Yi-Shiuan Liu

In search of the pivot point of mechanotransduction: Mechanosensing of stem cells.

Stem cells are undifferentiated cells with the ability to self-renew and to differentiate into diverse specialized cell types; hence, they have great potential in tissue engineering and cell therapies. In addition to biochemical regulation, the physical properties of the microenvironments, such as scaffold topography, substrate stiffness, and mechanical forces, including fluid shear stress, compression, and tensile strain, can also regulate the proliferation and differentiation of stem cells. Upon physical stimuli, cytoskeleton rearrangements are expected to counterbalance the extracellular mechanical forces, trigger signaling cascades, and eventually cause epigenetic modifications. This article mainly focuses on the mechanosensing, which is the upstream event of stem cell mechanotransduction and the downstream one of physical stimuli. Putative mechanosensors such as ion channels, integrins, and cell membrane as well as primary cilia are discussed. Because mechanical environment is an important stem cell niche, identification of mechanosensors not only can elucidate the mechanisms of mechanotransduction and fate commitments but also bring new prospects of the mechanical control as well as drug development for clinical application.

Mechanosensitive TRPM7 mediates shear stress and modulates osteogenic differentiation of mesenchymal stromal cells through Osterix pathway.

Microenvironments that modulate fate commitments of mesenchymal stromal cells (MSCs) are composed of chemical and physical cues, but the latter ones are much less investigated. Here we demonstrate that intermittent fluid shear stress (IFSS), a potent and physiologically relevant mechanical stimulus, regulates osteogenic differentiation of MSCs through Transient receptor potential melastatin 7 (TRPM7)-Osterix axis. Immunostaining showed the localization of TRPM7 near or at cell membrane upon IFSS, and calcium imaging analysis demonstrated the transient increase of cytosolic free calcium. Expressions of osteogenic marker genes including Osterix, but not Runx2, were upregulated after three-hour IFSS. Phosphorylation of p38 and Smad1/5 was promoted by IFSS as well. TRPM7 gene knockdown abolished the promotion of bone-related gene expressions and phosphorylation. We illustrate that TRPM7 is mechanosensitive to shear force of 1.2 Pa, which is much lower than 98 Pa pressure loading reported recently, and mediates distinct mechanotransduction pathways. Additionally, our results suggest the differential roles of TRPM7 in endochondral and intramembranous ossification. Together, this study elucidates the mechanotransduction in MSCs fate commitments and displays an efficient mechano-modulation for MSCs osteogenic differentiation. Such findings should be taken into consideration when designing relevant scaffolds and microfluidic devices for osteogenic induction in the future.

Osteocalcin Mediates Biomineralization during Osteogenic Maturation in Human Mesenchymal Stromal Cells

There is a growing interest in cell therapies using mesenchymal stromal cells (MSCs) for repairing bone defects. MSCs have the ability to differentiate into osteoprogenitors and osteoblasts as well as to form calcified bone matrix. However, the molecular mechanisms governing mineralization during osteogenic differentiation remain unclear. Non-collagenous proteins in the extracellular matrix are believed to control different aspects of the mineralization. Since osteocalcin is the most abundant non-collagenous bone matrix protein, the purpose of this study is to investigate the roles of osteocalcin in mineral species production during osteogenesis of MSCs. Using Raman spectroscopy, we found that the maturation of mineral species was affected by osteocalcin expression level. After osteocalcin was knocked down, the mineral species maturation was delayed and total hydroxyapatite was lower than the control group. In addition, the expression of osteogenic marker genes, including RUNX2, alkaline phosphatase, type I collagen, and osteonectin, was downregulated during osteogenic differentiation compared to the control group; whereas gene expression of osterix was upregulated after the knockdown. Together, osteocalcin plays an essential role for the maturation of mineral species and modulates osteogenic differentiation of MSCs. The results offer new insights into the enhancement of new bone formation, such as for the treatments of osteoporosis and fracture healing.

Matrix dimensionality and stiffness cooperatively regulate osteogenesis of mesenchymal stromal cells

UNLABELLED Osteogenic potential of mesenchymal stromal cells (MSCs) is mechanosensitive. Its affected by the mechanical properties of the cellular microenvironment, particularly its mechanical modulus. To explore the effect of mechanical modulus on osteogenesis in the third dimension (3D), this study used a novel polyacrylamide (PA) scaffold whose pores are monodisperse and spherical, the mechanical moduli of which can be tuned across a wide range. It was found that MSCs have similar proliferation rates in PA scaffolds independent of the matrix stiffness. The contractile force exerted by MSCs inside PA scaffolds was strong enough to deform the pores of scaffolds made of more compliant PAs (whose shear modulus, Gscaffold<4 kPa). Only scaffolds of the highest stiffness (Gscaffold=12 kPa) can withhold the contraction from MSCs. After osteogenic induction for 21 days, the expression profiles of marker genes showed that PA scaffolds of Gscaffold=12 kPa promoted osteogenesis of MSCs. Confocal image analysis demonstrated that there are more F-actin cytoskeletons and bundled stress fibers at higher matrix moduli in 2D and 3D. Moreover, the 3D porous structure promotes osteogenesis of MSCs more than 2D flat substrates. Together, the differences of cellular behaviors when cultured in 2D and 3D systems are evident. The PA scaffolds developed in the present study can be used for further investigation into the mechanism of MSC mechanosensing in the 3D context. STATEMENT OF SIGNIFICANCE Mechanical properties of the microenvironment affect cellular behaviors, such as matrix stiffness. Traditionally, cell biological investigations have mostly employed cells growing on 2D substrates. The 3D porous PA scaffolds with the same topological conformation and pore sizes but different stiffness generated in this study showed that the differences of cellular behaviors in 2D and 3D systems are evident. Our 3D scaffolds provide insights into tissue engineering when stem cells incorporated with 3D scaffolds and support the future studies of cellular mechanobiology as well as the elucidation the role mechanical factor plays on the physiology and fate determination of MSCs in the 3D context.

