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Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.

ABSTRACT: Cell-matrix and cell-biomolecule interactions play critical roles in a diversity of biological events including cell adhesion, growth, differentiation, and apoptosis. Evidence suggests that a concise crosstalk of these environmental factors may be required to direct stem cell differentiation toward matured cell type and function. However, the culmination of these complex interactions to direct stem cells into highly specific phenotypes in vitro is still widely unknown, particularly in the context of implantable biomaterials. In this study, we utilized tunable hydrogels based on a simple high pressure CO2 method and silk fibroin (SF) the structural protein of Bombyx mori silk fibers. Modification of SF protein starting water solution concentration results in hydrogels of variable stiffness while retaining key structural parameters such as matrix pore size and ?-sheet crystallinity. To further resolve the complex crosstalk of chemical signals with matrix properties, we chose to investigate the role of 3D hydrogel stiffness and transforming growth factor (TGF-?1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Our data revealed the potential to upregulate matured vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Overall, our observations suggest that chemical and physical stimuli within the cellular microenvironment are tightly coupled systems involved in the fate decisions of hMSCs. The production of tunable scaffold materials that are biocompatible and further specialized to mimic tissue-specific niche environments will be of considerable value to future tissue engineering platforms.This article investigates the role of silk fibroin hydrogel stiffness and transforming growth factor (TGF-?1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Specifically, we demonstrate the upregulation of mature vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Moreover, we demonstrate the potential to direct specialized hMSC differentiation by modulating stiffness and growth factor using silk fibroin, a well-tolerated and -defined biomaterial with an impressive portfolio of tissue engineering applications. Altogether, our study reinforce the fact that complex differentiation protocols may be simplified by engineering the cellular microenvironment on multiple scales, i.e. matrix stiffness with growth factor.

SUBMITTER: Floren M

PROVIDER: S-EPMC4728007 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.

Floren Michael M Bonani Walter W Dharmarajan Anirudh A Motta Antonella A Migliaresi Claudio C Tan Wei W

Acta biomaterialia 20151124

Cell-matrix and cell-biomolecule interactions play critical roles in a diversity of biological events including cell adhesion, growth, differentiation, and apoptosis. Evidence suggests that a concise crosstalk of these environmental factors may be required to direct stem cell differentiation toward matured cell type and function. However, the culmination of these complex interactions to direct stem cells into highly specific phenotypes in vitro is still widely unknown, particularly in the contex ...[more]

PMID: 26621695

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Project description:Background:Hydrogels with tuneable mechanical properties are an attractive material platform for 3D bioprinting. Thus far, numerous studies have confirmed that the biophysical cues of hydrogels, such as stiffness, are known to have a profound impact on mesenchymal stem cell (MSC) differentiation; however, their differentiation potential within 3D-bioprinted hydrogels is not completely understood. Here, we propose a protocol for the exploration of how the stiffness of alginate-gelatin (Alg-Gel) composite hydrogels (the widely used bioink) affects the differentiation of MSCs in the presence or absence of differentiation inducing factors. Methods:Two types of Alg-Gel composite hydrogels (Young's modulus: 50 kPa vs. 225 kPa) were bioprinted independently of porosity. Then, stiffness-induced biases towards adipogenic and osteogenic differentiation of the embedded MSCs were analysed by co-staining with alkaline phosphatase (ALP) and oil red O. The expression of specific markers at the gene level was detected after a 3-day culture. Results:Confocal microscopy indicated that all tested hydrogels supported MSC growth and viability during the culture period. Higher expression of adipogenic and osteogenic markers (ALP and lipoprotein lipase (LPL)) in stiffer 3D-bioprinted matrices demonstrated a more significant response of MSCs to stiffer hydrogels with respect to differentiation, which was more robust in differentiation-inducing medium. However, the LPL expression in stiffer 3D-bioprinted constructs was reduced at day 3 regardless of the presence of differentiation-inducing factors. Although MSCs embedded in softer hydrogels to some extent proceeded toward adipogenic and osteogenic lineages within a few days, their differentiation seemed to be slower and more limited. Interestingly, the hydrogel itself (without differentiation-inducing factors) exhibited a slight effect on whether MSCs differentiated towards an adipogenic or an osteogenic fate. Considering that the mechano-regulated protein Yes-associated protein (YAP) is involved in MSC fate decisions, we further found that inhibition of YAP significantly downregulated the expression of ALP and LPL in MSCs in stiffer constructs regardless of the induced growth factors present. Conclusions:These results demonstrate that the differentiation of MSCs in 3D-bioprinted matrices is dependent on hydrogel stiffness, which emphasizes the importance of biophysical cues as a determinant of cellular behaviour.

