Recent advances in three‐dimensional bioprinting of stem cells

In spite of being a new field, three‐dimensional (3D) bioprinting has undergone rapid growth in the recent years. Bioprinting methods offer a unique opportunity for stem cell distribution, positioning, and differentiation at the microscale to make the differentiated architecture of any tissue while maintaining precision and control over the cellular microenvironment. Bioprinting introduces a wide array of approaches to modify stem cell fate. This review discusses these methodologies of 3D bioprinting stem cells. Fabricating a fully operational tissue or organ construct with a long life will be the most significant challenge of 3D bioprinting. Once this is achieved, a whole human organ can be fabricated for the defect place at the site of surgery.     

Effects of resveratrol on enrichment of adipose‐derived stem cells and their differentiation to osteoblasts in two‐and three‐dimensional cultures

The goal of this study was to develop a method for increasing the yield of multipotent adipose‐derived mesenchymal stem cells (ASCs) and osteoprogenitor cells (OPCs) from subcutaneous fat. After removing mature adipocytes and haematopoietic cells from rat inguinal fat, ASCs in the remaining cell population were verified by their attachment to plastic, surface marker profile (CD271⁺, CD73⁺ and CD45^–) and ability to differentiate into adipocytes, chondrocytes and osteoblasts. OPCs were defined as E11⁺ and OCN⁺. Adherent cells were cultured in growth medium (GM) or osteogenic medium (OM) and treated with resveratrol (0, 12.5, and 25 µ m ) for 7 days; ASCs and OPCs were assessed by flow cytometry. Osteogenic potential was determined in two‐dimensional (2D) cultures as a function of alkaline phosphatase‐specific activity and osteocalcin production. In addition, cells were seeded onto three‐dimensional (3D) poly‐ε‐caprolactone scaffolds and cultured under dynamic conditions; mineralization was quantified by micro‐CT at 4, 8 and 12 weeks. Resveratrol increased the percentage of ASCs in the population (population%) and number of ASCs in both GM and OM, but increased only the number of OPCs in GM. In both media types resveratrol increased alkaline phosphatase activity and osteocalcin levels. In 3D cultures, resveratrol‐treated cells significantly increased mineralized matrix volume at early time points. Resveratrol exerted a biphasic effect on adherent cells by enriching the ASC and OPC populations and enhancing osteogenic differentiation. Resveratrol pretreatment induced more mineralization at earlier time points and represents a clinically viable technique for orthopaedic and dental applications for autologous stem cell therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

In vivo cartilage repair using adipose‐derived stem cell‐loaded decellularized cartilage ECM scaffolds

We have previously reported a natural, human cartilage ECM (extracellular matrix)‐derived three‐dimensional (3D) porous acellular scaffold for in vivo cartilage tissue engineering in nude mice. However, the in vivo repair effects of this scaffold are still unknown. The aim of this study was to further explore the feasibility of application of cell‐loaded scaffolds, using autologous adipose‐derived stem cells (ADSCs), for cartilage defect repair in rabbits. A defect 4 mm in diameter was created on the patellar groove of the femur in both knees, and was repaired with the chondrogenically induced ADSC–scaffold constructs (group A) or the scaffold alone (group B); defects without treatment were used as controls (group C). The results showed that in group A all defects were fully filled with repair tissue and at 6 months post‐surgery most of the repair site was filled with hyaline cartilage. In contrast, in group B all defects were partially filled with repair tissue, but only half of the repair tissue was hyaline cartilage. Defects were only filled with fibrotic tissue in group C. Indeed, histological grading score analysis revealed that an average score in group A was higher than in groups B and C. GAG and type II collagen content and biomechanical property detection showed that the group A levels approached those of normal cartilage. In conclusion, ADSC‐loaded cartilage ECM scaffolds induced cartilage repair tissue comparable to native cartilage in terms of mechanical properties and biochemical components. Copyright © 2012 John Wiley & Sons, Ltd.     

