Determination of key events in mouse hepatocyte maturation at the single-cell level.

Hepatocytes, the liver's predominant cells, perform numerous essential biological functions. However, crucial events and regulators during hepatocyte maturation require in-depth investigation. In this study, we performed single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq) to explore the precise hepatocyte development process in mice. We defined three maturation stages of postnatal hepatocytes, each of which establishes specific metabolic functions and exhibits distinct proliferation rates. Hepatic zonation is gradually formed during hepatocyte maturation. Hepatocytes or their nuclei with distinct ploidies exhibit zonation preferences in distribution and asynchrony in maturation. Moreover, by combining gene regulatory network analysis with in vivo genetic manipulation, we identified critical maturation- and zonation-related transcription factors. This study not only delineates the comprehensive transcriptomic profiles of hepatocyte maturation but also presents a paradigm to identify genes that function in the development of hepatocyte maturation and zonation by combining genetic manipulation and measurement of coordinates in a single-cell developmental trajectory.

Merits and challenges of iPSC-derived organoids for clinical applications.

Induced pluripotent stem cells (iPSCs) have entered an unprecedented state of development since they were first generated. They have played a critical role in disease modeling, drug discovery, and cell replacement therapy, and have contributed to the evolution of disciplines such as cell biology, pathophysiology of diseases, and regenerative medicine. Organoids, the stem cell-derived 3D culture systems that mimic the structure and function of organs in vitro, have been widely used in developmental research, disease modeling, and drug screening. Recent advances in combining iPSCs with 3D organoids are facilitating further applications of iPSCs in disease research. Organoids derived from embryonic stem cells, iPSCs, and multi-tissue stem/progenitor cells can replicate the processes of developmental differentiation, homeostatic self-renewal, and regeneration due to tissue damage, offering the potential to unravel the regulatory mechanisms of development and regeneration, and elucidate the pathophysiological processes involved in disease mechanisms. Herein, we have summarized the latest research on the production scheme of organ-specific iPSC-derived organoids, the contribution of these organoids in the treatment of various organ-related diseases, in particular their contribution to COVID-19 treatment, and have discussed the unresolved challenges and shortcomings of these models.

Exosomes derived from umbilical cord mesenchymal stem cells protect cartilage and regulate the polarization of macrophages in osteoarthritis.

Background: Osteoarthritis (OA) is one of the most common joint diseases and a major global public health concern. Mesenchymal stem cells (MSCs) have been widely used for the treatment of OA owing to their paracrine secretion of trophic factors, a phenomenon in which exosomes may play a major role. Here, we investigate the potential of exosomes from human umbilical cord-derived MSCs (hUC-MSCs-Exos) in alleviating OA. Methods: The hUC-MSCs-Exos were harvested from hUC-MSC-conditioned medium using ultracentrifugation. Rats with surgically-induced OA were intra-articularly injected with hUC-MSCs-Exos. The effect of hUC-MSCs-Exos in repairing osteoarticular cartilage was assessed using hematoxylin and eosin (HE) staining, safranin-O and fast green staining and immunohistochemistry. The in vitro experiments were further carried out to verify the therapeutic effect. The effects of hUC-MSCs-Exos on the proliferation and migration of human chondrocytes were evaluated using the cell counting kit-8, EdU-555 cell proliferation kit, and transwell assays. Annexin V-FITC/PI staining were used to evaluate the effect of exosomes on chondrocyte apoptosis. An in vitro model of human articular chondrocytes treated with interleukin 1 beta (IL-1ß) was used to evaluate the effects of exosomes, analyses involved using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting. The role of hUC-MSCs-Exos in macrophage polarization was examined in the monocyte cell line, Tohoku Hospital Pediatrics-1 (THP-1) by qRT-PCR and immunofluorescence. Results: The results showed that hUC-MSCs-Exos prevented severe damage to the knee articular cartilage in the rat OA model. We confirmed the high efficacy of hUC-MSCs-Exos in promoting chondrocyte proliferation and migration and inhibiting chondrocyte apoptosis. Additionally, hUC-MSCs-Exos could reverse IL-1ß-induced injury of chondrocytes and regulate the polarization of macrophages in vitro. Conclusions: There is potential for hUC-MSCs-Exos to be used as a treatment strategy for OA.

