Human Tendon-derived Stem Cells Research Articles

Tenogenic differentiation of human tendon-derived stem cells induced by long non-coding RNA LINCMD1 via miR-342-3p/EGR1 axis

ABSTRACT Background Tendon-derived stem cells (TDSCs) are proposed as a potential cell-seed for the treatment of tendon injury due to their tenogenic differentiation potential. In this work, we defined the action of long non-coding RNA (lncRNA) muscle differentiation 1 (LINCMD1) in tenogenic differentiation of human TDSCs (hTDSCs). Methods Quantitative real-time PCR (qRT-PCR) was used to assess the levels of LINCMD1, microRNA (miR)-342-3p, and early growth response-1 (EGR1) mRNA. Cell proliferation was detected by the XTT colorimetric assay. Protein expression was quantified by western blot. hTDSCs were grown in an osteogenic medium to induce osteogenic differentiation, and the extent of osteogenic differentiation was assessed by Alizarin Red Staining (ARS). The activity of alkaline phosphatase (ALP) was measured by the ALP Activity Assay Kit. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to evaluate the direct relationship between miR-342-3p and LINCMD1 or EGR1. Results Our results showed that enforced expression of LINCMD1 or suppression of miR-342-3p accelerated the proliferation and tenogenic differentiation and reduced osteogenic differentiation of hTDSCs. LINCMD1 regulated miR-342-3p expression by binding to miR-342-3p. EGR1 was identified as a direct and functional target of miR-342-3p, and knockdown of EGR1 reversed the effects of miR-342-3p suppression on cell proliferation and tenogenic and osteogenic differentiation. Furthermore, the miR-342-3p/EGR1 axis mediated the regulation of LINCMD1 on hTDSC proliferation and tenogenic and osteogenic differentiation. Conclusion Our study suggests the induction of LINCMD1 in tenogenic differentiation of hTDSCs through miR-342-3p/EGR1 axis.

LncRNA MALAT1 promotes tenogenic differentiation of tendon-derived stem cells via regulating the miR-378a-3p/MAPK1 axis

ABSTRACT Tendinopathy is a type of chronic injury caused by repeated pulling. Previous studies have reported that long non-coding RNA MALAT1 (MALAT1) regulates a variety of genes affecting bone metabolism. This study aimed to explore the role of the MALAT1 in tendon injury in vivo and in vitro. Human tendon-derived stem cells (TDSCs) were treated with TGF β1. Eighteen Sprague-Dawley rats were used to establish the tendinopathy animal model. Sirius Red staining and colorimetric assays were conducted to analyze the collagen content. RT-qPCR was performed to measure the mRNA levels. Western blotting was performed to measure the MAPK1 protein levels. Additionally, hematoxylin and eosin (HE) and immunohistochemical staining were used to analyze the cell number and the content of collagen type 1 and Thbs, respectively. MALAT1 expression was upregulated in TGF β1 treated TDSCs, and MALAT1 knockdown downregulated Scleraxis, Mohawk homeobox, Collagen 1A1, Fibromodulin, Matrix metallopeptidase 3, and Thrombospondin 4 in TGF β1 treated TDSCs. Bioinformatics analysis showed that miR-378a-3p was the target of MALAT1 and MAPK1, and dual-luciferase reporter assay indicated that both MALAT1 and MAPK1 could bind to miR-378a-3p. Furthermore, miR-378a-3p knockdown reversed the effect of si-MALAT1, whereas overexpression of MAPK1 reversed the effect of the miR-378a-3p mimic. Finally, MALAT1 expression was downregulated in tendinopathy rats, and MALAT1 overexpression healed tendon injury in them. MALAT1 regulated the tenogenic differentiation of TDSCs by regulating the miR-378a-3p/MAPK1 axis. Our results therefore indicate that targeting the MALAT1/miR-378a-3p/MAPK1 axis may be a promising avenue for the treatment of tendinopathy.

Nudt21-mediated alternative polyadenylation of HMGA2 3'-UTR impairs stemness of human tendon stem cell.

