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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.

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.

Coordinated organ behavior is crucial for an effective response to environmental stimuli. By studying regeneration of hair follicles in response to patterned hair plucking, we demonstrate that organ-level quorum sensing allows coordinated responses to skin injury. Plucking hair at different densities leads to a regeneration of up to five times more neighboring, unplucked resting hairs, indicating activation of a collective decision-making process. Through data modeling, the range of the quorum signal was estimated to be on the order of 1 mm, greater than expected for a diffusible molecular cue. Molecular and genetic analysis uncovered a two-step mechanism, where release of CCL2 from injured hairs leads to recruitment of TNF-α-secreting macrophages, which accumulate and signal to both plucked and unplucked follicles. By coupling immune response with regeneration, this mechanism allows skin to respond predictively to distress, disregarding mild injury, while meeting stronger injury with full-scale cooperative activation of stem cells.

Hair cycling is modulated by factors both intrinsic and extrinsic to hair follicles. Cycling defects lead to conditions such as aging-associated alopecia. Recently, we demonstrated that mouse skin exhibits regenerative hair waves, reflecting a coordinated regenerative behavior in follicle populations. Here, we use this model to explore the regenerative behavior of aging mouse skin. Old mice (>18 months) tracked over several months show that with progressing age, hair waves slow down, wave propagation becomes restricted, and hair cycle domains fragment into smaller domains. Transplanting aged donor mouse skin to a young host can restore donor cycling within a 3 mm range of the interface, suggesting that changes are due to extracellular factors. Therefore, hair stem cells in aged skin can be reactivated. Molecular studies show that extra-follicular modulators Bmp2, Dkk1, and Sfrp4 increase in early anagen. Further, we identify follistatin as an extra-follicular modulator, which is highly expressed in late telogen and early anagen. Indeed, follistatin induces hair wave propagation and its level decreases in aging mice. We present an excitable medium model to simulate the cycling behavior in aging mice and illustrate how the interorgan macroenvironment can regulate the aging process by integrating both "activator" and "inhibitor" signals.

Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases.

Mesenchymal stem cells (MSCs) can differentiate toward various lineages, including the osteogenic lineage, and thus hold great potential for clinic purposes. By using pharmacological inhibitors, protein kinase C (PKC) signaling has been shown to either negatively or positively regulate differentiation of bone, however, due to the low transfection efficiency in MSCs, the role of individual PKC isoforms is still not fully understood. In this study, we established a TAT peptide-mediated transduction system that efficiently delivered PKCα proteins into MSCs in a non- invasive fashion. The increased PKCα protein levels significantly promoted osteogenic differentiation in the murine mesenchymal C3H10T1/2 cells and in primary MSCs from both human and mouse, as demonstrated by the enhanced activity of the osteoblast marker, alkaline phosphatase, and the enhanced expression of the key transcription factor runx2. Mineralization is an important functional indication for bone differentiation. Our results further showed that PKCα promoted expression of the important osteocalcin gene and the accumulation of calcium minerals. Taken together, this study provides direct evidence showing that PKCα positively regulates osteogenic differentiation and demonstrates that the TAT peptide-mediated method enables functional study of specific PKC isoforms in MSCs without using viral infection. This may promote the application of PKCs in therapeutic treatment. Key words: protein kinase Cα, mesenchymal stem cells, osteogenic differentiation, TAT-mediated protein transduction. Resume : Les cellules souches mesenchymateuses (SCM) peuvent se differencier en differents lignages, notamment en lignage osteogene, possedant de ce fait un grand potentiel clinique. L'utilisation d'inhibiteurs pharmacologiques a montre que la signalisation impliquant la proteine kinase C (PKC) regule soit positivement ou negativement la differenciation de l'os, toutefois, a cause de la faible efficacite de transfection des SCM, le role des isoformes individuelles de PKC n'est toujours pas pleinement compris. Dans cette etude, nous avons mis en place un systeme de transduction au moyen d'un peptide TAT qui a permis d'introduire efficacement la PKCα dans les SCM de maniere non invasive. Les niveaux accrus de PKCα favorisaient significativement la differenciation osteogene de la lignee de cellules mesenchymateuses de souris C3H10T1/2 et de SCM primaires chez l'humain et la souris, tel que demontre par l'accroissement de l'activite de la phosphatase alcaline, un marqueur des osteoblastes, et de l'expression accrue du facteur de transcription cle runx2. La mineralisation constitue une importante indication fonctionnelle de differenciation de l'os. Nos resultats ont montre en outre que la PKCα favorisait l'expression du gene de l'osteocalcine, un gene important, et l'accumulation de calcium. Dans l'ensemble, cette etude montre directement que la PKCα regule positivement la differenciation osteogene et demontre que la methode qui fait appel au peptide TAT permet de realiser des etudes fonctionnelles d'isoformes specifiques de la PKC chez les SCM sans utiliser l'infection virale. Ceci pourrait favoriser l'application therapeutique des PKC. [Traduit par la Redaction] Mots-cles : proteine kinase Cα, cellules souches mesenchymateuses, differenciation osteogene, transduction proteique par TAT., Introduction Mesenchymal stem cells (MSCs) can differentiate toward osteogenic, chondrogenic, adipogenic, and myogenic lineages upon appropriate induction (Liu et al. 2009). A number of factors, including bone morphogenetic protein (BMP)-2 [...]

