Tianyi Wu

An improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow

Summary Mesenchymal stem cells (MSCs) from bone marrow are main cell source for tissue repair and engineering, and vehicles of cell-based gene therapy. Unlike other species, mouse bone marrow derived MSCs (BM-MSCs) are difficult to harvest and grow due to the low MSCs yield. We report here a standardised, reliable, and easy-to-perform protocol for isolation and culture of mouse BM-MSCs. There are five main features of this protocol. (1) After flushing bone marrow out of the marrow cavity, we cultured the cells with fat mass without filtering and washing them. Our method is simply keeping the MSCs in their initial niche with minimal disturbance. (2) Our culture medium is not supplemented with any additional growth factor. (3) Our method does not need to separate cells using flow cytometry or immunomagnetic sorting techniques. (4) Our method has been carefully tested in several mouse strains and the results are reproducible. (5) We have optimised this protocol, and list detailed potential problems and trouble-shooting tricks. Using our protocol, the isolated mouse BM-MSCs were strongly positive for CD44 and CD90, negative CD45 and CD31, and exhibited tri-lineage differentiation potentials. Compared with the commonly used protocol, our protocol had higher success rate of establishing the mouse BM-MSCs in culture. Our protocol may be a simple, reliable, and alternative method for culturing MSCs from mouse bone marrow tissues.

Stepwise Differentiation of Mesenchymal Stem Cells Augments Tendon-Like Tissue Formation and Defect Repair In Vivo

Tendon injuries are common and present a clinical challenge, as they often respond poorly to treatment and result in long‐term functional impairment. Inferior tendon healing responses are mainly attributed to insufficient or failed tenogenesis. The main objective of this study was to establish an efficient approach to induce tenogenesis of bone marrow‐derived mesenchymal stem cells (BMSCs), which are the most common seed cells in tendon tissue engineering. First, representative reported tenogenic growth factors were used as media supplementation to induce BMSC differentiation, and the expression of teno‐lineage transcription factors and matrix proteins was compared. We found that transforming growth factor (TGF)‐β1 significantly induced teno‐lineage‐specific gene scleraxis expression and collagen production. TGF‐β1 combined with connective tissue growth factor (CTGF) elevated tenomodulin and Egr1 expression at day 7. Hence, a stepwise tenogenic differentiation approach was established by first using TGF‐β1 stimulation, followed by combination with CTGF for another 7 days. Gene expression analysis showed that this stepwise protocol initiated and maintained highly efficient tenogenesis of BMSCs. Finally, regarding in situ rat patellar tendon repair, tendons treated with induced tenogenic BMSCs had better structural and mechanical properties than those of the control group, as evidenced by histological scoring, collagen I and tenomodulin immunohistochemical staining, and tendon mechanical testing. Collectively, these findings demonstrate a reliable and practical strategy of inducing tenogenesis of BMSCs for tendon regeneration and may enhance the effectiveness of cell therapy in treating tendon disorders.

The roles of mesenchymal stem cells in tissue repair and disease modification.

Mesenchymal stem cells (MSCs) are multi-potent cells which have been widely used for tissue regeneration and immunomodulation. The infusion of autologous and allogenic MSCs has been proved to be safe and effective in tissue repair and disease modulation. The inherent homing ability of MSCs ensures the transplanted cells migrating into the damaged tissue areas, but only a small percentage of the transplanted (allogenic) MSCs survive for long. However, the beneficial effects of MSCs transplantation could be noted within 1-2 days that are unlikely due to their proliferation and differentiation. The regulatory roles of MSCs in tissue repair are rather more important than their direct involvement of repair processes. The most important effect of transplanted MSCs is their immunomodulation function through crosstalk with the immune cells or the paracrine actions. The active factor secreted by MSCs may vary in the different disease conditions or tissue niches, and are under dynamic changes in various local environments. To understand and define the MSCs secretion factors in various disease settings could be a future research direction, and the findings could lead to potential new MSCs-based therapeutic products.

Sulfated hyaluronic acid hydrogels with retarded degradation and enhanced growth factor retention promote hMSC chondrogenesis and articular cartilage integrity with reduced hypertrophy

