This research was supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (grant number: HI14C2116) and by Research Fund of Seoul St. Mary's Hospital, The Catholic University of Korea.

This study protocol was approved by the institutional review board of The Catholic University of Korea (KC16EAME0162) and performed according to NIH guidelines. All tissues were obtained with written informed consent.
1. Centrifugal Gravity Loading and Pellet Culture
<ol>
	<li>Cell culture and harvest
	<ol>
		<li>Culture ASCs (P2-P3; see List of Materials) in Dulbecco&#39;s Modified Eagle&#39;s Medium-low glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/stre...

<ol>
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F., Schedl, A., de Crombrugghe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+Sox9+has+essential+roles+in+successive+steps+of+the+chondrocyte+differentiation+pathway+and+is+required+for+expression+of+Sox5+and+Sox6.">The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6.</a> Genes Dev. 16 (21), 2813-2828 (2002).</li><li>Lefebvre, V., Huang, W., Harley, V. R., Goodfellow, P. N., de Crombrugghe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX9+is+a+potent+activator+of+the+chondrocyte-specific+enhancer+of+the+pro+alpha1(II)+collagen+gene.">SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene.</a> Mol Cell Biol. 17 (4), 2336-2346 (1997).</li><li>Jang, Y., 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=Characterization+of+adipose+tissue-derived+stromal+vascular+fraction+for+clinical+application+to+cartilage+regeneration.">Characterization of adipose tissue-derived stromal vascular fraction for clinical application to cartilage regeneration.</a> In Vitro Cell Dev Biol Anim. 51 (2), 142-150 (2015).</li><li>Chen, J., 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=Simultaneous+regeneration+of+articular+cartilage+and+subchondral+bone+in+vivo+using+MSCs+induced+by+a+spatially+controlled+gene+delivery+system+in+bilayered+integrated+scaffolds.">Simultaneous regeneration of articular cartilage and subchondral bone in vivo using MSCs induced by a spatially controlled gene delivery system in bilayered integrated scaffolds.</a> Biomaterials. 32 (21), 4793-4805 (2011).</li><li>Janzen, V., 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=Stem-cell+ageing+modified+by+the+cyclin-dependent+kinase+inhibitor+p16INK4a.">Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a.</a> Nature. 443 (7110), 421-426 (2006).</li><li>Muraglia, A., Cancedda, R., Quarto, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+mesenchymal+progenitors+from+human+bone+marrow+differentiate+in+vitro+according+to+a+hierarchical+model.">Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model.</a> J Cell Sci. 113, 1161-1166 (2000).</li></ol>

The stemness state of cells is very important for CG-induced overexpression of SOX9. In our study, SOX9 expression could be induced by CG in early-passage ASCs (2-3), but not in later-passage ASCs. It has been reported that, during cultivation, ASCs contain CD34+ cells until 3 passages16. ASCs tend to lose the expression of CD34 as the cells are passaged, resulting in a low response to CG.
With centrifugal gravity force, hydrostatic pressure can be loaded onto cells dur...

Defects in articular cartilage do not heal naturally. Consequently, stem cell transplantation has been proposed as a promising approach for the repair of impaired cartilage. However, this method requires both the acquisition of a sufficient number of stem cells and the induction of these cells to undergo chondrogenic differentiation. Bone marrow (BM) has been widely used as a source of stem cells, but cell isolation from BM has two major disadvantages: invasiveness and insufficient yield. Because of its ease of acquisition, adipose tissue is a preferable source of stem cells. Previous studies demonstrated the feasibility of isolating stem cells from adipose tissue and inducing chondrogenic differentiation in these cells using cytokines, such as TGF-&#946;11,2. These methods are effective but expensive.
As a lower-cost alternative to cytokines, mechanical stress can be used to induce chondrogenic differentiation. Mechanical loading plays a critical role in maintaining the health of articular cartilage3, and it can induce chondrogenic phenotypes in various cells. For example, hydrostatic pressure induces chondrogenic phenotypes in synovium-derived progenitor cells via the MAP kinase/JNK pathway4, and mechanical compression induces chondrogenesis in human mesenchymal stem cells (MSCs) by upregulating chondrocytic genes5. In addition, shear stress contributes to the expression of chondrogenesis-related extracellular matrix (ECM) in human MSCs6. Centrifugal gravity (CG), an easily applied and controlled mechanical stress generated by centrifugation, can induce differential gene expression in cells7. For example, in lung epithelial carcinoma cells, the expression of interleukin (IL)-1b is upregulated by centrifugation8. Therefore, as an experimentally inducible mechanical stress, CG can be used to induce chondrocytic gene expression in stem cells. However, it remains unclear whether CG can induce the chondrogenic differentiation of stem cells.
In this study, we found that CG induced the upregulation of SOX9, a master regulator of chondrogenesis, in human ASCs, resulting in the overexpression of chondrocytic genes. In addition, we compared the effects of CG on chondrogenesis with those of TGF-&#946;1, the growth factor most commonly used to induce in vitro chondrogenesis in stem cells.

