Adult mesenchymal stem cells (MSCs) derived from bone marrow contribute to the regeneration of multiple types of mesenchymal tissues. Here we describe the functional role of a novel form of ...cross-talk between the transforming growth factor β1 (TGF-β1) and Wnt signaling pathways in regulating the activities of human MSCs. We show that TGF-β1 induces rapid nuclear translocation of β-catenin in MSCs in a Smad3-dependent manner. Functionally, this pathway is required for the stimulation of MSC proliferation and the inhibition of MSC osteogenic differetiation by TGF-β1, likely through the regulation of specific downstream target genes. These results provide evidence for a new mode of cooperation between the TGF-β and Wnt signaling pathways in this specific cellular context and suggest a potentially important role for this distinct signaling pathway in the control of self-renewal and differentiation of a specific type of MSCs.
Encapsulated stem cells in various biomaterials have become a potentially promising cell transplantation strategy in the treatment of various neurologic disorders. However, there is no ideal cell ...delivery material and method for clinical application in brain diseases. Here we show silk fibroin (SF)-based hydrogel encapsulated engineered human mesenchymal stem cells (hMSCs) to overproduce brain-derived neurotrophic factor (BDNF) (BDNF-hMSC) is an effective approach to treat brain injury through trans-septal cell transplantation in the rat model. In this study, we observed SF induced sustained BDNF production by BDNF-hMSC both in 2D (9.367 ± 1.969 ng/ml) and 3D (7.319 ± 0.1025 ng/ml) culture conditions for 3 days. Through immunohistochemistry using α-tubulin, BDNF-hMSCs showed a significant increased average neurite length of co-cultured neuro 2a (N2a) cells, suggested that BDNF-hMSCs induced neurogenesis in vitro. Encapsulated BDNF-hMSC, pre-labeled with the red fluorescent dye PKH-26, exhibited intense fluorescence up to 14 days trans-septal transplantation, indicated excellent viability of the transplanted cells. Compared to the vehicle-treated, encapsulated BDNF- hMSC demonstrated significantly increased BDNF level both in the sham-operated and injured hippocampus (Hip) through immunoblot analysis after 7 days implantation. Transplantation of the encapsulated BDNF-hMSC promoted neurological functional recovery via significantly reduced neuronal death in the Hip 7 days post-injury. Using magnetic resonance imaging (MRI) analysis, we demonstrated that encapsulated BDNF-hMSC reduced lesion area significantly at 14 and 21 days in the damaged brain following trans-septal implantation. This stem cell transplantation approach represents a critical set up towards brain injury treatment for clinical application.
Phosphate is indispensable in osteogenesis and mineralization. However, mechanisms by which phosphate enhances osteogenic differentiation are not fully understood. In this study, we studied the ...effect of phosphate on osteogenic differentiation as well as signaling pathways induced by phosphate in the process.
Induced human bone marrow-derived mesenchymal stem cells differentiation into osteoblasts by the change of media containing β-glycerophosphate (GP), 1 mM inorganic phosphate, or 3 mM inorganic phosphate (Pi). The differentiation of osteoblasts was verified by the expression of osteoblast differentiation markers and calcium deposition. RNA sequencing was performed to assess transcriptome in the early stage of osteogenic differentiation.
Osteogenic differentiation and mineralization were promoted in the 3 mM Pi group compared to those in the GP and 1 mM Pi groups on day 7 of culture. RNA sequencing revealed that the gene expressions involved in osteogenesis and the components in the Wnt signaling pathway was increased in 3 mM Pi group compared with those in the GP on day 7. Analysis with qPCR and Western blot suggested upregulation of components in the non-canonical Wnt signaling pathway, including WNT5b and phosphorylated-c-Jun in the 3 mM Pi group on day 7. WNT11 mRNA expression was increased in the 2 induction groups on day 7. Inhibition of WNT5b by siRNA experiment attenuated the components in non-canonical Wnt signaling expression, including WNT5b, WNT11 and ROR2 mRNA expression and phosphorylated-c-Jun protein expression. In addition, osteogenic differentiation and mineralization were partly decreased in 3 mM Pi group on day 7 by the inhibition of WNT5b.
