Evaluation of in vitro spermatogenesis using poly(D,L‐lactic‐co‐glycolic acid) (PLGA)‐based macroporous biodegradable scaffolds

Successful in vitro differentiation of spermatogenic cells into spermatids appears to offer extremely attractive potential for the treatment of impaired spermatogenesis and male infertility. Experimental evidence indicates that biocompatible polymers may improve in vitro reconstitution and regeneration of tissues of various origins. Here, we fabricated highly porous biodegradable poly(D,L ‐lactic‐co‐glycolic acid) or PLGA co‐polymer scaffolds by combining the gas‐foaming and salt‐leaching methods, using ammonium bicarbonate as a porogen, which allowed us to generate polymer scaffolds with a high density of interconnected pores of 400–500 µm in average diameter, concomitant with a high malleability to mould a wide range of temporal tissue scaffolds requiring a specific shape and geometry. The PLGA scaffolds were biocompatible and biodegradable, as evidenced by the fact that they survived almost 3 month long subcutaneous xenografting into immunodeficient host mice and became easily destroyable after recovery. Immature rat testicular cells that were seeded onto the surface of the scaffold exhibited about 65% seeding efficiency and up to 75% viability after 18 days in culture. Furthermore, our scaffolds enhanced the proliferation and differentiation of spermatogenic germ cells to a greater extent than conventional in vitro culture methods, such as monolayer or organ culture. Taken together, an implication of the present findings is that the PLGA‐based macroporous scaffold may provide a novel means by which spermatocytes could be induced to differentiate into presumptive spermatids. Copyright © 2010 John Wiley & Sons, Ltd.     

Three‐dimensional polymer scaffolds for enhanced differentiation of human mesenchymal stem cells to hepatocyte‐like cells: a comparative study

Stem cell‐based tissue engineering has emerged as a promising avenue for the treatment of liver diseases and as drug metabolism and toxicity models in drug discovery and development. The in vitro simulation of a micro‐environmental niche for hepatic differentiation remains elusive, due to lack of information about crucial factors for the stem cell niche. For generation of functional hepatocytes, an in vivo three‐dimensional (3D) micro‐environment and architecture should be reproduced. Towards this, we fabricated three scaffolds as dextran–gelatin (DG1), chitosan–hyaluronic acid (CH1) and gelatin–vinyl acetate (GEVAC). Hepatic differentiation of human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) was induced by culturing hUC‐MSCs on these scaffolds. The scaffolds support hepatic differentiation by mimicking the native extracellular matrix (ECM) micro‐environment and architecture to facilitate 3D cell–cell and cell–matrix interactions. The expression of hepatic markers, glycogen storage, urea production, albumin secretion and cytochrome P450 (CYP450) activity indicated the hepatic differentiation of hUC‐MSCs. The differentiated hUC‐MSCs on the 3D scaffolds formed hepatospheroids (3D hepatocyte aggregates), as illustrated by scanning electron microscopy (SEM), confocal microscopy and cytoskeleton organization. It was observed that the 3D scaffolds supported improved cell morphology, expression of hepatic markers and metabolic activities, as compared to Matrigel‐coated plates. To the best of our knowledge, this is the first report demonstrating the use of a well‐characterized scaffold (GEVAC) for enhanced differentiation of hUC‐MSCs to hepatocyte‐like cells (HLCs). Copyright © 2016 John Wiley & Sons, Ltd.     

Design of semi-degradable hydrogels based on poly(vinyl alcohol) and poly(lactic-co-glycolic acid) for cartilage tissue engineering

Articular cartilage damage is a persistent challenge in biomaterials and tissue engineering. Poly(vinyl alcohol) (PVA) hydrogels have shown promise as implants, but their lack of integration with surrounding cartilage prevents their utility. We sought to combine the advantages of PVA hydrogels with poly(lactic-co-glycolic acid) (PLGA) scaffolds, which have been successful in facilitating the integration of neocartilage with surrounding tissue. Through a novel double-emulsion technique, PLGA microparticles and a high level of porosity were simultaneously incorporated into PVA hydrogels. The porosity, average pore size and swelling properties of the hydrogels were controlled by varying initial processing parameters, such as the relative amounts of PLGA and solvent. Average pore sizes were in the ranged 50-100 μm. The PLGA microparticles degraded within the hydrogels over time in aqueous conditions, resulting in increases in porosity and pore size. After 4 weeks in cell culture, immature cartilage tissue filled many of the pores of the hydrogels that initially contained PLGA, and proteoglycan production was proportional to the amount of PLGA. In contrast, there was little cell attachment and no proteoglycan production in control hydrogels without PLGA. The compressive moduli of the hydrogels were similar to that of healthy cartilage and increased over time from 0.05-0.1 to 0.3-0.7 MPa. The generation of a hybrid cartilage-hydrogel construct using this technique may finally allow the integration of PVA hydrogels with surrounding cartilage.     