Integrated PPI- and WGCNA-retrieval of hub gene signatures for soft substrates inhibition of human fibroblasts proliferation and differentiation.

Fibroblasts (FBs) are the most important functional cells in the process of wound repair, and their functions can be activated by different signals at the pathological site. Although wound repair is associated with microenvironmental stiffness, the effect of matrix stiffness on FBs remains elusive. In this study, TGF-ß1 was used to mimic the fibrotic environment under pathological conditions. We found that the soft substrates made FBs slender compared with tissue culture plastic, and the main altered biological function was the inhibition of proliferation and differentiation ability. Through PPI and WGCNA analysis, 63 hub genes were found, including GADD45A, CDKN3, HIST2H3PS2, ACTB, etc., which may be the main targets of soft substrates affecting the proliferation and differentiation of FBs. Our findings not only provide a more detailed report on the effect of matrix stiffness on the function of human skin FBs, but also may provide new intervention ideas for improving scars and other diseases caused by excessive cell proliferation, with potential clinical application prospects.

Glycyrrhizic acid suppresses atopic dermatitis-like symptoms by regulating the immune balance.

BACKGROUND: Glycyrrhiza is one of the most widely used traditional Chinese medicines in China. Its main bioactive ingredient glycyrrhizic acid (GA) has the potential to be used as a treatment for atopic dermatitis (AD) because it has similar actions to steroids, but with relatively few side effects. AIMS: The objective of this study was to explore the potential mechanisms of GA on AD mice model. METHODS: Calcipotriol, a vitamin D3 analogue (MC903) was applied topically to establish AD mouse model. Mice were intraperitoneally administrated with 2 mg/kg dexamethasone (DEX), 25 or 50 mg/kg GA for 15 days. After mice were executed, skin tissues were collected and detected the expression levels of IL-4, IFN-γ, TNF-α and thymic stromal lymphopoietin (TSLP). The percentages of Th1, Th2, Th17, langerhans cells (LCs) in draining lymph nodes (dLNs) were measured by flow cytometry. RESULTS: Our data demonstrated that GA improved the symptoms of AD by exerting anti-inflammatory and anti-allergic functions in vivo. We found that GA treatment decreased the level of total IgE in serum, suppressed ear swelling, reduced the infiltration of mast cells in skin lesions and decreased expressions of IL-4, IFN-γ, TNF-α and TSLP in skin lesions. Furthermore, our experimental results demonstrated that GA suppressed the Th1/Th2/Th17-immune responses in the dLNs, inhibited the migration of LCs in dLNs. CONCLUSIONS: In conclusion, our findings suggested potential therapeutic effects of GA against MC903-induced AD-like skin lesions in mice.

Soft substrates promote direct chemical reprogramming of fibroblasts into neurons.