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			<td>Plasticware</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>100mm Dish</td>
			<td>TPP</td>
			<td>93100</td>
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			<td>60mm Dish</td>
			<td>TPP</td>
			<td>93060</td>
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			<td>50 mL Cornical Tube</td>
			<td>SPL</td>
			<td>50050</td>
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		<tr>
			<td>15 mL Cornical Tube</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
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		<tr>
			<td>10 mL Disposable Pipette</td>
			<td>Falcon</td>
			<td>7551</td>
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		</tr>
		<tr>
			<td>5 mL Disposable Pipette</td>
			<td>Falcon</td>
			<td>7543</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>ASC Culture Media Materials</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>DPBS</td>
			<td>Life Technologies</td>
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			<td>DMEM Low glucose</td>
			<td>Life Technologies</td>
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			<td>growth base media</td>
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			<td>Penicilin Streptomycin</td>
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			<td>P4333</td>
			<td>1%</td>
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			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>PBS/1 mM EDTA</td>
			<td>Life Technologies</td>
			<td>12604-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
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			<td>Chondrogenic Differentiation Media Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM High glucose</td>
			<td>Life Technologies</td>
			<td>11995</td>
			<td>chondrogenic differentiation base media</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>Dexamethasone</td>
			<td>Sigma Aldrich</td>
			<td>D2915</td>
			<td>100nM</td>
		</tr>
		<tr>
			<td>Penicilin Streptomycin</td>
			<td>Life Technologies</td>
			<td>P4333</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Ascorbate-2-phosphate</td>
			<td>Sigma Aldrich</td>
			<td>A8960</td>
			<td>50ug/ml</td>
		</tr>
		<tr>
			<td>L-proline</td>
			<td>Sigma Aldrich</td>
			<td>P5607</td>
			<td>50ug/ml</td>
		</tr>
		<tr>
			<td>ITS</td>
			<td>BD</td>
			<td>354352</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Human TGF&#946;1</td>
			<td>Peprotech</td>
			<td>100-21</td>
			<td>10ng/ml</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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			<td>Materials</td>
			<td></td>
			<td></td>
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			<td>18 mm Cover Glass</td>
			<td>Superior</td>
			<td>HSU-0111580</td>
			<td></td>
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		<tr>
			<td>4% Paraformaldyhyde</td>
			<td>Tech &#38; Innovation</td>
			<td>BPP-9004</td>
			<td></td>
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		<tr>
			<td>Tween 20</td>
			<td>BIOSESANG</td>
			<td>T1027</td>
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		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Vector Lab</td>
			<td>SP-5050</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Collagen II antibody</td>
			<td>abcam&#160;</td>
			<td>ab34712</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>&#160;Goat anti-Rabbit IgG (H+L) Secondary Antibody, 
			&#160;Alexa Fluor 594 conjugate&#160;</td>
			<td>Molecular Probe</td>
			<td>&#160;A-11037</td>
			<td>1:200</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>
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		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
			<td></td>
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			<td>Chloroform</td>
			<td>Sigma Aldrich</td>
			<td>366919</td>
			<td></td>
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		<tr>
			<td>Isoprypylalcohol</td>
			<td>Millipore</td>
			<td>109634</td>
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		<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>
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		<tr>
			<td>1kb (+) DNA ladder marker</td>
			<td>Enzynomics</td>
			<td>DM003</td>
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		</tr>
		<tr>
			<td>Human adipose-derived stem cells (ASCs)</td>
			<td>&#160;Catholic MASTER Cells</td>
			<td></td>
			<td></td>
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	</tbody>
</table>

chondrogenic differentiation induction of adipose-derived stem cells by centrifugal gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Centrifugal gravity induces the overexpression of chondrogenic differentiation markers in adipose-derived stem cells.
To determine the degree of centrifugal gravity force that is suitable to induce chondrogenic differentiation, ASCs were stimulated with different degrees of CG (0, 300, 600, 1,200, and 2,400 x g) for 15 min. After stimulation, the ASCs were re-seeded onto culture plates and cultured for 24 h. As shown in ...