Tendon-derived stem cells (TSCs) play a primary role in tendon physiology, pathology, as well as tendon repair and regeneration after injury. TSCs are often exposed to mechanical loading-related cellular stresses such as oxidative stress, resulting in loss of stemness and multipotent differentiation potential. Cytoprotective autophagy has previously been identified as an important mechanism to protect human TSCs (hTSCs) from oxidative stress induced impairments. In this study, we found that high-mobility AT-hook 2 (HMGA2) overexpression protects hTSCs against H2O2-induced loss of stemness through autophagy activation. Evidentially, H2O2 treatment increases the expression of Nudt21, a protein critical to polyadenylation site selection in alternative polyadenylation (APA) of mRNA transcripts. This leads to increased cleavage and polyadenylation of HMGA2 3′-UTR at the distal site, resulting in increased HMGA2 silencing by the microRNA let-7 and reduced HMGA2 expression. In conclusion, Nudt21-regulated APA of HMGA2 3′-UTR and subsequent HMGA2 downregulation mediates oxidative stress induced hTSC impairments.

Influence of Supplements and Drugs used for the Treatment of Musculoskeletal Disorders on Adult Human Tendon-Derived Stem Cells

Influence of Supplements and Drugs used for the Treatment of Musculoskeletal Disorders on Adult Human Tendon-Derived Stem Cells

Fibroblast growth factor-2 promotes the function of tendon-derived stem cells in Achilles tendon restoration in an Achilles tendon injury rat model

The prognosis of Achilles tendon rupture is often unsatisfactory. Proliferative fibrous tissues and disordered collagen bundles make it difficult to guarantee normal biomechanical properties. The present study aimed to investigate the role of fibroblast growth factor-2 (FGF-2) in promoting the ability of human tendon-derived stem cells (hTDSCs) to treat Achilles tendon injury. hTDSCs were isolated from fetal Achilles tendon tissue and verified using fluorescence activated cell sorting analysis and multi-directional differentiation. The cells were then transfected with a lentivirus carrying the FGF2 gene. In vitro, FGF2 overexpression increased the expression of Collagen Type III Alpha 1 Chain (collagen-III) and scleraxis BHLH transcription factor (SCXA) significantly. Additionally, FGF-2-hTDSCs were transplanted into a rat Achilles tendon defect model. The in vivo results showed that the Achilles tendon tissue in the FGF-2 group secreted more extracellular matrix and produced collagen fibers that showed a more orderly arrangement. The expression of collagen-I and III in the FGF-2 group was significantly increased at 4 weeks postoperatively compared with the control group. Moreover, biomechanical tests showed that the failure load of FGF-2 group was higher at 4 and 8 weeks postoperatively than that of the controls. FGF-2 group had the highest stiffness in the early postoperative period, but showed no significant difference in the middle and late postoperative periods compared with that of the controls. In conclusion, FGF2 gene-modified hTDSCs promoted healing of Achilles tendon injury more effectively than hTDSCs alone.

Ferulic acid improves self-renewal and differentiation of human tendon-derived stem cells by upregulating early growth response 1 through hypoxia.

We aimed to investigate the potential beneficial effect of ferulic acid (FA) on stemness of human tendon-derived stem cells (hTSCs) in vitro and to elucidate the underlying molecular mechanism. The self-renewal ability of hTSCs was evaluated by colony formation and cell proliferation was determined by CCK-8 kit. Adipogenesis, osteogenesis, and chondrogenesis were determined by Oil Red O, Alizarin Red, and Alcian Blue stainings, respectively. Relative mRNA levels of PPARγ, Col2A1, Acan, Runx2, HIF1α, and EGR1 were measured with real-time PCR. Protein levels of HIF1α and EGR1 were detected by western blot. Direct binding of HIF1α with EGR1 promoter was analyzed by ChIP assay. Hypoxia-induced expression of EGR1 was interrogated by luciferase reporter assay. We demonstrated that FA treatment improved both self-renewal ability and multi-differentiation potential of hTSCs. FA induced hypoxia which in turn upregulated EGR1 expression via direct association with its hypoxia response element consensus sequence. Furthermore, we showed that both HIF1α and EGR1 were required for the enhancing effects of FA on hTSC self-renewal and differentiation. We hereby characterize the beneficial effect of FA on the stemness of hTSCs and highlight the critical role of HIF1α-EGR1 axis in this process.

3D uniaxial mechanical stimulation induces tenogenic differentiation of tendon‐derived stem cells through a PI3K/AKT signaling pathway