Fibroblasts can be directly reprogrammed via a combination of small molecules to generate induced neurons (iNs), bypassing intermediate stages. This method holds great promise for regenerative medicine; however, it remains inefficient. Recently, studies have suggested that physical cues may improve the direct reprogramming of fibroblasts into neurons, but the underlying mechanisms remain to be further explored, and the physical factors reported to date do not exhibit the full properties of the extracellular matrix (ECM). Previous in vitro studies mainly used rigid polystyrene dishes, while one of the characteristics of the native in-vivo environment of neurons is the soft nature of brain ECM. The reported stiffness of brain tissue is very soft ranging between 100 Pa and 3 kPa, and the effect of substrate stiffness on direct neuronal reprogramming has not been explored. Here, we show for the first time that soft substrates substantially improved the production efficiency and quality of iNs, without needing to co-culture with glial cells during reprogramming, producing more glutamatergic neurons with electrophysiological functions in a shorter time. Transcriptome sequencing indicated that soft substrates might promote glutamatergic neuron reprogramming through integrins, actin cytoskeleton, Hippo signalling pathway, and regulation of mesenchymal-to-epithelial transition, and competing endogenous RNA network analysis provided new targets for neuronal reprogramming. We demonstrated that soft substrates may promote neuronal reprogramming by inhibiting microRNA-615-3p-targeting integrin subunit beta 4. Our findings can aid the development of regenerative therapies and help improve our understanding of neuronal reprogramming. STATEMENT OF SIGNIFICANCE: First, we have shown that low stiffness promotes direct reprogramming on the basis of small molecule combinations. To the best of our knowledge, this is the first report on this type of method, which may greatly promote the progress of neural reprogramming. Second, we found that microRNA (miR)-615-3p may interact with integrin subunit beta 4 (ITGB4), and the soft substrates may promote neural reprogramming by inhibiting miR-615-3p targeting ITGB4. We are the first to report on this mechanism. Our findings will provide more functional neurons for subsequent basic and clinical research in neurological regenerative medicine, and will help to improve the overall understanding of neural reprogramming. This work also provides new ideas for the design of medical biomaterials for nerve regeneration.

Therapeutic effects of myricetin on atopic dermatitis in vivo and in vitro.

BACKGROUND: Myricetin (Myr) is a flavonoid compound that exist widely in many natural plants. Myr has been proven to have multiple biological functions, including immunomodulatory and anti-inflammatory effects. PURPOSE: In this study, we investigated the therapeutic effect of Myr on calcipotriol (MC903) induced atopic dermatitis (AD) mouse model and tumor necrosis factor (TNF)-α/interferon (IFN)-γ stimulated human immortal keratinocyte line (HaCaT) in vivo and in vitro. METHODS: MC903 was applied topically to the left ears of mice to establish AD mouse model. After the AD model established successfully, the cream base, dexamethasone (DEX) cream or Myr cream were applied on the lesions of mice for 8 days. Through measuring ear thickness and scoring dermatitis severity, we evaluated the therapeutic effect of Myr, the draining lymph nodes (DLNs) and ears of the mice were collected for mechanistic study. In addition, TNF-α and IFN-γ-activated HaCaT cells were used to investigate the underlying mechanism. RESULTS: Our data demonstrated that Myr alleviated the symptoms of AD by exerting anti-inflammatory and anti-allergic functions in vivo. We found that Myr treatment suppressed ear swelling and IgE level in the serum, reduced the infiltration of mast cells in skin lesions, decreased expressions of thymus and activation regulated chemokine (TARC), IL-4, IFN-γ and thymic stromal lymphopoietin (TSLP) in ear lesions, increased the expressions of filaggrin (FLG). Furthermore, our experimental results demonstrated that Myr down-regulated the mRNA expressions of T-bet and GATA-3 in DLNs. In vitro, Myr treatment decreased MDC and TARC expressions in IFN-γ and TNF-α-induced HaCaT cells by blocking the NF-κB and STAT1 signal pathway. CONCLUSION: The present study is the first to investigate the anti-atopic effects of Myr. Our findings suggested the therapeutic effects of Myr against MC903-induced AD-like skin lesions in mice. Therefore, Myr may be a potential therapeutic agent for AD.

Extramedullary Osseointegration-A Novel Design of Percutaneous Osseointegration Prosthesis for Amputees.