Sustained release of parathyroid hormone via in situ cross‐linking gelatin hydrogels improves the therapeutic potential of tonsil‐derived mesenchymal stem cells for hypoparathyroidism

Biomimetic parathyroid regeneration with sustained release of parathyroid hormone (PTH) into the blood stream is a considerable challenge in hypoparathyroidism treatment. We recently reported that tonsil‐derived mesenchymal stem cells (TMSCs), if these cells were both differentiated in vitro before implantation and incorporated into a scaffold Matrigel, are a good cell source for parathyroid regeneration in a parathyroidectomized (PTX) animal model. Here, we present a new strategy for improved clinical application that enhances the sustained release of PTH by controlling mechanical stiffness using in situ‐forming gelatin‐hydroxyphenyl propionic acid (GH) hydrogels (GHH). Differentiated TMSCs (dTMSCs) embedded in a GHH with a strength of 4.4 kPa exhibited the best sustained release of PTH and were the most effective in hypoparathyroidism treatment, showing improved blood calcium homeostasis compared with Matrigel‐embedded dTMSCs. Interestingly, undifferentiated control TMSCs (cTMSCs) also released PTH in a sustained manner if incorporated into GHH. Collectively, these findings may establish a new paradigm for parathyroid regeneration that could ultimately evolve into an improved clinical application. Copyright © 2017 John Wiley & Sons, Ltd.     

Multilineage co‐culture of adipose‐derived stem cells for tissue engineering

Stem cell interactions through paracrine cell signalling can regulate a range of cell responses, including metabolic activity, proliferation and differentiation. Moving towards the development of optimized tissue‐engineering strategies with adipose‐derived stem cells (ASCs), the focus of this study was on developing indirect co‐culture models to study the effects of mature adipocytes, chondrocytes and osteoblasts on bovine ASC multilineage differentiation. For each lineage, ASC differentiation was characterized by histology, gene expression and protein expression, in the absence of key inductive differentiation factors for the ASCs. Co‐culture with each of the mature cell populations was shown to successfully induce or enhance lineage‐specific differentiation of the ASCs. In general, a more homogeneous but lower‐level differentiation response was observed in co‐culture as compared to stimulating the bovine ASCs with inductive differentiation media. To explore the role of the Wnt canonical and non‐canonical signalling pathways within the model systems, the effects of the Wnt inhibitors WIF‐1 and DKK‐1 on multilineage differentiation in co‐culture were assessed. The data indicated that Wnt signalling may play a role in mediating ASC differentiation in co‐culture with the mature cell populations. Copyright © 2012 John Wiley & Sons, Ltd.     

Efficient generation of functional hepatocyte‐like cells from menstrual blood‐derived stem cells

In recent years, the advantages of menstrual blood‐derived stem cells (MenSCs), such as minimal ethical considerations, easy access and high proliferative ability, have inspired scientists to investigate the potential of MenSCs in cell therapy of different diseases. In order to characterize the potency of these cells for future cell therapy of liver diseases, we examined the potential of MenSCs to differentiate into hepatocytes, using different protocols. First, the immunophenotyping properties and potential of MenSCs to differentiate into osteoblasts, adipocytes and chondrocytes were evaluated. Thereafter, the differentiation protocols developed by two concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM), in combination with other components in serum‐supplemented or serum‐free culture media, were also investigated. The sequential differentiation was monitored by real‐time PCR, immunostaining and functional assays. Our primary data revealed that the isolated MenSCs exhibited mesenchymal stem cell markers in parallel to OCT‐4 as an embryonic marker. Regardless of differentiation procedures, the developed cells expressed mature hepatocyte markers, such as albumin, tyrosine aminotransferase and cytokeratin‐18 at the mRNA and protein levels. They also showed functional properties of hepatocytes, including albumin secretion, glycogen storage and cytochrome P450 7A1 expression. However, the degree of differentiation was dependent on the concentrations of HGF and OSM. Indeed, omission of serum during the differentiation process caused typical improvement in hepatocyte‐specific functions. This study is a novel report demonstrating the differentiation potential of MenSCs into hepatocyte‐like cells. We recommend a complementary serum‐free differentiation protocol for enrichment of in vitro production of functional MenSC‐derived hepatocyte‐like cells that could lead to a major step toward applied stem cell therapy of chronic liver diseases. Copyright © 2013 John Wiley & Sons, Ltd.     

TGFβ3 secretion by three‐dimensional cultures of human dental apical papilla mesenchymal stem cells