The initial step of interaction of some pathogens with the host is driven by the interaction of glycoproteins of either side via endcaps of their glycans. These end caps consist of sialic acids or sugar molecules. Coronaviruses (CoVs), including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are found to use this route of interaction. The strength and spatial interactions on the single molecule level of sialic acids with either the spike (S) protein of SARS coronaviruses, or human angiotensin-converting enzyme 2 (ACE2) and furin are probed and compared to the binding modes of those sugar molecules which are present in glycans of glycoproteins. The protocol of using single molecules is seen as a simplified but effective mimic of the complex mode of interaction of the glycans. Averaged estimated binding energies from a docking approach result in preferential binding of the sialic acids to a specific binding site of the S protein of human coronavirus OC43 (HCoV-OC43). Furin is proposed to provide better binding sites for sialic acids than ACE2, albeit outweighed by sites for other sugar molecules. Absolute minimal estimated binding energies indicate weak binding affinities and are indifferent to the type of sugar molecules and the proteins. Neither the proposed best binding sites of the sialic acids nor those of the sugar molecules overlap with any of the cleavage sites at the S protein and the active sites of the human proteins. [ABSTRACT FROM AUTHOR]

Abstract Background Spinocerebellar ataxia (SCA) refers to a disease entity in which polyglutamine aggregates are over-produced in Purkinje cells (PCs) of the cerebellum as well as other neurons in the central nervous system, and the formation of intracellular polyglutamine aggregates result in the loss of neurons as well as deterioration of motor functions. So far there is no effective neuroprotective treatment for this debilitating disease although numerous efforts have been made. Mesenchymal stem cells (MSCs) possess multi-lineage differentiation potentials as well as immuno-modulatory properties, and are theoretically good candidates for SCA treatment. The purpose of this study is to investigate whether transplantation of human MSCs (hMSCs) can rescue cerebellar PCs and ameliorate motor function deterioration in SCA in a pre-clinical animal model. Method Transgenic mice bearing poly-glutamine mutation in ataxin-2 gene (C57BL/6J SCA2 transgenic mice) were serially transplanted with hMSCs intravenously or intracranially before and after the onset of motor function loss. Motor function of mice was evaluated by an accelerating protocol of rotarod test every 8 weeks. Immunohistochemical stain of whole brain sections was adopted to demonstrate the neuroprotective effect of hMSC transplantation on cerebellar PCs and engraftment of hMSCs into mice brain. Results Intravenous transplantation of hMSCs effectively improved rotarod performance of SCA2 transgenic mice and delayed the onset of motor function deterioration; while intracranial transplantation failed to achieve such neuroprotective effect. Immunohistochemistry revealed that intravenous transplantation was more effective in the preservation of the survival of cerebellar PCs and engraftment of hMSCs than intracranial injection, which was compatible to rotarod performance of transplanted mice. Conclusion Intravenous transplantation of hMSCs can indeed delay the onset as well as improve the motor function of SCA2 transgenic mice. The results of this preclinical study strongly support further exploration of the feasibility to transplant hMSCs for SCA patients.