Parsing the early cytoskeletal and nuclear organizational cues that demarcate stem cell lineages

Abstract

O ur recent report suggests that subtle changes in early cytoskeletal protein-level organization correlate with long-term stem cell lineage commitment. 1 In this extra-view, we dissect changes in the expression of both cytoskeletal and nuclear-regulating genes that may precede and, possibly, govern the formative lineage-specific organizational cues. Human mesenchymal stem cells cultured on glass under basal, osteogenic and adipogenic induction media were analyzed for gene expression profiles within the first 24 hours. Several key actin organization regulating genes and nuclear and cell cycle regulatory genes were found to be upregulated in osteogenic media compared to adipogenic and basal conditions. Given the role of both cytoskeletal and nuclear genes, we examined the possibility of classifying stem cell subpopulations using high content imaging approaches based on the organization of both actin, as previously proposed, as well as nuclear organization and distribution of a nuclear organizational protein, the nuclear mitotic apparatus (NuMA). A pool of combined cytoskeletal and nuclear descriptors were merged into a composite feature space via dimension reduction, data fusion and classification methodologies. This composite approach enabled feature-based identification of specific lineage committed as well as non-differentiating cell populations. Using the improved classification of this high-content imagingbased profiling tool, we demonstrate that MSCs induced to differentiate to either Parsing the early cytoskeletal and nuclear organizational cues that demarcate stem cell lineages

Interconnected contribution of tissue morphogenesis and the nuclear protein NuMA to the DNA damage response

“…5C). To analyze precisely the behavior of NuMA in polarized and nonpolarized flat monolayers of cells, we performed high-content analysis of nuclear morphometric descriptors (Liu et al, 2010) in the absence or presence of BLM treatment (see supplementary material Fig. S6A).…”

“…Optical sections (,20 mm thickness) were taken, with a step size of 0.5 mm. Average projection images were generated and used for image analysis (Liu et al, 2010) (see schematic in supplementary material Fig. S5).…”

Organizational metrics of interchromatin speckle factor domains: integrative classifier for stem cell adhesion & lineage signaling

“…We propose the premise that the organization of subcellular proteins involved in outside-in signaling, particularly of those mediating mechanotransductive signaling, is regulated by microenvironmental cues during differentiation, and that these minute differences can be captured by enhanced content imaging. As a major evidence of this premise, our laboratory advanced an imaging-based profiling methodology that allows for the dissection of morphologic signatures in the cytoskeleton unique to a particular culture condition 19–22 . Using this approach, early (24 hour) actin morphologic signatures of hMSCs were identified and used to predict downstream osteogenic differentiation 20 .…”

Application of Theranostics to Measure and Treat Cell Heterogeneity in Cancer

“…Classification of NuMA distribution with LBF analysis on a per-cell basis into three patterns corresponding to normal, preinvasive cancer, and invasive cancer phenotypes on biopsy sections of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) breast tissues revealed cell heterogeneity within tumors (Knowles and Lelièvre, unpublished data). High-content analysis of NuMA and cytoskeletal morphometric descriptors is another computational approach that was used to monitor cell differentiation [68]. Interestingly, NuMA controls chromatin organization [19,67] confirming that chromatin features are important markers to measure cellular heterogeneity in cancer.…”