Geometric Cues in Bioengineered Scaffold Enhance Podocyte Differentiation in Vitro

Title: Geometric cues in bioengineered scaffold
enhance podocyte differentiation in vitro

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Session: Differentiation/morphogenesis

Words: 484

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This
work reports on a novel, biomimetic, microfabricated platform that has been
engineered to induce functional differentiation of podocytes, a cell type found
in the kidney nephron.

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The
kidney nephron is the biological unit in which waste is filtered from the blood
into the urine. In the nephron�s glomerulus, podocytes wrap around a dense
cluster of capillaries and along with the glomerular basement membrane and
fenestrated endothelium, forms the functional filtration unit. Podocytes have an
arborized morphology with interdigitated processes that wrap around the
capillaries and connect to other podocytes. It has been demonstrated that the
slit diaphragm on these processes, marked by the gene nephrin, is the barrier
structure for filtration in the glomerulus¹, and a hallmark
of differentiation. �

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The
in vitro study of these cells is driven by a need for mechanistic
understanding of podocyte barrier function, which is an essential common pathway
in proteinuric diseases of the kidney. Many kidney diseases are associated with
dedifferentiation/dysfunctions of podocytes, where they lose the specialized
features required for their function. Thus, the study of podocytes in vitro
is focused on understanding the cell�s biology and the hallmarks of
differentiation, which has been done by exploring the chemical environment,
co-cultures and perfusion conditions. These studies are currently conducted on
plated cells in a regular two-dimensional (2D) culture, where it is difficult
to reach appreciable levels of nephrin expression. A cell culture system that
incorporates a 3D physical environment has not yet been investigated.

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In
the glomerulus in vivo, the podocyte experiences out-of-plane curvature.
We hypothesized that a biomimetic topography, that mimics the curving glomerular
capillary structures, would provide a physical stimulus that promotes podocyte
differentiation in vitro, leading to more appreciable levels of nephrin
expression.

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We
engineered a culture platform that resembles the rounded and densely arranged
capillary structures of the glomerulus by using spherical glass beads in a
modified microfabrication technique. 100 �m diameter glass beads were cured
onto a silicon wafer to create a mold for a platform that has out-of-plane
curvature as its topography. This topography presents cells with curvature akin
to curved glomerular capillaries.

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Podocytes
were cultured on the topographic platform and compared to a flat platform.
After 9 days of differentiation, cell samples were either fixed for imaging, or
RNA was collected for reverse transcription and subsequent qPCR to quantify
gene expression. In all experiments, the topographic platform consistently
yielded cells with more arborized morphologies and greater upregulation of nephrin,
than in the flat platform. While the trend was present in experiments with
non-supplemented culture media, it was more pronounced where cells were
cultured with a hormone and vitamin supplement.

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This
work presents a new parameter, that of 3D physical environment, to consider and
incorporate into the culture of podocytes for improved differentiation. It shows
the effectiveness of biomimicry toward the engineering of higher-fidelity cells,
and contributes to the development of physiologically relevant biological
models for the study of pathology.

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