The tendon is a mechanosensitive tissue, but little is known about how mechanical stimulation selectively signals tenogenic differentiation and neo-tendon formation. In this study, we compared the impact of uniaxial and biaxial mechanical loading on tendon-derived stem cells (TDSCs). Our data show that there are variations in cell signaling and cell differentiation of mouse TDSCs in response to uniaxial and biaxial loading in monolayer culture. Whereas uniaxial loading induced TDSCs toward tenogenic and osteogenic differentiation, biaxial loading induced osteogenic, adipogenic, and chondrogenic differentiation of TDSCs. Furthermore, by applying uniaxial loading on 3-dimensional (3D) TDSC constructs, tenogenic-specific differentiation and neo-tendon formation were observed, results that were replicated in human TDSCs. We also showed that uniaxial loading induced PKB (AKT) phosphorylation (pAKT), whereas biaxial loading induced pERK. Most importantly, we found that inhibition of the PI3K/AKT signaling pathway could attenuate tenogenic differentiation and tendon formation in 3D TDSC constructs subjected to uniaxial loading. Taken together, our study highlights the importance of appropriate mechanobiological stimulation in 3D cell niches on tendon-like tissue formation and demonstrates that uniaxial mechanical loading plays an essential role in tenogenic differentiation and tendon formation by activating the PI3K/AKT signaling pathway.-Wang, T., Thien, C., Wang, C., Ni, M., Gao, J., Wang, A., Jiang, Q., Tuan, R. S., Zheng, Q., Zheng, M. H. 3D uniaxial mechanical stimulation induces tenogenic differentiation of tendon-derived stem cells through a PI3K/AKT signaling pathway.

Celastrol improves self-renewal and differentiation of human tendon-derived stem cells by suppressing Smad7 through hypoxia

BackgroundWe aimed to evaluate the potential enhancing effect of celastrol on the stemness of human tendon-derived stem cells (hTSCs) in vitro and the underlying molecular mechanisms.MethodsThe capability of hTSC self-renewal was assessed by cell proliferation and colony formation as determined with the CCK-8 kit. Adipogenesis, chondrogenesis, and osteogenesis were determined by Oil Red O, Alcian Blue, and Alizarin Red staining, respectively. The relative mRNA levels of Sox9, PPARγ, Runx2, Smad7, and HIF1α were determined by real-time polymerase chain reaction (PCR). The levels of Smad7 and HIF1α protein were measured by immunoblotting. The chromatin immunoprecipitation (ChIP) assay was used to assess the direct binding of HIF1α to the Smad7 promoter. Suppression of Smad7 induced by hypoxia was examined using the luciferase reporter assay.ResultsWe found that treatment with celastrol resulted in improvement in both the multi-differentiation potential and self-renewal capability of hTSCs. Celastrol elicited hypoxia and subsequently suppressed the expression of Smad7 through direct association with the hypoxia response element consensus sequence. Further, we demonstrated that both Smad7 and HIF1α were involved in the beneficial effects of celastrol on the differentiation and self-renewal of hTSCs.ConclusionsWe demonstrated the positive effect of celastrol on the stemness of hTSCs and elucidated the essential role of the HIF1α-Smad7 pathway in this process.

Effect of Hypoxia on Self-Renewal Capacity and Differentiation in Human Tendon-Derived Stem Cells

BackgroundHypoxic conditions play roles in functioning of human tendon-derived stem cells (hTSCs). The goal of this study was to investigate the effect of various hypoxic conditions in self-renewal capacity and differentiation of TSCs.Material/MethodshTSCs was obtain from supraspinatus tendon donors. Colony formation and cell proliferation assay were used to assess the self-renewal of hTSCs. qRT-PCT and Western blot analysis were used to examine stemness and multi-differentiation potential of hTSCs.ResultsWe found that culturing at 5% O2 is more beneficial for the self-renewal of hTSCs than the other 3 culture conditions, with larger colony size and numbers. The proliferation of hTSCs in 5%, 10%, and 20% O2 cultures increased after seeding. The number of cells in the 5% O2 condition was higher than that in other culture; however, self-renewal capacity of hTSCs in 0.5% O2 was inhibited. The expression levels of stem cell markers, including NS, Nanog, Oct-4, and SSEA-4, were highest in 0.5% O2 culture. Furthermore, hTSCs cultured in 20% O2 exhibited significantly higher expression of the 3 markers (PPAR-γ, Sox-9, and Runx-2).ConclusionsHypoxic condition of culture encouraged self-renewal capacity of hTSCs, but inhibited their multi-differentiation potential, compared to normoxic condition of culture. Moreover, excessively low oxygen concentration impaired the capacity of hTSCs.