Immunomodulatory effect of human bone marrow‐derived mesenchymal stromal/stem cells on peripheral blood T cells from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a Th1/Th17‐mediated autoimmune disease whose current treatment, consisting in the blockage of inflammatory cytokines by disease‐modifying antirheumatic drugs, is not effective for all patients. The therapeutic potential of mesenchymal stromal/stem cells' (MSCs) immunomodulatory properties is being explored in RA. Here, we investigate the effect of human bone marrow (BM)‐MSCs on the expression of cytokines involved in RA physiopathology by the distinct functional compartments of CD4⁺ and CD8⁺ T cells from RA patients. Peripheral blood mononuclear cells from healthy individuals (n = 6) and RA patients (n = 12) were stimulated with phorbol myristate acetate plus ionomycin and cultured in the presence/absence of BM‐MSCs. The expression of (interleukin) IL‐2, tumor necrosis factor alpha (TNF‐α), and interferon‐gamma (IFN‐γ) was evaluated in naive, central memory, effector memory, and effector CD4⁺ and CD8⁺ T cells, whereas IL‐6, IL‐9, and IL‐17 expression was measured in total CD4⁺ and CD8⁺ T cells. mRNA expression of IL‐4, IL‐10, transforming growth factor beta (TGF‐β), cytotoxic T‐lymphocyte‐associated antigen 4, and/or forkhead box P3 was quantified in fluorescence‐activated cell sorting‐purified CD4⁺ T cells, CD8⁺ T cells, and CD4⁺ Treg. BM‐MSCs inhibited the production of TNF‐α, IL‐17, IL‐6, IL‐2, IFN‐γ, and IL‐9 by T cells from RA patients, mainly by reducing the percentage of cells producing cytokines. This inhibitory effect was transversal to all T cell subsets analyzed. At mRNA level, BM‐MSCs increased expression of IL‐10 and TGF‐β by CD4⁺ and CD8⁺ T cells. BM‐MSCs displayed a striking inhibitory action over T cells from RA patients, reducing the expression of cytokines involved in RA physiopathology. Remarkably, BM‐MSC‐derived immunomodulation affected either naive, effector, and memory T cells.     

Improved expansion of human bone marrow‐derived mesenchymal stem cells in microcarrier‐based suspension culture

Human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) have potential clinical utility in the treatment of a multitude of ailments and diseases, due to their relative ease of isolation from patients and their capacity to form many cell types. However, hBM‐MSCs are sparse, and can only be isolated in very small quantities, thereby hindering the development of clinical therapies. The use of microcarrier‐based stirred suspension bioreactors to expand stem cell populations offers an approach to overcome this problem. Starting with standard culture protocols commonly reported in the literature, we have successfully developed new protocols that allow for improved expansion of hBM‐MSCs in stirred suspension bioreactors using CultiSpher‐S microcarriers. Cell attachment was facilitated by using intermittent bioreactor agitation, removing fetal bovine serum, modifying the stirring speed and manipulating the medium pH. By manipulating these parameters, we enhanced the cell attachment efficiency in the first 8 h post‐inoculation from 18% (standard protocol) to 72% (improved protocol). Following microcarrier attachment, agitation rate was found to impact cell growth kinetics, whereas feeding had no significant effect. By serially subculturing hBM‐MSCs using the new suspension bioreactor protocols, we managed to obtain cell fold increases of 10³ within 30 days, which was superior to the 200‐fold increase obtained using the standard protocol. The cells were found to retain their defining characteristics after several passages in suspension. This new bioprocess represents a more efficient approach for generating large numbers of hBM‐MSCs in culture, which in turn should facilitate the development of new stem cell‐based therapies. Copyright © 2012 John Wiley & Sons, Ltd.     