Mesenchymal stem cells (MSCs) can be isolated from dental tissues, such as pulp and periodontal ligament; the dental apical papilla (DAP) is a less‐studied MSC source. These dental‐derived MSCs are of great interest because of their potential as an accessible source for cell‐based therapies and tissue‐engineering (TE) approaches. Much of the interest regarding MSCs relies on the trophic‐mediated repair and regenerative effects observed when they are implanted. TGFβ3 is a key growth factor involved in tissue regeneration and scarless tissue repair. We hypothesized that human DAP‐derived MSCs (hSCAPs) can produce and secrete TGFβ3 in response to micro‐environmental cues. For this, we encapsulated hSCAPs in different types of matrix and evaluated TGFβ3 secretion. We found that dynamic changes of cell–matrix interactions and mechanical stress that cells sense during the transition from a monolayer culture (two‐dimensional, 2D) towards a three‐dimensional (3D) culture condition, rather than the different chemical composition of the scaffolds, may trigger the TGFβ3 secretion, while monolayer cultures showed almost 10‐fold less secretion of TGFβ3. The study of these interactions is provided as a cornerstone in designing future strategies in TE and cell therapy that are more efficient and effective for repair/regeneration of damaged tissues. Copyright © 2015 John Wiley & Sons, Ltd.     

Influence of self‐assembling peptide nanofibre scaffolds on retinal differentiation potential of stem/progenitor cells derived from ciliary pigment epithelial cells

Aim of the study was to investigate the influence of the self‐assembling peptide nanofibre scaffolds (SAPNs) on the growth, proliferation and retinal neuronal differentiation of the stem/progenitor cells (SCs) derived from the ciliary pigment epithelium (CPE) of human cadaveric eye. Here SAPNs (RADA16‐I, PM), which is well described in previous studies, commercially available and xeno‐free. The CPE cells isolated were cultured in DMEM/F12 supplemented with N2 and growth factors such as basic fibroblast growth factor and epidermal growth factor, encapsulated in the scaffolds. The entrapped SCs actively expanded and formed clone‐like clusters in the scaffolds. Many cells in the cluster were proliferating, as revealed by 5‐bromo‐2‐deoxyuridine uptake and could be maintained for up to 6 days and expressed neural progenitor markers such as β‐III tubulin, Nestin, Pax6 and Musashi1. Upon differentiation of these cells in conditioned medium, the cells exhibited retinal neuronal markers such as s‐Opsin, rhodopsin and Recoverin. The RT² profiler polymerase chain reaction array experiments showed selective gene expression, possibly involved in neural stem/progenitor cell adhesion and differentiation. These findings suggest the suitability of the three‐dimensional culture system for the proliferation and maintenance of CPE stem/progenitor cells (CPE‐NS) and for possible use in ex vivo studies of small molecules, drug deliveries for retinal diseases and for use in combination with directed stem/progenitor cell differentiation. and ultimately for tissue replacement therapies. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparison of several attachment methods for human iPS,embryonic and adipose‐derived stem cells for tissue engineering

As actual stem cell application quickly approaches tissue engineering and regenerative medicine, aspects such as cell attachment to scaffolds and biomaterials become important and are often overlooked. Here, we compare the effects of several attachment proteins on the adhesion, proliferation and stem cell identity of three promising human stem cell types: human adipose‐derived stem cells (hASCs), human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Traditional tissue culture polystyrene plates (TCPS), Matrigel (Mat), laminin (Lam), fibronectin (FN) and poly‐ l ‐lysine (PLL) were investigated as attachment protein surfaces. For hASCs typically cultured on TCPS, laminin resulted in the greatest cell attachment and proliferation with largest cell areas, indicating favourability by cell spreading. However, mesenchymal stem cell markers indicative of hASCs were slightly more expressed on surfaces with lowest cell attachment, corresponding to increased cell roundness, a newly observed attribute in hASCs possibly indicating a more stem cell‐like character. hESCs preferred Matrigel as a feeder‐free culture surface. Interestingly, hiPSCs favoured laminin over Matrigel for colony expansion, shown by larger cell colony area and perimeter lengths, although cell numbers and stem cell marker expression level remained highest on Matrigel. These data provide a practical reference guide for selecting a suitable attachment method for using human induced pluripotent, embryonic or adipose stem cells in tissue engineering and regenerative medicine applications. Copyright © 2012 John Wiley & Sons, Ltd.     

The suitability of human adipose‐derived stem cells for the engineering of ligament tissue

