脂肪干细胞的软骨细胞分化诱导离心重力
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
在关节软骨缺损不自然愈合。因此，干细胞移植已被提出作为削弱的软骨的修复一个有前途的方法。然而，该方法需要有足够数量的干细胞的同时获取和这些细胞的诱导来进行软骨细胞分化。骨髓（BM）已被广泛使用作为干细胞的来源，但是从骨髓细胞分离有两个主要的缺点：侵袭和产量不足。因为它易于获得的，脂肪组织是干细胞的一个优选来源。以往的研究表明，从脂肪组织中分离干细胞和诱导用细胞因子，如TGF-β11,2这些细胞软骨分化的可行性。这些方法是有效的，但价格昂贵。
作为一个成本较低的替代细胞因子，机械应力可以用来诱导软骨细胞分化。机械加载在维持关节软骨3的健康至关重要的作用，并且它可以诱导各种细胞软骨表型。例如，静水压经由MAP激酶/ JNK途径4诱导滑膜衍生的祖细胞的软骨形成的表型，和机械压缩通过上调软骨基因5诱导软骨在人类间质干细胞（MSCs）。此外，剪切应力有助于软骨相关的细胞外基质（ECM）的人MSC 6的表达。离心重力（CG），离心产生很容易地应用和控制的机械应力，可诱导细胞7差异表达基因。例如，在肺上皮癌细胞，白细胞介素（IL）-1b的表达通过离心8上调。 ŧherefore，作为实验诱导机械应力，CG可用于诱导干细胞软骨基因表达。然而，它的CG能否诱导干细胞的软骨细胞分化还不清楚。
在这项研究中，我们发现，CG诱导SOX9的上调，软骨的主要调控，在人类携带者，导致软骨细胞基因的过度表达。此外，我们比较的CG上与TGF-β1的软骨的影响，生长因子最常用于在体外诱导软骨中的干细胞。
Protocol
Representative Results
Discussion
细胞的干性状态为SOX9的CG诱导过表达非常重要的。在我们的研究中，SOX9的表达可以通过CG在早期传代的ASC（2-3）引起的，而不是在后面通道的ASC。已经报道的是，在培养过程中，携带者包含CD34 +细胞，直至3代16。的ASCs往往失去的CD34的表达作为将细胞传代，从而产生至CG低响应。
离心重力，静水压力可以刺激CG过程中加载到细胞。因此，介质的体积可能是影?…
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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