Micropatterned matrix directs differentiation of human mesenchymal stem cells towards myocardial lineage.

Stem cell response can be influenced by a multitude of chemical, topological and mechanical physiochemical cues. While extensive studies have been focused on the use of soluble factors to direct stem cell differentiation, there are growing evidences illustrating the potential to modulate stem cell differentiation via precise engineering of cell shape. Fibronectin were printed on poly(lactic-co-glycolic acid) (PLGA) thin film forming spatially defined geometries as a means to control the morphology of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs that were cultured on unpatterned substrata adhered and flattened extensively (approximately 10,000 microm(2)) while cells grown on 20 microm micropatterend wide adhesive strips were highly elongated with much smaller area coverage of approximately 2000 microm(2). Gene expression analysis revealed up-regulation of several hallmark markers associated to neurogenesis and myogenesis for cells that were highly elongated while osteogenic markers were specifically down-regulated or remained at its nominal level. Even though there is clearly upregulated levels of both neuronal and myogenic lineages but at the functionally relevant level of protein expression, the myogenic lineage is dominant within the time scale studied as determined by the exclusive expression of cardiac myosin heavy chain for the micropatterned cells. Enforced cell shape distortion resulting in large scale rearrangement of cytoskeletal network and altered nucleus shape has been proposed as a physical impetus by which mechanical deformation is translated into biochemical response. These results demonstrated for the first time that cellular shape modulation in the absence of any induction factors may be a viable strategy to coax lineage-specific differentiation of stem cells.

Mechanical behavior of human mesenchymal stem cells during adipogenic and osteogenic differentiation.

Human mesenchymal stem cells (hMSCs) have gained widespread attention in the field of tissue engineering but not much is known about the changes of mechanical properties during the process of cell lineage commitment and the mechanisms of these behaviors. It is believed that exploring the inter-relations between stem cells mechanical properties and lineage commitment will shed light on the mechanobiology aspect of differentiation. hMSCs were cultured in adipogenic and osteogenic mediums and the elastic moduli were monitored using micropipette aspiration. It was found that hMSCs undergoing osteogenesis have an instantaneous Young's modulus of 890 +/- 219 Pa and an equilibrium Young's modulus of 224 +/- 40 Pa, each is about 2-fold higher than the control group. Interestingly, cells cultured in adipogenic medium exhibited a slight increase in the cellular modulus followed by a decrease relative to that of the control group. Gene expression study was employed to gain insights into this phenomenon. Concomitant up regulation of actin binding filamin A (FLNa) and gamma-Tubulin with the cellular elastic modulus indicated their important role in mechanical regulation during hMSCs differentiation. Statistical results showed that cell shape and cell area changed with cellular mechanical properties, which means that cell morphology has a close relation with cell elastic modulus in the initial stage of differentiation. Collectively, these results provide a quantitative description of hMSCs mechanical behavior during the process of differentiation as well as the possible accompanying mechanism at the biomolecular level.

Thickness sensing of hMSCs on collagen gel directs stem cell fate.

Mechanically compliant substrate provides crucial biomechanical cues for multipotent stem cells to regulate cellular fates such as differentiation, proliferation and maintenance of their phenotype. Effective modulus of which cells sense is not only determined by intrinsic mechanical properties of the substrate, but also the thickness of substrate. From our study, it was found that interference from underlying rigid support at hundreds of microns away could induce significant cellular response. Human mesenchymal stem cells (hMSCs) were cultured on compliant biological gel, collagen type I, of different thickness but identical ECM composition and local stiffness. The cells sensed the thin gel (130 µm) as having a higher effective modulus than the thick gel (1440 µm) and this was reflected in their changes in morphology, actin fibers structure, proliferation and tissue specific gene expression. Commitment into neuronal lineage was observed on the thin gel only. Conversely, the thick gel (1440 µm) was found to act like a substrate with lower effective modulus that inhibited actin fiber polymerization. Stem cells on the thick substrate did not express tissue specific genes and remained at their quiescent state. This study highlighted the need to consider not only the local modulus but also the thickness of biopolymer gel coating during modulation of cellular responses.

A bio-inspired platform to modulate myogenic differentiation of human mesenchymal stem cells through focal adhesion regulation.

The use of human mesenchymal stem cells (hMSCs) in cardiac-tissue engineering has gained widespread attention and many reports have shown that matrix compliance, micro/nano-patterns could be some of the important biophysical cues that drive hMSCs differentiation. Regardless of the type of biophysical induction cues, cells mainly explore their environment via focal adhesion (FA) and FA plays an important role in many cellular behaviours. Therefore, it is hypothesized that FA modulation through materials manipulation could be an important cue for modulation that would result in the stem cell lineage commitment. In this work, the FA of hMSCs is modulated by a novel microcontact printing method using polyvinyl alcohol as a trans-print media which can successfully print proteins on soft polydimethylsiloxane (PDMS). The FA is successfully modified into dense FA and elongated FA by micropatterning square and rectangular patterns on 12.6 kPa PDMS respectively. Additionally, the combined effects of stiffness of PDMS substrates (hard (308 kPa), intermediate (12.6 kPa)) and FA patterning on hMSCs differentiation are studied. The results indicate that dense FA does not induce myogenesis while elongated FA can promote cytoskeleton alignment and further myogenesis on PDMS with intermediate stiffness of 12.6 kPa. However, on stiff substrate (308 kPa), with or without patterns, the cytoskeleton alignment and myogenesis are not obvious. This demonstrates for the first time that it is possible to induce the differentiation of hMSCs by regulating the FA using a materials platform even in the absence of any biochemical factors. It also shows that there is a synergistic effect between FA regulation and matrix stiffness that results in a more specific and higher up-regulated myogenesis. This platform presents a new chemical/biological-free method to engineer the myogenic differentiation of hMSCs.

