Human pluripotent stem cells (hPSCs;
both embryonic and induced
pluripotent) rapidly proliferate in adherent culture to maintain their
undifferentiated state. However, for mammals exhibiting delayed gestation
(diapause), mucin-coated embryos can remain dormant for days or months in utero, with their constituent PSCs remaining pluripotent
under these conditions. Here we report cellular stasis for both hPSC
colonies and preimplantation embryos immersed in a wholly synthetic
thermoresponsive gel comprising poly(glycerol monomethacrylate)-poly(2-hydroxypropyl
methacrylate) [PGMA55-PHPMA135] diblock copolymer
worms. This hydroxyl-rich mucin-mimicking nonadherent 3D gel maintained
PSC viability and pluripotency in the quiescent G0 state
without passaging for at least 14 days. Similarly, gel-coated human
embryos remain in a state of suspended animation (diapause) for up
to 8 days. The discovery of a cryptic cell arrest mechanism for both
hPSCs and embryos suggests an important connection between the cellular
mechanisms that evoke embryonic diapause and pluripotency. Moreover,
such synthetic worm gels offer considerable utility for the short-term
(weeks) storage of either pluripotent stem cells or human embryos
without cryopreservation.
Synthetic hydrogel-amorphous calcium phosphate composites are promising candidates to substitute biologically sourced scaffolds for bone repair. While the hydrogel matrix serves as a template for stem cell colonisation, amorphous calcium phosphate s provide mechanical integrity with the potential to stimulate osteogenic differentiation. Here, we utilise composites of poly(ethylene glycol)-based hydrogels and differently stabilised amorphous calcium phosphate to investigate potential effects on attachment and osteogenic differentiation of human mesenchymal stem cells. We found that functionalisation with integrin binding motifs in the form of RGD tripeptide was necessary to allow adhesion of large numbers of cells in spread morphology. Slow dissolution of amorphous calcium phosphate mineral in the scaffolds over at least 21 days was observed, resulting in the release of calcium and zinc ions into the cell culture medium. While we qualitatively observed an increasingly mineralised extracellular matrix along with calcium deposition in the presence of amorphous calcium phosphate-loaded scaffolds, we did not observe significant changes in the expression of selected osteogenic markers.
Strategies that enable hydrogel substrates to support cell attachment typically incorporate either entire extracellular matrix proteins or synthetic peptide fragments such as the RGD (arginine-glycine-aspartic acid) motif. Previous studies have carefully analysed how material characteristics can affect single cell morphologies. However, the influence of substrate stiffness and ligand presentation on the spatial organisation of human mesenchymal stem cells (hMSCs) have not yet been examined. In this study, we assessed how hMSCs organise themselves on soft (E = 7.4-11.2 kPa) and stiff (E = 27.3-36.8 kPa) poly(ethylene glycol) (PEG) hydrogels with varying concentrations of RGD (0.05-2.5 mM). Our results indicate that hMSCs seeded on soft hydrogels clustered with reduced cell attachment and spreading area, irrespective of RGD concentration and isoform. On stiff hydrogels, in contrast, cells spread with high spatial coverage for RGD concentrations of 0.5 mM or higher. In conclusion, we identified that an interplay of hydrogel stiffness and the availability of cell attachment motifs are important factors in regulating hMSC organisation on PEG hydrogels. Understanding how cells initially interact and colonise the surface of this material is a fundamental prerequisite for the design of controlled platforms for tissue engineering and mechanobiology studies.
Platelet lysates
(PL) contain a selection of proteins and growth
factors (GFs) that are known to mediate cell activity. Many of these
biomolecules have been identified as chemoattractants with the capacity
to induce cell migration. In order to effectively deliver and retain
these biomolecules to the site of injury, a scaffold containing PL
could be an option. We use poly(ethylene glycol) (PEG) hydrogels consisting
of 90 vol % PL to investigate their migratory potential on human mesenchymal
stem cells (hMSCs). Cells exposed to these hydrogels were tracked,
resulting in cell trajectories and detailed migratory parameters (velocity,
Euclidean distance, directness, and forward migration index). Volumetric
swelling ratios, hydrogel mechanical properties, and the release kinetics
of proteins and GFs from hydrogels were also assessed. Furthermore,
hMSC spheroids were encapsulated within the hydrogels to qualitatively
assess cell invasion by means of sprouting and disintegration of the
spheroid. Cell spheroids encapsulated within the PL-PEG gels exhibited
initial outgrowths and eventually colonized the 3D matrix successfully.
Results from this study confirmed that hMSCs exhibit directional migration
toward the PL-loaded hydrogel with increased velocity and directness,
compared to the controls. Overall, the incorporation of PL renders
the PEG hydrogel bioactive. This study demonstrates the capacity of
PL-loaded hydrogel constructs to attract stem cells for endogenous
tissue engineering purposes.
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