Pi promoted osteogenic differentiation through the up-regulation of the non-canonical Wnt signaling pathway, including WNT5b, WNT11, p-c-Jun/c-Jun, in the early stage of differentiation. These findings provide a new perspective into the association of Pi and the non-canonical Wnt signaling pathway during osteogenic differentiation.
•High phosphate promotes the non-canonical Wnt signaling including WNT5b at the early stage of osteoblast differentiation from human mesenchymal stem cells.•WNT5b is a new player involved in osteoblast differentiation from mesenchymal stem cells induced by phosphate.•Inhibition of WNT5b attenuated mineralization and osteogenic differentiation.
Human mesenchymal stem cells (MSCs) have attracted attention as a major cellular source for regenerative medicine and preclinical studies. Three-dimensional (3D) cell culture systems can be used to ...construct physiologically relevant MSC spheroids with enhanced tissue regeneration capacity. However, what aspect of the MSC spheroids retains the regeneration capacity is not yet fully understood. Here, we induced osteoblast differentiation of MSC spheroids and investigated their respiratory activity using electrochemiluminescence (ECL) imaging developed in our previous study. In parallel, the ability of MSC spheroids to induce angiogenesis was investigated in a microfluidic device with a co-culture of endothelial cells. We found that the osteogenic differentiation of MSCs induced changes in their metabolic activity (respiratory activity of the spheroids decreased during differentiation) and lead to the loss of their angiogenesis-inducing ability. In other words, it is suggested that maintaining the stem cell nature with in the MSC spheroids is an important factor for tissue regeneration. In this study, we demonstrated the effective use of ECL imaging and microfluidic systems for the evaluation of MSC spheroids, providing new insights into stem cell tissue engineering.
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Sodium alginate (SA)/hydroxyethylcellulose (HEC)/hydroxyapatite (HA) composite scaffolds were explored for enhanced in vitro bone regeneration. The SA/HEC/HA composites were synthesized using the ...lyophilization technique and further cross-linked in the presence of calcium ions to form composite hydrogel networks. The physicochemical, thermal behavior and morphology properties of the prepared scaffolds were characterized through XRD, DSC/TGA, FTIR and SEM. Furthermore, the mechanical behavior of the under investigated scaffolds was determined using texture analyzer. The in vitro bioactivity in SBF and adsorption of bovine serum albumin as well as cell viability for all the prepared scaffolds were also tested. The results indicated that the higher HA concentration (40wt%) enhanced the mechanical properties (23.9MPa), bioactivity and protein adsorption. Cell viability of the tested scaffolds confirmed the non-toxicity of the fabricated systems on the human mesenchymal stem cells (hMSCs). Proliferation capability was also confirmed for the tested scaffolds after 3 and 7days, but the higher HA-containing scaffold showed increased cell populations specially after 7days compared to HA-free scaffolds. This novel composite material could be used in bone tissue engineering as a scaffold material to deliver cells and biologically active molecules.
•Series of alginate/HEC/HA scaffolds were prepared by freeze-drying technique.•Presence of HEC controlled the scaffold degradation, swelling and porosity.•The higher HA concentration the higher mechanical behavior, adequate porosity and bioactivity were achieved.•The designed scaffolds possessed good biodegradability and protein adsorption.•Higher Cells viability and proliferation for Human MSCs were recorded.
Stem cell therapeutics has emerged as a novel regenerative therapy for tissue repair in the last decade. However, dynamically tracking the transplanted stem cells in vivo remains a grand challenge ...for stem cell‐based regeneration medicine in full understanding the function and the fate of the stem cells. Herein, Ag2S quantum dots (QDs) in the second near‐infrared window (NIR‐II, 1.0–1.4 μm) are employed for dynamically tracking of human mesenchymal stem cells (hMSCs) in vivo with high sensitivity and high spatial and temporal resolution. As few as 1000 Ag2S QDs‐labeled hMSCs are detectable in vivo and their fluorescence intensity can maintain up to 30 days. The in situ translocation and dynamic distribution of transplanted hMSCs in the lung and liver can be monitored up to 14 days with a temporal resolution of 100 ms. The in vivo high‐resolution imaging indicates the heparin‐facilitated translocation of hMSCs from lung to liver as well as the long‐term retention of hMSCs in the liver contribute to the treatment of liver failure. The novel NIR‐II imaging offers a possibility of tracking stem cells in living animals with both high spatial and temporal resolution, and encourages the future clinical applications in imaging‐guided cell therapies.