Osteoarthritic human chondrocytes proliferate in 3D co‐culture with mesenchymal stem cells in suspension bioreactors

Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage‐generating cells. Previous studies in static culture have shown that hACs co‐cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co‐culture of hMSCs and OA hACs under serum‐free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125‐ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75‐fold increase over 16 days. Whereas the apparent growth rate was lower than that achieved during co‐culture as a 2D monolayer in static culture flasks, bioreactor co‐culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5 μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum‐free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell‐based cartilage repair.     

Effective expansion of human adipose‐derived stromal cells and bone marrow‐derived mesenchymal stem cells cultured on a fragmin/protamine nanoparticles‐coated substratum with human platelet‐rich plasma

Fragmin/protamine nanoparticles (F/P NPs) can be stably coated onto plastic surfaces and used as a substratum for the absorption and controlled release of growth factors (GFs) secreted from human platelet‐rich plasma (PRP). In this study, we investigated the capability of F/P NP‐coated plates to act as a substratum for the proliferation of human adipose‐derived stromal cells (ASCs) and bone marrow‐derived mesenchymal stem cells (BMSCs) with GFs in PRP. Both cell types adhered well to the F/P NP‐coated plates and grew optimally, with a doubling time of 30 and 32 h in low‐concentration PRP (0.5%) medium supplemented with 5 ng/ml fibroblast growth factor‐2 (FGF‐2) on the F/P NP‐coated plates. These cells maintained their multilineage potential for differentiation into adipocytes or osteoblasts. Furthermore, ASCs and BMSCs grew well in medium without PRP and FGF‐2 on F/P NP‐coated plates pretreated with PRP and FGF‐2 in a concentration‐dependent manner. Thus, F/P NP‐coated plates are a useful substratum for the adherence and proliferation of ASCs and BMSCs in low‐concentration PRP medium supplemented with FGF‐2. No xenogeneic serum is required. Copyright © 2012 John Wiley & Sons, Ltd.     

Transplantation of human bone marrow‐derived clonal mesenchymal stem cells reduces fibrotic scar formation in a rat spinal cord injury model

This study aimed to evaluate the therapeutic effect on tissue repair and scar formation of human bone marrow‐derived clonal mesenchymal stem cells (hcMSCs) homogeneously isolated by using a subfractionation culturing method, in comparison with the non‐clonal MSCs (hMSCs), in a rat spinal cord injury (SCI) model. The SCI was made using a vascular clip at the T9 level. Cells were transplanted into the lesion site 3 days after injury. A functional test was performed over 4 weeks employing a BBB score. Rats were killed for histological analysis at 3 days, 1 week and 4 weeks after injury. The transplantation of hMSCs and hcMSCs significantly reduced lesion size and the fluid‐filled cavity at 4 weeks in comparison with the control group injected with phosphate buffered saline (PBS) (p < 0.01). Transplantation of hcMSCs showed more axons reserved than that of hMSCs in the lesion epicentre filled with non‐neuronal tissues. In addition, hMSCs and hcMSCs clearly reduced the inflammatory reaction and intraparenchymal hemorrhaging, compared with the PBS group. Interestingly, hcMSCs largely decreased Col IV expression, one of the markers of fibrotic scars. hcMSCs yielded therapeutic effects more than equal to those of hMSCs on the SCI. Both hMSCs and hcMSCs created an increase in axon regeneration and reduced scar formation around the SCI lesion. Copyright © 2017 John Wiley & Sons, Ltd.     

Accelerating bone defects healing in calvarial defect model using 3D cultured bone marrow‐derived mesenchymal stem cells on demineralized bone particle scaffold

Bone defects are usually difficult to be regenerated due to pathological states or the size of the injury. Researchers are focusing on tissue engineering approaches in order to drive the regenerative events, using stem cells to regenerate bone. The purpose of this study is to evaluate the osteogenic differentiation of bone marrow‐derived mesenchymal stem cells (BMSCs) on biologically derived Gallus gallus domesticus‐derived demineralized bone particle (GDD) sponge. The sponges were prepared by freeze‐drying method using 1, 2, and 3 wt% GDD and cross‐linked with glutaraldehyde. The GDD sponge was characterized using scanning electron microscopy, compressive strength, porosity, and Fourier transform infrared. The potential bioactivity of the sponge was evaluated by osteogenic differentiation of BMSCs using 3(4, dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide assay and quantifying alkaline phosphatase (ALP) activity. in vivo experiments were evaluated through a micro‐computerized tomography (μ ‐CT) and histological assays. The analysis confirmed that an increase in the concentration of the GDD in the sponge leads to a higher bone formation and deposition in rat calvarial defects. Histological assay results were in line with μ ‐CT. The results reported in this study demonstrated the potential application of GDD sponges as osteoinductor in bone tissue engineering in pathological or nonunion bone defects.     

