Applicability of the Dyes CFSE, CM-DiI and PKH26 for Tracking of Human Preadipocytes to Evaluate Adipose Tissue Engineering

Abstract

Adipose tissue engineering with preadipocyte-loaded scaffolds requires adequate tracking of preadipocytes to allow evaluation and quantification of cell proliferation, expansion and differentiation in three-dimensional systems. To differentiate between graft and host cells, labeling of preadipocytes before implantation and tracking of these cells until harvest would be useful. Immunohistochemistry enables the differentiation between cells of different species but is time-consuming, expensive, elaborate, and not applicable for autologous transplantation. So far, there is no published method to use externally applied dyes for tracking of human preadipocytes in adipose tissue engineering. We tested the cell dyes PKH26, CM-DiI, and CFSE to analyze their applicability for labeling human preadipocytes. CM-DiI had toxicity levels of 45–70%, while 3–4% proliferating cells were stained on day 35. CFSE revealed clear cytoplasmic coloring in proliferating cells with 5–6% stained cells after 35 days and toxicity ranging from 55 to 90% dead cells. PKH26 demonstrates lowest levels of toxicity and best labeling results after 4 weeks in proliferating preadipocytes in monolayer. Although none of the dyes showed long-lasting labeling during proliferation, all three dyes demonstrated permanent staining in differentiated cells. The results reveal the problems of preadipocyte tracking with fluorescent dyes but justify the dye application for limited time periods.

A Comparative Study of Proliferation and Hepatic Differentiation of Human Adipose-Derived Stem Cells

“…Recently, it has been reported by Banas et al [2007] that TTR is expressed in ADSC CD105+ differentiated into hepatic lineage. In our previous study [Raposio et al, 2008], we verified that MTT test, DNA assay and CFSE staining were suitable analyses to evaluate cell proliferation in undifferentiated ADSC, in accordance with other data in the literature [Hemmrich et al, 2006;Liu et al, 2008;Zaminv et al, 2008]. So, we apply the above analyses to evaluate the proliferation rate of HA and HB cells.…”

Stereological Analysis on Migration of Human Neural Stem Cells in the Brain of Rats Bearing Glioma

“…Multipotent NSCs are found in the developing and adult mammalian CNS, including that of humans. 15,27,28 The human NSC line F3 carries the normal human karyotype (46XX) and has the ability to selfrenew, differentiate into cells of neuronal or glial lineage, and integrate into the damaged CNS on transplantation in animal locally converts 5-fluorocytosine into 5-fluorouracil, which interferes with DNA synthesis and results in the death of dividing cells. 27,30,31 Similarly, the TK gene converts the nontoxic drug ganciclovir into a phosphorylated metabolite that acts as a potent killer of dividing cancer cells.…”

DiI Labeling of Human Adipose-Derived Stem Cells: Evaluation of DNA Damage, Toxicity and Functional Impairment

“…These include luciferase transfection [Bereziat et al, 2005;Wolbank et al, 2007;Bai et al, 2011], green fluorescent protein [Bereziat et al, 2005;Lin et al, 2006;Yan et al, 2007;Wolbank et al, 2007;Bai et al, 2011], fluorescence in situ hybridization [Li et al, 2008], various fluorochromes [Rieck and Schlaak, 2003;Hemmrich et al, 2006;Yan et al, 2007;Li et al, 2008;Li et al, 2009;Lequeux et al, 2011], iron oxide labeling for MRI [Heymer et al, 2008;Kraitchman and Bulte, 2008;Yan et al, 2007] as well as radionuclide labeling strategies [Hofmann et al, 2005;Yan et al, 2007].…”

“…Fluorochromes such as PKH26, a fluorescent surface marker [Rieck and Schlaak, 2003;Hemmrich et al, 2006], BrdU (5-bromo-2-deoxyuridine) which replaces thymidine and incorporates into DNA strands during cell proliferation [Lequeux et al, 2011] and CSFE (Vybrant ® CFDA-SE cell tracer kit) which diffuses passively into the cells [Hemmrich et al, 2006] were analyzed for their applicability to ASCs. In addition, gene transfection procedures resulting in the expression of green fluorescence protein or luciferase were also used to label ASCs [Béréziat et al, 2005;Wolbank et al, 2007].…”