Biological characteristics and karyotiping of a new isolation method for human adipose mesenchymal stem cells in vitro

Li, Jun; Huang, He; Xu, Ximing

doi:10.1016/j.tice.2017.04.005

Cited by 17 publications

(14 citation statements)

References 12 publications

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“…In addition to sterility tests, the methods of expansion must guarantee and maintain the phenotypic and functional characteristics of the cells as well as their genomic stability during the in vitro culture period. Few studies have focused on the safety of cultured human ASCs by molecular evaluation of genomic stability [ 30 , 31 ]. Due to the lack of international indications about genetic stability assessments, the karyotype technique is the most frequently used analysis [ 27 ].…”

Section: Culture and Expansion Of Ascsmentioning

confidence: 99%

“…ASCs expanded in both media show unaltered phenotype as assayed by flow cytometry and no genetic lesion is observed [ 27 ]. Li et al [ 30 ] have performed an explant method to isolate and expand in a serum-free medium ASCs according to current GMP guidelines. The biological characteristics, such as cell morphology, karyotype, cell cycle, immunophenotype and growth factors, are assessed at different culture passages (n. 1, 3, 5, 10, 15, 20).…”

Section: Culture and Expansion Of Ascsmentioning

confidence: 99%

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Methods of Isolation, Characterization and Expansion of Human Adipose-Derived Stem Cells (ASCs): An Overview

Palumbo

Lombardi

Siragusa

et al. 2018

IJMS

111

102

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Considering the increasing interest in adipose-derived stem cells (ASCs) in regenerative medicine, optimization of methods aimed at isolation, characterization, expansion and evaluation of differentiation potential is critical to ensure (a) the quality of stem cells also in terms of genetic stability; (b) the reproducibility of beneficial effects; and (c) the safety of their use. Numerous studies have been conducted to understand the mechanisms that regulate ASC proliferation, growth and differentiation, however standard protocols about harvesting and processing techniques are not yet defined. It is also important to note that some steps in the procedures of harvesting and/or processing have been reported to affect recovery and/or the physiology of ASCs. Even considering the great opportunity that the ASCs provide for the identification of novel molecular targets for new or old drugs, the definition of homogeneous preparation methods that ensure adequate quality assurance and control, in accordance with current GMPs (good manufacturing practices), is required. Here, we summarize the literature reports to provide a detailed overview of the methodological issues underlying human ASCs isolation, processing, characterization, expansion, differentiation techniques, recalling at the same time their basilar principles, advantages and limits, in particular focusing on how these procedures could affect the ASC quality, functionality and plasticity.

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Section: Culture and Expansion Of Ascsmentioning

confidence: 99%

Section: Culture and Expansion Of Ascsmentioning

confidence: 99%

Methods of Isolation, Characterization and Expansion of Human Adipose-Derived Stem Cells (ASCs): An Overview

Palumbo

Lombardi

Siragusa

et al. 2018

IJMS

111

102

View full text Add to dashboard Cite

show abstract

“…Therefore, there is a critical need to assess the long-term culture influence on the phenotype and differentiation potential of ASCs. Recent literature suggest that long-term cell culture does not cause any chromosome aberrations 4 . Nevertheless, the debate regarding the impact of cell culture on the characteristic ASCs markers and their differentiation potential tend to constantly emerge 2 .…”

Section: Introductionmentioning

confidence: 99%

Immunophenotyping and transcriptional profiling of in vitro cultured human adipose tissue derived stem cells

Mieczkowska

Schumacher

Filipowicz

et al. 2018

Sci Rep

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Adipose-derived stem cells (ASCs) have become an important research model in regenerative medicine. However, there are controversies regarding the impact of prolonged cell culture on the ASCs phenotype and their differentiation potential. Hence, we studied 10 clinical ASCs replicates from plastic and oncological surgery patients, in six-passage FBS supplemented cultures. We quantified basic mesenchymal cell surface marker transcripts and the encoded proteins after each passage. In parallel, we investigated the differentiation potential of ASCs into chondrocytes, osteocytes and adipocytes. We further determined the effects of FBS supplementation and subsequent deprivation on the whole transcriptome by comprehensive mRNA and miRNA sequencing. Our results show that ASCs maintain differentiation potential and consistent profile of key mesenchymal markers, with apparent expression of distinct isoforms, in long-term cultures. No significant differences were observed between plastic and oncological surgery cohorts. ASCs in FBS supplemented primary cultures are almost committed to mesenchymal lineages as they express key epithelial-mesenchymal transition genes including early mesenchymal markers. Furthermore, combined mRNA/miRNA expression profiling strongly supports a modulatory role for the miR-30 family in the commitment process to mesenchymal lineages. Finally, we propose improvements to existing qPCR based assays that address alternative isoform expression of mesenchymal markers.

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“…During adipogenic differentiation of hASCs, LDs increase in number and size. The adipogenic potential of hASCs tends to decline with consecutive passages under standard culture conditions (Li et al, 2017 ). To induce this process in vitro , we used low passage (up to 6) hASCs with an optimized drug cocktail (Gojanovich et al, 2016 ), based on a comparison of previously used formulations (Gimble and Guilak, 2003 ; Lee et al, 2004 ; Rodriguez et al, 2004 ; Dicker et al, 2005 ; Bunnell et al, 2008 ; Zhu et al, 2008 ; da Silva Meirelles and Covas, 2011 ; Kim et al, 2016 ).…”

Section: Anticipated Resultsmentioning

confidence: 99%

Human Adipose-Derived Mesenchymal Stem/Stromal Cells Handling Protocols. Lipid Droplets and Proteins Double-Staining

Gojanovich

Giménez

Masone

et al. 2018

Front. Cell Dev. Biol.

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Human Adipose-derived mesenchymal stem/stromal cells (hASCs) are of great interest because of their potential for therapeutic approaches. The method described here covers every single step necessary for hASCs isolation from subcutaneous abdominal adipose tissue, multicolor phenotyping by flow cytometry, and quantitative determination of adipogenic differentiation status by means of lipid droplets (LDs) accumulation, and Western blot analysis. Moreover, to simultaneously analyze both LDs accumulation and cellular proteins localization by fluorescence microscopy, we combined Oil Red O (ORO) staining with immunofluorescence detection. For LDs quantification we wrote a program for automatic ORO-stained digital image processing implemented in Octave, a freely available software package. Our method is based on the use of the traditional low cost neutral lipids dye ORO, which can be imaged both by bright-field and fluorescence microscopy. The utilization of ORO instead of other more expensive lipid-specific dyes, together with the fact that the whole method has been designed employing cost-effective culture reagents (standard culture medium and serum), makes it affordable for tight-budget research laboratories. These may be replaced, if necessary or desired, by defined xeno-free reagents for clinical research and applications.

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Biological characteristics and karyotiping of a new isolation method for human adipose mesenchymal stem cells in vitro

Cited by 17 publications

References 12 publications

Methods of Isolation, Characterization and Expansion of Human Adipose-Derived Stem Cells (ASCs): An Overview

Methods of Isolation, Characterization and Expansion of Human Adipose-Derived Stem Cells (ASCs): An Overview

Immunophenotyping and transcriptional profiling of in vitro cultured human adipose tissue derived stem cells

Human Adipose-Derived Mesenchymal Stem/Stromal Cells Handling Protocols. Lipid Droplets and Proteins Double-Staining

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