Name: chondrogenic differentiation induction of adipose-derived stem cells by centrifugal gravity
Uploaded: 2017-02-24T12:30:00
Description: Watch this Scientific Journal Video about Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity at JoVE.com

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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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>60mm Dish</td>
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			<td>50 mL Cornical Tube</td>
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			<td>15 mL Cornical Tube</td>
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			<td>10 mL Disposable Pipette</td>
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			<td>5 mL Disposable Pipette</td>
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			<td>ASC Culture Media Materials</td>
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			<td>DPBS</td>
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			<td>Chondrogenic Differentiation Media Materials</td>
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			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Life Technologies</td>
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			<td>Dexamethasone</td>
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			<td>100nM</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>
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			<td>100-21</td>
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			<td>Anti-Collagen II antibody</td>
			<td>abcam&#160;</td>
			<td>ab34712</td>
			<td>1:100</td>
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			<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>
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			<td>Prolong gold antifade reagent</td>
			<td>Invitrogen</td>
			<td>P36934</td>
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			<td>Slide Glass, Coated</td>
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			<td>Trizol</td>
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			<td>RevertAid First Strand cDNA Synthesis kit</td>
			<td>Thermo Scientfic</td>
			<td>K1622</td>
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			<td>i-Taq DNA Polymerase</td>
			<td>iNtRON BIOTECH</td>
			<td>25021</td>
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			<td>UltraPure 10X TBE Buffer</td>
			<td>Life Technologies</td>
			<td>15581-044</td>
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			<td>loading star</td>
			<td>Dyne Bio</td>
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			<td>Agarose</td>
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			<td>9012-36-6</td>
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			<td>1kb (+) DNA ladder marker</td>
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			<td>Human adipose-derived stem cells (ASCs)</td>
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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 ...

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