Name: isolation and cellular phenotyping of mesenchymal stem cells derived from synovial fluid and bone marrow of minipigs
Uploaded: 2016-07-02T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs at JoVE.com

We gratefully acknowledge the financial support provided by the Next-Generation BioGreen 21 Program (No. PJ007969), Rural Development Administration, and the National Research Foundation (grant no. NRF-2015R1D1A1A01056639) of the Republic of Korea.

Animal experiments were authorized by the Animal Center for Biomedical Experimentation at Gyeongsang National University.
1. Preparations for the Animal Procedure
<ol>
	<li>Prepare the adult female minipigs for the non-invasive collection of MSCs from bone marrow and synovial fluid. Perform the clinical examination of the minipigs one day prior to anesthesia and sample collection.
	<ol>
		<li>Provide clinically normal animals for physical examinations, which include body temperature, respira...

<ol>
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W., Niemann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+perspectives+for+porcine-to-human+xenografts.">The perspectives for porcine-to-human xenografts.</a> Comp Immunol Micro biol Infect Dis. 32 (2), 91-105 (2009).</li><li>Zeng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+adult+progenitor+cells+from+swine+bone+marrow.">Multipotent adult progenitor cells from swine bone marrow.</a> Stem Cells. 24 (11), 2355-2366 (2006).</li><li>Zeng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+and+functional+consequences+of+bone+marrow-derived+multipotent+progenitor+cell+transplantation+in+hearts+with+postinfarction+left+ventricular+remodeling.">Bioenergetic and functional consequences of bone marrow-derived multipotent progenitor cell transplantation in hearts with postinfarction left ventricular remodeling.</a> Circulation. 115 (14), 1866-1875 (2007).</li><li>Sekiya, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells+in+synovial+fluid+increase+in+the+knee+with+degenerated+cartilage+and+osteoarthritis.">Human mesenchymal stem cells in synovial fluid increase in the knee with degenerated cartilage and osteoarthritis.</a> J Orthop Res. 30 (6), 943-949 (2012).</li><li>Mochizuki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+chondrogenic+potential+of+fibrous+synovium-+and+adipose+synovium-derived+cells+compared+with+subcutaneous+fat-derived+cells:+Distinguishing+properties+of+mesenchymal+stem+cells+in+humans.">Higher chondrogenic potential of fibrous synovium- and adipose synovium-derived cells compared with subcutaneous fat-derived cells: Distinguishing properties of mesenchymal stem cells in humans.</a> Arthritis Rheum. 54 (3), 843-853 (2006).</li></ol>

Minipigs were used to establish MSCs isolation from bone marrow and synovial fluid. To eliminate various physiological conditions, such as age, gender, and disease, minipigs from isogenic background donors were chosen to accurately evaluate the cell source dependent characterization. Pigs are known to be anatomically, physiologically, and genetically similar to humans, and in particular, minipigs can produce size-matched organs; therefore, they can be used as a substitute donor species for xenotransplantation14,15</...

Multipotent mesenchymal stem cells (MSCs) can be classified into mesenchymal cell lineages, and MSCs have been established and isolated from various tissue sources, such as from bone marrow, umbilical cords, placentas, adipose tissue, dermal skin, skeletal muscle, hair follicles, synovial membranes, and teeth1-5. Currently, attention has been given to synovial-derived MSCs because these cells may help treat joint diseases, such as bone fraction, osteoarthritis, and rheumatoid arthritis (RA), and due to the regenerative potential of MSCs in damaged cartilage or bone, they may also help treat immune modulation or autoimmune diseases6-8. The majority of research of MSCs derived from synovial sources have employed MSCs from the synovial membrane rather than from synovial fluid2,9; thus, the biological understanding of synovial-fluid-derived MSCs is limited.
Synovial fluid is an easily accessible source of MSCs, which can be aspirated during the diagnoses or treatments of patients and during diagnostic confirmations of various arthritis conditions that do not require invasive procedures, such as osteoarthritis or RA. The population of synovial-fluid-derived MSCs in the synovial cavity is significantly increased in arthritic patients compared to non-arthritic patients10-12. Therefore, synovial fluid is an excellent source candidate for MSCs, particularly for autologous stem cell therapy in patients with inflamed or injured joints. Additionally, synovial-fluid-derived MSCs have a chondrogenic capacity, immunomodulation abilities, and a high rate of proliferation. This study established protocols for bone marrow extraction and synovial fluid aspiration from minipigs using non-invasive methods and for the in vitro expansion of isolated cells and analysis of cell phenotypes.

