(760d) Exploring Structural Variations of Cytoskeleton to Understand hESC Differentiation to Insulin Producing Phenotype

Embryonic stem cells (ESCs) have the ability to differentiate into cells of all three germ layer designations: mesoderm, endoderm, and ectoderm. As ESCs differentiate along these various lineages they undergo changes in cell phenotype, both structurally and chemically.  There are various methods used to categorize the changes in cell behavior apparent in the protein expression, gene expression, and cell morphology.  Currently many of the tools available to study changes in ESC morphology and structure, especially that of the cytoskeletal components, are purely qualitative.  Evidence has suggested that as a cell differentiates it undergoes changes in structure and expression of these cytoskeletal components.  Also, cell shape and cytoskeletal tension have a direct effect on stem cell lineage fate [1].  The cell cytoskeleton components contribute to various cellular functions, including cellular adhesion, movement, intracellular transport and signaling, and cell mechanics so it makes sense that differentiation to a target cell phenotype would include mimicking its cytoskeletal traits. 

Our current focus is harnessing the pluripotent nature of human ESCs to produce a therapeutic tool in controlling the disease pathogenesis associated with the loss of function of insulin producing pancreatic Beta cells due to type I diabetes.  The actin cytoskeleton plays a role in proper insulin exocytosis. It has been demonstrated that proper insulin production in response to glucose levels in beta cells is linked to cytoskeletal organization, specifically F-actin [2, 3].   In this study we use a previously established model to quantify the features of induced stem cells at different stages along the lineage towards a mature insulin producing cell, by fluorescently labeling components of the cytoskeleton and imaging at various stages of differentiation towards mature insulin producing cells.

Methods:

Human embryonic stem cells grown in culture were differentiated by chemical signaling in media with a well-established protocol first towards definitive endoderm phenotype, later to a pancreatic precursor phenotype, and finally to a mature insulin producing cell.  After each stage of differentiation a population of cells were fixed and stained for the cytoskeletal components F-actin using rhodamine phalloidin and B-tubulin using anti-tubulin AlexaFlour 488.  Additionally, the differentiated cells were costained for a marker indicative of a cell in the desired phenotype.  For undifferentiated embryonic stem cells we stained for the pluripotent marker OCT4, definitive endoderm cells for SOX17, pancreatic precursor cells for PDX1, and finally mature insulin producing cell for C-Peptide.  Cells were chosen for analysis based on a positive signal from the marker labeling.  After fluorescently imaging the cytoskeletal components we used an established FiberScore algorithm to quantify the structural properties of the cells at all three stages of development [4, 5].

Results:

We were successfully able to locate and image both the actin and beta-tubulin cellular structure for cell phenotypes from the pluripotent hESCs, definitive endoderm, pancreatic precursor, and mature insulin producing cell.  Qualitatively we noted an increase in overall cell size, most notably when compared to the pluripotent ESC stage.  We also noted a trend regarding the formation of stress fibers, specifically during the definitive endoderm and pancreatic precursor stage.  The cells were analyzed by the FiberScore algorithm and structural properties such as fiber intensity, polarity, average width, and total fiber length were found for each group.  For instance, we also found a change in the quantitative measurements of the differentiating cells, most notably an increase in beta-tubulin intensity and width between the definitive endoderm and pancreatic precursor stage and fiber intensity in the later stages of differentiation for the actin components. 

Conclusion:

This study demonstrates the feasibility of utilizing an established technique to better quantify structural changes that occur during the differentiation of hESCS towards an insulin producing phenotype.  While there are various established methods to differentiate towards such a cell type, evidence has suggested that variances in the cytoskeleton and its downstream effects of exocytosis of insulin in response to glucose sitimuli are linked to the cytoskeleton.  Gaining a better understanding of how the cytoskeleton changes during differentiation can be used to possibly elucidate a more beneficial pathway to attain the overall goal of a glucose responsive insulin producing cell. This knowledge would also be useful in designing and fine tuning materials substrates for inducing desired differentiation of hESCs towards an insulin producing phenotype.     

1.         McBeath, R., et al., Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell, 2004. 6(4): p. 483-95.

2.         Hammar, E., et al., Role of the Rho-ROCK (Rho-associated kinase) signaling pathway in the regulation of pancreatic beta-cell function. Endocrinology, 2009. 150(5): p. 2072-9.

3.         Tomas, A., et al., Regulation of pancreatic beta-cell insulin secretion by actin cytoskeleton remodelling: role of gelsolin and cooperation with the MAPK signalling pathway. J Cell Sci, 2006. 119(Pt 10): p. 2156-67.

4.         Lichtenstein, N., B. Geiger, and Z. Kam, Quantitative analysis of cytoskeletal organization by digital fluorescent microscopy. Cytometry A, 2003. 54(1): p. 8-18.

5.         Shah, S.A., P. Santago, and B.K. Rubin, Quantification of biopolymer filament structure. Ultramicroscopy, 2005. 104(3-4): p. 244-54.