The percutaneous osseointegrated (OI) prostheses have greatly improved the overall quality of life for amputees. However, the long-term maintenance of the OI prostheses is still challenging. A major problem is bone resorption around the bone-implant-skin interface, which might cause implant loosening or osteomyelitis. Another problem is the breakage of connecting components between the intramedullary implant and external prosthesis due to excessive stress. We designed a novel osseointegration implant by changing the bone-implant contact from the inner cortex to the outer surface of cortical bone. In the current study, we compared the extramedullary cap-shaped implants with the intramedullary screw-type implants in rabbits. Osteointegration was confirmed at the interface of bone to implant contact (BIC) in both implant types. The external implant induced intramedullary bone regeneration in the medullary canal and increased the cortical bone density at the end of the stump. This study provides a new perspective on the design of osseointegration implants which might prevent the currently reported complications of the intramedullary OI systems.

Influence of biased feedback on performance in a Vernier discrimination task.

The influence of feedback on performance is a topic of ongoing debate, with some previous studies finding it to be ineffective, while others have discovered that it can be helpful or harmful. One possible reason for these inconsistent results may be that feedback can create a conflict between a person's beliefs and the sensory information they receive. In the present study, we used a Vernier discrimination task to examine the influence of biased feedback on performance, as this type of feedback is most likely to create conflict. Biased feedback refers to feedback that does not align with the subjects' choices. The Vernier discrimination task is a type of psychophysical task that is often used to measure an individual's ability to perceive differences in the position or orientation of two visual stimuli. The task involves presenting two stimuli, one of which is slightly offset from the other, and asking the individual to determine the direction and magnitude of the offset. In Experiment 1, feedback was provided after each trial using large-offset verniers as guidance. The large-offset verniers always received correct feedback, but the small and medium-offset verniers might receive biased feedback. In Experiment 2, feedback was provided after each block of eight verniers. In Experiment 3, we removed the large offset vernier to investigate the influence of block feedback on the signal and noise. The results showed that the accuracy for the target vernier decreased due to biased feedback in both the trial feedback (Experiment 1) and the block feedback (Experiment 2). However, in Experiments 1 and 2, performance improved when feedback was absent. Moreover, if the difference between the two types of stimuli is great, the individual will engage in encoding learning rather than decision learning (Experiments 1 and 2). If the discrimination between the two types of stimuli is low, an individual's ability to discriminate noise is more vulnerable to the influence of biased feedback than the ability to discriminate the signal (Experiment 3). These results are discussed in relation to the mechanism of biased feedback, the process of encoding learning, the monitoring of internal feedback, and the generalization of false decisions.

CCM2L (Cerebral Cavernous Malformation 2 Like) Deletion Aggravates Cerebral Cavernous Malformation Through Map3k3-KLF Signaling Pathway.

[Figure: see text].

Single-cell patterning and axis characterization in the murine and human definitive endoderm.

Defining the precise regionalization of specified definitive endoderm progenitors is critical for understanding the mechanisms underlying the generation and regeneration of respiratory and digestive organs, yet the patterning of endoderm progenitors remains unresolved, particularly in humans. We performed single-cell RNA sequencing on endoderm cells during the early somitogenesis stages in mice and humans. We developed molecular criteria to define four major endoderm regions (foregut, lip of anterior intestinal portal, midgut, and hindgut) and their developmental pathways. We identified the cell subpopulations in each region and their spatial distributions and characterized key molecular features along the body axes. Dorsal and ventral pancreatic progenitors appear to originate from the midgut population and follow distinct pathways to develop into an identical cell type. Finally, we described the generally conserved endoderm patterning in humans and clear differences in dorsal cell distribution between species. Our study comprehensively defines single-cell endoderm patterning and provides novel insights into the spatiotemporal process that drives establishment of early endoderm domains.

Matrix stiffness regulates myocardial differentiation of human umbilical cord mesenchymal stem cells.