<table border="0" cellpadding="0" cellspacing="0" width="499">
	<tbody>
		<tr height="70">
			<td height="70" style="height:70px;width:136px;">Advanced Dulbecco&#8217;s modified Eagle medium (ADMEM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">12491-023</td>
			<td style="width:143px;">MSC culture medium</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:136px;">Dulbecco&#8217;s phosphate-buffered saline (DPBS)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">14190-144</td>
			<td style="width:143px;">Cell washing medium, free of Ca2+/Mg2+,</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:136px;">Fetal bovine serum (FBS)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">16000-044</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:136px;">Glutamax</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">35050-061</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:136px;">Penicillin/streptomycin</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">10378-016</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Basic fibroblast growth factor (bFGF)</td>
			<td style="width:143px;">Simga</td>
			<td style="width:79px;">F0291</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Bovine serum albumin (BSA)</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">A6003</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">Trypsin-EDTA</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">25200-072</td>
			<td style="width:143px;">Cell dissociation reagent&#160;</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:136px;">&#946;-mercaptoethanol</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">M7522</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:136px;">Isotype antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 550616</td>
			<td style="width:143px;">Isotype Control</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:136px;">CD29 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 552369</td>
			<td style="width:143px;">Integrin beta-1 MSCs marker</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:136px;">CD44 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 553133</td>
			<td style="width:143px;">Cell surface glycoprotein MSCs marker</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">CD45 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 340664</td>
			<td style="width:143px;">Hematopoietic stem cells marker</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">CD34 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 555821</td>
			<td style="width:143px;">Hematopoietic stem cells marker</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:136px;">CD90 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 555595</td>
			<td style="width:143px;">Thy-1 membrane glycoprotein MSCs marker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:136px;">MHC Class II antibody</td>
			<td style="width:143px;">Santa Cruz</td>
			<td style="width:79px;">SC-32247</td>
			<td style="width:143px;">Antigen presenting cells marker</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Vimentin antibody</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">Sigma-S6389</td>
			<td style="width:143px;">Type III intermediate filament MSCs marker</td>
		</tr>
		<tr height="115">
			<td height="115" style="height:115px;width:136px;">Alkaline phosphatase</td>
			<td style="width:143px;">Promega</td>
			<td style="width:79px;">S3841</td>
			<td style="width:143px;">Mixture of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT)</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:136px;">Ficoll-Paque</td>
			<td style="width:143px;">GE Healthcare</td>
			<td style="width:79px;">17-1440-02</td>
			<td style="width:143px;">Density gradient centrifugation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Cell strainer</td>
			<td style="width:143px;">BD Falcon</td>
			<td style="width:79px;">352340</td>
			<td style="width:143px;">40&#181;m nylon cell strainer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">15-mL polystyrene conical tube</td>
			<td style="width:143px;">BD Falcon</td>
			<td style="width:79px;">351095</td>
			<td style="width:143px;">Sample collection and cell isolation</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:136px;">35 mm dishes</td>
			<td style="width:143px;">Nunc</td>
			<td style="width:79px;">153066</td>
			<td style="width:143px;">Cell culture dish</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:136px;">Bone marrow extractor</td>
			<td style="width:143px;">GmbH Medizintechnologie, Germany</td>
			<td style="width:79px;">1145-1W010</td>
			<td style="width:143px;">TrokaBone, 3.0x100mm</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Hematocritchamber</td>
			<td style="width:143px;">Marinfeld</td>
			<td style="width:79px;">640130</td>
			<td style="width:143px;">Cell counting chamber</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:136px;">Atropine</td>
			<td style="width:143px;">Jeil Pharmaceutical Co, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Atropine sulfate Hydrate</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:136px;">Acepromazine</td>
			<td style="width:143px;">Samu Median, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Pre-anaesthetic sedative and antiemetic drug</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">Medetomidine</td>
			<td style="width:143px;">Pfizer</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Anesthetic and analgesic drug</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:136px;">Enrofloxacin</td>
			<td style="width:143px;">Bayer Healthcare, Germany</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Anti-biotics</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">Meloxicam</td>
			<td style="width:143px;">Over Veterinary Medicine, Argentina</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Anti-inflammatory and analgesic drug</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Isoflurane</td>
			<td style="width:143px;">Hana pharm, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Inhalational anesthesia</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:136px;">Povidone iodine&#160;</td>
			<td style="width:143px;">Korea Pharma, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Sterilization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:136px;">Ethanol</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">E7023</td>
			<td style="width:143px;">Sterilization agent</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Heparin</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">H3393</td>
			<td style="width:143px;">Anti-coagulating agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:136px;">Formaldehyde</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">F8775</td>
			<td style="width:143px;">Cell fixation agent</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:136px;">Ophthalmologic ointment&#160;</td>
			<td style="width:143px;">Pfizer</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Oxytetracycline HCL with polymyxin B sulfate</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Circulating water blanket</td>
			<td style="width:143px;">Gaymar Industries</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Warming system during and after anesthesia</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:136px;">Skin stapler</td>
			<td style="width:143px;">Covidien, USA</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Suture skin closure</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">CO2 Incubator</td>
			<td style="width:143px;">Thermo Forma</td>
			<td style="width:79px;">3111</td>
			<td style="width:143px;">Cell culture Equipment</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:136px;">Flow cytometer</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Cell analyzer</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:136px;">Minipig</td>
			<td style="width:143px;">PWG Genetics Korea, Ltd.</td>
			<td style="width:79px;">T-type</td>
			<td style="width:143px;">Miniature pig</td>
		</tr>
	</tbody>
</table>

