Profiling of human neural crest chemoattractant activity as replacement of fetal bovine serum for in vitro chemotaxis assays

Abstract

Fetal bovine serum (FBS) is the only known stimulus for migration of human neural crest cells (NCCs). Non-animal chemoattractants are desirable for the optimization of chemotaxis assays to be incorporated in a test battery for reproductive and developmental toxicity. We confirmed here in an optimized transwell assay that FBS triggers directed migration along a concentration 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 chemotactic 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 in a broad screening approach, whether human cells would produce any factor able to trigger NCC migration. We found that HepG2 hepatoma 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 direct source, independent of any cell culture manipulations. A CTA was enriched from platelet lysates several thousand-fold. Its temperature and protease-sensitivity suggested 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.

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).…”

Use of metabolic glycoengineering and pharmacological inhibitors to assess lipid and protein sialylation on cells

“…Neural crest cells were differentiated from the induced pluripotent stem cell (iPSC) line IMR90_clone_#4 (; WiCell) exactly as described earlier (Dolde et al, 2021). To initiate differentiation in neural crest cells, iPSCs were plated on MatrigelTM coated 6‐well plates at a density of 100,000 cells/cm 2 in E8 medium (cat.no.…”