rinse the cells with rinse buffer.
7. Cover the cells with PBS to prevent drying and verify expression using a bright-field microscope.

结果

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 ...

讨论

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...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
100 mm 培养皿	TPP	93100
6 孔板	TPP	92006
50 ml 角质管	SPL	50050
15 mlL 角质管	SPL	50015
10 ml 一次性移液器	Falcon	7551
5 ml 一次性移液器	Falcon	7543
12 孔板	TPP	92012
FLS 隔离材料
手术剪刀
手术钳
DPBS	Life Technologies	14190-144
DMEM	Life Technologies	11995-073
青霉素链霉素	Sigma Aldrich	P4333
胎牛血清 （FBS）	Life Technologies	16000-044
胶原酶	Sigma Aldrich	C6885-100MG
封口膜	Sigma Aldrich	54956
PBS/1 mM EDTA	Life Technologies	12604-039
iPSC 生成材料
DMEM	Life Technologies	11885
MEM 非必需氨基酸溶液 （100x）	Life Technologies	11140-050
β-巯基乙醇	Sigma Aldrich	M3148
聚凝	胺化学图标	TR-1003-G
青霉素链霉素	Life Technologies	P4333
胎牛血清 （FBS）	Life Technologies	16000-044
DPBS	Life Technologies	14190-144
慢病毒
DMEM/F12，HEPES	Life Technologies	11330-057	iPSC 培养基成分（500 ml）
碳酸氢钠	Life Technologies	25080-094	iPSC 培养基成分（浓度：543 &μ;g/ml）
亚硒酸钠	Sigma Aldrich	S5261	iPSC 培养基成分 （浓度：14 ng/mL）
人转食蛋白	Sigma Aldrich	T3705	iPSC 培养基成分（浓度：10.7 &μ;g/ml）
碱性 FGF2	Peprotech	100-18B	iPSC 培养基成分 （浓度：100 ng/ml）
人胰岛素	Life Technologies	12585-014	iPSC 培养基成分（浓度：20 &μ;g/ml）
人 TGF&β;1	Peprotech	100-21	iPSC 培养基成分（浓度：2 ng/ml）
抗坏血酸	Sigma Aldrich	A8960	iPSC 培养基成分 （浓度： 64 μg/ml）
聚凝	胺 Chemicon	TR-1003
丁酸钠	Sigma Aldrich	B5887
玻连蛋白	Life Technologies	A14700
ROCK 抑制剂	Sigma Aldrich	Y0503
<强> 质量控制材料
18 mm 盖玻片	Superior	HSU-0111580
4% 多聚甲醛 （PFA）	技术 &创新	BPP-9004
Triton X-100	BIOSESANG	9002-93-1
牛血清白蛋白 （BSA）	载体实验室	SP-5050
抗 SSEA4 抗体	MAB4304
抗 Oct4 抗体	Santa Cruz	SC9081
抗 TRA-1-60 抗体	Millipore	MAB4360
抗 Sox2 抗体	Biolegend	630801
抗 TRA-1-81 抗体	Millipore	MAB4381
抗 Klf4 抗体	Abcam	ab151733
Alexa Fluor 488 山羊抗小鼠 IgG （H+L） 抗体	分子探针	A11029
Alexa Fluor 594 山羊抗兔 IgG （H+L） 抗体	探针	A11037
DAPI	分子探针	D1306
延长金抗淬灭试剂	Invitrogen	P36934
载玻片，涂层	Hyun Il Lab-Mate	HMA-S9914
Trizol	Invitrogen	15596-018
氯仿	Sigma Aldrich	366919
异丙醇	Millipore	109634
乙醇	Duksan	64-17-5
RevertAid 第一链 cDNA 合成试剂盒	Thermo Scientfic	K1622
i-Taq DNA 聚合酶	iNtRON BIOTECH	25021
UltraPure 10x TBE 缓冲液	Life Technologies	15581-044
加载星	形 Dyne Bio	A750
琼脂糖	Sigma-Aldrich	9012-36-6
1 kb （+） DNA 分子量标准物	Enzynomics	DM003
碱性磷酸酶	Millipore	SCR004