Profiling of Human Neural Crest Chemoattractant Activity as a Replacement of Fetal Bovine Serum for In Vitro Chemotaxis Assays

Abstract

Fetal bovine serum (FBS) is the only known stimulus for the migration of human neural crest cells (NCCs). Non-animal chemoattractants are desirable for the optimization of chemotaxis as-says to be incorporated in a test battery for reproductive and developmental toxicity. We con-firmed here in an optimized transwell assay that FBS triggers directed migration along a con-centration gradient. The responsible factor was found to be a protein in the 30–100 kDa size range. In a targeted approach, we tested a large panel of serum constituents known to be chem-otactic for NCCs in animal models (e.g., VEGF, PDGF, FGF, SDF-1/CXCL12, ephrins, endothelin, Wnt, BMPs). None of the corresponding human proteins showed any effect in our chemotaxis assays based on human NCCs. We then examined, whether human cells would produce any fac-tor able to trigger NCC migration in a broad screening approach. We found that HepG2 hepa-toma cells produced chemotaxis-triggering activity (CTA). Using chromatographic methods and by employing the NCC chemotaxis test as bioassay, the responsible protein was enriched by up to 5000-fold. We also explored human serum and platelets as a direct source, independent of any cell culture manipulations. A CTA was enriched from platelet lysates several thousand-fold. Its temperature and protease sensitivity suggested also a protein component. The capacity of this factor to trigger chemotaxis was confirmed by single-cell video-tracking analysis of migrating NCCs. The human CTA characterized here may be employed in the future for the setup of assays testing for the disturbance of directed NCC migration by toxicants.

Targeting Acid Ceramidase Inhibits Glioblastoma Cell Migration through Decreased AKT Signaling

“…ASAH1 mRNA was significantly decreased when cells were transduced with lentivirus expressing ASAH1-directed shRNA ( Figure 3 B). Cells were allowed to migrate toward 10% FBS as a chemoattractant similar to previous reports for Boyden chamber assays [ 36 , 37 , 38 , 39 , 40 ]. Representative images of Boyden chamber inserts show less cell migration (stained in purple) when ASAH1 was knocked down ( Figure 3 C).…”

Quantification of cell migration: metrics selection to model application

“…Bias and errors in tracking exist whether cells are manually tracked or tracked using automation; additional specifics to avoid biased results are detailed in a review for immune cell migration ( Beltman et al, 2009 ; Svensson et al, 2018 ), which can be readily extended to other cell types. Images can be computationally expensive, particularly with long duration tracking, and tracking individual paths is time consuming; therefore, where fewer cells are tracked, care must be taken in generalizing the results ( Dolde et al, 2021 ; Cavanaugh et al, 2022 ). Faster moving cells may require shorter tracking durations than slow moving cells, but ultimately, similar amounts of data are collected ( Gorelik and Gautreau, 2014 ).…”

Identification of the bacterial metabolite aerugine as potential trigger of human dopaminergic neurodegeneration

“…HeLa were acquired from ATCC (CCL-2). The cell culture was performed as described previously ( Dolde et al, 2021 ). Briefly, the cell lines were kept in DMEM Glutamax with 10% FCS and 1% penicillin/streptomycin at 37 °C with 5% CO 2 .…”