isolation and cellular phenotyping of mesenchymal stem cells derived from synovial fluid and bone marrow of minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

Establishment of MSCs Derived from the Bone Marrow and Synovial Fluid of Minipigs
MSCs were successfully isolated from the bone marrow and articular synovial joints of the minipigs and expanded in vitro (Figure 2). Syringe aspiration of synovial fluid is simple, and it is possible to obtain sufficient adherent cells during in vitro cultivation of the primary cells. The morpholo...

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Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

The overall goal of this procedure is the non-invasive collection of synovial fluid and bone marrow from minipigs to collect mesenchymal stem cells to be used in regeneration and immunomodulation studies. This method can help answer key questions in the otologist MSC therapy field, such as how to establish the synovial fluid in MSC, and how to characterize them in comparison to bone marrow MSC. The main advantage of this technique is to effectively establish the key properties of synovial fluid and bone marrow-derived MSCs from minipigs, which will be useful for application in patients.The implications of this technique extend toward therapy of various joint diseases, because the population of the synovial fluid-derived MSC in the synovial cavity is significantly increased in athletic patients. Though this method can provide insight into regenerative medicine, it can also be applied to other studies such as autoimmune disease research. Because minipig MSCs are autologous, immunosuppression might interest humans.In addition to the other scientists, demonstrating the procedure will be Hyeon-Jeong Lee and Won-Jae Lee from my level three. To begin, orient an anesthetized minipig to access the iliac crest. Next, depilate a 15-square-centimeter region on the iliac crest, using a razor or clippers.Then, alternately scrub the exposed skin three times with povidone-iodine and 70%ethanol. Once completely dried, apply sterile drapes. Now, pre-coat a ten-milliliter syringe with heparin, using a three-to four-milliliter rinse.This will prevent the bone marrow from coagulating while it is extracted. Then, precisely at the iliac bone crest, tightly attach a bone marrow extractor to the heparin-coated syringe, and extract at least five milliliters of bone marrow. To isolate the cells from the extracted bone marrow, first dilute it with an equal volume of DPBS in a 15-milliliter conical tube at room temperature.Then, load three milliliters of density centrifugation media into a second 15-milliliter conical tube. Next, carefully transfer four milliliters of the diluted bone marrow onto the density centrifugation media to create two-solution layers. Now, centrifuge the tube at 400 Gs for 40 minutes at room temperature.The desired MNCs are collected in the central white layer. Aspirate just this layer. Eject the MNC layer into a 15-milliliter conical tube, and dilute it with seven to eight milliliters of DPBS.Then, centrifuge the cells at 500 Gs for ten minutes at room temperature. Discard the supernatant, and wash the palleted cells twice with ten milliliters of DPBS, repeating the previous centrifugation cycle. After the two washes, we suspend the MNCs in two milliliters of ADMEM.Then, load the cells onto a 35-millimeter tissue culture dish, and incubate them at 38.5 degrees Celsius in a humidified 5%carbon dioxide incubator. After 48 hours, aspirate the medium along with the unattached MNCs. Then, add back two milliliters of fresh ADMEM, and continue the