In situ translocation and dynamic distribution of transplanted hMSCs in the lung and liver of mice are clearly visualized up to 14 days by using Ag2S QDs with emission of 1200 nm in the second near‐infrared window (NIR‐II). The novel NIR‐II fluorescence imaging promises the future cell tracking and clinical imaging‐guided cell therapy.
Human mesenchymal stem cells (hMSCs) are widely used to model osteogenic differentiation in vitro, yet few studies compare the numerous available protocols. The poor translation between in vitro and ...in vivo results in bone regeneration highlights the need for improved methodologies to assess osteogenic phenotype. Omics technologies generate vast biological data but are complex for comparing several osteogenic differentiation strategies. Conversely, conventional methods like Alizarin red S stains or alkaline phosphatase assays do not provide enough data. Here, we propose a targeted protein multiplex assay to characterize and compare several models of osteogenic differentiation in hMSCs, containing markers for osteogenesis, angiogenesis, and inflammation—critical processes in bone regeneration. To induce osteogenic differentiation, hMSCs were stimulated with dexamethasone, with bone morphogenetic protein 2, supplemented with calcium or phosphate ions, or seeded on a calcium phosphate–based coating. Based on mineralization status determined by Alizarin red S stain, conditions were classified into non-mineralizing, late-mineralizing, and early-mineralizing groups. Protein patterns associated with these groups revealed distinct mechanisms of osteogenic differentiation for early- and late-mineralizing hMSCs. While non-mineralizing hMSCs did not undergo osteogenic differentiation, they exhibited increased angiogenic and inflammatory marker expression compared to the control. Late-mineralizing hMSCs had limited potential to produce angiogenesis-related factors, while early-mineralizing hMSCs showed an increased effect. Overall, we compared commonly used in vitro models of osteogenic differentiation in hMSCs to establish a comprehensive roadmap of osteogenic differentiation.
Hypervirulent Klebsiella pneumoniae (hvKP) causes severe infections even in healthy individuals by escaping surveillance and killing from polymorphonuclear neutrophils (PMNs), the first-line ...leukocytes in bacterial infections; moreover, the emergence of multidrug-resistant strains further limits treatment options. We therefore assess whether multilineage mesenchymal stem cells (MSCs), best known for immunomodulation toward T cells, could be therapeutic for highly virulent bacterial infections via modulation of PMNs. We find that both bone marrow MSCs and placental MSCs (PMSCs) preserve in vitro PMN survival, but only PMSCs significantly enhance multiple PMN bactericidal functions, including phagocytosis, through secretion of interleukin-1β (IL-1β). PMSC treatment of hvKP-infected mice suppresses T and natural killer (NK) cell responses as expected but can preferentially recruit PMNs and enhance antibacterial functions to allow for disease survival; IL-1β knockdown in PMSCs significantly decreases hvKP clearance, worsening survival and resulting in 100% lethality. Our data strongly implicate the possible use of PMSCs for infections of PMN-resistant hvKP strains.
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•Human PMSCs preserve PMN viability while suppressing T and NK lymphocyte responses•Human PMSC-secreted IL-1β is involved in enhancing PMN bactericidal functions•Human PMSCs have a therapeutic role for infections of PMN-resistant hvKP strains•Human PMSC treatment improves survival of mice with abdominal hvKP infection
Wang et al. demonstrate that human PMSC-secreted IL-1β is critical in the preferential recruitment of PMNs during hvKP-induced intra-abdominal infection. This results in enhanced PMN antibacterial functions, including improved hepatic and peritoneal cavity bacterial clearance, without eliciting excessive T and NK lymphocyte responses, which allow for survival against absolute lethality.
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