Download Identifying Geometry Directed Stem Cell Differentiation with RNA

Towards Bio-Instructive Scaffolds: Identifying Geometry Directed Stem Cell Differentiation with RNA-Sequencing
Brittany Banik,* Evan Witmer,* Lila Rieber,+ Shaun Mahony, Ph.D.,+ and Justin Brown, Ph.D.*
*
Department of Biomedical Engineering, +Department of Biochemistry & Molecular Biology
The Pennsylvania State University
Statement of Purpose: The stem cell niche is a dynamic
environment that serves to provide various cues to cells for
adherence, proliferation, development, migration, and
differentiation.1 Nanofibers have been of particular interest
due to similarities to the native extracellular matrix. The
importance of understanding how cells respond to fiber
architectures and the effect on cell phenotype is critical for
the advancement of basic biology and regenerative
medicine. It has been shown throughout literature that
fibers drive differentiation. The presented research
emphasizes the opportunity to harness biomaterial
scaffolds through fiber alignment and size (e.g. diameter)
to affect change on cellular response and phenotype.
Future investigations will consider synergistic effects
between GDF7 growth factor stimulation and scaffold
characteristics of fiber size and alignment to in turn guide
the differentiation down a very specific tissue route,
tendon. It is anticipated that the scaffold properties will
reduce the amount of growth factor required to accelerate
differentiation along the tendon lineage. The significance
of this work lies in the knowledge advancement of better
understanding how to regulate mesenchymal stem cells.
Methods: Glass coverslips were etched with a potassium
hydroxide bath and spincoated with polyvinyl alcohol to
prevent cells from attaching to the underlying surface.
Electrospinning was utilized to create three groups of
varying
fiber
surfaces:
large
aligned
fibers
(475.3+108.3nm), small aligned fibers (213+37.3 nm), and
small random fibers (189.4+33.8 nm). The control or
baseline surfaces were tissue cultured polystyrene without
any geometric constraints. Poly-ϵ-caprolactone (PCL) (Mn
80,000) was used to generate the fibrous surfaces; various
electrospinning conditions were used to produce different
sizes and alignment. Human mesenchymal stem cells
(hMSCs), passage 9, were seeded onto coverslips for a
period of 7 days with media changes every two days.
Following 7 days, cells were lysed with a strong lysis
buffer, and the RNA was isolated and purified with
Qiagen’s RNeasy Mini Kit (Cat No.: 74104). RNAsequencing reads were reported in fastq format and
FastQC, quality control, was evaluated. To analyze the
RNA-Sequencing files, HTSeq-count, part of the Python
module version 2.7.5, was used to count the number of
reads per gene in the BAM files. DESeq, an R package,
was then used to test the count data for differential
expression analysis. Gene outputs and their respective
responses were categorized into tissue groups.
Results: RNA-sequencing distinguishes how genes are
expressed (e.g. turned on or off, the level of expression) at
a specific time point.
Identifying changes at the
transcriptome level allow researchers to gain a complex
understanding of the genome and the downstream effects
of various factors and system inputs, such as scaffold
architecture and design. Using this high throughput, deep
sequencing tool, quantitative data was obtained to show
phenotypic trends based on fiber alignment and size,
Figure 1. The following trends appear from preliminary
RNA-sequencing data analysis: Tendon factors were up
regulated for aligned fibers, with less dependence on size.
Differing from the tendon results, bone elements were
influenced strongly by random fiber orientation, without a
strong correlation to the size of the fibers. For muscle, a
greater upregulation is noted based on the smaller fiber
diameter sizes. Lastly, it appears that the trend for nerve
tissue depends on both alignment and small sizes. On
average, 25% of the genes were significantly different than
the control. These generalizations begin to provide insight
into critical biomaterial scaffold considerations for
regenerative medicine applications.
Figure 1. Fiber alignment and size drives specific tissue
differentiation. Human mesenchymal stem cells on three
fibrous surfaces differing in alignment and size illustrate
varying tissue expression levels.
Conclusions: Cells without supplementation on fibrous
surfaces demonstrate different phenotypic lineage
commitment. Overall, small, aligned fibers tend to be
driven towards musculoskeletal tissues—nerve, muscle,
and tendon. This trend matches expectations, since tendon,
muscle, and nerve tissues are composed of organized
matrix and aligned cells. In future work, it is planned to
down-select and give consideration to biochemical
signaling as a mechanism that can be used to
synergistically affect differentiation. The family of
growth/differentiation factors (GDFs) are particularly
important in the development of musculoskeletal and
nervous systems. Therefore, it is hypothesized that by
supplementing with GDF7, differentiation can be regulated
to promote differentiation towards tendon tissue.
References: 1Li X, et al. J. Biomed. Mater. Res.
2014;102(5):1580–94.