incubation, changing the medium every three days.When the cells reach 70 to 80%confluency, wash the culture with fresh DPBS. Then, dissociate the cells, using one milliliter of 0.25%Trypsin-EDTA for three to five minutes at 37 degrees Celsius. Collect the cells in ten milliliters of ADMEM, and transfer them into a 15-milliliter conical tube.Then, spin the cells down at 300 Gs for five minutes at room temperature, and discard the supernatant. Now, suspend the harvested MNCs in one milliliter of fresh ADMEM, and measure their density. Finish with plating between 1/2 and three-quarter million cells into T25 flasks with four milliliters of ADMEM.Incubate the flasks just as the plates, and change the medium every third day. Prepare a 15x10 centimeter section of skin over the femoral joint, as was done when preparing for bone marrow collections. Then, palpate for the patella and tibia crests below the skin to identify the synovial joint, and insert a three-milliliter syringe with a 23-gauge needle, precisely into the joint.Using gentle suction, extract between 1/2 and one milliliter of fluid, and immediately withdraw the needle. If the synovial fluid volume is too small, then flush the cavity with DPBS to collect synovial fluid with DPBS. The volume of DPBS to use varies with the size of the synovial cavity.The optimal volume of synovial fluid is limited in healthy joint. The volume varies with the size and pathological condition of its joint. To isolate the cells from the synovial fluid, dilute the fluid with an equal volume of DPBS in a 15-milliliter conical tube at room temperature.Next, we move the debris by filtering the dilution through a 40-micron nylon cell strainer. Then, add ten more milliliters of DPBS, and centrifuge the suspension at 400 Gs for ten minutes at room temperature. Next, discard the supernatant, and wash the cells two times, using DPBS.We suspend the washed cell pallet in two milliliters of ADMEM. Then, proceed with culturing the cells as was demonstrated with the MSCs. MSCs were successfully isolated from the bone marrow and articular synovial joints of the minipigs and expanded in vitro.The morphology of synovial fluid-derived MSCs is similar to that of bone marrow-derived MSCs:both have a fibroblastic spindle shape and are homogeneously adherent. The MSCs showed positive expression for alkaline-phosphatase staining after the third subculture was completed. Next, surface markers were analyzed in both cultures.As expected, both types of MSCs significantly expressed CD29, CD44, and Vimentin. By contrast, expression of CD34 and CD45 were negative and low. These results were identical to those obtained through distinctive MSCs, including cells isolated from synovial fluid.After watching this video, you should have a good understanding of how to non-invasively aspirate synovial fluid and bone marrow in order to isolate their MSC from minipigs and how to perform the comparison and phenotyping of this MSC. While attempting this procedure, it's important to remember to locate the objective point on the iliac crest and the synovial, determined by careful palpation. Following this procedure, other methods like in models of osteoarthritis or rheumatoid arthritis can be performed in order the answer additional questions like whether not only from marrow MSC but also synovial fluid MSCs have a same kind capacity for cartilage or bone initiation and immunosuppressions.After its development, this technique advanced autologous therapy of joint diseases by helping to characterize MSCs derived from attacked or inflamed joint in arthritis patients.

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