Cell Fate, Morphogenesis and Biophysical Dynamics within Pluripotent Microenvironments

Cell fate, morphogenesis and
biophysical dynamics within pluripotent microenvironments

M. A. Kinney¹, R.
Saeed¹, and T. C. McDevitt^1,2

¹The Wallace H. Coulter Department of Biomedical
Engineering

²The Parker H. Petit
Institute for Bioengineering and Bioscience

Georgia Institute of Technology & Emory University,
Atlanta, GA, USA

Stem cell fate and function are
dynamically modulated by the interdependent relationships between biochemical
and biophysical signals constituting the local 3D microenvironment. While
approaches to recapitulate the stem cell niche have been explored for directing
stem cell differentiation, a quantitative relationship between embryonic stem
cell (ESC) morphogenesis and intrinsic biophysical cues within
three-dimensional microtissues has not been established.  The objective of this
study, therefore, was to define the intrinsic cellular biophysical
characteristics and phenotypic changes that arise simultaneously during cell
fate specification and morphogenesis of three-dimensional pluripotent stem cell
microenvironments.

Spheroids were formed from murine
embryonic stem cells (D3) via forced aggregation (1000 cells per well in 400 µm
diameter PDMS microwells) for 24 hours and were subsequently transferred to
rotary orbital suspension (45 rpm), which together enable precise control of
aggregation kinetics and spheroid size. ESCs were differentiated in serum free
media (N2B27) in the presence or absence of BMP4, in order to direct
differentiation trajectories toward mesoderm or ectoderm lineages,
respectively.  Biophysical characteristics were calculated using the linear
viscoelastic model of creep compression, which was measured via a micron-scale
material testing platform (Microsquisher, CellScale) that employs cantilever deformation
to measure the force on individual aggregates. The multiparametric dynamics of
morphogenesis was analyzed via histological, gene expression, and biomechanical
changes in 3D microtissues, and correlative associations were assessed using
multivariate data modeling.

Consistent with results from
monolayer differentiation, pluripotent spheroids supplemented with BMP4
increased the expression of genes related to mesoderm lineages (Hba-x, Gata2),
in contrast to the increased endoderm (Foxa2) and ectoderm (Pax6)
expression in basal, serum-free biochemical environments. In addition,
mesodermal aggregates exhibited loss of traditional epithelial characteristics,
resulting in elongated, mesenchymal cells expressing alpha smooth muscle actin
(α-SMA), suggesting that ESCs differentiated in 3D
undergo an epithelial-to-mesenchymal transition (EMT) during mesoderm
specification. Interestingly, the stiffness of stem cell spheroids (10¹-10²
Pa) was several orders of magnitude less than that of native tissues (10³-10⁴
Pa), and was dynamically increased during differentiation, with increased
stiffness (1.5 fold; p=0.004) concomitant with increasing mesoderm cell fate
and mesenchymal morphogenesis. Microtissue stiffness was significantly
(p<0.03 for all conditions) decreased upon inhibition of ROCK-mediated
tension (Y27632; 10 µM) at all stages of differentiation, indicating that the
mechanical properties were largely (~50%) attributed to structural
characteristics of the actomyosin cytoskeleton. Moreover, while individual
mechanical properties, such as stiffness, were not directly related to gene
expression, the phenotypic profiles were accurately predicted by the combination
of several biophysical properties, thus indicating that multiparameteric
viscoelastic characteristics can describe pluripotent tissue composition.

Together, the dynamic remodeling
during 3D pluripotent stem cell differentiation motivates the development of
engineering approaches for controlling spatiotemporal administration of
physical and chemical cues. Understanding biochemical and physical tissue
morphogenesis, including the relationships between remodeling of cytoskeletal
elements and the physical properties of the EB structure will inform approaches
for the development of complex, functional tissue structures for regenerative
therapies.