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Cited by 112Mesenchymal stem cell researchPluripotent Stem Cells ResearchAdipose Tissue and Metabolism

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High efficient isolation and systematic identification of human adipose-derived mesenchymal stem cells

Open access
Yang, Xu-Fang, He, Xu, He, Jian, Zhang, Li-Hong, Su, Xue-Jin, Dong, Zhi-Yong, Xu, Yun-Jian, Li, Yan, Li, Yu-Lin·2011·Journal of Biomedical Science
DOI 10.1186/1423-0127-18-59PMC PMC3175156
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Abstract

BACKGROUND: Developing efficient methods to isolate and identify human adipose-derived mesenchymal stem cells (hADSCs) remains to be one of the major challenges in tissue engineering. METHODS: We demonstrate here a method by isolating hADSCs from abdominal subcutaneous adipose tissue harvested during caesarian section. The hADSCs were isolated from human adipose tissue by collagenase digestion and adherence to flasks. RESULTS: The yield reached around 1 × 10(6) hADSCs per gram adipose tissue. The following comprehensive identification and characterization illustrated pronounced features of mesenchymal stem cells (MSCs). The fibroblast-like hADSCs exhibited typical ultrastructure details for vigorous cell activities. Karyotype mapping showed normal human chromosome. With unique immunophenotypes they were positive for CD29, CD44, CD73, CD105 and CD166, but negative for CD31, CD34, CD45 and HLA-DR. The growth curve and cell cycle analysis revealed high capability for self-renewal and proliferation. Moreover, these cells could be functionally induced into adipocytes, osteoblasts, and endothelial cells in the presence of appropriate conditioned media. CONCLUSION: The data presented here suggest that we have developed high efficient isolation and cultivation methods with a systematic strategy for identification and characterization of hADSCs. These techniques will be able to provide safe and stable seeding cells for research and clinical application.

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Figures

Figure 1 The morphological features and karyotype of hADSCs. The hADSCs are typical fibroblast-like cells with fusiform shape from the 3rd passage (P3) (A) to the 12th passage (P12) (B) (Bars = 100 μm). Under transmission electron microscope, hADSCs exhibited irregular nuclear morphology and abundant organelles (C), abundant microvilli with some inclusion body-like structures (arrow) (Bars = 500 nm) (D). (E) One of two reports from G-banding karyotype analysis at P12 showed normal female chromosome type: 46, XX.
Figure 1 The morphological features and karyotype of hADSCs. The hADSCs are typical fibroblast-like cells with fusiform shape from the 3rd passage (P3) (A) to the 12th passage (P12) (B) (Bars = 100 μm). Under transmission electron microscope, hADSCs exhibited irregular nuclear morphology and abundant organelles (C), abundant microvilli with some inclusion body-like structures (arrow) (Bars = 500 nm) (D). (E) One of two reports from G-banding karyotype analysis at P12 showed normal female chromosome type: 46, XX.CC BY · PubMed Central
Figure 2 The hADSCs expressed a unique set of CD markers. (A) Flow cytometry analysis disclosed that the 3rd passage (P3) were positive for CD29, CD44, CD73, CD105 and CD166 with expression rates all up to 95%, but negative for CD31, CD34, CD45 and HLA-DR. (B) This immunophenotype was consistent among different passages. (C) Merged images from immunofluorescent staining of CD antigens (green) and propidium iodide (PI) staining of nuclei (red) demonstrated the same phenotype (Bars = 10 μm).
Figure 2 The hADSCs expressed a unique set of CD markers. (A) Flow cytometry analysis disclosed that the 3rd passage (P3) were positive for CD29, CD44, CD73, CD105 and CD166 with expression rates all up to 95%, but negative for CD31, CD34, CD45 and HLA-DR. (B) This immunophenotype was consistent among different passages. (C) Merged images from immunofluorescent staining of CD antigens (green) and propidium iodide (PI) staining of nuclei (red) demonstrated the same phenotype (Bars = 10 μm).CC BY · PubMed Central
Figure 3 Growth kinetics and cell cycle analysis. (A) The growth curves showed no significant difference in the growth rate among different passages. (B) 15.1 ± 2.9% of the cells was in S+G2/M phase (active proliferative phase) (pink area) with the remaining cells in G0/G1 phase (quiescent phase, 84.9% ± 2.9%) (blue area).
Figure 3 Growth kinetics and cell cycle analysis. (A) The growth curves showed no significant difference in the growth rate among different passages. (B) 15.1 ± 2.9% of the cells was in S+G2/M phase (active proliferative phase) (pink area) with the remaining cells in G0/G1 phase (quiescent phase, 84.9% ± 2.9%) (blue area).CC BY · PubMed Central
Figure 4 Adipogenic differentiation of hADSCs. The lipid was detected by Oil-red O staining after induced for 1 week (A) and 2 weeks (B) (Bars = 100 μm). (C) Quantification of the adipogenesis was done by extraction of the Oil red O retention. *P < 0.01. (D) The expression of adipogenic specific marker PPAR-γ was detected by sqRT-PCR, lane 1: non-induced hADSCs control; lane 2: hADSCs induced for 1 week; lane 3: hADSCs induced for 2 weeks.
Figure 4 Adipogenic differentiation of hADSCs. The lipid was detected by Oil-red O staining after induced for 1 week (A) and 2 weeks (B) (Bars = 100 μm). (C) Quantification of the adipogenesis was done by extraction of the Oil red O retention. *P < 0.01. (D) The expression of adipogenic specific marker PPAR-γ was detected by sqRT-PCR, lane 1: non-induced hADSCs control; lane 2: hADSCs induced for 1 week; lane 3: hADSCs induced for 2 weeks.CC BY · PubMed Central
Figure 5 Osteogenic differentiation of hADSCs. Compared to non-induced control (A), Alkaline phosphatase staining was increased after being induced for 2 weeks (B) (Bars = 100 μm) and ALP activity was quantified by PNPP analysis (C). *P < 0.01. Calcium nodule formation was demonstrated by Alizarin red staining (D: non-induced control; E: induced for 4 weeks) (Bars = 100 μm). (F) The expression of the osteogenic specific marker osteopontin (OPN) was detected by sqRT-PCR, lane 1: non-induced hADSCs control; lane 2: hADSCs induced for 2 weeks; lane 3: hADSCs induced for 4 weeks.
Figure 5 Osteogenic differentiation of hADSCs. Compared to non-induced control (A), Alkaline phosphatase staining was increased after being induced for 2 weeks (B) (Bars = 100 μm) and ALP activity was quantified by PNPP analysis (C). *P < 0.01. Calcium nodule formation was demonstrated by Alizarin red staining (D: non-induced control; E: induced for 4 weeks) (Bars = 100 μm). (F) The expression of the osteogenic specific marker osteopontin (OPN) was detected by sqRT-PCR, lane 1: non-induced hADSCs control; lane 2: hADSCs induced for 2 weeks; lane 3: hADSCs induced for 4 weeks.CC BY · PubMed Central
Figure 6 Immunocytochemical analysis and ultrastructure of hADSCs under endothelial differentiation. (A) The expression of endothelial-specific protein vascular endothelial growth factor receptor-2 (KDR), CD34 and CD31 were detected by diaminobenzidine staining of the secondary antibody (Bars = 50 μm). (B) Ultrastructural images showed clear specific endothelial granule, the Weibel-Palade body (arrow) (Bars = 200 nm).
Figure 6 Immunocytochemical analysis and ultrastructure of hADSCs under endothelial differentiation. (A) The expression of endothelial-specific protein vascular endothelial growth factor receptor-2 (KDR), CD34 and CD31 were detected by diaminobenzidine staining of the secondary antibody (Bars = 50 μm). (B) Ultrastructural images showed clear specific endothelial granule, the Weibel-Palade body (arrow) (Bars = 200 nm).CC BY · PubMed Central

