Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal 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

Defects in articular cartilage do not heal naturally. Consequently, stem cell transplantation has been proposed as a promising approach for the repair of impaired cartilage. However, this method requires both the acquisition of a sufficient number of stem cells and the induction of these cells to undergo chondrogenic differentiation. Bone marrow (BM) has been widely used as a source of stem cells, but cell isolation from BM has two major disadvantages: invasiveness and insufficient yield. Because of its ease of acquisition, adipose tissue is a preferable source of stem cells. Previous studies demonstrated the feasibility of isolating stem cells from adipose tissue and inducing chondrogenic differentiation in these cells using cytokines, such as TGF-β1^1,2. These methods are effective but expensive.

As a lower-cost alternative to cytokines, mechanical stress can be used to induce chondrogenic differentiation. Mechanical loading plays a critical role in maintaining the health of articular cartilage³, and it can induce chondrogenic phenotypes in various cells. For example, hydrostatic pressure induces chondrogenic phenotypes in synovium-derived progenitor cells via the MAP kinase/JNK pathway⁴, and mechanical compression induces chondrogenesis in human mesenchymal stem cells (MSCs) by upregulating chondrocytic genes⁵. In addition, shear stress contributes to the expression of chondrogenesis-related extracellular matrix (ECM) in human MSCs⁶. Centrifugal gravity (CG), an easily applied and controlled mechanical stress generated by centrifugation, can induce differential gene expression in cells⁷. For example, in lung epithelial carcinoma cells, the expression of interleukin (IL)-1b is upregulated by centrifugation⁸. Therefore, as an experimentally inducible mechanical stress, CG can be used to induce chondrocytic gene expression in stem cells. However, it remains unclear whether CG can induce the chondrogenic differentiation of stem cells.

In this study, we found that CG induced the upregulation of SOX9, a master regulator of chondrogenesis, in human ASCs, resulting in the overexpression of chondrocytic genes. In addition, we compared the effects of CG on chondrogenesis with those of TGF-β1, the growth factor most commonly used to induce in vitro chondrogenesis in stem cells.

Protocol

This study protocol was approved by the institutional review board of The Catholic University of Korea (KC16EAME0162) and performed according to NIH guidelines. All tissues were obtained with written informed consent.

1. Centrifugal Gravity Loading and Pellet Culture

1. Cell culture and harvest
   1. Culture ASCs (P2-P3; see List of Materials) in Dulbecco's Modified Eagle's Medium-low glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37 °C in a humidified incubator containing 5% CO₂.
   2. When the cells reach 80% confluence, discard the medium and wash the cells with 5 mL of 1x phosphate-buffered saline (PBS).
   3. Add 1 mL of PBS containing 1 mM EDTA and incubate for 2 min at 37 °C in a humidified incubator containing 5% CO₂.
   4. Tap the culture plate gently, add 4 mL of fresh medium, transfer the cells to a 15-mL conical tube, and centrifuge the cells at 250 × g for 2 min at room temperature (RT).
2. Centrifugal gravity loading
   1. After centrifugation (step 1.1.4), remove the supernatant without disturbing the pellet and resuspend the pellet in 10 mL of DMEM-LG with 10% FBS. Count the cells using a hemocytometer.
   2. For CG loading, transfer 2.5 × 10⁵ cells to a new 15-mL conical tube and centrifuge at 2,400 × g for 15 min.
3. Pellet culture
   1. Immediately after centrifugation, aspirate the supernatant and add 500 µL of defined chondrogenic differentiation medium (CDM, high-glucose DMEM supplemented with 1% FBS, 1% ITS+Premix, 100 nM dexamethasone, 1x MEM non-essential amino acid solution, 50 µg/mL L-proline, and 1% penicillin/streptomycin). Add 50 µg/mL of freshly prepared L-ascorbic acid 2-phosphate at every medium change. As a positive control, induce chondrogenic differentiation in uncentrifuged cells by adding CDM containing 10 ng/mL TGF-β1.
   2. Place the loosely capped tubes containing the pellets in a standing position and incubate at 37 °C in a humidified incubator containing 5% CO₂.
   3. Change the medium every other day for 3 weeks.
4. Micromass culture
   1. Immediately after centrifugation (step 1.2.2; 2,400 × g for 15 min), aspirate the supernatant and resuspend the pellet in 10 µL of CDM.
   2. To form a micromass, place the resuspended cells in the center of a well of a 24-well plate.
   3. After 2 h, carefully add 1 mL of CDM to the well, pipetting against the wall of the plate to avoid disrupting the micromass. As a positive control, perform a micromass culture with uncentrifuged cells by adding CDM containing 10 ng/mL TGF-β1.
   4. Incubate the micromass at 37 °C in a humidified incubator containing 5% CO₂.
   5. Change the medium every other day for 3 weeks.

2. Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR) to Detect Transcriptional Upregulation of Chondrogenic Differentiation Markers

1. On day 14, use a pipette to transfer the spheroid pellets to a new 1.5-mL tube and wash them in 1x PBS.
2. Extract the total RNA from the pellets using the guanidinium thiocyanate-phenol-chloroform extraction method⁹.
3. Synthesize cDNA from 2 µg of total RNA using reverse transcriptase according to the manufacturer's protocol (incubate at 42 °C for 1 h and then inactivate the enzyme at 70 °C for 5 min).
4. Perform PCR with primers specific for chondrogenic differentiation markers¹⁰.