Myocardial infarction is a cardiovascular disease with high mortality. Human umbilical cord mesenchymal stem cells (hUC-MSCs) with strong self-renewal capacity and multipotency, provide the possibility of replacing injured cardiomyocytes. hUC-MSCs were cultured on polyacrylamide hydrogels with stiffnesses corresponding to Young's modulus of 13-16kPa and 62-68kPa which mimic the stiffnesses of healthy heart tissue and fibrotic myocardium. The expression of early myocardial markers Nkx2.5, GATA4, Mesp1 and the mature myocardial markers cTnT, cTnI, α-actin were detected by RT-PCR and Western Blot, which showed that soft matrix (13-16 kPa) tended to induce the differentiation of hUC-MSCs into myocardium, compared with stiff matrix (62-68 kPa). Piezos are mechanically sensitive non-selective cation channels. The expression of Piezo1 increased with the stiffness gradient of 1-10kPa, 13-16kPa, 35-38kPa and 62-68kPa on the 1st day, but Piezo2 expression was irregular. The expression of integrin ß1 and calcium ions were also higher on stiff substrate than on soft substrate. hUC-MSCs tend to differentiate into myocardium on the matrix stiffness of 13-16 kPa. The relationship among matrix stiffness, Piezo1 and myocardial differentiation needs further validation.

Large-scale Generation of Functional and Transplantable Hepatocytes and Cholangiocytes from Human Endoderm Stem Cells.

The ever-increasing therapeutic and pharmaceutical demand for liver cells calls for systems that enable mass production of hepatic cells. Here we describe a large-scale suspension system that uses human endoderm stem cells (hEnSCs) as precursors to generate functional and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos). hEnSC-derived hepatic populations are characterized by single-cell transcriptomic analyses and compared with hESC-derived counterparts, in-vitro-maintained or -expanded primary hepatocytes and adult cells, which reveals that hepatic differentiation of hEnSCs recapitulates in vivo development and that the heterogeneities of the resultant populations can be manipulated by regulating the EGF and MAPK signaling pathways. Functional assessments demonstrate that E-heps and E-chos possess properties comparable with adult counterparts and that, when transplanted intraperitoneally, encapsulated E-heps were able to rescue rats with acute liver failure. Our study lays the foundation for cell-based therapeutic agents and in vitro applications for liver diseases.

How to reprogram human fibroblasts to neurons.

Destruction and death of neurons can lead to neurodegenerative diseases. One possible way to treat neurodegenerative diseases and damage of the nervous system is replacing damaged and dead neurons by cell transplantation. If new neurons can replace the lost neurons, patients may be able to regain the lost functions of memory, motor, and so on. Therefore, acquiring neurons conveniently and efficiently is vital to treat neurological diseases. In recent years, studies on reprogramming human fibroblasts into neurons have emerged one after another, and this paper summarizes all these studies. Scientists find small molecules and transcription factors playing a crucial role in reprogramming and inducing neuron production. At the same time, both the physiological microenvironment in vivo and the physical and chemical factors in vitro play an essential role in the induction of neurons. Therefore, this paper summarized and analyzed these relevant factors. In addition, due to the unique advantages of physical factors in the process of reprogramming human fibroblasts into neurons, such as safe and minimally invasive, it has a more promising application prospect. Therefore, this paper also summarizes some successful physical mechanisms of utilizing fibroblasts to acquire neurons, which will provide new ideas for somatic cell reprogramming.

Soft Matrix Combined With BMPR Inhibition Regulates Neurogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells.

Stem cells constantly encounter as well as respond to a variety of signals in their microenvironment. Although the role of biochemical factors has always been emphasized, the significance of biophysical signals has not been studied until recently. Additionally, biophysical elements, like extracellular matrix (ECM) stiffness, can regulate functions of stem cells. In this study, we demonstrated that soft matrix with 1-10 kPa can induce neural differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs). Importantly, we used a combination of soft matrix and bone morphogenetic protein receptor (BMPR) inhibition to promote neurogenic differentiation of hUC-MSCs. Furthermore, BMPR/SMADs occurs in crosstalk with the integrinß1 downstream signaling pathway. In addition, BMPR inhibition plays a positive role in maintaining the undifferentiated state of hUC-MSCs on the hydrogel substrate. The results provide further evidence for the molecular mechanisms via which stem cells convert mechanical inputs into fateful decisions.