Watch this Scientific Journal Video about Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity at JoVE.com

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

The overall goal of this procedure is to induce chondrogenic differentiation of ASC's for defective cartilage repair. This method is to induce the prechondrogenic differentiation of ASC's using centrifugal gravity force. Though this method can provide insight into chondrogenic differentiation of ASC's, it can also be applied to other systems, such as osteogenic and fibrogenic differentiation.The main advantage of this technique is that chondrogenic differentiation can be easily induced from stem cells without treating any cytokines. Demonstrating the procedure will be Yeri Alice Rim and Yoojun Nam from my laboratory. Begin by harvesting 80%confluent adipose-derived stem cells from a 100 milliliter culture dish by discarding the medium and washing with five milliliters of PBS.Then, add one milliliter of PBS containing one millimolar EDTA, and incubate for two minutes at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide. After two minutes, tap the culture plate gently then add four milliliters of fresh medium and transfer the cells to a 15 milliliter conical tube. Centrifuge the cells at 250 times g for two minutes at room temperature.After centrifugation, remove the supernatant without disturbing the pellet. Then, re-suspend the pellet in 10 milliliters of medium. Count the cells using a hemocytometer.For centrifugal gravity loading, transfer 2.5 times ten to the fifth cells to a new 15 milliliter conical tube, and centrifuge at 2400 times g for 15 minutes. Following the centrifugation, proceed to either pellet or micromass culture. Immediately after centrifugation, aspirate the supernatant and at 500 microliters of chondrogenic differentiation medium, or CDM, and 50 micrograms per milliliter of freshly-prepared L-ascorbic-acid 2-phosphate to the pellet.A positive control, induce chondrogenic differentiation and collect its cells by adding CDM containing 10 nanograms per milliliter TGF-beta-1. Loosely cap the tubes and incubate at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide for three weeks. Immediately after the gravity-loading centrifugation, aspirate the supernatant and re-suspend the pellet in 10 microliters of CDM.To form a micromass, pipette the re-suspended cells into the center of a well of a 24 well plate and then place the plate into the incubator. After two hours, carefully add 500 microliters of CDM to the well, pipetting against the wall of the plate to avoid disrupting the micromass. As a positive control, perform a micromass culture with uncentrifuged cells by adding CDM containing 10 nanograms per milliliter TGF-beta-1.Incubate the micromass for three weeks. On day 21 after micromass formation, use a pipette to harvest the spheroid pellets, and then wash with 1X PBS. Fix the spheroid pellets by immersion in 4%paraformaldehyde for 24 hours.The next day, discard the fixative and rinse the cell pellets with deionized water. Place one layer of gauze onto the tissue cassette and transfer the fixed spheroid pellets using a pipette. Cover the pellet by folding the gauze and close the cassette lid.Next, dehydrate the spheroid pellets by immersion in 70%ethanol for five minutes at room temperature. Then, continue through a series of ethanol concentrations for five minutes each. Then immerse the pellets in 100%xylene for 15 minutes.Immerse the spheroid pellets in paraffin at 56 degrees Celsius for embedding. Next, cut five micron-thick sections from the paraffin-embedded cell pellet using a rotary microtome. Float the sections in 50%ethanol, and then transfer them to a 50 degree Celsius water bath.Mount the floating sections onto slides, and then proceed to safranin o and alcian blue for immuno-flourescent staining. To determine a suitable centrifugal gravity force, ASC's were spun at centrifugal gravity of zero, 300, 600, 1, 200, and 2, 400 times g for fifteen minutes. SOX9 mRNA expression was evaluated at 24 hours after CG stimulation.A CG of 2, 400 times g significantly induced SOX9 mRNA expression. To evaluate the expression of chondrogenesis-related genes, total RNA was extracted from unstimulated ASC's, ASC's that had been centrifuged, or treated with TGF-beta-one. As seen here, CG stimulation induces collagen type-2 and aggrecan expression.over-expression of collagen type-2 in ASC's treated with CG was confirmed by immunoflourescence using an antibody against collagen type-2 in an Alexa Fluor 594 conjugated secondary antibody. To determine the effect of CG on chondrogenesis-related ECM over-expression, ASC's were treated with TGF-beta-one as a positive control. The negative control was a pellet culture not subjected to CG loading, or TGF-beta-one treatment.The over-expression of chondrogenesis-related ECM was evaluated in spheroid pellets consisting of centrifuged ASC's through the use of safranin o and alcian blue staining. Glycosaminoglycan, a major component of articular cartilage, was stained blue by alcian blue, or stained orange by safranin o. After 14 days of micromass culture, chondrocyte condensation was evaluated by the size of the aggregates.The size of each aggregate, generated by CG, was around 0.5 millimeters. Once mastered, this technique can be done in 30 minutes, if it is performed properly. After watching this video, you should have a good understanding of how induce chondogenic differentiation of stem cells using centrifugal gravity force.

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Research

JoVE Journal

Developmental Biology

9.4K vistas.  The Catholic University of Korea. The overall goal of this procedure is to induce chondrogenic differentiation of ASC's for defective cartilage repair. This method is to induce the prechondrogenic differentiation of ASC's using centrifugal gravity force. Though this method can provide insight into chondrogenic differentiation of ASC's, it can also be applied to other systems, such as osteogenic and fibrogenic differentiation.The main advantage of this technique is that chondrogenic differentiation can be easily induced...