Isolation and Characterization of a Spontaneously Immortalized Multipotent Mesenchymal Cell Line Derived from Mouse Subcutaneous Adipose Tissue

Abstract

The emerging field of tissue engineering and regenerative medicine is a multidisciplinary science that is based on the combination of a reliable source of stem cells, biomaterial scaffolds, and cytokine growth factors. Adult mesenchymal stem cells are considered important cells for applications in this field, and adipose tissue has revealed to be an excellent source of them. Indeed, adipose-derived stem cells (ASCs) can be easily isolated from the stromal vascular fraction (SVF) of adipose tissue. During the isolation and propagation of murine ASCs, we observed the appearance of a spontaneously immortalized cell clone, named m17.ASC. This clone has been propagated for more than 180 passages and stably expresses a variety of stemness markers, such as Sca-1, c-kit/CD117, CD44, CD106, islet-1, nestin, and nucleostemin. Furthermore, these cells can be induced to differentiate toward osteogenic, chondrogenic, adipogenic, and cardiogenic phenotypes. m17.ASC clone displays a normal karyotype and stable telomeres; it neither proliferates when plated in soft agar nor gives rise to tumors when injected subcutaneously in NOD/SCID-γ (null) mice. The analysis of gene expression highlighted transcriptional traits of SVF cells. m17.ASCs were genetically modified by lentiviral vectors carrying green fluorescent protein (GFP) as a marker transgene and efficiently engrafted in the liver, when injected in the spleen of NOD/SCID-γ (null) monocrotaline-treated mice. These results suggest that this non-tumorigenic spontaneously immortalized ASC line may represent a useful tool (cell model) for studying the differentiation mechanisms involved in tissue repair as well as a model for pharmacological/toxicological studies.

Electrical conditioning of adipose‐derived stem cells in a multi‐chamber culture platform

“…In this work, we present the design of a bioreactor capable of delivering controlled electrical stimulations to the cells and tailored to the needs of a standard biological laboratory. The device was used to study the effect of the electrical stimulation on stem cells, in particular on the m17.ASC clonal cell line, which spontaneously stabilized from murine subcutaneous adipose tissue (Zamperone et al, ). As a validation test, two different electrical regimes were investigated.…”

“…The m17.ASC line was obtained from inguinal subcutaneous adipose tissue of fvb/n strain mice by limiting diluition after immunomagnetic selection and enrichment for Sca‐1 positive cells (Zamperone et al, ) These cells were cultured in Claycomb medium (Sigma–Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Lonza‐Cambrex, Basel, Switzerland), 50 IU/mL penicillin, and 100 µg/mL streptomycin (Lonza‐Cambrex), at 37°C in a humidified atmosphere of 5% CO 2 . m17.ASC were maintained in culture for more than 2 years and were passaged twice per week at subconfluency.…”

“…The result of the proposed design is a robust, easy‐to‐use, cost‐effective, and reproducible device able to apply different electrical stimulation protocols on cells. To validate the bioreactor tool, we explored the possibility of inducing cardiac differentiation on a mouse adipose‐derived stem cell line (Zamperone et al, ) utilizing two different electrical stimulation protocols, that is, monophasic and biphasic stimulation, and we compared the results of these two strategies in terms of the cell viability, proliferation and global gene expression. Finally, we evaluated the levels of Cx‐43 (Hahn et al, ; Mills et al, ) in order to compare control culture samples to samples stimulated electrically within the bioreactor device.…”

Injured cardiomyocytes promote dental pulp mesenchymal stem cell homing

“…Observation that mesenchymal stem cells (MSCs) of both homo-and hetero-topic origins can differentiate in-vitro towards cardiac lineage [6,[9][10][11] provoked increasing interest and perspective about the possibility to restore damaged myocardium with MSCs. Since cells "implanted in" or "delivered to" the target site may frequently vanish within few days, some Authors support the hypothesis that MSCs promote positive actions in terms of cardiac functionality due to production of trophic factors, induction of vasculogenesis, and differentiation into CM [5,6].…”

Recent advances in the understanding of how neuropeptide Y andα-melanocyte stimulating hormone function in adipose physiology

“…20 Another example would be isolating cells from the stromal vascular fraction (SVF), the cells isolated after completion of enzymatic digestion, centrifugation, cellular filtration, and red blood cell lysis of the adipose tissue, 79 a mixture of cells that include mesenchymal stem cells, preadipocytes, endothelial cells, and immune cells. [80][81][82] This technique has been applied in mice, 83 humans, 84 and chickens.…”