3D Spheroids Derived from Human Lipedema ASCs Demonstrated Similar Adipogenic Differentiation Potential and ECM Remodeling to Non-Lipedema ASCs In Vitro
Abstract
:1. Introduction
2. Results
2.1. ASC Spheroid Formation and Stemness Characterization
2.2. Adipogenic Differentiation of ASC-Derived Spheroids
2.3. Levels of Inflammatory Gene Expression in ASC Spheroids
2.4. Fibrosis in ASC Spheroids
2.5. Expression of ECM Components in 3D Differentiated Spheroids
2.6. Retention of Stemness Properties in ASC-Derived from 3D Spheroids
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Formation of ASC Spheroids
4.3. Adipogenic Differentiation of ASC Spheroids
4.4. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR)
4.5. Preparation of Frozen Sections and Immunofluorescence
4.6. Characterizing ASCs Derived from 3D Spheroids
4.7. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
ASCs | Adipose tissue-derived stem cells |
AT | Adipose tissue |
ECM | Extracellular matrix |
MMP | Matrix metalloproteinase |
LN | Laminin |
FN | Fibronectin |
SVF | Stromal vascular fraction |
CD | Cluster of differentiation |
2D | Two-dimensional |
3D | Three-dimensional |
ADIPOQ | Adiponectin |
LPL | Lipoprotein lipase |
PPAR-γ | Peroxisome proliferator-activated receptor gamma |
Glut4 | Glucose transporter type 4 |
Lip | Lipedema |
IL | Interleukin |
VEGF | Vascular endothelial growth factor |
TNFα | Tumor necrosis factor |
Col | Collagen |
CFU-F | Colony-forming unit fibroblast |
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Characteristics | Healthy | Lipedema |
---|---|---|
N | 8 | 8 |
Sex | Female | Female |
Age | 47.86 ± 2.28 | 44.57 ± 3.99 |
BMI | 27.30 ± 1.042 | 30.43 ± 1.163 |
Stage 1 | − | 12% |
Stage 2 | − | 63% |
Stage 3 | − | 25% |
Name | Forward (5′–3′) | Reverse (5′–3′) |
---|---|---|
IL-6 | GTAGCCGCCCCACACAGACAGCC | GCCATCTTTGGAAGGTTC |
LPL | GAGATTTCTCTGTATGGCACC | CTGCAAATGAGACACTTTCTC |
IL-1 β | TCCCCAGCCCTTTTGTTGA | TTAGAACCA AATGTGGCCGTG |
VEGF | AGGCCCACAGGGATTTTCTT | ATCAAACCTCACCAAGGCCA |
Glut-4 | AGC AGC TCT CTG GCA TCA AT | CAA TGG AGA CGT AGC ACA TG |
Leptin | GAAGACCACATCCACACACG | AGCTCAGCCAGACCCATCTA |
PPAR-γ | AGGCGAGGGCGATCTTG | CCCATCATTAAGGAATTCATGTCATA |
GAPDH | CGCTGAGTACGTCGTGGAGTC | GCAGGAGGCATTGCAGATGA |
TNF-α | GAG CCA GCT CCC TCT ATT TA | GGG AAC AGC CTA TTG TTC AG |
Oct4 | GACAGGGGGAGGGGAGGAGCTAGG | CTTCCCTCCAACCAGTTGCCCCAAAC |
Nanog | AGTCCCAAAGGCAAACAACCCACTTC | TGCTGGAGGCTGAGGTATTTCTGTCTC |
Col1 A1 | CATGTTCAGCTTTGTGGACCTC, | AGGTGATTGGTGGGATGTCTT |
Col6 A1 | GACCTCGGACCTGTTGGGTAC | TACCCCATCTCCCCCTTCAC |
Col6 A2 | CTGCGACAAGCCACAGCAG | GGGCACACGATCTGAGGGT |
Col6 A3 | GAGCAGCTTGACAACATTGCC | GCCCAGAGCACTTGCAGG |
ITAGA5 | GACAGGGAAGAGCGGGCACTATGG | GTCCCTTCCCGGCCGGTAAAACTC |
MMP-2 | TTGACGGTAAGGACGGACTC | ACTTGCAGTACTCCCCATCG |
MMP-9 | TTGACAGCGACAAGAAGTGG | GCCATTCACGTCGTCCTTAT |
MMP-11 | ATTTGGTTCTTCCAAGGTGCTCAGT | CCTCGGAAGAAGTAGATCTTGTTCT |
ADIPOQ | AACATGCCCATTCGCTTTAC | AGAGGCTGACCTTCACATCC |
Fibronectin | TTCCTTGCTGGTATCATGGCA | TATTCGGTTCCCGGTTCCA |
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Al-Ghadban, S.; Pursell, I.A.; Diaz, Z.T.; Herbst, K.L.; Bunnell, B.A. 3D Spheroids Derived from Human Lipedema ASCs Demonstrated Similar Adipogenic Differentiation Potential and ECM Remodeling to Non-Lipedema ASCs In Vitro. Int. J. Mol. Sci. 2020, 21, 8350. https://doi.org/10.3390/ijms21218350
Al-Ghadban S, Pursell IA, Diaz ZT, Herbst KL, Bunnell BA. 3D Spheroids Derived from Human Lipedema ASCs Demonstrated Similar Adipogenic Differentiation Potential and ECM Remodeling to Non-Lipedema ASCs In Vitro. International Journal of Molecular Sciences. 2020; 21(21):8350. https://doi.org/10.3390/ijms21218350
Chicago/Turabian StyleAl-Ghadban, Sara, India A. Pursell, Zaidmara T. Diaz, Karen L. Herbst, and Bruce A. Bunnell. 2020. "3D Spheroids Derived from Human Lipedema ASCs Demonstrated Similar Adipogenic Differentiation Potential and ECM Remodeling to Non-Lipedema ASCs In Vitro" International Journal of Molecular Sciences 21, no. 21: 8350. https://doi.org/10.3390/ijms21218350