Chondrogene differentiatie Inductie van vetcellen afgeleide stamcellen door Centrifugaal Zwaartekracht
Summary
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.
Abstract
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 × 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.
Introduction
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's) van genen opwaarts reguleren chondrocytische 5. Bovendien afschuifspanning bij tot de expressie van chondrogenese gerelateerde extracellulaire matrix (ECM) in humane MSC'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.
Protocol
Representative Results
Discussion
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'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 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C2116) and by Research Fund of Seoul St. Mary’s Hospital, The Catholic University of Korea.
Materials
| Plasticware | |||
| 100mm Dish | TPP | 93100 | |
| 60mm Dish | TPP | 93060 | |
| 50 mL Cornical Tube | SPL | 50050 | |
| 15 mL Cornical Tube | SPL | 50015 | |
| 10 mL Disposable Pipette | Falcon | 7551 | |
| 5 mL Disposable Pipette | Falcon | 7543 | |
| Name | Company | Catalog Number | Comments |
| ASC Culture Media Materials | |||
| DPBS | Life Technologies | 14190-144 | |
| DMEM Low glucose | Life Technologies | 11885-084 | growth base media |
| Penicilin Streptomycin | Sigma Aldrich | P4333 | 1% |
| Fetal Bovine Serum | Life Technologies | 16000-044 | 10% |
| PBS/1 mM EDTA | Life Technologies | 12604-039 | |
| Name | Company | Catalog Number | Comments |
| Chondrogenic Differentiation Media Materials | |||
| DMEM High glucose | Life Technologies | 11995 | chondrogenic differentiation base media |
| MEM Non-Essential Amino Acids Solution (100X) | Life Technologies | 11140-050 | |
| Dexamethasone | Sigma Aldrich | D2915 | 100nM |
| Penicilin Streptomycin | Life Technologies | P4333 | 1% |
| Fetal Bovine Serum | Life Technologies | 16000-044 | 1% |
| Ascorbate-2-phosphate | Sigma Aldrich | A8960 | 50ug/ml |
| L-proline | Sigma Aldrich | P5607 | 50ug/ml |
| ITS | BD | 354352 | 1% |
| Human TGFβ1 | Peprotech | 100-21 | 10ng/ml |
| Name | Company | Catalog Number | Comments |
| Materials | |||
| 18 mm Cover Glass | Superior | HSU-0111580 | |
| 4% Paraformaldyhyde | Tech & Innovation | BPP-9004 | |
| Tween 20 | BIOSESANG | T1027 | |
| Bovine Serum Albumin | Vector Lab | SP-5050 | |
| Anti-Collagen II antibody | abcam | ab34712 | 1:100 |
| Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate |
Molecular Probe | A-11037 | 1:200 |
| DAPI | Molecular Probe | D1306 | |
| Prolong gold antifade reagent | Invitrogen | P36934 | |
| Slide Glass, Coated | Hyun Il Lab-Mate | HMA-S9914 | |
| Trizol | Invitrogen | 15596-018 | |
| Chloroform | Sigma Aldrich | 366919 | |
| Isoprypylalcohol | Millipore | 109634 | |
| Ethanol | Duksan | 64-17-5 | |
| RevertAid First Strand cDNA Synthesis kit | Thermo Scientfic | K1622 | |
| i-Taq DNA Polymerase | iNtRON BIOTECH | 25021 | |
| UltraPure 10X TBE Buffer | Life Technologies | 15581-044 | |
| loading star | Dyne Bio | A750 | |
| Agarose | Sigma-Aldrich | 9012-36-6 | |
| 1kb (+) DNA ladder marker | Enzynomics | DM003 | |
| Human adipose-derived stem cells (ASCs) | Catholic MASTER Cells |
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