Long noncoding RNA H19 accelerates tenogenic differentiation and promotes tendon healing through targeting miR‐29b‐3p and activating TGF‐β1 signaling

Tendon injures are common orthopedic conditions, but tendon development and the pathogenesis of tendon injures, such as tendinopathy, remain largely unknown and have limited the development of clinical therapy. Studies on tenogenic differentiation at the molecular level may help in developing novel therapeutic strategies. As novel regulators, long noncoding RNAs (lncRNAs) have been found to have widespread biological functions, and emerging evidence demonstrates that lncRNAs may play important regulatory roles in cell differentiation and tissue regeneration. In this study, we found that lncRNA H19 stimulated tenogenesis of human tendon-derived stem cells. Stable overexpression of H19 significantly accelerated TGF-β1-induced tenogenic differentiation in vitro and accelerated tendon healing in a mouse tendon defect model. H19 directly targeted miR-29b-3p, which is considered to be a negative regulator of tenogenesis. Furthermore, miR-29b-3p directly suppressed the expression of TGF-β1 and type I collagen, thereby forming a novel regulatory feedback loop between H19 and TGF-β1 to mediate tenogenic differentiation. Our study demonstrated that H19 promotes tenogenic differentiation both in vitro and in vivo by targeting miR-29b-3p and activating TGF-β1 signaling. Regulation of the TGF-β1/H19/miR-29b-3p regulatory loop may be a new strategy for treating tendon injury.-Lu, Y.-F., Liu, Y., Fu, W.-M., Xu, J., Wang, B., Sun, Y.-X., Wu, T.-Y., Xu, L.-L, Chan, K.-M., Zhang, J.-F., Li, G. Long noncoding RNA H19 accelerates tenogenic differentiation and promotes tendon healing through targeting miR-29b-3p and activating TGF-β1 signaling.

MiR124 suppresses collagen formation of human tendon derived stem cells through targeting egr1

Collagen formation is used as a crucial indicator of tenogenic differentiation of human tendon derived stem cell (hTDSC). Early growth response-1(egr1), a transcriptional factor, has been demonstrated to regulate tendon differentiation and promote tendon repair. Considering that the therapeutic options for tendon injuries remain limited, investigating the regulation of egr1 could facilitate the understanding of tendon development at molecular level so as to find a promising therapeutic target. MicroRNAs (miRNA) have been considered as epigenetic regulators to mediate multiple biological activities including stem cell differentiation. In the present study, biological experiments confirmed the prediction that miR124–3p (miR124) could have direct binding with egr1. We also found that miR124 suppressed collagen formation during the tendon differentiation of hTDSC while anti-miR124 promoted it. Furthermore, egr1 knockdown abolished the promotive effect of anti-miR124, suggesting that miR124 prevents tendon differentiation via suppressing egr1 expression. Therefore, miR124 may be a promising therapeutic target for tendon injury.

Mir-X suppresses tenogenic differentiation of human tendon derived stem cells through targeting Egr1

Mir-X suppresses tenogenic differentiation of human tendon derived stem cells through targeting Egr1

Hypoxia-Mediated Efficient Expansion of Human Tendon–Derived Stem CellsIn Vitro

Tendons regenerate and repair slowly and inefficiently after injury. Tendon-derived stem cells (TDSCs) have been isolated recently and have been shown to promote tendon repair. The ability to achieve sufficient numbers of cells for transplantation is essential for their clinical application. In this study, we aimed to study the effect of low oxygen (O(2)) tension (2%) on the clonogenicity, metabolic rate, DNA incorporation, population doubling time, β-galactosidase activity, immunophenotypes, multilineage differentiation potential, and tenocyte-like properties of human TDSCs (hTDSCs). hTDSCs were isolated from patellar tendon and characterized according to their adherence to plastic; colony-forming ability; multilineage differentiation potential; and high expression level of CD44, CD73, CD 90, and CD105 but low CD34, CD45, CD146, and Stro-1 at 20% O(2) tension. Low O(2) tension increased DNA incorporation but not metabolic rate of hTDSCs. It increased cell number 25% and the number of colonies but reduced the osteogenic, adipogenic, and chondrogenic differentiation potential of hTDSCs. The reduction in differentiation potential was associated with lower messenger RNA (mRNA) expression ratios of some lineage-related markers, including BGLAP, ALP, C/EBPα, PPARγ2, ACAN, and SOX9; the expression of a tendon-related marker, TNMD, was greater. There was no significant difference in the production of collagenous to noncollagenous protein ratio; the immunophenotypes and β-galactosidase activity were similar at 2% and 20% O(2) tension. Hypoxia-preconditioned hTDSCs could successfully differentiate at 20% O(2) tension, as shown by the return of the mRNA expression ratios of lineage-related markers to levels comparable to cells pre-incubated and differentiated at 20% O(2) tension. In conclusion, hypoxia is advantageous for efficient expansion of hTDSCs in vitro for tendon tissue engineering.