Condrogenico Differenziazione induzione di cellule staminali derivate da tessuto adiposo per gravità centrifuga
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
Difetti di cartilagine articolare non guariscono naturalmente. Di conseguenza, trapianto di cellule staminali è stata proposta come un approccio promettente per la riparazione della cartilagine compromessa. Tuttavia, questo metodo richiede sia l'acquisizione di un numero sufficiente di cellule staminali e l'induzione di queste cellule a subire differenziazione condrogenico. Il midollo osseo (BM) è stato ampiamente utilizzato come fonte di cellule staminali, ma l'isolamento delle cellule da BM ha due principali svantaggi: invasività e di rendimento insufficiente. A causa della sua facilità di acquisizione, tessuto adiposo è fonte preferibile di cellule staminali. Studi precedenti hanno dimostrato la fattibilità di isolare cellule staminali dal tessuto adiposo e indurre la differenziazione condrogenica in queste cellule con citochine, come TGF-β1 1, 2. Questi metodi sono efficaci ma costosi.
Come un più basso costo alle citochine, stress meccanico può essere utilizzato perindurre la differenziazione condrogenico. Carico meccanico gioca un ruolo fondamentale nel mantenimento della salute della cartilagine articolare 3, e può indurre fenotipi condrogeniche in varie cellule. Ad esempio, la pressione idrostatica induce fenotipi condrogeniche in cellule progenitrici derivate dal sinovio attraverso la via MAP chinasi / JNK 4, e la compressione meccanica induce condrogenesi nelle cellule staminali mesenchimali umane (MSC) per upregulating geni chondrocytic 5. Inoltre, shear stress contribuisce alla espressione della matrice extracellulare chondrogenesis legati (ECM) di MSC umani 6. Gravità centrifugo (CG), uno stress meccanico facilmente applicata e controllata generato per centrifugazione, può indurre l'espressione genica differenziale in cellule 7. Ad esempio, nelle cellule di carcinoma epiteliale polmonare, l'espressione di interleuchina (IL) -1 ter è upregulated per centrifugazione 8. Therefore, come sollecitazioni meccaniche sperimentalmente inducibile, CG può essere utilizzato per indurre l'espressione genica in cellule staminali chondrocytic. Tuttavia, non è chiaro se CG può indurre la differenziazione delle cellule staminali condrogenica.
In questo studio, abbiamo scoperto che CG ha indotto la upregulation di SOX9, un regolatore maestro di condrogenesi, in ASC umani, causando la sovraespressione di geni chondrocytic. Inoltre, abbiamo confrontato gli effetti di CG su chondrogenesis con quelli di TGF-β1, il fattore di crescita più comunemente utilizzati per indurre in vitro condrogenesi nelle cellule staminali.
Protocol
Representative Results
Discussion
Lo stato stemness delle cellule è molto importante per la sovraespressione CG-indotta SOX9. Nel nostro studio, espressione SOX9 potrebbe essere indotta da CG ASC precoce passaggio (2-3), ma non in ASC dopo-passaggio. E 'stato riportato che, durante la coltivazione, ASC contengono cellule CD34 + fino a 3 passaggi 16. ASC tendono a perdere l'espressione di CD34 come le cellule sono diversi passaggi, con conseguente bassa risposta al CG.
Con forza di gravità ce…
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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