Automated Single Cell Tracking of In Vitro Multi-Cellular Morphogenesis Predicts Pluripotent Stem Cell Fate Decisions
Joy, D.; Libby, A.; McDevitt, T.
Journal of biomolecular techniques JBT 30(Suppl): S6
ISSN/ISBN: 1524-0215 PMID: 31892872 Accession: 069670853
During stem cell differentiation, individual cells must undergo a series of lineage commitment decisions to reach their final cell fate, analogous to the transitions that occur during early development. While many transcriptional and microscopic techniques exist to assess the quality of differentiation from fixed samples, there are few approaches capable of providing non-destructive monitoring of stem cell cultures. To enable live assessment of in-vitro differentiation, we have developed an assay to detect changes in human pluripotent stem cell behavior by imaging nuclear labeled cells using confocal microscopy. By developing a cell tracking algorithm based on an ensemble of neural nets, we are able to detect changes to cell and colony parameters such as migration velocity, cell density, and persistence of migration at single cell resolution. Using this platform, we explored changes to cell behavior during the early stages of common differentiation protocols to induce multilineage (using BMP4) or directed differentiation (using CHIR or dual SMAD inhibition). We show that single cell tracking behavior assessment strongly correlates with canonical loss of pluripotency (OCT4 and SOX2) and acquisition of early lineage markers (EOMES). Furthermore, whole colony analysis reveals regions where cell behavior changes in advance of canonical lineage marker expression, enabling prediction of localized differentiation within the larger stem cell population. These results indicate that assessing single cell behavior through whole colony tracking provides a rapid and effective tool for non-destructively assaying pluripotent stem cell fate decisions.