Bio- chemical and physical characterizations of mesenchymal stromal cells along the time course of directed differentiation.

Cellular biophysical properties are novel biomarkers of cell phenotypes which may reflect the status of differentiating stem cells. Accurate characterizations of cellular biophysical properties, in conjunction with the corresponding biochemical properties could help to distinguish stem cells from primary cells, cancer cells, and differentiated cells. However, the correlated evolution of these properties in the course of directed stem cells differentiation has not been well characterized. In this study, we applied video particle tracking microrheology (VPTM) to measure intracellular viscoelasticity of differentiating human mesenchymal stromal/stem cells (hMSCs). Our results showed that osteogenesis not only increased both elastic and viscous moduli, but also converted the intracellular viscoelasticity of differentiating hMSCs from viscous-like to elastic-like. In contrast, adipogenesis decreased both elastic and viscous moduli while hMSCs remained viscous-like during the differentiation. In conjunction with bio- chemical and physical parameters, such as gene expression profiles, cell morphology, and cytoskeleton arrangement, we demonstrated that VPTM is a unique approach to quantify, with high data throughput, the maturation level of differentiating hMSCs and to anticipate their fate decisions. This approach is well suited for time-lapsed study of the mechanobiology of differentiating stem cells especially in three dimensional physico-chemical biomimetic environments including porous scaffolds.

Glucocorticoid receptor and Histone deacetylase 6 mediate the differential effect of dexamethasone during osteogenesis of mesenchymal stromal cells (MSCs).

Lineage commitment and differentiation of mesenchymal stromal cells (MSCs) into osteoblasts in vitro is enhanced by a potent synthetic form of glucocorticoid (GC), dexamethasone (Dex). Paradoxically, when used chronically in patients, GCs exert negative effects on bone, a phenomenon known as glucocorticoid-induced osteoporosis in clinical practice. The mechanism on how GC differentially affects bone precursor cells to become mature osteoblasts during osteogenesis remains elusive. In this study, the dose and temporal regulation of Dex on MSC differentiation into osteoblasts were investigated. We found that continuous Dex treatment led to a net reduction of the maturation potential of differentiating osteoblasts. This phenomenon correlated with a decrease in glucocorticoid receptor (GR) expression, hastened degradation, and impaired sub cellular localization. Similarly, Histone Deacetylase 6 (HDAC6) expression was found to be regulated by Dex, co-localized with GR and this GR-HDAC6 complex occupied the promoter region of the osteoblast late marker osteocalcin (OCN). Combinatorial inhibition of HDAC6 and GR enhanced OCN expression. Together, the cross-talk between the Dex effector molecule GR and the inhibitory molecule HDAC6 provided mechanistic explanation of the bimodal effect of Dex during osteogenic differentiation of MSCs. These findings may provide new directions of research to combat glucocorticoid-induced osteoporosis.

Intermittent Administration of Parathyroid Hormone 1–34 Enhances Osteogenesis of Human Mesenchymal Stem Cells by Regulating Protein Kinase Cδ

Human mesenchymal stem cells (hMSCs) can differentiate into osteoblasts and are regulated by chemical cues. The recombinant N-terminal (1–34 amino acids) fragment of the parathyroid hormone (PTH (1–34)) is identified to promote osteogenesis. The osteoanabolic effects of intermittent PTH (1–34) treatment are linked to a complex consisting of signaling pathways; additionally, protein kinase C (PKC) act as mediators of multifunctional signaling transduction pathways, but the role of PKC δ (PKCδ), a downstream target in regulating osteoblast differentiation during intermittent administration of PTH (1–34) is less studied and still remains elusive. The purpose of this study is to examine the role of PKCδ during intermittent and continuous PTH (1–34) administration using osteoblast-lineage-committed hMSCs. Relative gene expression of osteoblast-specific genes demonstrated significant upregulation of RUNX2, type I Collagen, ALP, and Osterix and increased alkaline phosphatase activity in the presence of PTH (1–34). Intermittent PTH (1–34) administration increased PKC activity at day 7 of osteogenic differentiation, whereas inhibition of PKC activity attenuated these effects. In addition, the specific isoform PKCδ was activated upon treatment. These findings demonstrate that intermittent PTH (1–34) treatment enhances the osteogenesis of hMSCs by upregulating osteoblast-specific genes via PKCδ activation.