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 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.
Defekter i ledbrusk ikke heler naturligt. Derfor celle transplantation er blevet foreslået som en lovende tilgang til reparation af forringet brusk stammer. Men denne metode kræver både erhvervelse af et tilstrækkeligt antal af stamceller og induktion af disse celler til at undergå chondrogen differentiering. Knoglemarv (BM) er blevet almindeligt anvendt som en kilde til stamceller, men celleisolering fra BM har to store ulemper: invasionsevne og utilstrækkelig udbytte. På grund af den lette erhvervelse, fedtvæv er en foretrukken kilde til stamceller. Tidligere undersøgelser demonstrerede gennemførligheden af at isolere stamceller fra fedtvæv og inducere chondrogen differentiering i disse celler ved anvendelse cytokiner, såsom TGF-β1 1, 2. Disse fremgangsmåder er effektive, men dyre.
Som en billigere alternativ til cytokiner, kan mekanisk belastning anvendes tilinducere chondrogen differentiering. Mekanisk belastning spiller en kritisk rolle i opretholdelsen af sundhed ledbrusken 3, og det kan inducere chondrogene fænotyper i forskellige celler. For eksempel, hydrostatisk tryk fremkalder chondrogene fænotyper i synovium progenitorceller via MAP-kinase / JNK-banen 4, og mekanisk kompression inducerer chondrogenese i humane mesenkymale stamceller (MSC'er) ved opregulering chondrocytisk gener 5. Desuden forskydningsspænding bidrager til ekspressionen af chondrogenese-relateret ekstracellulær matrix (ECM) i humane MSC'er 6. Centrifugal tyngdekraft (CG), en let anvendt og kontrolleret mekanisk spænding genereret ved centrifugering, kan inducere differentiel genekspression i celler 7. For eksempel i lungeepitelceller carcinomceller er ekspressionen af interleukin (IL) -1b opreguleres ved centrifugering 8. Therefore, som en eksperimentelt inducerbar mekanisk stress, CG kan anvendes til at inducere chondrocytisk genekspression i stamceller. Men det er uklart, om CG kan fremkalde den chondrogene differentiering af stamceller.
I denne undersøgelse fandt vi, at CG inducerede opregulering af Sox9, en master regulator af chondrogenese, i humane ASC'er, hvilket resulterer i overekspression af chondrocytisk gener. Desuden har vi sammenlignede virkningerne af CG på chondrogenese med de TGF-β1, vækstfaktor mest almindeligt anvendte til at fremkalde in vitro chondrogenese i stamceller.
Den stemness tilstand af celler er meget vigtigt for CG-induceret overekspression af Sox9. I vores undersøgelse kunne Sox9 ekspression induceres af CG i tidlig passage ASC'er (2-3), men ikke i senere passage ASC'er. Det er blevet rapporteret, at under dyrkning, ASC'er indeholder CD34 + celler indtil 3 passager 16. ASC'er tendens til at miste ekspressionen af CD34 som cellerne passeres, hvilket resulterer i en lav reaktion på CG.
Med centrifugal…
The authors have nothing to disclose.
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.
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 |