Rupture of the anterior cruciate ligament (ACL) is the one of the most common sports‐related injuries. With its poor healing capacity, surgical reconstruction using either autografts or allografts is currently required to restore function. However, serious complications are associated with graft reconstructions and the number of such reconstructions has steadily risen over the years, necessitating the search for an alternative approach to ACL repair. Such an approach may likely be tissue engineering. Recent engineering approaches using ligament‐derived fibroblasts have been promising, but the slow growth rate of such fibroblasts in vitro may limit their practical application. More promising results are being achieved using bone marrow mesenchymal stem cells (MSCs). The adipose‐derived stem cell (ASC) is often proposed as an alternative choice to the MSC and, as such, may be a suitable stem cell for ligament engineering. However, the use of ASCs in ligament engineering still remains relatively unexplored. Therefore, in this study, the potential use of human ASCs in ligament tissue engineering was initially explored by examining their ability to express several ligament markers under growth factor treatment. ASC populations treated for up to 4 weeks with TGFβ1 or IGF1 did not show any significant and consistent upregulation in the expression of collagen types 1 and 3, tenascin C and scleraxis. While treatment with EGF or bFGF resulted in increased tenascin C expression, increased expression of collagens 1 and 3 were never observed. Therefore, simple in vitro treatment of human ASC populations with growth factors may not stimulate their ligament differentiative potential. Copyright © 2011 John Wiley & Sons, Ltd.     

Matrilin‐3 codelivery with adipose‐derived mesenchymal stem cells promotes articular cartilage regeneration in a rat osteochondral defect model

Matrilin‐3 is an essential extracellular matrix component present only in cartilaginous tissues. Matrilin‐3 exerts chondroprotective effects by regulating an anti‐inflammatory function and extracellular matrix components. We hypothesized that the codelivery of matrilin‐3 with infrapatellar adipose‐tissue‐derived mesenchymal stem cells (Ad‐MSCs) may enhance articular cartilage regeneration. Matrilin‐3 treatment of Ad‐MSCs in serum‐free media induced collagen II and aggrecan expression, and matrilin‐3 in chondrogenic media also enhanced in vitro chondrogenic differentiation. Next, the in vivo effect of matrilin‐3 codelivery with Ad‐MSCs on cartilage regeneration was assessed in an osteochondral defect model in Sprague Dawley rats: Ad‐MSCs and hyaluronic acid were implanted at the defect site with or without matrilin‐3 (140, 280, and 700 ng). Safranin O staining revealed that matrilin‐3 (140 and 280 ng) treatment significantly improved cartilage regeneration and glycosaminoglycan accumulation. In the animals treated with 140‐ng matrilin‐3, in particular, the defect site exhibited complete integration with surrounding tissue and a smooth glistening surface. The International Cartilage Repair Society macroscopic and O'Driscoll microscopic scores for regenerated cartilage were furthermore shown to be considerably higher for this group (matrilin‐3; 140 ng) compared with the other groups. Furthermore, the defects treated with 140‐ng matrilin‐3 revealed significant hyaline‐like cartilage regeneration in the osteochondral defect model; in contrast, the defects treated with 700‐ng matrilin‐3 exhibited drastically reduced cartilage regeneration with mixed hyaline–fibrocartilage morphology. Codelivery of matrilin‐3 with Ad‐MSCs significantly influenced articular cartilage regeneration, supporting the potential use of this tissue‐specific protein for a cartilage‐targeted stem cell therapy.     

Development of decellularized scaffolds for stem cell‐driven tissue engineering

Organ transplantation is an effective treatment for chronic organ dysfunctioning conditions. However, a dearth of available donor organs for transplantation leads to the death of numerous patients waiting for a suitable organ donor. The potential of decellularized scaffolds, derived from native tissues or organs in the form of scaffolds has been evolved as a promising approach in tissue‐regenerative medicine for translating functional organ replacements. In recent years, donor organs, such as heart, liver, lung and kidneys, have been reported to provide acellular extracellular matrix (ECM)‐based scaffolds through the process called ‘decellularization’ and proved to show the potential of recellularization with selected cell populations, particularly with stem cells. In fact, decellularized stem cell matrix (DSCM) has also emerged as a potent biological scaffold for controlling stem cell fate and function during tissue organization. Despite the proven potential of decellularized scaffolds in tissue engineering, the molecular mechanism responsible for stem cell interactions with decellularized scaffolds is still unclear. Stem cells interact with, and respond to, various signals/cues emanating from their ECM. The ability to harness the regenerative potential of stem cells via decellularized ECM‐based scaffolds has promising implications for tissue‐regenerative medicine. Keeping these points in view, this article reviews the current status of decellularized scaffolds for stem cells, with particular focus on: (a) concept and various methods of decellularization; (b) interaction of stem cells with decellularized scaffolds; (c) current recellularization strategies, with associated challenges; and (iv) applications of the decellularized scaffolds in stem cell‐driven tissue engineering and regenerative medicine. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro and in vivo co‐culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL–PEG–PCL hydrogels enhances cartilage formation

Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co‐culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL–PEG–PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co‐cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co‐culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co‐culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co‐culture system in rabbit models. The co‐culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co‐culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

Application of marrow mesenchymal stem cell‐derived extracellular matrix in peripheral nerve tissue engineering

To advance molecular and cellular therapy into the clinic for peripheral nerve injury, modification of neural scaffolds with the extracellular matrix (ECM) of peripheral nerves has been established as a promising alternative to direct inclusion of support cells and/or growth factors within a neural scaffold, while cell‐derived ECM proves to be superior to tissue‐derived ECM in the modification of neural scaffolds. Based on the fact that bone marrow mesenchymal stem cells (BMSCs), just like Schwann cells, are adopted as support cells within a neural scaffold, in this study we used BMSCs as parent cells to generate ECM for application in peripheral nerve tissue engineering. A chitosan nerve guidance conduit (NGC) and silk fibroin filamentous fillers were respectively prepared for co‐culture with purified BMSCs, followed by decellularization to stimulate ECM deposition. The ECM‐modified NGC and lumen fillers were then assembled into a chitosan–silk fibroin‐based, BMSC‐derived, ECM‐modified neural scaffold, which was implanted into rats to bridge a 10 mm‐long sciatic nerve gap. Histological and functional assessments after implantation showed that regenerative outcomes achieved by our engineered neural scaffold were better than those achieved by a plain chitosan–silk fibroin scaffold, and suggested the benefits of BMSC‐derived ECM for peripheral nerve repair. Copyright © 2016 John Wiley & Sons, Ltd.     

Hyaline cartilage next generation implants from adipose‐tissue–derived mesenchymal stem cells: Comparative study on 3D‐printed polycaprolactone scaffold patterns

We used additive manufacturing to fabricate 3D‐printed polycaprolactone scaffolds of different geometry topologies and porosities. We present a comparative analysis of hyaline cartilage development from adipose‐tissue–derived mesenchymal stem cells (ADMSCs) on three different, newly designed scaffold geometry patterns. The first scaffold design (MESO) was based on a rectilinear layer pattern. For the second pattern (RO45), we employed a 45° rotational layer loop. The design for the third scaffold (3DHC) was a three‐dimensional honeycomb‐like pattern with a hexagonal cellular distribution and small square shapes. We examined cell proliferation, colonization, and differentiation, in relation to the scaffold's structure, as well as to the mechanical properties of the final constructs. We gave emphasis on the scaffolds, both microarchitecture and macroarchitecture, for optimal and enhanced chondrogenic differentiation, as an important parameter, not well studied in the literature. Among the three patterns tested, RO45 was the most favourable for chondrogenic differentiation, whereas 3DHC better supported cell proliferation and scaffold penetration, exhibiting also the highest rate of increase onto the mechanical properties of the final construct. We conclude that by choosing the optimal scaffold architecture, the resulting properties of our cartilaginous constructs can better approximate those of the physiological cartilage.     

Optimal administration routes for adipose‐derived stem cells therapy in ischaemic flaps

Improvement of flap survival represents an ongoing challenge in reconstructive surgery. The angiogenic potential of adipose‐derived stem cells (ASCs) offers a promising approach to improve the viability of random pattern flaps. Recently, to maximize the therapeutic effects of ASCs, increasing focus is being placed on how to deliver the stem cells to target lesions. The purpose of the present study was to compare the effectiveness of different administration routes of ASCs to improve the viability of the random pattern skin flap. ASCs labelled with PKH26 were applied via four methods to the cranially‐based random pattern skin flaps of rats: (a) intravenous injection; (b) subcutaneous injection; (c) application with collagen sponge seeding; and (d) application with fibrin glue seeding. ASCs led to a significant increase in flap viability in the subcutaneous injection group and the collagen sponge group. Cutaneous blood flow was increased in the intravenous injection, subcutaneous injection and collagen sponge groups. Capillary density in the intravenous injection group and collagen sponge group was significantly greater than in the control group (no treatment). PKH26‐positive cells via the collagen sponge were distributed more densely within the flap than in other groups. This study demonstrated that the collagen sponge method delivered ASCs most effectively within the flap and increased flap vascularity. The clinical therapeutic effects of ASCs can therefore be maximized when the optimal delivery route is chosen. Copyright © 2012 John Wiley & Sons, Ltd.     

