(193b) Alginate Encapsulated Activina Doped Chitosan Nanoparticles (CNP) to Promote Endodermal Differentiation in Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have the ability differentiate along pathways of all three primary germ layers: endoderm, ectoderm, and mesoderm.  This ability renders them especially useful as a possible therapeutic solution to a variety of disease pathogenesis.  hESCs can be directed along these differential pathways via both internal and external stimuli, such as genetic manipulation, chemical signaling, surface morphology, and the stiffness of the immediate microenvironment.  A current challenge of significant interest is in driving hESCs to become a sustainable and viable source of beta-like insulin producing cells as a replacement for cellular function lost with type I diabetes, which currently affects over 1 million people in the United States alone. The initial step along the pathway of viable beta-like insulin producing cells is to first produce a definitive endoderm phenotype, which has been induced in embryonic stem cells in the presence of Activin A [1]. 

In addition to lineage fate signaling, in some therapeutic applications it may also be facilitative and even a necessity to protect a differentiating cell from the larger system while allowing the passage of waste, nutrients, and other chemical signals to and from the immediate cell microenvironment.  This is of special importance in the transplantation of beta and beta-like insulin producing cells to combat diabetes. A larger goal application of such a system is deriving a source of functional insulin producing cells from hESCs while protecting them from the body’s natural immune pathway.  In this study we report a novel alginate/chitosan system to encapsulate the hESC while providing a source of Activin A chemical signaling for the purpose of inducing a definitive endoderm phenotype, an important first step towards attaining this larger goal.

Methods:

In our system we have used the ionic gelation method to create Activin A loaded chitosan nanoparticles (CNPs) by combining a solution of sodium tripolyphosphate (TPP) and Activin A with a solution of dissolved chitosan, a natural biocompatible polymer derived from sea shells.  Chitosan nanoparticles have been utilized as a delivery method for both organic and inorganic particles.  Activin A was chosen for the explicit purpose of inducing endodermal lineage in the hESCs.  The size of the nanoparticles before and after Activin A loading was determined by both dynamic light scattering and atomic force microscopy.  Further characterization of the loading efficiency and release dynamics of Activin A loaded in the CNPs were investigated by ELISA assay, targeted for Activin A only.  Endoderm differentiation of the hESCs was induced by introducing the Activin loaded CNP to an otherwise signal and serum free media in 2D cell culture for 4 days.  We used quantitative PCR to determine the changes in the hESC gene expression of FOXA2 and SOX17, markers indicative of endodermal gene expression.

After loading, the CNPs were encapsulated along with hESC colonies in a 3D alginate bead by the external gelation method.  Alginate, also a well known natural polymer has been utilized as a non-biodegradable and biologically compatible encapsulation method for a variety of applications.   Briefly, the CNPs and hESCs were suspended in a 1.1% medium viscosity sodium alginate solution and then extruded in droplets from a syringe into a calcium chloride bath.  The size of the encapsulated beads was determined by light microscopy, and the release dynamics were determined for the alginate encapsulated CNPs by ELISA assay.  Cell viability was qualitatively monitored after 4 days using a Calcein AM and ethidium homodimer-1 based live/dead assay.  After 4 days of differentiation quantitative PCR analysis was used to determine the changes in FOXA2 and SOX17 endodermal gene expression of the hESCs in our 3D encapsulated system.

Results:

We successfully loaded Activin A into nanoparticles with a size range of 200-300 nm diameter and a high loading efficiency of ~87 to 90%.  The size of the CNPs did not vary significantly before and after loading with Activin A.  Release profiles determined by ELISA showed an initial “burst” release of 18% of the loaded Activin A after 1 day and ~30% release after 4 days in cell culture media.  We also observed an increase in the endodermal gene markers SOX17 and FOXA2 in hESCs grown in 2D culture with the loaded CNPs when compared to hESCs spontaneously differentiated in a signal free environment.  These results verified the feasibility of using Activin A doped chitosan nanoparticles to drive hESC differentiation to a definitive endoderm cell type.

The Activin A loaded CNPs were also successfully encapsulated along with hESC cells in alginate beads of a diameter in the range of 1 to 2 mm.  A live/dead cell assay demonstrated cell survival in this environment after 4 days in culture.  Activin A release in the media was determined to be reduced by the alginate encasement when compared to release in media only.  Quantitative PCR analysis on the 3D system still demonstrated an increase in endodermal genetic markers SOX17 and FOXA2 after 4 days of culture in the alginate beads, demonstrating the feasibility of our system as a whole. 

Conclusion:

This study verifies the feasibility of creating a system of Activin A loaded chitosan nanoparticles encapsulated along with human embryonic stem cells in an alginate microbead.  We demonstrated both a high efficiency in loading of Activin A and a controlled release into the media.  Gene expression consistent with an endodermal phenotype was found in both 2D cell culture and 3D alginate encapsulated cell culture when Activin A loaded CNPs were introduced.  Based on the results produced in this study, we have demonstrated a closed system useful for both protecting and signaling human embryonic stem cells towards the definitive endodermal cell fate, and an important first step in producing insulin producing beta-like cells while containing in a closed system for protection and possible downstream implementation in vivo studies.

1.         Kubo, A., et al., Development of definitive endoderm from embryonic stem cells in culture. Development, 2004. 131(7): p. 1651-62.