Objective: Our goal is to generate new knowledge of how cell division occurs in normal, diverse human cell types. Division must be carefully controlled to ensure that daughter cells have the proper fate and ploidy. Cytokinesis occurs due to the ingression of a RhoA-dependent contractile ring, which pinches in the cell to form two daughters. Cytokinesis is regulated by multiple pathways associated with the mitotic spindle, composed of astral microtubules and the central spindle, and chromatin. Most of our knowledge of cytokinesis in human cells comes from studies using transformed or cancer cell lines. Given the diversity of cell types in the body, we lack knowledge of how most cells divide before they become fully differentiated.

Methods: We are using a split mNeonGreen (mNG) system to endogenously tag genes in human iPSCs. We engineered a parental cell line expressing the large fragment (mNG1-10), which reconstitutes fluorescence when the short fragment (mNG-11) is expressed. We then used CRISPR-Cas9 to integrate the short fragment into RhoA, tubulin, actin and histone, and the cytokinesis protein anillin, which regulates ring position. Live-cell imaging was used to characterize cytokinesis in tagged iPSCs before and after differentiation into endoderm, and after drug treatment to perturb the spindle.

Results: We found that cytokinesis occurs differently in iPSCs compared to HeLa cells. In iPSCs, the astral microtubules are large and extend equatorially, while the central spindle is small. Further, anillin and RhoA localization are uncoupled in iPSCs while they are similar in HeLa cells. Perturbation of the astral microtubules showed that they restrict anillin to a narrow zone at the equatorial cortex. Differentiating iPSCs into endoderm cells revealed that anillin localization is similar to iPSCs. We created additional iPSC lines to explore the roles of chromatin-sensing pathways, and will determine how cytokinesis changes as endoderm cells are further differentiated into hepatoblasts.

Conclusions: Revealing how cell division changes occur in diverse cell types is critical for understanding diseases. For example, hepatocytes are the major cell type in the liver and gain ploidy by failing mitosis. Hepatocellular carcinoma (HCC, liver cancer) cells have upregulated anillin and have reduced ploidy. Our findings could reveal how changes in cytokinesis occur during normal hepatocyte differentiation to better understand HCC.