Pressure‐activated microsyringe (PAM) fabrication of bioactive glass–poly(lactic‐co‐glycolic acid) composite scaffolds for bone tissue regeneration

The aim of this work was the fabrication and characterization of bioactive glass–poly(lactic‐co‐glycolic acid) (PLGA) composite scaffolds mimicking the topological features of cancellous bone. Porous multilayer PLGA–CEL2 composite scaffolds were innovatively produced by a pressure‐activated microsyringe (PAM) method, a CAD/CAM processing technique originally developed at the University of Pisa. In order to select the optimal formulations to be extruded by PAM, CEL2–PLGA composite films (CEL2 is an experimental bioactive SiO₂–P₂O₅–CaO–MgO–Na₂O–K₂O glass developed at Politecnico di Torino) were produced and mechanically tested. The elastic modulus of the films increased from 30 to > 400 MPa, increasing the CEL2 amount (10–50 wt%) in the composite. The mixture containing 20 wt% CEL2 was used to fabricate 2D and 3D bone‐like scaffolds composed by layers with different topologies (square, hexagonal and octagonal pores). It was observed that the increase of complexity of 2D topological structures led to an increment of the elastic modulus from 3 to 9 MPa in the composite porous monolayer. The elastic modulus of 3D multilayer scaffolds was intermediate (about 6.5 MPa) between the values of the monolayers with square and octagonal pores (corresponding to the lowest and highest complexity, respectively). MG63 osteoblast‐like cells and periosteal‐derived precursor cells (PDPCs) were used to assess the biocompatibility of the 3D bone‐like scaffolds. A significant increase in cell proliferation between 48 h and 7 days of culture was observed for both cell phenotypes. Moreover, qRT–PCR analysis evidenced an induction of early genes of osteogenesis in PDPCs. Copyright © 2015 John Wiley & Sons, Ltd.     

Interaction of material stiffness and negative pressure to enhance differentiation of bone marrow‐derived stem cells and osteoblast proliferation

Negative pressure wound therapy (NPWT) results in improved wound repair and the combined use of NPWT with elastomeric materials may further stimulate and accelerate tissue repair. No firmly established treatment modalities using both NPWT and biomaterials exist for orthopedic application. The goal of this study was to investigate the response of osteoblasts and bone marrow‐derived mesenchymal stem cells to negative pressure and to determine whether a newly developed elastic osteomimetic bone repair material (BRM), a blend of type I collagen, chondroitin 6‐sulfate, and poly (octanediol citrate) could enhance the osteoblastic phenotype. The results indicate that proliferation and alkaline phosphatase activity of hFOB1.19 osteoblasts were significantly increased with exposure to 12 hr of negative pressure (?125 mmHg). Follow‐on studies with rat and human mesenchymal stem cells confirmed that negative pressure enhanced osteoblastic maturation. In addition, a significant interaction of negative pressure and electrospun BRM resulted in increased mRNA expression of alkaline phosphatase, osteopontin, collagen1α2, and HIF1α, whereas little or no effect on these genes was observed on electrospun collagen or tissue culture plastic. Together, these results suggest that the use of this novel biomaterial, BRM, with NPWT may ultimately translate into a safe and cost‐effective clinical application to accelerate bone repair.     

