8 research outputs found
Modulating Human Mesenchymal Stem Cell Plasticity Using Micropatterning Technique
<div><p>In our previous work, we have reported that enforced elongation of human mesenchymal stem cells (hMSCs) through micropatterning promoted their myocardial lineage commitment. However, whether this approach is robust enough to retain the commitment when subsequently subjected to different conditions remains unsolved. This de-differentiation, if any, would have significant implication on the application of these myocardial-like hMSCs either as tissue engineered product or in stem cell therapy. Herein, we investigated the robustness of micropatterning induced differentiation by evaluating the retention of myocardial differentiation in patterned hMSCs when challenged with non-myocardial differentiation cues. Altogether, we designed four groups of experiments; 1) Patterned hMSCs cultured in normal growth medium serving as a positive control; 2) Patterned hMSCs cultured in normal growth medium for 14 days followed by osteogenic and adipogenic media for next 7 days (to study the robustness of the effect of micropatterning); 3) Patterned hMSCs (initially grown in normal growth medium for 14 days) trypsinized and recultured in different induction media for next 7 days (to study the robustness of the effect of micropatterning without any shape constrain) and 4) Patterned hMSCs cultured in osteogenic and adipogenic media for 14 days (to study the effects of biochemical cues versus biophysical cues). It was found that hMSCs that were primed to commit to myocardial lineage (Groups 2 and 3) were able to maintain myocardial lineage commitment despite subsequent culturing in osteogenic and adipogenic media. However, for hMSCs that were not primed (Group 4), the biochemical cues seem to dominate over the biophysical cue in modulating hMSCs differentiation. It demonstrates that cell shape modulation is not only capable of inducing stem cell differentiation but also ensuring the permanent lineage commitment.</p></div
Immunostaining of β-MHC and osteocalcin after 21 days of hMSCs culture (14 days in normal growth medium +7 days in osteogenic medium).
<p>Patterned cells showed positive expression of β-MHC (<b>a</b>), whereas unpatterned cells stained negatively for β-MHC expression (<b>c</b>). Patterned cells showed negligible signs of osteocalcin expression (<b>b</b>) but significant increase in the level of osteocalcin expression was visualized in unpatterned cells (<b>d</b>). The scale bar is 100 µm.</p
Validation of tissue-lineage commitment of hMSCs.
<p>Tissue-lineage specific markers; osteocalcin and β-MHC along with oil red O dye were used to check hMSCs commitment grown in osteogenic (<b>a, b, e, f</b>) and adipogenic medium (<b>c, d, g, h</b>) for 14 days. Immunostaining results revealed that patterned cells displayed osteocalcin expression (<b>a</b>) and oil globules formation (<b>c</b>) cultured in osteo- and adipogenic medium respectively, while patterned cells grown in osteo- and adipogenic medium failed to express cardiac marker (<b>b, d</b>). Unpatterned cells followed similar trend by showing osteocalcin expression (<b>e</b>), distinct oil globules formation (<b>g</b>) and no indication of β-MHC expression (<b>f, h</b>). The scale bar is 100 µm.</p
Immunostaining of β-MHC and osteocalcin along with oil droplets detection after 2 weeks of hMSCs culture in normal growth medium.
<p>Distinct up-regulation of β-MHC, a cardiac marker (green) was observed in the patterned cells (<b>a</b>) but not for the unpatterned cells (<b>d</b>). No signs of osteocalcin expression (<b>b</b>) and oil droplet formation (<b>c</b>) were observed in cells from patterned group. Unpatterned cells expressed osteocalcin abundantly (red) (<b>e</b>), while no oil droplets formation was observed in unpatterned cells (<b>f</b>). The scale bar is 100 µm.</p
Verification of tissue-lineage commitment of trypsinized and re-cultured hMSCs (re-cultured in normal growth medium).
<p>Trypsinized hMSCs were re-cultured in normal growth medium for 7 days and subjected to immunostaining procedure. Trypsinized cells maintained their myocardial lineage commitment and it was confirmed by β-MHC positive immuno-labeling (<b>a</b>) and no osteocalcin expression (<b>b</b>). Contrary to that, trypsinized unpatterned cells showed down regulation of β-MHC (<b>c</b>) and predominant expression of osteocalcin (<b>d</b>).</p
Investigation of tissue-lineage specificity of trypsinized and re-cultured hMSCs (re-cultured in osteo- and adipogenic induction medium).