Comparative analysis of cell lineage differentiation during hepatogenesis in humans and mice at the single-cell transcriptome level.

During embryogenesis, the liver is the site of hepatogenesis and hematopoiesis and contains many cell lineages derived from the endoderm and mesoderm. However, the characteristics and developmental programs of many of these cell lineages remain unclear, especially in humans. Here, we performed single-cell RNA sequencing of whole human and mouse fetal livers throughout development. We identified four cell lineage families of endoderm-derived, erythroid, non-erythroid hematopoietic, and mesoderm-derived non-hematopoietic cells, and defined the developmental pathways of the major cell lineage families. In both humans and mice, we identified novel markers of hepatic lineages and an ID3+ subpopulation of hepatoblasts as well as verified that hepatoblast differentiation follows the "default-directed" model. Additionally, we found that human but not mouse fetal hepatocytes display heterogeneity associated with expression of metabolism-related genes. We described the developmental process of erythroid progenitor cells during human and mouse hematopoiesis. Moreover, despite the general conservation of cell differentiation programs between species, we observed different cell lineage compositions during hematopoiesis in the human and mouse fetal livers. Taken together, these results reveal the dynamic cell landscape of fetal liver development and illustrate the similarities and differences in liver development between species, providing an extensive resource for inducing various liver cell lineages in vitro.

Analysis of differentially expressed genes among human hair follicle-derived iPSCs, induced hepatocyte-like cells, and primary hepatocytes.

BACKGROUND: Differentiation of human induced pluripotent stem cells (hiPSCs) into hepatocytes has important clinical significance in providing a new stem cell source for cell therapy of terminal liver disease. The differential gene expression analysis of hiPSCs, induced hepatocyte-like cells (HLCs), and primary human hepatocytes (PHHs) provides valuable information for optimization of an induction scheme and exploration of differentiation mechanisms. METHODS: Human hair follicle-derived iPSCs (hHF-iPSCs) were induced in vitro by mimicking the environment of a developing liver for 19 days. Expression of specific proteins was determined by immunofluorescence staining; the function of HLCs in storage and metabolism was identified by detecting periodic acid-Schiff, indocyanine green, and low-density lipoprotein. Based on the transcriptomics data, the differential gene expression profiles of hHF-iPSCs, HLCs, and PHHs were analyzed by Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway, FunRich, and network analysis methods. RESULTS: HLCs were able to express albumin (ALB), alpha-fetoprotein, CYP3A4, and CYP7A1, and exhibited matured liver cell functions such as glycogen synthesis and storage. Complement and coagulation cascades and metabolic pathways ranked top in the downregulated list of HLCs/PHHs, while the cell cycle ranked top in the upregulated list of HLCs/PHHs. In the protein-protein interaction network, according to the degree rankings, TOP2A, CDK1, etc. were the important upregulated differentially expressed genes (DEGs), while ALB, ACACB, etc. were the major downregulated DEGs in HLCs/PHHs; the module analysis indicated that CDCA8, AURKB, and AURKA were the top upregulated DEGs in HLCs/PHHs. CONCLUSIONS: We presented the differences in gene expression among hHF-iPSCs, HLCs, and PHHs through transcriptome array data and provided new ideas for the optimization of induction.

Single-cell transcriptomic analyses reveal distinct dorsal/ventral pancreatic programs.

