Chondrogenic Differensiering Induksjon av adipose-avledet stamceller ved Sentrifugal 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
Defekter i leddbrusk ikke gro naturlig. Derfor stamcelletransplantasjon har blitt foreslått som en lovende metode for reparasjon av svekket brusk. Imidlertid krever denne metoden både oppkjøp av et tilstrekkelig antall stamceller og induksjon av disse cellene til å gjennomgå chondrogenic differensiering. Benmarg (BM) har blitt mye brukt som en kilde av stamceller, men celleisolasjon fra BM har to store ulemper: invasivitet og utilstrekkelig kapasitet. På grunn av sin enkle oppkjøpet, er fettvev en foret kilde til stamceller. Tidligere studier demonstrert muligheten for å isolere stamceller fra fettvev og indusere differensiering chondrogenic i disse cellene ved anvendelse av cytokiner, slik som TGF-β1 1, 2. Disse fremgangsmåtene er effektive, men kostbare.
Som et rimeligere alternativ til cytokiner, kan mekanisk stress brukes til åindusere chondrogenic differensiering. Mekanisk belastning spiller en avgjørende rolle i å opprettholde helsen til leddbrusk tre, og det kan indusere chondrogenic fenotyper i forskjellige celler. For eksempel, hydrostatiske trykket induserer chondrogenic fenotyper i synovialhinne-avledet stamceller via MAP kinase / JNK vei 4, og mekanisk kompresjon induserer chondrogenesis i humane stamceller (MSC) ved oppregulering chondrocytic gener 5. I tillegg bidrar skjærspenning til ekspresjon av chondrogenesis relaterte ekstracellulære matriks (ECM) i humane MSC 6. Sentrifugal gravitasjon (CG), en lett brukt og kontrollert mekanisk stress generert ved sentrifugering, kan indusere differensial genuttrykk i celler 7. For eksempel, i lunge epiteliale karcinomceller, blir ekspresjon av interleukin (IL) -1b oppregulert ved sentrifugering 8. Therefore, som et eksperimentelt induserbar mekaniske påkjenninger, cg kan anvendes for å indusere chondrocytic genekspresjon i stamceller. Imidlertid er det fortsatt uklart om CG kan indusere chondrogenic differensiering av stamceller.
I denne studien fant vi at CG indusert oppregulering av SOX9, en mester regulator av chondrogenesis, i menneskelige ASCs, noe som resulterer i overekspresjon av chondrocytic gener. I tillegg, sammenlignet vi effektene av CG på chondrogenesis med de til TGF-β1, vekstfaktor som oftest anvendt for å indusere in vitro chondrogenesis i stamceller.
Protocol
Representative Results
Discussion
Den stemness tilstand av celler er svært viktig for CG-indusert overekspresjon av SOX9. I vår studie kunne SOX9 uttrykk induseres av CG tidlig-passasjen ASCs (2-3), men ikke i senere-passasjen ASCs. Det har blitt rapportert at, under dyrking, ASCer inneholde CD34 + celler til 3 passasjer 16. ASC har en tendens til å miste ekspresjon av CD34 som cellene er passert, noe som resulterer i en lav respons til CG.
Med sentrifugal tyngdekraften, kan det hydrostatiske trykk…
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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