<p>Trypsinized hMSCs (group 3) were re-cultured in osteogenic (<b>a, b, e, f</b>) and adipogenic medium (<b>c, d, g, h</b>) for 7 days and lineage commitment was validated by detecting the tissue-lineage specific markers; β-MHC, osteocalcin along with oil red O dye. Immunostaining results proved that trypsinized and re-cultured patterned cells exhibited cardiac MHC expression (<b>a, c</b>) in osteo- and adipogenic medium respectively, whereas these re-cultured patterned cells failed to express osteogenic marker (<b>b</b>) or to produce oil globules (<b>d</b>). Trypsinized and re-cultured unpatterned cells exhibited no traces of cardiac marker expression (<b>e, g</b>) but showed osteocalcin expression (<b>f</b>) and tiny oil globules formation (<b>h</b>). The scale bar is 100 µm.</p
Investigating the Spatial Distribution of Integrin β<sub>1</sub> in Patterned Human Mesenchymal Stem Cells Using Super-Resolution Imaging
Lineage commitment of human mesenchymal
stem cells (hMSCs) could
be directed through micro/nanopatterning of the extracellular matrix
(ECM) between cells and substrate. Integrin receptors, integrator
of the ECM and cell cytoskeleton, function as molecular bridges linking
cells to different biophysical cues translated from patterned ECM.
Here we report the distinct recruitment of active integrin β<sub>1</sub> (ITG-β<sub>1</sub>) in hMSCs when they were committed
toward the cardiomyogenic lineage on a micropatterned surface. In
addition, a systematic study of the distribution of ITG-β<sub>1</sub> was performed on focal adhesions (FAs) using a direct stochastic
optical reconstruction microscopy (dSTORM) technique, a super-resolution
imaging technique to establish the relationship between types of integrin
expression and its distribution pattern that are associated with cardiomyogenic
differentiation of hMSCs. We ascertained that elongated FAs of ITG-β<sub>1</sub> expressed in patterned hMSCs were more prominent than FAs
expressed in unpatterned hMSCs. However, there was no significant
difference observed between the widths of FAs from both experimental
groups. It was found in patterned hMSCs that the direction of FA elongation
coincides with cell orientation. This phenomenon was however not observed
in unpatterned hMSCs. These results showed that the biophysical induction
methods like FAs patterning could selectively induce hMSCs lineage
commitment via integrin-material interaction
Induction of Myogenic Differentiation of Human Mesenchymal Stem Cells Cultured on Notch Agonist (Jagged-1) Modified Biodegradable Scaffold Surface
Engineered scaffold surface provides
stem cells with vital cues that could determine the eventual fate
of stem cells. In this work, biodegradable poly(l-lactide-<i>co</i>-ε-caprolactone) (PLCL) scaffold conjugated with
Notch agonist-Jagged-1(JAG) peptide (2.1 kDa) was prepared to initiate
myogenic differentiation of human mesenchymal stem cells (hMSCs).
The scaffold surface was activated with oxygen plasma and acrylic
acid was engrafted via UV polymerization to form a surface bearing
carboxylic groups. JAG peptide was subsequently immobilized onto the
carboxylated scaffold surface. Surface chemistry and topography were
examined using attenuated total reflection Fourier transform infrared,
X-ray photoelectron spectroscopy, and atomic force microscopy. Quantitative
real time polymerase chain reaction analysis revealed activation of
the Notch pathway; furthermore, several specific markers associated
with myogenic but not osteogenic differentiation were shown to be
up-regulated in hMSCs cultured on the engineered surface. The pro-myocardial
effect of surface bound JAG peptide was further affirmed via immunodetection
of the distinct myocardial marker, cardiac troponin T. Collectively,
our results suggest that PLCL conjugated JAG peptide is a viable strategy
to enhance the functional potential of scaffolds to be used as a bioengineered
cardiac patch in myocardial infarction repair