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.

Deze studie protocol werd goedgekeurd door de institutionele review board van de Katholieke Universiteit van Korea (KC16EAME0162) en uitgevoerd volgens de NIH richtlijnen. Alle weefsels werden verkregen met schriftelijke informed consent. 1. Centrifugaal Zwaartekracht laden en Pellet Cultuur <ol><li> Celcultuur en oogst <ol><li> Cultuur ASC (P2-P3, zie Lijst van Materials) in Dulbecco&#39;s gemodificeerd Eagle&#39;s medium-laag glucose (DMEM-LG) aangevuld met 10% foetaal runderserum (FBS) ...

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Stemness de toestand van cellen is zeer belangrijk voor CG-geïnduceerde overexpressie van SOX9. In onze studie konden SOX9 expressie worden geïnduceerd door CG in vroege passage ASC&#39;s (2-3), maar niet in latere doorgang ASC. Het is gerapporteerd dat, tijdens de teelt, ASC bevatten CD34 + -cellen tot 3 passages 16. ASC neiging om de expressie van CD34 verliezen de cellen gepasseerd, waardoor een lage respons CG. Met centrifugale zwaartekracht, kan hydrostatische ...

Defecten in gewrichtskraakbeen niet genezen natuurlijk. Bijgevolg stamcel is voorgesteld als een veelbelovende benadering voor het herstel van aangetast kraakbeen. Deze werkwijze vereist zowel het verkrijgen van een voldoende aantal stamcellen en de inductie van deze cellen chondrogene differentiatie ondergaan. Beenmerg (BM) is op grote schaal gebruikt als bron van stamcellen, maar celisolatie van BM heeft twee belangrijke nadelen: invasiviteit en onvoldoende opbrengst. Vanwege het gemak van verwerving, vetweefsel is een voorkeur bron van stamcellen. Eerdere studies toonden de haalbaarheid van het isoleren van stamcellen uit vetweefsel en het induceren van chondrogene differentiatie van deze cellen met behulp cytokines zoals TGF-β1 1, 2. Deze werkwijzen zijn effectief maar duur. Als goedkopere alternatief voor cytokinen, kunnen mechanische belasting worden gebruiktchondrogene differentiatie induceren. Mechanische belasting speelt een cruciale rol bij het handhaven van de gezondheid van gewrichtskraakbeen 3, en kan chondrogene fenotypes in verschillende cellen. Bijvoorbeeld, hydrostatische druk veroorzaakt chondrogene fenotypen in synovium afgeleide progenitorcellen via de MAP kinase / JNK pathway 4 en mechanische compressie veroorzaakt chondrogenese van menselijke mesenchymale stamcellen (MSC&#39;s) van genen opwaarts reguleren chondrocytische 5. Bovendien afschuifspanning bij tot de expressie van chondrogenese gerelateerde extracellulaire matrix (ECM) in humane MSC&#39;s 6. Centrifugale zwaartepunt (CG), een gemakkelijk worden gecontroleerd spanning gegenereerd door centrifugeren, kan differentiële genexpressie in cellen 7 induceren. Bijvoorbeeld, in longepitheelcellen carcinoomcellen, de expressie van interleukine (IL) -1 ter opgereguleerd door centrifugeren 8. Therefore, als experimenteel induceerbare spanning, CG kan worden gebruikt om chondrocyte genexpressie te induceren in stamcellen. Het blijft echter onduidelijk of CG het chondrogene differentiatie van stamcellen kunnen induceren. In deze studie hebben we vastgesteld dat CG induceerde de opregulatie van SOX9, regulering van de chondrogenese in menselijk ASCs, wat resulteert in de overexpressie van chondrocyte genen. Verder vergeleken we de effecten van CG op chondrogenese met die van TGF-β1, de groeifactor meest gebruikte in vitro chondrogenese te induceren in stamcellen.

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			<td>Plasticware</td>
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			<td>100mm Dish</td>
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			<td>93100</td>
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			<td>60mm Dish</td>
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			<td>93060</td>
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			<td>50 mL Cornical Tube</td>
			<td>SPL</td>
			<td>50050</td>
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			<td>15 mL Cornical Tube</td>
			<td>SPL</td>
			<td>50015</td>
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			<td>10 mL Disposable Pipette</td>
			<td>Falcon</td>
			<td>7551</td>
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			<td>5 mL Disposable Pipette</td>
			<td>Falcon</td>
			<td>7543</td>
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			<td>Name</td>
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			<td>DPBS</td>
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			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
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			<td>PBS/1 mM EDTA</td>
			<td>Life Technologies</td>
			<td>12604-039</td>
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			<td>Name</td>
			<td>Company</td>
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			<td>Chondrogenic Differentiation Media Materials</td>
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			<td>DMEM High glucose</td>
			<td>Life Technologies</td>
			<td>11995</td>
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			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
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			<td>Dexamethasone</td>
			<td>Sigma Aldrich</td>
			<td>D2915</td>
			<td>100nM</td>
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			<td>Penicilin Streptomycin</td>
			<td>Life Technologies</td>
			<td>P4333</td>
			<td>1%</td>
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			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td>1%</td>
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			<td>Ascorbate-2-phosphate</td>
			<td>Sigma Aldrich</td>
			<td>A8960</td>
			<td>50ug/ml</td>
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			<td>L-proline</td>
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			<td>ITS</td>
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			<td>4% Paraformaldyhyde</td>
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			<td>Tween 20</td>
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			<td>Bovine Serum Albumin</td>
			<td>Vector Lab</td>
			<td>SP-5050</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>DAPI</td>
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			<td>D1306</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>HMA-S9914</td>
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			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
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			<td>Chloroform</td>
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			<td>366919</td>
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			<td>Isoprypylalcohol</td>
			<td>Millipore</td>
			<td>109634</td>
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			<td>Ethanol</td>
			<td>Duksan</td>
			<td>64-17-5</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>
			<td>A750</td>
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			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>9012-36-6</td>
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			<td>1kb (+) DNA ladder marker</td>
			<td>Enzynomics</td>
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			<td>Human adipose-derived stem cells (ASCs)</td>
			<td>&#160;Catholic MASTER Cells</td>
			<td></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.

Centrifugale zwaartekracht induceert de overexpressie van chondrogene differentiatie merkers in adipeus afgeleide stamcellen. Om de mate van centrifugale zwaartekracht die geschikt is voor chondrogene differentiatie te induceren, werden ASC gestimuleerd met verschillende graden van CG (0, 300, 600, 1200 en 2400 x g) gedurende 15 min. Na stimulatie, het ASC werden opnieuw gezaaid in kweekplaten en gekweekt gedurende 24 uur. Zoa...

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.

Chondrogene differentiatie Inductie van vetcellen afgeleide stamcellen door Centrifugaal Zwaartekracht

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

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Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

Immunomagnetische Scheiding van Fat Depot-specifieke SCA1<sup&gt; hoog</sup&gt;-Vetcellen afgeleide stamcellen (ASC)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Inductie van Acute Skeletal Muscle Regeneration door cardiotoxine Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In vitro differentiatie van humane pluripotente stamcellen in trofoblastcellen

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Chondrogenische Pelletvorming van Koord Bloed afgeleide geïnduceerde Pluripotente Stamcellen

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Snelle, gerichte differentiatie van de epitheliale cellen van de retinale Pigment van menselijke embryonale of geïnduceerde pluripotente stamcellen

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computationele analyse van de Caenorhabditis elegans Germline te bestuderen van de distributie van kernen, eiwitten en het cytoskelet

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Afleiding en differentiatie van Canine ovariële mesenchymale stamcellen

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...