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

Abstract

Stem cell fates on biomaterials are influenced by the complex confluence of microenvironmental cues emanating from soluble growth factors, cell-to-cell contacts, and biomaterial properties. Cell-microenvironment interactions influence the cell fate by initiating a series of outside-in signaling events that traverse from the focal adhesions to the nucleus via the cytoskeleton and modulate the sub-nuclear protein organization and gene expression. Here, we report a novel imaging-based framework that highlights the spatial organization of sub-nuclear proteins, specifically the splicing factor SC-35 in the nucleoplasm, as an integrative marker to distinguish between minute differences of stem cell lineage pathways in response to stimulatory soluble factors, surface topologies, and microscale topographies. This framework involves the high resolution image acquisition of SC-35 domains and imaging-based feature extraction to obtain quantitative nuclear metrics in tandem with machine learning approaches to generate a predictive cell state classification model. The acquired SC-35 metrics led to > 90% correct classification of emergent human mesenchymal stem cell (hMSC) phenotypes in populations of hMSCs exposed for merely 3 days to basal, adipogenic, or osteogenic soluble cues, as well as varying levels of dexamethasone-induced alkaline phosphatase (ALP) expression. Early osteogenic cellular responses across a series of surface patterns, fibrous scaffolds, and micropillars were also detected and classified using this imaging-based methodology. Complex cell states resulting from inhibition of RhoGTPase, β-catenin, and FAK could be classified with > 90% sensitivity on the basis of differences in the SC-35 organizational metrics. This indicates that SC-35 organization is sensitively impacted by adhesion-related signaling molecules that regulate osteogenic differentiation. Our results show that diverse microenvironment cues affect different attributes of the SC-35 organizational metrics and lead to distinct emergent organizational patterns. Taken together, these studies demonstrate that the early organization of SC-35 domains could serve as a “fingerprint” of the intracellular mechanotransductive signaling that governs growth factor- and topography-responsive stem cell states.

Architecture in 3D cell culture: An essential feature for in vitro toxicology

“…Structural proteins such as CTCF, NuMA and lamins organize chromatin (Abad et al, 2007; Dechat et al, 2008; Kim et al, 2015), and by doing so they might redistribute when the phenotype of cells changes. Proteins like NuMA and splicing factor speckle SC35 have been used with high-content analysis of nuclear morphometric descriptors to identify changes in differentiation stages, notably of stem cells, before standard markers are modified (Liu et al, 2010; Vega et al 2015; Vega et al, 2017. More simply, changes in nuclear morphology such as shape (circularity) and size (area) might be sufficient to indicate a drastic impact of the microenvironment on cell behavior.…”

Convergence of Highly Resolved and Rapid Screening Platforms with Dynamically Engineered, Cell Phenotype-Prescriptive Biomaterials

“…Recently incorporation of high content image analysis approaches to evaluate cell response to engineered biomaterial platforms is gaining momentum. The Moghe lab at Rutgers has advanced the application of high content image informatics (HCII) to deduce biomarker profiles at the single cell level indicative of emergent phenotypes on a variety of platforms (Treiser et al 2010; Liu et al 2009; Vega et al 2015; Treiser et al 2007; Liu et al 2010). This approach is based on acquiring high resolution images of cellular reporters and performing high content analysis to acquire features quantifying reporter organization, in tandem with machine learning approaches to model sub-cellular reporter organization.…”

“…Specifically, the organizational metrics of interchromatin speckle factor SC-35 were used to profile hMSC lineage commitment and adhesion signaling in response to soluble cues and on varied platforms including patterned surfaces, fibrous scaffolds and micropillars (Vega et al 2015). In this approach, texture features capturing minute high-order variations in the sub-nuclear spatial organization of SC-35 were computed and utilized with machine learning approaches to generate a predictive cell-state classification model.…”

“…In this approach, texture features capturing minute high-order variations in the sub-nuclear spatial organization of SC-35 were computed and utilized with machine learning approaches to generate a predictive cell-state classification model. Using this methodology, differences in osteogenesis induced by varied topographies were quantitatively identified and classified for hMSCs cultured on a subset of micropillars chosen from a library of 2176 distinct, randomly designed surface topographies (Vega et al 2015; Unadkat et al 2011). These studies, therefore, illustrate that high content image informatics based approaches provide complementary insights as they allow screening relevant materials in high throughput and dynamic platforms by evaluating cell response at single cell level and at early time-points.…”

Profiling stem cell states in three-dimensional biomaterial niches using high content image informatics

“…Furthermore, cells often need to be cultured at high densities to induce differentiation or co-cultured for tissue engineering approaches, and in such cases it becomes difficult to segment cells on the basis of cytoplasmic actin. To overcome these limitations, we developed more sensitive and robust high content image informatics approaches that seek to classify cell states based on various nuclear reporters [8, 9]. In a recent study, we screened several nuclear reporters and demonstrated that the two dimensional sub-nuclear organization of splicing factor SC-35 served as an integrative marker to profile early osteogenic cellular responses across a series of surface patterns, fibrous scaffolds, and micropillars [8].…”