Role of cell–matrix interactions on VIC phenotype and tissue deposition in 3D PEG hydrogels

Valvular interstitial cells (VICs) respond to 3D matrix interactions in a complex manner, but understanding these effects on VIC function better is important for applications ranging from valve tissue engineering to studying valve disease. Here, we encapsulated VICs in poly(ethylene glycol) (PEG) hydrogels modified with three different adhesive ligands, derived from fibronectin (RGDS), elastin (VGVAPG) and collagen‐1 (P15). By day 14, VICs became significantly more elongated in RGDS‐containing gels compared to VGVAPG or P15. This difference in cell morphology appeared to correlate with global matrix metalloproteinase (MMP) activity, as VICs encapsulated in RGDS‐functionalized hydrogels secreted higher levels of active MMP at day 2. VIC activation to a myofibroblast phenotype was also characterized by staining for α‐smooth muscle actin (αSMA) at day 14. The percentage of αSMA⁺ VICs in the VGVAPG gels was the highest (56%) compared to RGDS (33%) or P15 (38%) gels. Matrix deposition and composition were also characterized at days 14 and 42 and found to depend on the initial hydrogel composition. All gel formulations had similar levels of collagen, elastin and chondroitin sulphate deposited as the porcine aortic valve. However, the composition of collagen deposited by VICs in VGVAPG‐functionalized gels had a significantly higher collagen‐X:collagen‐1 ratio, which is associated with stenotic valves. Taken together, these data suggest that peptide‐functionalized PEG hydrogels are a useful system for culturing VICs three‐dimensionally and, with the ability to systematically alter biochemical and biophysical properties, this platform may prove useful in manipulating VIC function for valve regeneration. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of adiponectin secreted from adipose‐derived stem cells on bone‐fat balance and bone defect healing

The efficacy of adiponectin (APN) in regulating bone metabolism remains controversial. This study aimed to investigate the role of APN secreted from adipose‐derived stem cells on adipogenesis and osteogenesis. Human APN gene was transfected via recombinant adenovirus into adipose derived stem cells (ASCs) in vitro and were cocultured with bone marrow mesenchymal stem cells (BMSCs) in using a transwell chamber. Adipogenesis was inhibited in APN‐transfected ASCs; in BMSCs, adipogenesis was inhibited, but osteogenesis was promoted in coculture with APN‐transfected ASCs. Next, the same adenovirus construct was transfected into the abdominal adipose tissue of a Sprague Dawley rat in vivo, and then a tibia defect was established in the same rat. We confirmed there was higher gene and protein expression of APN in ASCs and the abdominal adipose tissue of these rat models. Development of adipocytes in abdominal adipose tissue was suppressed, and less new bone was formed in the bone defect area. In conclusion, APN secreted from ASCs could directly inhibit adipogenesis in ASCs and BMSCs and promote osteogenesis in the latter. However, APN overexpression in adipose tissue was inversely associated with bone formation in tibia defects potentially due to decreased levels of circulating bone‐activating hormones.     

Control the fate of human umbilical cord mesenchymal stem cells with dual‐enzymatically cross‐linked gelatin hydrogels for potential applications in nerve regeneration

Stem‐cell‐based therapy is a promising strategy to treat challenging neurological diseases, while its application is hindered primarily by the low viability and uncontrolled differentiation of stem cell. Hydrogel can be properly engineered to share similar characteristics with the target tissue, thus promoting cell viability and directing cell differentiation. In this study, we proposed a new dual‐enzymatically cross‐linked and injectable gelatin hydrogel for regulating survival, proliferation, and differentiation of human umbilical cord mesenchymal stem cells (hUC‐MSCs) in a three‐dimensional matrix. This injectable gelatin hydrogel was formed by oxidative coupling of gelatin–hydroxyphenyl acid conjugates catalyzed by hydrogen horseradish peroxidase (HRP) and choline oxidase (ChOx). Modulus and H₂O₂ release can be well controlled by ChOx activity. Results from calcein‐AM/PI staining and Ki67 immunofluorescence tests demonstrated that the survival and proliferation behavior of hUC‐MSCs were highly enhanced in HRP_1UChOx_0.25U hydrogel with lower modulus and less H₂O₂ release compared with other groups. Attractively, the expression of neuron‐specific markers β‐III tubulin, neurofilament light chain (NFL), and synapsin‐1 was significantly increased in HRP_1UChOx_0.25U hydrogel as well. Additionally, in vitro hemolysis test and in vivo HE staining data highlighted the good biocompatibility. Undoubtedly, this injectable gelatin hydrogel's ability to control hUC‐MSCs' fate holds enormous potentials in nervous disorders' therapy and nerve regeneration.