The pancreas of vertebrates is separately derived from both the dorsal and ventral endodermal domains. However, the difference between these two programs has been unclear. Here, using a pancreatic determination gene, Pdx1, driven GFP transgenic mouse strain, we identified Pdx1-GFP highly expressing cells (Pdx1high) and Pdx1-GFP lowly expressing cells (Pdx1low) in both embryonic dorsal Pdx1-expressing region (DPR) and ventral Pdx1-expressing region (VPR). We analyzed the transcriptomes of single Pdx1low and Pdx1high cells from the DPR and VPR. In the VPR, Pdx1low cells have an intermediate progenitor identity and can generate hepatoblasts, extrahepatobiliary cells, and Pdx1high pancreatic progenitor cells. In the DPR, Pdx1high cells are directly specified as pancreatic progenitors, whereas Pdx1low cells are precocious endocrine cells. Therefore, our study defines distinct road maps for dorsal and ventral pancreatic progenitor specification. The findings provide guidance for optimization of current ß-cell induction protocols by following the in vivo dorsal pancreatic specification program.

Extracellular matrix stiffness controls osteogenic differentiation of mesenchymal stem cells mediated by integrin α5.

BACKGROUND: Human mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury, and the stiffness of the extracellular matrix (ECM) has been shown to be an important regulatory factor for hMSC differentiation. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through integrin α5/ß1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs. METHODS: hMSCs were cultured on tunable polyacrylamide hydrogels coated with fibronectin with stiffness corresponding to a Young's modulus of 13-16 kPa and 62-68 kPa. After hMSCs were cultured on gels for 1 week, gene expression of alpha-1 type I collagen, BGLAP, and RUNX2 were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin α5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3ß, p-GSK-3ß, and ß-catenin) were evaluated by western blot analysis. RESULTS: Osteogenic differentiation was increased on 62-68 kPa ECM, as evidenced by alpha-1 type I collagen, BGLAP, and RUNX2 gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin α5/ß1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3ß, p-GSK-3ß, and ß-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin α5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular, alpha-1 type I collagen, RUNX2, and BGLAP were significantly downregulated, indicating that integrin α5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and ß-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3ß, and p-GSK-3ß were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin α5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin α5 antibody groups. At the same time, expression of GSK-3ß and p-GSK-3ß was upregulated and ß-catenin levels showed no difference between the groups with or without anti-integrin α5 antibody. Since Akt, p-Akt, GSK-3ß, and p-GSK-3ß displayed the same changes between the groups with or without anti-integrin α5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3ß was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3ß, and ß-catenin were unchanged. These results suggested that expression of p-GSK-3ß was regulated by p-Akt on 62-68 kPa ECM. CONCLUSIONS: Taken together, our results provide evidence that matrix stiffness (62-68 kPa) affects the osteogenic outcome of hMSCs through mechanotransduction events that are mediated by integrin α5.

Effects of Matrix Stiffness on the Morphology, Adhesion, Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells.

BMMSCs have drawn great interest in tissue engineering and regenerative medicine attributable to their multi-lineage differentiation capacity. Increasing evidence has shown that the mechanical stiffness of extracellular matrix is a critical determinant for stem cell behaviors. However, it remains unknown how matrix stiffness influences MSCs commitment with changes in cell morphology, adhesion, proliferation, self-renewal and differentiation. We employed fibronectin coated polyacrylamide hydrogels with variable stiffnesses ranging from 13 to 68 kPa to modulate the mechanical environment of BMMSCs and found that the morphology and adhesion of BMMSCs were highly dependent on mechanical stiffness. Cells became more spread and more adhesive on substrates of higher stiffness. Similarly, the proliferation of BMMSCs increased as stiffness increased. Sox2 expression was lower during 4h to 1 week on the 13-16 kPa and 62-68 kPa, in contrast, it was higher during 4h to 1 week on the 48-53 kPa. Oct4 expression on 13-16 kPa was higher than 48-53 kPa at 4h, and it has no significant differences at other time point among three different stiffness groups. On 62-68 kPa, BMMSCs were able to be induced toward osteogenic phenotype and generated a markedly high level of RUNX2, ALP, and Osteopontin. The cells exhibited a polygonal morphology and larger spreading area. These results suggest that matrix stiffness modulates commitment of BMMSCs. Our findings may eventually aid in the development of novel, effective biomaterials for the applications in tissue engineering.