Chondrogene Differenzierung Induktion von Fettgewebe gewonnene Stammzellen durch Zentrifugalkraft Gravity
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
Defekte im Gelenkknorpel nicht heilen natürlich. Folglich Stammzelltransplantation hat sich als vielversprechender Ansatz für die Reparatur von Knorpel beeinträchtigt vorgeschlagen worden. Jedoch erfordert dieses Verfahren sowohl die Übernahme von einer ausreichenden Anzahl von Stammzellen und die Induktion dieser Zellen chondrogene Differenzierung zu unterziehen. Knochenmark (BM) wurde als Quelle für Stammzellen weit verbreitet, aber Zellisolation aus BM hat zwei wesentliche Nachteile: Invasivität und ungenügender Ausbeute. Aufgrund seiner Leichtigkeit des Erwerbs, Fettgewebe ist eine bevorzugte Quelle für Stammzellen. Frühere Studien zeigten die Machbarkeit Stammzellen aus Fettgewebe zur Isolierung und chondrogene Differenzierung in diesen Zellen Zytokine, wie TGF-β1 1 unter Verwendung von 2 zu induzieren. Diese Verfahren sind wirksam, aber teuer.
Als preiswertere Alternative zu Cytokine können mechanische Belastungen verwendet werden,induzieren die chondrogene Differenzierung. Mechanische Belastung spielt eine kritische Rolle bei der Gesundheit von Gelenkknorpel Aufrechterhaltung 3, und es kann chondrogenen Phänotyp in verschiedenen Zellen induzieren. Beispielsweise induziert hydrostatischem Druck chondrogenen Phänotyp in Synovium abgeleiteten Vorläuferzellen über die MAP kinase / JNK – Weg 4 und mechanische Kompression induziert die Chondrogenese in mesenchymalen Stammzellen (MSCs) durch chondrozytische Gene 5 upregulating. Zusätzlich trägt Scherspannung auf die Expression von Chondrogenese bezogenen extrazellulären Matrix (ECM) in humanen MSCs 6. Kreiselschwerkraft (CG), ein leicht angewendet und kontrolliert mechanischer Beanspruchung durch Zentrifugation erzeugt, kann Differential Gen induzieren Expression in Zellen 7. Beispielsweise in Karzinomzellen Lungenepithelzellen, wird die Expression von Interleukin (IL) -1b durch Zentrifugation 8 nach oben reguliert. Therefore als experimentell induzierbaren mechanische Beanspruchung kann CG verwendet werden chondrozytische Genexpression in Stammzellen zu induzieren. Es bleibt jedoch unklar, ob CG die chondrogene Differenzierung von Stammzellen induzieren können.
In dieser Studie haben wir festgestellt, dass CG die Hochregulation von SOX9, einen Master-Regulator der Chondrogenese, in der menschlichen ASCS induziert, was zu einer Überexpression von chondrozytische Gene. Zusätzlich verglichen wir die Wirkungen von CG auf Chondrogenese mit denen von TGF-β1, die meisten der Wachstumsfaktor im Allgemeinen in vitro – Chondrogenese in Stammzellen zu induzieren , verwendet.
Protocol
Representative Results
Discussion
Die stemness Zustand von Zellen ist sehr wichtig für CG-induzierten Überexpression von SOX9. In unserer Studie konnte SOX9 Expression von CG in frühen Passage ASCs (2-3), aber nicht in späteren Durchgang ASCs induziert werden. Es wurde berichtet , dass während der Kultivierung ASCs 16 CD34 + -Zellen bis 3 Passagen enthalten. ASCS neigen dazu, die Expression von CD34 zu verlieren, wie die Zellen passagiert werden, in einer geringen Reaktion auf CG führt.
Mit zent…
Declarações
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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