Cyclic tensile loading regulates human mesenchymal stem cell differentiation into neuron‐like phenotype

Mechanical loading has been utilized as an effective tool to direct mesenchymal stem cells (MSCs) commitment into cell lineages of mesodermal origin. However, the use of this tool to induce transdifferentiation of MSCs into the neural lineage has never been attempted. In this study, we examined the potential of uniaxial cyclic tensile loading in promoting neuronal differentiation of human MSCs (hMSCs) on modified biodegradable poly(ε‐caprolactone) (PCL). The stem cell morphology, tissue‐specific gene and protein expression, microfilament structure and, subsequently, Rho GTPase activity were analysed after cyclically stretching the cells at a range of amplitudes (0.5%, 2% or 3.5%) and frequencies (0.5, 1 or 1.5 Hz) for 8 h. hMSCs responded to these stimuli and displayed distinctly different microfilament organization. However, only those stretched at 0.5% strain amplitude and 0.5 Hz frequency showed promoted outgrowth of filopodia with significant upregulation of neurogenic genes expression. Positive staining of the neurogenic protein markers Nestin and Tuj1 suggested that the hMSCs had been committed to early neuronal progenitors. In addition, Rac1 but not RhoA was activated at this particular loading parameter. Furthermore, inhibition of Rac1 activity with NSC23766 disrupted the effect of cyclic loading. The results suggest that cyclic tensile loading at low amplitude and frequency is capable of triggering neuron‐like differentiation through the regulation of Rho GTPases activity, even in the absence of neurogenic induction medium. Copyright © 2012 John Wiley & Sons, Ltd.     

Influence of in vitro biomimicked stem cell ‘niche’ for regulation of proliferation and differentiation of human bone marrow‐derived mesenchymal stem cells to myocardial phenotypes: serum starvation without aid of chemical agents and prevention of spontaneous stem cell transformation enhanced by the matrix environment

Niche appears important for preventing the spontaneous differentiation or senescence that cells undergo during in vitro expansion. In the present study, it was revealed that human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) undergo senescence‐related differentiation into the myocardial lineage in vitro without any induction treatment. This phenomenon occurred over the whole population of MCSs, much different from conventional differentiation with limited frequency of occurrence, and was accompanied by a change of morphology into large, flat cells with impeded proliferation, which are the representative indications of MSC senescence. By culturing MSCs under several culture conditions, it was determined that induction treatment with 5‐azacytidine was not associated with the phenomenon, but the serum‐starvation condition, under which proliferation is severely hampered, caused senescence progression and upregulation of cardiac markers. Nevertheless, MSCs gradually developed a myocardial phenotype under normal culture conditions over a prolonged culture period and heterogeneous populations were formed. In perspectives of clinical applications, this must be prevented for fair and consistent outcomes. Hence, the biomimetic 'niche' was constituted for hBM‐MSCs by cultivating on a conventionally available extracellular matrix (ECM). Consequently, cells on ECM regained a spindle‐shape morphology, increased in proliferation rate by two‐fold and showed decreased expression of cardiac markers at both the mRNA and protein levels. In conclusion, the outcome indicates that progression of MSC senescence may occur via myocardial differentiation during in vitro polystyrene culture, and this can be overcome by employing appropriate ECM culture techniques. Copyright © 2013 John Wiley & Sons, Ltd.     

A mixed co‐culture of mesenchymal stem cells and transgenic chondrocytes in alginate hydrogel for cartilage tissue engineering

To regenerate articular cartilage tissue from degeneration and trauma, synovial mesenchymal stem cells (SMSCs) were used in this study as therapeutic progenitor cells to induce therapeutic chondrogenesis. To accomplish this, chondrocytes pre‐transduced with adenoviral vectors carrying the transforming growth factor (TGF) β3 gene were selected as transgenic companion cells and co‐cultured side‐by‐side with SMSCs in a 3D environment to provide chondrogenic growth factors in situ. We adopted a mixed co‐culture strategy for this purpose. Transgenic delivery of TGF‐β3 in chondrocytes was performed via recombinant adenoviral vectors. The mixed co‐culture of SMSCs and transgenic chondrocytes was produced in alginate gel constructs. Gene expression in both SMSCs and chondrocytes were characterized. Biochemical assays in vitro and in vivo showed that release of TGF‐ß3 from transgenic chondrocytes not only induced SMSC differentiation into chondrocytic cells but also preserved the chondrocytic phenotype of chondrocytes from suspected dedifferentiation. As a result, this mixed co‐culture strategy in conjunction with TGF‐ß3 gene delivery could be a promising approach in cartilage tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Reconstruction of hepatic stellate cell‐incorporated liver capillary structures in small hepatocyte tri‐culture using microporous membranes

In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC‐incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri‐culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell–cell interactions, which are essential to form HSC–EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d , l ‐lactide‐co‐glycolide) (PLGA) microporous membranes in SH–HSC–EC tri‐culture to reconstruct the HSC‐incorporated liver capillary structures in vitro. First, the formation of EC capillary‐like structures was induced on Matrigel‐coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary‐like structures. This process was mediated in part by platelet‐derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary‐like structures with their long cytoplasmic processes. In the HSC‐incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro. Copyright © 2012 John Wiley & Sons, Ltd.     