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Metadata

Authors
Yang, X.-F., He, X., He, J., Zhang, L.-H., Su, X.-J., Dong, Z.-Y., Xu, Y.-J., Li, Y., & Li, Y.-L.
Affiliations
Jilin University (CN)
Corresponding author
  • He, Xu
  • Li, Yan
ORCID
Venue
Journal of Biomedical Science
Publisher
Springer Science and Business Media LLC
Published
2011-08-19
Volume
18
Issue
1
Pages
59-59
ISSN
  • 1021-7770 (ISSN-L)
  • 1423-0127
DOI
10.1186/1423-0127-18-59
PMC
PMC3175156
OpenAlex
W2087905124
Cited by
112
References
25
Citations by year
2026: 1 · 2025: 6 · 2024: 1 · 2023: 2 · 2022: 7 · 2021: 7 · 2020: 8 · 2019: 6 · 2018: 17 · 2017: 9 · 2016: 9 · 2015: 12 · 2014: 8 · 2013: 15 · 2012: 4
FWCI
4.37
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Journal of Biomedical Science (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Natural Science Foundation of China (863 Program)
  • National High-tech Research and Development Program
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Health Sciences › Medicine › Physiology (medical) › Adipose Tissue and Metabolism
Keywords
human adipose-derived mesenchymal stem cells, adipose tissue harvesting, collagenase digestion, cell isolation, immunophenotyping, cell cycle analysis, self-renewal, multilineage differentiation, adipogenesis, bone formation, endothelial differentiation, karyotype
MeSH terms
Adult, Cell Cycle, Cell Differentiation, Cell Separation, Female, Humans, Immunohistochemistry, Karyotyping, Immunophenotyping, Collagenases, Fluorescent Antibody Technique, Indirect, Tissue Engineering, Microscopy, Electron, Transmission, Cell Proliferation, Subcutaneous Fat, Mesenchymal Stem Cells, Real-Time Polymerase Chain Reaction

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