Abstract
Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into cells from all germ lineages, making them a potentially robust cell source for regenerative medicine therapies, but difficulties in predicting and controlling ESC differentiation currently limit the development of therapies and applications from such cells. A common approach to induce the differentiation of ESCs in vitro is via the formation of multicellular aggregates known as embryoid bodies (EBs), yet cell fate specification within EBs is generally considered an ill-defined and poorly controlled process. Thus, the objective of this study was to use rules-based cellular modeling to provide insight into which processes influence initial cell fate transitions in 3-dimensional microenvironments. Mouse embryonic stem cells (D3 cell line) were differentiated to examine the temporal and spatial patterns associated with loss of pluripotency as measured through Oct4 expression. Global properties of the multicellular aggregates were accurately recapitulated by a physics-based aggregation simulation when compared to experimentally measured physical parameters of EBs. Oct4 expression patterns were analyzed by confocal microscopy over time and compared to simulated trajectories of EB patterns. The simulations demonstrated that loss of Oct4 can be modeled as a binary process, and that associated patterns can be explained by a set of simple rules that combine baseline stochasticity with intercellular communication. Competing influences between Oct4+ and Oct4− neighbors result in the observed patterns of pluripotency loss within EBs, establishing the utility of rules-based modeling for hypothesis generation of underlying ESC differentiation processes. Importantly, the results indicate that the rules dominate the emergence of patterns independent of EB structure, size, or cell division. In combination with strategies to engineer cellular microenvironments, this type of modeling approach is a powerful tool to predict stem cell behavior under a number of culture conditions that emulate characteristics of 3D stem cell niches.
Highlights
Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into cells of the three germ lineages that form all of the tissues and organs of a mature organism
One of the most widely used techniques relies upon the formation of multicellular aggregates composed of undifferentiated ESCs in suspension culture, commonly referred to as embryoid bodies (EBs) [1,2], that spontaneously induce the differentiation of ESCs within the 3D aggregate [3,4]
Pluripotent embryonic stem cells can differentiate into all cell types making up the adult body; this process occurs in a complex three dimensional environment with many different parameters present that are capable of influencing cell fate decisions
Summary
Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into cells of the three germ lineages that form all of the tissues and organs of a mature organism. Differentiation of pluripotent ESCs can be induced in vitro via a variety of existing approaches to emulate aspects of the developmental program. One of the most widely used techniques relies upon the formation of multicellular aggregates composed of undifferentiated ESCs in suspension culture, commonly referred to as embryoid bodies (EBs) [1,2], that spontaneously induce the differentiation of ESCs within the 3D aggregate [3,4]. Due to the fact that EBs mimic the physical structure and cellular composition of the morphogenic embryonic microenvironment, they have been used to study aspects of development in vitro as well as the formation of primitive tissue complexes [3,4,5]. The development of techniques to control ESC differentiation in vitro requires an improved understanding of cellular cues that regulate differentiation and the means to precisely control these complex signals
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