3. Staining to Detect the Overexpression of Chondrogenic Differentiation Marker Proteins

1. Paraffin-embedding cell pellets
   1. On day 21, use a pipette to harvest the spheroid pellets and wash them with 1x PBS.
   2. Fix the spheroid pellets by immersion in 4% paraformaldehyde for 24 h.
      Caution: Paraformaldehyde is highly toxic. Avoid contact with eyes, skin, or mucous membranes. Minimize exposure and avoid inhalation during preparation. Wear appropriate personal protective equipment.
   3. Discard the fixative and rinse the cell pellets with deionized water (DW).
   4. Place one layer of gauze onto the cassette and transfer the fixed pellets using a pipette. Cover the pellet by folding the gauze and close the cassette lid.
   5. Dehydrate the pellets.
      1. Immerse the pellets in 70% ethanol (EtOH) for 5 min at RT.
      2. Immerse the pellets in 80% EtOH for 5 min at RT.
      3. Immerse the pellets in 95% EtOH for 5 min at RT.
      4. Immerse the pellets in 100% EtOH for 5 min at RT. Repeat twice.
      5. Immerse the pellets in 100% xylene for 15 min at RT. Repeat twice.
   6. Embed the fixed cell pellets in paraffin at 56 °C in a mold; the pellets can be embedded into paraffin using specialized automated tissue processing systems.
   7. Cut 5 µm-thick sections from the paraffin-embedded cell pellet using a rotary microtome.
   8. Float the sections in 50% EtOH and then transfer them to a 50 °C water bath using a slide.
   9. Place the floating sections onto slides.
2. Safranin O and Alcian blue staining
   1. Rehydrate the paraffin sections.
      1. Immerse the slides in xylene for 15 min. Repeat twice.
      2. Immerse the slides in 100% EtOH for 5 min. Repeat twice.
      3. Immerse the slides in 90% EtOH for 5 min.
      4. Immerse the slides in 80% EtOH for 5 min.
      5. Immerse the slides in 70% EtOH for 5 min, and then wash them in 1x PBS.
   2. Stain the rehydrated sections with 1% Safranin O solution and 3% Alcian blue for 30 min.
   3. Discard the staining solution and rinse the sections with DW.
   4. Stain the sections with Hematoxylin/Eosin solution (counterstain) for 1 min.
   5. Discard the staining solution and rinse the sections with DW.
   6. Place a drop of mounting medium on a coverslip after removing any residual water. Carefully place the slide (cell-side down) on the coverslip containing the mounting medium.
   7. Allow the slides to dry for 2-3 h at RT.
   8. Capture images using a bright-field microscope (automated upright microscope, 50X and 200X).
3. Immunofluorescence assay
   1. Rehydrate the sections as described in section 3.2.1.
   2. Immerse the slides in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0), and then maintain them at a sub-boiling temperature for 10 min using a microwave.
   3. Cool the slides for 20 min at RT and then wash them with tap water.
   4. Incubate the slides in 3% H₂O₂ (in 1x PBS) for 15 min, and then wash them with tap water for 15 min¹¹.
   5. Block the slides with blocking buffer (10% normal goat serum in PBS) for 1 h.
   6. Aspirate the blocking solution, and then apply a primary antibody diluted with 5% normal goat serum onto the slides.
   7. Incubate the slides overnight at 4 °C.
   8. Wash the slides three times in 1x PBS for 5 min each.
   9. Incubate the slides in fluorochrome-conjugated secondary antibody diluted with 5% normal goat serum for 1 h at RT in the dark.
   10. Wash the slides three times in 1x PBS for 5 min each in the dark.
   11. Incubate the slides with DAPI (1 µg/mL) for 10 min, and then wash them two times in 1x PBS for 5 min each.
   12. Place a drop of mounting medium on a coverslip after removing any residual water. Carefully place the slide (cell-side down) on the mounting medium.
   13. Allow the slides to dry for 2-3 h in the dark at RT.
   14. Visualize the slides using fluorescence microscopy (Rhod: 594 nm, DAPI: 340 nm; 60X and 200X magnification)¹².

Results

Centrifugal gravity induces the overexpression of chondrogenic differentiation markers in adipose-derived stem cells.

To determine the degree of centrifugal gravity force that is suitable to induce chondrogenic differentiation, ASCs were stimulated with different degrees of CG (0, 300, 600, 1,200, and 2,400 x g) for 15 min. After stimulation, the ASCs were re-seeded onto culture plates and cultured for 24 h. As shown in ...

Discussion

The stemness state of cells is very important for CG-induced overexpression of SOX9. In our study, SOX9 expression could be induced by CG in early-passage ASCs (2-3), but not in later-passage ASCs. It has been reported that, during cultivation, ASCs contain CD34+ cells until 3 passages¹⁶. ASCs tend to lose the expression of CD34 as the cells are passaged, resulting in a low response to CG.

With centrifugal gravity force, hydrostatic pressure can be loaded onto cells dur...

Materials

Name	Company	Catalog Number	Comments
Plasticware
100 mm Dish	TPP	93100
60 mm 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
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
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	100 nM
Penicilin Streptomycin	Life Technologies	P4333	1%
Fetal Bovine Serum	Life Technologies	16000-044	1%
Ascorbate-2-phosphate	Sigma Aldrich	A8960	50 μg/mL
L-proline	Sigma Aldrich	P5607	50 μg/mL
ITS	BD	354352	1%
Human TGFβ1	Peprotech	100-21	10 ng/mL
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
1 kb (+) DNA ladder marker	Enzynomics	DM003
Human adipose-derived stem cells (ASCs)	Catholic MASTER Cells