Recently, hyaluronic acid (HA) hydrogels have been extensively researched for delivering cells and drugs to repair damaged tissues, particularly articular cartilage. However, the in vivo degradation of HA is fast, thus limiting the clinical translation of HA hydrogels. Furthermore, HA cannot bind proteins with high affinity because of the lack of negatively charged sulfate groups. In this study, we conjugated tunable amount of sulfate groups to HA. The sulfated HA exhibits significantly slower degradation by hyaluronidase compared to the wild type HA. We hypothesize that the sulfation reduces the available HA octasaccharide substrate needed for the effective catalytic action of hyaluronidase. Moreover, the sulfated HA hydrogels significantly improve the protein sequestration, thereby effectively extending the availability of the proteinaceous drugs in the hydrogels. In the following in vitro study, we demonstrate that the HA hydrogel sulfation exerts no negative effect on the viability of encapsulated human mesenchymal stem cells (hMSCs). Furthermore, the sulfated HA hydrogels promote the chondrogenesis and suppresses the hypertrophy of encapsulated hMSCs both in vitro and in vivo. Moreover, intra-articular injections of the sulfated HA hydrogels avert the cartilage abrasion and hypertrophy in the animal osteoarthritic joints. Collectively, our findings demonstrate that the sulfated HA is a promising biomaterial for the delivery of therapeutic agents to aid the regeneration of injured or diseased tissues and organs. STATEMENT OF SIGNIFICANCE In this paper, we conjugated sulfate groups to hyaluronic acid (HA) and demonstrated the slow degradation and growth factor delivery of sulfated HA. Furthermore, the in vitro and in vivo culture of hMSCs laden HA hydrogels proved that the sulfation of HA hydrogels not only promotes the chondrogenesis of hMSCs but also suppresses hypertrophic differentiation of the chondrogenically induced hMSCs. The animal OA model study showed that the injected sulfated HA hydrogels significantly reduced the cartilage abrasion and hypertrophy in the animal OA joints. We believe that this study will provide important insights into the design and optimization of the HA-based hydrogels as the scaffold materials for cartilage regeneration and OA treatment in clinical setting.

Multifunctional Quantum Dot Nanoparticles for Effective Differentiation and Long‐Term Tracking of Human Mesenchymal Stem Cells In Vitro and In Vivo

Human mesenchymal stem cells (hMSCs) hold great potential for regenerative medicine. Efficient induction of hMSC differentiation and better understanding of hMSCs behaviors in vitro and in vivo are essential to the clinical translation of stem cell therapy. Here a quantum dots (QDs)-based multifunctional nanoparticle (RGD-β-CD-QDs) is developed for effective enhancing differentiation and long-term tracking of hMSCs in vitro and in vivo. The RGD-β-CD-QDs are modified with β-cyclodextrin (β-CD) and Cys-Lys-Lys-Arg-Gly-Asp (CKKRGD) peptide on the surface. The β-CD can harbor hydrophobic osteogenic small molecule dexamethasone (Dex) and the RGD peptide not only facilitates the complexation of siRNA and delivers siRNA into hMSCs but also leads to cellular uptake of nanoparticles by RGD receptor. Co-delivery of Dex and siRNA by RGD-β-CD-QDs nanocarrier significantly expedites and enhances the osteogenesis differentiation of hMSCs in vitro and in vivo by combined effect of small molecule and RNAi. Furthermore, the RGD-β-CD-QDs can be labeled with hMSCs for a long-term tracking (3 weeks) in vivo to observe the behaviors of implanted hMSCs in animal level. These findings demonstrate that the RGD-β-CD-QDs nanocarrier provides a powerful tool to simultaneously enhance differentiation and long-term tracking of hMSCs in vitro and in vivo for regenerative medicine.

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‐b1‐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‐b1 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. FASEB J. 31, 954–964 (2017). www.fasebj.org

Silver Nanoparticles/Ibuprofen-Loaded Poly(l-lactide) Fibrous Membrane: Anti-Infection and Anti-Adhesion Effects

Infection caused by bacteria is one of the crucial risk factors for tendon adhesion formation. Silver nanoparticles (AgNP)-loaded physical barriers were reported to be effective in anti-infection and anti-adhesion. However, high silver load may lead to kidney and liver damages. This study was designed for Ibuprofen (IBU)-loaded poly(l-lactide) (PLLA) electrospun fibrous membranes containing a low dosage of Ag to evaluate its potential in maintaining suitable anti-infection and good anti-adhesion effects. The in vitro drug release study showed a sustained release of Ag ions and IBU from the membrane. Inferior adherence and proliferation of fibroblasts were found on the Ag4%–IBU4%-loaded PLLA electrospun fibrous membranes in comparison with pure PLLA and 4% Ag-loaded PLLA membranes. In the antibacterial test, all Ag-loaded PLLA electrospun fibrous membranes prevented the adhesion of Staphylococcus aureus and Staphylococcus epidermidis. Taken together, these results demonstrate that Ibuprofen is effective in enhancing the anti-adhesion and anti-proliferation effects of 4% Ag-loaded PLLA fibrous membrane. The medical potential of infection reduction and adhesion prevention of Ag4%–IBU4%-loaded PLLA electrospun fibrous membrane deserves to be further studied.