Insights into the role of focal adhesion modulation in myogenic differentiation of human mesenchymal stem cells.

We report the establishment of a novel platform to induce myogenic differentiation of human mesenchymal stem cells (hMSCs) via focal adhesion (FA) modulation, giving insights into the role of FA on stem cell differentiation. Micropatterning of collagen type I on a polyacrylamide gel with a stiffness of 10.2 kPa efficiently modulated elongated FA. This elongated FA profile preferentially recruited the ß(3) integrin cluster and induced specific myogenic differentiation at both transcription and translation levels with expression of myosin heavy chain and α-sarcomeric actin. This was initiated with elongation of FA complexes that triggered the RhoA downstream signaling toward a myogenic lineage commitment. This study also illustrates how one could partially control myogenic differentiation outcomes of similar-shaped hMSCs by modulating FA morphology and distribution. This technology increases our toolkit choice for controlled differentiation in muscle engineering.

A novel and simple microcontact printing technique for tacky, soft substrates and/or complex surfaces in soft tissue engineering.

Microcontact printing (µCP) has attracted much interest due to its simplicity and wide range of applications. However, when conventional µCP is applied to soft and/or tacky substrates, substrate sagging and difficulty in stamp removal cause non-conformance in the patterns. Moreover, it is almost impossible to apply conventional µCP on complex or wavy surfaces. In this study, we developed a novel yet simple trans-print method to create efficient micropatterning on soft and/or tacky substrates such as polydimethylsiloxane and polyacrylamide gel, and also on curved surfaces, by introducing polyvinyl alcohol film as a trans-print media. This technique is simple as it only involves one trans-print step and is also cost-effective. Most importantly, this technique is also versatile and we have proven this by printing various designs on more complex non-flat surfaces using various proteins as inks. The quality of the trans-printed pattern was excellent with high reproducibility and resolution as verified by immunostaining. Human mesenchymal stem cells cultured on these patterns displayed good conformance on the soft and tacky substrates printed using this technique. These results suggest that this novel trans-print technique can be extended to a potentially generic methodology for µCP of other proteins and biomolecules, other shapes and sizes, and cells, and will also be useful in three-dimensional micropatterning for soft tissue engineering.

Cyclic tensile loading regulates human mesenchymal stem cell differentiation into neuron-like phenotype.

Mechanical loading has been utilized as an effective tool to direct mesenchymal stem cells (MSCs) commitment into cell lineages of mesodermal origin. However, the use of this tool to induce transdifferentiation of MSCs into the neural lineage has never been attempted. In this study, we examined the potential of uniaxial cyclic tensile loading in promoting neuronal differentiation of human MSCs (hMSCs) on modified biodegradable poly(ε-caprolactone) (PCL). The stem cell morphology, tissue-specific gene and protein expression, microfilament structure and, subsequently, Rho GTPase activity were analysed after cyclically stretching the cells at a range of amplitudes (0.5%, 2% or 3.5%) and frequencies (0.5, 1 or 1.5 Hz) for 8 h. hMSCs responded to these stimuli and displayed distinctly different microfilament organization. However, only those stretched at 0.5% strain amplitude and 0.5 Hz frequency showed promoted outgrowth of filopodia with significant upregulation of neurogenic genes expression. Positive staining of the neurogenic protein markers Nestin and Tuj1 suggested that the hMSCs had been committed to early neuronal progenitors. In addition, Rac1 but not RhoA was activated at this particular loading parameter. Furthermore, inhibition of Rac1 activity with NSC23766 disrupted the effect of cyclic loading. The results suggest that cyclic tensile loading at low amplitude and frequency is capable of triggering neuron-like differentiation through the regulation of Rho GTPases activity, even in the absence of neurogenic induction medium.

Control of in vitro neural differentiation of mesenchymal stem cells in 3D macroporous, cellulosic hydrogels.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells that can be induced to differentiate into multiple cell lineages, including neural cells. They are a good cell source for neural tissue-engineering applications. Cultivation of human (h)MSCs in 3D scaffolds is an effective means for the development of novel neural tissue-engineered constructs, and may serve as a promising strategy in the treatment of nerve injury. AIM: This study presents the in vitro growth and neural differentiation of hMSCs in 3D macroporous, cellulosic hydrogels. RESULTS: The number of hMSCs cultivated in the 3D scaffolds increased by more than 14-fold after 7 days. After 2 days induction, most of the hMSCs in the 3D scaffolds were positive for nestin, a marker of neural stem cells. After 7 days induction, most of the hMSCs in the 3D scaffolds showed glial fibrillary acidic protein, tubulin or neurofilament M-positive reaction and a few hMSCs were positive for nestin. After 14 days induction, hMSCs in the 3D scaffolds could completely differentiate into neurons and glial cells. The neural differentiation of hMSCs in the 3D scaffolds was further demonstrated by real-time PCR. CONCLUSION: These results show that the 3D macroporous cellulosic hydrogel could be an appropriate substrate for neural differentiation of hMSCs and its possible applications in neural tissue engineering are discussed.