Elsevier

Biomaterials

Volume 30, Issue 13, May 2009, Pages 2507-2515
Biomaterials

Homogeneous and organized differentiation within embryoid bodies induced by microsphere-mediated delivery of small molecules

https://doi.org/10.1016/j.biomaterials.2009.01.007Get rights and content

Abstract

Cell specification and tissue formation during embryonic development are precisely controlled by the local concentration and temporal presentation of morphogenic factors. Similarly, pluripotent embryonic stem cells can be induced to differentiate in vitro into specific phenotypes in response to morphogen treatment. Embryonic stem cells (ESCs) are commonly differentiated as 3D spheroids referred to as embryoid bodies (EBs); however, differentiation of cells within EBs is typically heterogeneous and disordered. In this study, we demonstrate that in contrast to soluble morphogen treatment, delivery of morphogenic factors directly within EB microenvironments in a spatiotemporally controlled manner using polymer microspheres yields homogeneous, synchronous and organized ESC differentiation. Degradable PLGA microspheres releasing retinoic acid were incorporated directly within EBs and induced the formation of cystic spheroids uniquely resembling the phenotype and structure of early streak mouse embryos (E6.75), with an exterior of FOXA2+ visceral endoderm enveloping an epiblast-like layer of OCT4+ cells. These results demonstrate that controlled morphogen presentation to stem cells using degradable microspheres more efficiently directs cell differentiation and tissue formation than simple soluble delivery methods and presents a unique route to study the spatiotemporal effects of morphogenic factors on embryonic developmental processes in vitro.

Introduction

Pluripotent embryonic stem cells (ESCs) are a renewable cell source for studies of embryonic development, regenerative medicine and in vitro diagnostics. ESCs, derived from the inner cell mass of the blastocyst stage of development [1], [2], [3], can be induced to differentiate via aggregation into multi-cellular spheroids referred to as embryoid bodies (EBs) [4]. Cells within EBs differentiate in response to a variety of environmental stimuli, including cell–cell adhesions, cell–matrix interactions [5], cytokines [6], growth factors [7], and small molecules [8]. Because the EB microenvironment is comprised of a complex mixture of these extracellular stimuli, differentiation within EBs is typically heterogeneous and spatially disorganized. Efforts to manipulate the EB environment and subsequent ESC differentiation have focused primarily on controlling media composition [7] and assembly of EBs using different culture methods [9]; however, precise control over the molecular milieu within the interior of EBs has not been achieved by such approaches [10].

During development, morphogens are secreted locally and presented to embryonic cells in a spatially and temporally controlled manner to direct appropriate differentiation and tissue formation [11], [12], [13], [14]. In vitro strategies to deliver morphogenic factors to EBs, namely diffusion of supplemental media components, do not accurately replicate this process, with exogenous morphogens originating in the external fluid rather than within the cell spheroid. In addition, the diffusion of soluble factors into EBs may be restricted by the formation of an exterior shell composed of collagenous matrix and tight E-cadherin mediated cell–cell adhesions at the EB surface [15]. These fundamental challenges in morphogen presentation to ESCs limit the ability of EBs to accurately serve as controlled models of embryogenesis in vitro and perhaps limit the homogeneity of differentiated phenotypes that can be attained using EB differentiation methods. Engineering of biomaterials-based approaches has been used successfully to control the spatiotemporal presentation of morphogens to 3D assemblies of cells [16], [17]. Thus, the use of biodegradable microspheres to deliver morphogens directly within EBs may enable production of more homogeneous populations of differentiated cells.

In this study, we describe the differentiation effects of spatially and temporally controlled presentation of morphogenic factors to ESCs comprising EBs from degradable biomaterials. Microsphere incorporation within EBs was assessed as a function of initial mixing conditions, and the release and cellular uptake of a fluorescent dye from incorporated microspheres were evaluated. The morphology, gene and protein expression, and ultrastructure of EBs containing retinoic acid (RA)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres are reported. These studies demonstrate that biomaterials can be used to engineer the microenvironment within EBs to efficiently direct ESC differentiation, which may be applied in regenerative cell therapies, in vitro pharmaceutical screening and models of developmental biology.

Section snippets

Microsphere fabrication

PLGA (50:50, Absorbable Polymers International) microspheres were fabricated using a water-in-oil single emulsion, as described [18]. Briefly, 200 mg PLGA was dissolved in dichloromethane (DCM) containing 50 μg CellTracker Red (Molecular Probes, Invitrogen Corp., Carlsbad, CA) or 600 μg RA (all trans, Acros Organics, Geel, Belgium), added to 0.3% PVA, and homogenized at 3000 rpm (Polytron PT 3100, Kinematica Inc., Bohemia, NY). DCM was evaporated for 4 h and microspheres were collected by

Microsphere incorporation within EBs

Single cell suspensions of ESCs cultured in rotary suspension aggregated into spheroids in <24 h, and the resulting EBs developed a smooth, spherical appearance after ∼4 days of differentiation [19]. Scanning electron microscopy (SEM) revealed the formation of a smooth, epithelial cell layer on the surface of day 10 EBs (Fig. 1A). Cross-sectional views of bisected EBs revealed the formation of an exterior layer approximately 10 μm thick enveloping the entire EB (Fig. 1B, C). The appearance of a

Discussion

The efficient formation of highly organized, cystic EBs is significant for a number of reasons, including improved in vitro models of embryogenesis and biomanufacturing of differentiated cell types for regenerative medicine and in vitro diagnostic applications. Currently, directed differentiation strategies using EBs are limited in their ability to produce a homogeneous differentiated cell population, due largely to the fact that EBs consist of a population of cells exposed to a diverse set of

Conclusion

Our results demonstrate that the locally controlled presentation of morphogens from microspheres-to-cells comprising an EB more closely mimics characteristics of early embryogenesis than simple soluble delivery of signaling molecules to cell spheroids. In addition to developmental studies, microsphere-mediated presentation of morphogenic cues to stem cell spheroids represents a powerful enabling technology readily capable of being integrated into biomanufacturing strategies to obtain stem cell

Acknowledgements

We thank J. Phillips for assistance with the development of immunostaining methods and L. Matyunina for conducting microarray experiments. We are grateful to P. Ramaswami and R. Nair for assistance with manuscript preparation. This work was supported by funding from the NSF (CBET 0651739).

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