(323c) Mouse Embryonic Stem Cell Sensitivity to Compliant Alginate Substrates: A Promising Platform for Endoderm Leaning Differentiation

Embryonic stem cells (ESCs) have been implicated to have tremendous impact in regenerative therapeutics of various diseases, including Type 1 Diabetes. However to extract the full potential of ESCs a robust differentiation regime is required to generate functionally mature islet-like cells from the pluripotent ESCs. Upon generation of functionally mature ESC derived islet cell types, they need to be implanted into diabetic patients to restore the loss of islet activity. Encapsulation in alginate microcapsules is a promising route of implantation, which can protect the cells from the recipient’s immune system. While there has been a significant investigation into islet encapsulation over the past decade, the feasibility of encapsulation and differentiation of ESCs has been less explored. Also for islet encapsulation the primary features of interest are nutrient diffusion and immune protection, the use of ESCs adds an additional variability in the system. Research over the past few years has identified the cellular mechanical microenvironment to play a central role in phenotype commitment of stem cells, both adult and embryonic. Hence it will be important to design the encapsulation material to be supportive to cellular functionality and maturation.

As a first step towards achieving these objectives, we investigated the effect of stiffness of alginate substrate on initial differentiation and phenotype commitment of murine ESCs. The first crucial stage of pancreatic differentiation is the endoderm germ layer commitment. Herein we are focusing on investigating the effect of modulation of alginate substrate properties in inducing and/ or enhancing endoderm commitment of the differentiating ESCs.

Methods:

For this study we utilized the well established nano-indentation technique of the atomic force microscope to determine the biomechanical properties of both the native pancreatic tissue and the artificial alginate substrates.  The elastic modulus was measured on unfixed cryosectioned samples taken from a murine pancreas.  Alginate scaffolds were generated to match this stiffness range by varying the alginate concentration over a range of 6mg/ml to 15 mg/ml and varying the ionic calcium concentrations from 16 mg/ml to 48 mg/ml.  Fibronectin content for cell-adhesion to the scaffold was held constant throughout all scaffolds, and the distribution was verified by fluorescently tagging the fibronectin molecules in the alginate scaffolds.  A murine embryonic stem cell line (ESD3) grown in culture was seeded onto the alginate scaffolds of varying stiffness and allowed to spontaneously differentiate for 5 days in an otherwise signal free environment.  Cell morphology was tracked over this period by intermittently taking phase contrast light microscopy images over the entire course of the experiment.  After 5 days alamar blue assay was used to determine the cell proliferation for each of the alginate constructs.  Of most particular interest to our study were observing changes to the cell phenotype while spontaneously differentiating on the alginate substrates.  This was investigated by using quantitative RT-PCR to study changes in the differentiating embryonic stem cells by looking at the gene expression across all three germ layers. 

Results:

In order to properly tune the biomechanical characteristics of the alginate gels to that of the native pancreas environment we determined the elasticity of unfixed native mouse pancreatic tissue.  The Young’s modulus of the tissue was measured to be 1210 ± 77 Pa as determined by AFM nano-indentation.  We created alginate substrates of 12 different elastic moduli, ranging from 242 ± 16 Pa to 1337 ± 27 Pa.  A constant amount of fibronectin was saturated into the gel and we noted an even distribution throughout as verified by FITC tagging the adhesion proteins and fluorescently imaging the gels.  Phase contrast imaging of the cells did not demonstrate a significant variance in the cell spreading during the experiment, though it was noted that cells on the stiffer end of our spectrum did form larger clumps at a faster rate.  We also did not witness a significant difference in the cell proliferation across the alginate substrates.  To study the gene expression of the spontaneously differentiating murine ESCs after 5 days of culture on the alginate substrates we used quantitative RT-PCR to observe the markers for the three germ layers, as well as the pluripotency.  Across all substrate stiff nesses we noted either down-regulation or no up-regulation in the pluripotency markers.  There was no stiffness related difference noted.  We also observed little or no up-regulation in the ectoderm markers BMP4 and Nestin, however we did perceive very large up-regulation in the primitive ectoderm marker FGF5, which is also highly expressed in the mouse epiblast indicative of cells in a “gastrulation-like” state.  This high up-regulation was not sensitive to gel stiffness.  There was some up-regulation of the mesoderm marker FGF8 noted in the 700-950 Pa range of gel stiffness, which could be attributed to a portion of the cells mesodermal lineage characteristics.  The murine ESCs demonstrated the most up-regulation and sensitivity to changes in the substrate stiffness in markers relating to the developing endoderm.  HNF4 and CXCR4 demonstrated a 10-15 and 30-50 fold increase in the 450 Pa to 900 Pa range of gel elastic modulus. 

Conclusion:

In this study we verified that alginate gels are a suitable material to study the influence of passive changes in the biomechanical microenvironment of embryonic stem cell differentiation.  The system allows us to manipulate the stiffness of the microenviroment without changing the chemical signaling.  At a soft substrate stiffness range we noted spontaneous differentiation of pluripotent murine ESCs to lineage specific cell types.  Over this defined range of substrate stiffness we determined the endoderm lineage to be both the most prevalent and also the most responsive to small perturbations in the stiffness of the substrate.  These results demonstrate the importance of the mechanical microenvironment to cellular differentiation, even over a relatively small range of stiffness.  A better understanding of these biophysical phenomena would allow them to be harnessed along with other cues to enhance the differentiation of embryonic stem cells towards a specific lineage fate.