Isolation of Stem Cells from Human Adipose Tissue: Technique, Problems, and Pearls

Stocchero, Ithamar Nogueira; Stocchero, Guilherme Flosi

doi:10.1007/978-3-642-20012-0_2

Cited by 8 publications

(10 citation statements)

References 12 publications

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“…The CFU-F assay is recommended to define the number of progenitor cells and to predict the performance of any cell therapy product (Bourin et al 2013). In our hands, ADMSCs showed a clonal efficiency higher than that reported by others, thus testifying the high proliferative ability of multipotent cells obtained from intact tissue (Stocchero and Stocchero 2011). The multipotency of ADMSCs was further confirmed, since when cultured with proper inducers they were able to generate osteoblastic, chondrocytic and adipocytic lineages.…”

Section: Discussionsupporting

confidence: 71%

“…Based on literature data, we have chosen to obtain ADMSCs from intact adipose tissue, thus obtaining abundant amounts of material to store and recover over time. Some authors demonstrated that sliced fat tissue obtained through lipectomy generated a much higher CFU number when compared to fat samples obtained through liposuction (Stocchero and Stocchero 2011;Schreml et al 2009). The relative paucity of ADMSCs seems to depend on the distribution of multipotent stem cells, which are mainly located around large vessels and thus remain in the donors when the fat is aspirated.…”

Section: Discussionmentioning

confidence: 99%

“…Several methods have been employed to isolate and cultivate ADMSCs (Stocchero and Stocchero 2011;Bourin et al 2013). Most of the protocols involve enzymatic digestion of the sample, but there is a large variety in the kind and concentration of the enzyme used, time and conditions of incubation for the tissue digestion.…”

mentioning

confidence: 99%

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Hypoxia enhances proliferation and stemness of human adipose-derived mesenchymal stem cells

et al. 2014

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The aim of the study was to obtain the highest number of multipotent adipose-derived mesenchymal stem cells (ADMSCs) by using culture conditions which favour cell expansion without loss of mesenchymal stem cells (MSC)-like properties. Based on the assumption that stem cells reside in niches characterized by hypoxic condition, we investigated if the low oxygen tension may improve the proliferation and stemness of ADMSCs. Intact adipose tissue was resected from eight subjects, and the stromal vascular fraction was obtained by using type II collagenase. The heterogeneity of cellular lineages was confirmed by immunophenotypic analysis that showed the presence of leukocytes (CD45+), endothelial cells (CD34+), and pericytes (CD140+). The immunophenotype of confluent ADMSCs was similar to that of bone marrow-derived MSCs, except for the expression of CD34, which was variable (donor-dependent) and inversely correlated to the CD36 expression. ADMSCs showed a high clonal efficiency (94.5 ± 1 %) and were able to generate osteoblastic, chondrocytic and adipocytic lineages. ADMSCs were cultured under normoxic (21 % O2) and hypoxic (1 % O2) conditions, and we found that hypoxia significantly favoured ADMSC proliferation and preserved the expression of stemness genes, i.e. Nanog and Sox2. Since hypoxia reflects the microenvironment in which ADMSCs must proliferate and differentiate, the culture in hypoxic condition allows to better understand the biology of these cells and their regenerative potential. Low oxygen concentrations promote cell proliferation and stemness, thus enriching the pool of cells potentially able to differentiate into multi-lineages, and extending the possibility of a long-term expansion.

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Section: Discussionsupporting

confidence: 71%

Section: Discussionmentioning

confidence: 99%

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Hypoxia enhances proliferation and stemness of human adipose-derived mesenchymal stem cells

et al. 2014

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“…1,5 Despite being only multipotent, they demonstrate great therapeutic potential through the release of growth factors, chemokines, or antimicrobial signal molecules. 6,7 A sustainable labeling of hMSCs is relevant to track their way into damaged target tissue and as a forensic test in cases with occurring tumors after cell donation. Substantial manipulations of hMSCs often comprise an increased tendency for carcinogenesis and it may, therefore, be essential to discriminate these donated cells from native cells.…”

Section: Introductionmentioning

confidence: 99%

Electroporation: A Sustainable and Cell Biology Preserving Cell Labeling Method for Adipogenous Mesenchymal Stem Cells

Haar

Jonczyk

Lavrentieva

et al. 2019

BioResearch Open Access

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Human mesenchymal stem cells derived from adipose tissue (AD-hMSCs) represent a promising source for tissue engineering and are already widely used in cell therapeutic clinical trials. Until today, an efficient and sustainable cell labeling system for cell tracking does not exist. We evaluated transient transfection through electroporation for cell labeling and compared it with lentiviral transduction for AD-hMSCs. In addition, we tested whether nonsense DNA or a reporter gene such as enhanced green fluorescent protein (EGFP) is the more suitable label for AD-hMSCs. Using electroporation, the transfection efficiency reached a maximal level of 44.6 ± 1.1% EGFP-positive cells after selective and expansive cultivation of the mixed MSC population, and was 44.5 ± 1.4% after gene transfer with Cyanin3-marked nonsense-label DNA, which remained stable during 2 weeks of nonselective cultivation (37.2 ± 4.7% positive AD-hMSCs). Electroporation with both nonsense DNA and pEGFP-N1 led to a slight growth retardation of 45.2% and 59.1%, respectively. EGFP-transfected or transduced AD-hMSCs showed a limited adipogenic and osteogenic differentiation capacity, whereas it was almost unaffected in cells electroporated with the nonsense-label DNA. The nonsense DNA was detectable through quantitative real-time polymerase chain reaction for at least 5 weeks/10 passages and in differentiated AD-hMSCs. EGFP-labeled cells were trackable for 24 h in vitro and served as testing cells with new materials for dental implants for 7 days. In contrast, lentivirally transduced AD-hMSCs showed an altered natural immune phenotype of the AD-hMSCs with lowered expression of two cell type defining surface markers (CD44 and CD73) and a relevantly decreased cell growth by 71.8% as assessed by the number of colony-forming units. We suggest electroporation with nonsense DNA as an efficient and long-lasting labeling method for AD-hMSCs with the comparably lowest negative impact on the phenotype or the differentiation capacity of the cells, which may, therefore, be suitable for tissue engineering. In contrast, EGFP transfection by electroporation is efficient but may be more suitable for cell tracking within cell therapies without MSC differentiation procedures. Since current protocols of lentiviral gene transduction include the risk of cell biological alterations, electroporation seems advantageous and sustainable enough for hMSC labeling.

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“…A vascular stroma supports the fat, and mesenchymal stem cells within the blood vessels and the pericytes around them are responsible for producing fat cells. It is also relevant to point out that the stromal vascular fraction, which has been used in wound treatment and breast reconstruction, contains stem cells, but the volume required for this method to be effective is unknown 16 .…”

Section: Discussionmentioning

confidence: 99%

Lipoenxertia em reconstrução mamária

Fontenele

Gonçalves

2013

Rev. Bras. Cir. Plást.

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Isolation of Stem Cells from Human Adipose Tissue: Technique, Problems, and Pearls

Cited by 8 publications

References 12 publications

Hypoxia enhances proliferation and stemness of human adipose-derived mesenchymal stem cells

Hypoxia enhances proliferation and stemness of human adipose-derived mesenchymal stem cells

Electroporation: A Sustainable and Cell Biology Preserving Cell Labeling Method for Adipogenous Mesenchymal Stem Cells

Lipoenxertia em reconstrução mamária

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