Analysis of normalized, metabolite levels revealed MTX caused no switch in osteogenic or chondrogenic differentiation, while slightly increasing adipogenic response. nuclei as individual cells. These data confirm that our plate-based approach is comparable to the commonly used hemocytometer-based counting technique. Furthermore, the plate-based approach is usually potentially more accurate than the software-based approach, a less standard, but validated method. (*p < 0.001 between software-based and plate/hemocytometer-based counts) NIHMS610984-product-01.tif (6.0M) GUID:?8D26947C-36D5-4648-8017-4D31A21760C3 02: Supplemental Figure 2. To assist with cell counting, a simple fluorescence-based assay was Cholesteryl oleate developed in our lab. Standard curves for (A) ASCs and (B) NHFs were generated by plating known numbers of each cell type (ranging from 0-50,000 cells) in 96-well plates. The following day, nuclei were stained with Hoechst dye, and spectrofluorometry was used to quantify relative fluorescence models. Data were plotted to obtain an equation correlating relative fluorescence models with cell counts. This assay was extensively validated for each cell type and served as a reliable method for quantifying proliferation. While relative fluorescence models could also be used to assess proliferation, transforming to cell figures provided for more intuitive descriptions. NIHMS610984-product-02.tif (6.0M) GUID:?76513E6A-3C69-4CA1-8828-110444E6C45B 03: Supplemental Physique 3. To confirm that adipose-derived stem cell and normal human fibroblast media were not contributing to the effects of methotrexate, cells were treated with the drug in swapped media. (A) ASCs cultured and treated in NHF medium were Cholesteryl oleate resistant to methotrexate treatment. NHFs cultured and treated in ASC medium were inhibited by 2.5 and 50 M methotrexate. (B) Swapped media results were consistent with findings for cells treated in matched media conditions (from Fig. 1A). NIHMS610984-product-03.tif (3.4M) GUID:?772319E7-9937-4DEE-9F4F-743C8F26E1A2 Abstract In musculoskeletal tissues like bone, chemotherapy can impair progenitor cell differentiation and proliferation, resulting in decreased bone growth and mineralization throughout a patient’s lifetime. In the current study, we investigated the effects of chemotherapeutics on adipose-derived stem cell (ASC) function to determine whether this cell source could be a candidate for repairing, or even preventing, chemotherapy-induced tissue damage. Dose-dependent proliferation rates of ASCs and normal human fibroblasts (NHFs) were quantified after treatment with cytarabine (CY), etoposide (ETO), methotrexate (MTX), and vincristine (VIN) using a fluorescence-based assay. The influence of MTX around the multipotency TNFRSF9 of ASCs and freshly isolated stromal vascular portion (SVF) cells was also evaluated using lineage-specific staining and spectrophotometry. ASC and NHF proliferation were equally inhibited by exposure to CY and ETO; however, when treated with MTX and VIN, ASCs exhibited greater resistance. This was especially apparent for MTX-treated samples, with ASC proliferation showing no inhibition for clinically relevant MTX doses ranging from 0.1 to 50 M. Additional experiments revealed that this differentiation potential of ASCs was Cholesteryl oleate not affected by MTX treatment and that upregulation of dihydrofolate reductase possibly contributed to this response. Moreover, SVF cells, which include ASCs, exhibited comparable resistance to MTX impairment, with respect to cellular proliferation, clonogenicity, and differentiation capability. Therefore, we have shown Cholesteryl oleate that this regenerative properties of ASCs resist the cytotoxicity of MTX, identifying these cells as a potential important for fixing musculoskeletal damage in patients undergoing chemotherapy. exposure to common chemotherapeutics. We sought to identify resistance or susceptibility of ASCs to the tested drugs and improve upon our current understanding of chemotherapy effects. Furthermore, we aimed to investigate a potential mechanism behind any drug resistance to elucidate the phenomena observed in our results. Initial experiments used monolayer-expanded ASCs, which are more homogeneous than freshly isolated cells, to examine the effects of chemotherapeutics on regenerative properties. To investigate whether these effects were conserved for a more complex cell populace, subsequent experiments used heterogeneous, SVF cells to examine the proliferation and differentiation capabilities of drug-treated samples. To determine the effects of MTX, VIN, CY, and ETO on ASC and NHF proliferation, cells were counted on days 6-10 following treatment with specified drug concentrations. Most interestingly, we observed that ASC growth was not inhibited by MTX at any concentration (0.1-50 M). Conversely, NHF growth was inhibited after treatment with as low as 2.5 M,.