Project description:Silk hydrogels have shown potential for tissue engineering applications, but several gaps and challenges, such as a restricted ability to form hydrogels with tuned mechanics and structural features, still limit their utilisation. Here, Bombyx mori and Antheraea mylitta (Tasar) silk microfibres were embedded within self-assembling B. mori silk hydrogels to modify the bulk hydrogel mechanical properties. This approach is particularly attractive because it creates structured silk hydrogels. First, B. mori and Tasar microfibres were prepared with lengths between 250 and 500 μm. Secondary structure analyses showed high beta-sheet contents of 61% and 63% for B. mori and Tasar microfibres, respectively. Mixing either microfibre type, at either 2% or 10% (w/v) concentrations, into 3% (w/v) silk solutions during the solution-gel transition increased the initial stiffness of the resulting silk hydrogels, with the 10% (w/v) addition giving a greater increase. Microfibre addition also altered hydrogel stress relaxation, with the fastest stress relaxation observed with a rank order of 2% (w/v) > 10% (w/v) > unmodified hydrogels for either fibre type, although B. mori fibres showed a greater effect. The resulting data sets are interesting because they suggest that the presence of microfibres provided potential 'flow points' within these hydrogels. Assessment of the biological responses by monitoring cell attachment onto these two-dimensional hydrogel substrates revealed greater numbers of human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) attached to the hydrogels containing 10% (w/v) B. mori microfibres as well as 2% (w/v) and 10% (w/v) Tasar microfibres at 24 h after seeding. Cytoskeleton staining revealed a more elongated and stretched morphology for the cells growing on hydrogels containing Tasar microfibres. Overall, these findings illustrate that hydrogel stiffness, stress relaxation and the iPSC-MSC responses towards silk hydrogels can be tuned using microfibres.

Project description:Despite recent advances in tissue engineered heart valves (TEHV), a major challenge is identifying a cell source for seeding TEHV scaffolds. Native heart valves are durable because valve interstitial cells (VICs) maintain tissue homeostasis by synthesizing and remodeling the extracellular matrix. This study demonstrates that induced pluripotent stem cells (iPSC)-derived mesenchymal stem cells (iMSCs) can be derived from iPSCs using a feeder-free protocol and then further matured into VICs by encapsulation within 3D hydrogels. The differentiation efficiency was characterized using flow cytometry, immunohistochemistry staining, and trilineage differentiation. Using our feeder-free differentiation protocol, iMSCs were differentiated from iPSCs and had CD90+, CD44+, CD71+, αSMA+, and CD45- expression. Furthermore, iMSCs underwent trilineage differentiation when cultured in induction media for 21 days. iMSCs were then encapsulated in poly(ethylene glycol)diacrylate (PEGDA) hydrogels grafted with adhesion peptide (RGDS) to promote remodeling and further maturation into VIC-like cells. VIC phenotype was assessed by the expression of alpha-smooth muscle actin (αSMA), vimentin, and collagen production after 28 days. When MSC-derived cells were encapsulated in PEGDA hydrogels that mimic the leaflet modulus, a decrease in αSMA expression and increase in vimentin was observed. In addition, iMSCs synthesized collagen type I after 28 days in 3D hydrogel culture. Thus, the results from this study suggest that iMSCs may be a promising cell source for TEHV.Statement of significanceDeveloping a suitable cell source is a critical component for the success and durability of tissue engineered heart valves. The significance of this study is the generation of iPSCs-derived mesenchymal stem cells (iMSCs) that have the capacity to mature into valve interstitial-like cells when introduced into a 3D cell culture designed to mimic the layers of the valve leaflet. iMSCs were generated using a feeder-free protocol, which is one major advantage over other methods, as it is more clinically relevant. In addition to generating a potential new cell source for heart valve tissue engineering, this study also highlights the importance of a 3D culture environment to influence cell phenotype and function.

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Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.

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Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.

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