Angiogenic and osteogenic regeneration in rats via calcium phosphate scaffold and endothelial cell co‐culture with human bone marrow mesenchymal stem cells (MSCs), human umbilical cord MSCs,human induced pluripotent stem cell‐derived MSCs and human embryonic stem cell‐derived MSCs

Angiogenesis is a limiting factor in regenerating large bone defects. The objective of this study was to investigate angiogenic and osteogenic effects of co‐culture on calcium phosphate cement (CPC) scaffold using human umbilical vein endothelial cells (hUVECs) and mesenchymal stem cells (MSCs) from different origins for the first time. hUVECs were co‐cultured with four types of cell: human umbilical cord MSCs (hUCMSCs), human bone marrow MSCs (hBMSCs) and MSCs from induced pluripotent stem cells (hiPSC‐MSCs) and embryonic stem cells (hESC‐MSCs). Constructs were implanted in 8 mm cranial defects of rats for 12 weeks. CPC without cells served as control 1. CPC with hBMSCs served as control 2. Microcapillary‐like structures were successfully formed on CPC in vitro in all four co‐cultured groups. Microcapillary lengths increased with time (p < 0.05). Osteogenic and angiogenic gene expressions were highly elevated and mineralization by co‐cultured cells increased with time (p < 0.05). New bone amount and blood vessel density of co‐cultured groups were much greater than controls (p < 0.05) in an animal study. hUVECs co‐cultured with hUCMSCs, hiPSC‐MSCs and hESC‐MSCs achieved new bone and vessel density similar to hUVECs co‐cultured with hBMSCs (p > 0.1). Therefore, hUCMSCs, hiPSC‐MSCs and hESC‐MSCs could serve as alternative cell sources to hBMSCs, which require an invasive procedure to harvest. In conclusion, this study showed for the first time that co‐cultures of hUVECs with hUCMSCs, hiPSC‐MSCs, hESC‐MSCs and hBMSCs delivered via CPC scaffold achieved excellent osteogenic and angiogenic capabilities in vivo. The novel co‐culture constructs are promising for bone reconstruction with improved angiogenesis for craniofacial/orthopaedic applications. Copyright © 2017 John Wiley & Sons, Ltd.     

Tonsil‐derived mesenchymal stem cells (T‐MSCs) prevent Th17‐mediated autoimmune response via regulation of the programmed death‐1/programmed death ligand‐1 (PD‐1/PD‐L1) pathway

Our knowledge of the immunomodulatory role of mesenchymal stem cells (MSCs) in both the innate and adaptive immune systems has dramatically expanded, providing great promise for treating various autoimmune diseases. However, the contribution of MSCs to Th17‐dominant immune disease, such as psoriasis and its underlying mechanism remains elusive. In this study, we demonstrated that human palatine tonsil‐derived MSCs (T‐MSCs) constitutively express both the membrane‐bound and soluble forms of programmed death‐ligand 1 (PD‐L1), which enables T‐MSCs to be distinguished from MSCs originating from other organs (i.e. bone marrow or adipose tissue). We also found that T‐MSC‐derived PD‐L1 effectively represses Th17 differentiation via both cell‐to‐cell contact and a paracrine effect. Further, T‐MSCs increase programmed death‐1 (PD‐1) expression on T‐cells by secreting IFN‐β, which may enhance engagement with PD‐L1. Finally, transplantation of T‐MSCs into imiquimod‐induced psoriatic skin inflammation in mice significantly abrogated disease symptoms, mainly by blunting the Th17 response in a PD‐L1‐dependent manner. This study suggests that T‐MSCs might be a promising cell source to treat autoimmune diseases such as psoriasis, via its unique immunoregulatory features. Copyright © 2017 John Wiley & Sons, Ltd.