Cystic fibrosis transmembrane conductance regulator mediates tenogenic differentiation of tendon-derived stem cells and tendon repair: accelerating tendon injury healing by intervening in its downstream signaling

Tendons are a mechanosensitive tissue, which enables them to transmit to bone forces that are derived from muscle. Patients with tendon injuries, such as tendinopathy or tendon rupture, were often observed with matrix degeneration, and the healing of tendon injuries remains a challenge as a result of the limited understanding of tendon biology. Our study demonstrates that the stretch‐mediated activation channel, cystic fibrosis transmembrane conductance regulator (CFTR), was up‐regulated in tendon‐derived stem cells (TDSCs) during tenogenic differentiation under mechanical stretching. Tendon tissues in CFTR‐dysfunctional DF508 mice exhibited irregular cell arrangement, uneven fibril diameter distribution, weak mechanical properties, and less matrix formation in a tendon defect model. Moreover, both tendon tissues and TDSCs isolated from DF508 mice showed significantly decreased levels of tendon markers, such as scleraxis, tenomodulin, Col1A1 (collagen type I α 1 chain), and decorin. Furthermore, by RNA sequencing analysis, we demonstrated that Wnt/β‐catenin signaling was abnormally activated in TDSCs from DF508 mice, thereby further activating the pERK1/2 signaling pathway. Of most importance, we found that intervention in pERK1/2 signaling could promote tenogenic differentiation and tendon regeneration both in vitro and in vivo. Taken together, our study demonstrates that CFTR plays an important role in tenogenic differentiation and tendon regeneration by inhibiting the β‐catinin/pERK1/2 signaling pathway. The therapeutic strategy of intervening in the CFTR/β‐catenin/pERK1/2 regulatory axis may be helpful for accelerating tendon injury healing, which has implications for tendon injury management.—Liu, Y., Xu, J., Xu, L., Wu, T., Sun, Y., Lee, Y.‐W., Wang, B., Chan, H.‐C., Jiang, X., Zhang, J., Li, G. Cystic fibrosis transmembrane conductance regulator mediates tenogenic differentiation of tendon‐derived stem cells and tendon repair: accelerating tendon injury healing by intervening in its downstream signaling. FASEB J. 31, 3800–3815 (2017). www.fasebj.org—Liu, Yang, Xu, Jia, Xu, Liangliang, Wu, Tianyi, Sun, Yuxin, Lee, Yuk‐Wai, Wang, Bin, Chan, Hsiao‐Chang, Jiang, Xiaohua, Zhang, Jinfang, Li, Gang Cystic fibrosis transmembrane conductance regulator mediates tenogenic differentiation of tendon‐derived stem cells and tendon repair: accelerating tendon injury healing by intervening in its downstream signaling. FASEB J. 31, 3800–3815 (2017)

MicroRNA-144-3p inhibits bone formation in distraction osteogenesis through targeting Connexin 43

Distraction osteogenesis (DO), one of effective therapies for bone regeneration, has been received more attention in recent years. However, the underlying mechanism remains elusive. Recently, microRNAs (miRNAs) have been reported to play important roles in regulating osteogenesis and bone formation. We therefore provided the hypothesis that miRNAs could involve in the DO-mediated bone regeneration. After successfully established the DO model of rats, a miRNA microarray was performed to find the differently expressed miRNAs in DO and control groups in this study. As one of the most downregulated miRNAs, miR-144-3p was found to be decreased during osteogenic differentiation in mesenchymal stem cells of rats (rBMSCs) and DO model. And miR-144-3p overexpression suppressed the osteogenesis while its inhibitor promoted osteogenesis. Furthermore, Connexin-43, an essential regulator for osteogenesis, was validated to be a novel target for miR-144-3p. Finally, miR-144-3p inhibitor modified MSCs promoted mineralization of distracted bone in rat DO model. In conclusion, miR-144-3p was found to regulate osteogenesis and inhibition of miR-144-3p resulted in acceleration of mineralization of DO, which not only give clues to understanding the mechanism of DO but also provide a potential therapeutic target in clinical practice.

Staphylococcal enterotoxin C2 expedites bone consolidation in distraction osteogenesis.

Distraction osteogenesis (DO) technique could be used to manage large‐size bone defect successfully, but DO process usually requires long duration of bone consolidation. Innovative approaches for augmenting bone consolidation are of great need. Staphylococcal enterotoxin C2 (SEC2) has been found to suppress osteoclastogenesis of mesenchymal stem cells in vitro. In this study, we investigated the effect of SEC2 on proliferation and osteogenic differentiation of rat bone marrow derived mesenchymal stem cells (rBMSCs). Further, we locally administrated SEC2 (10 ng/ml) or PBS into the distraction gap in Sprague–Dawley male rat DO model every 3 days till termination at 3 and 6 weeks. The regenerates were subjected to X‐rays, micro‐computed tomography, mechanical testing, histology, and immunohischemistry examinations to assess new bone quality. SEC2 had no effect on cell viability. The calcium deposition was remarkably increased and osteogenic marker genes were significantly up‐regulated in rBMSCs treated with SEC2. In rat DO model, SEC2 group had higher bone volume/total tissue volume in the regenerates. At 6 weeks, mechanical properties were significantly higher in SEC2‐treated tibiae comparing to the control group. Histological analysis confirmed that the new bone had improved quality in SEC2 treated group, where the osteocalcin and osterix expression in the regenerates was up‐regulated, indicating faster bone formation. The current study demonstrated that SEC2 local injection promotes osteogenesis and enhanced bone consolidation in DO. The findings support application of SEC2 as a potential novel strategy to expedite bone consolidation in patients undergoing DO treatment.