Diverse cellular morphologies during lumen maturation in Anopheles gambiae larval salivary glands

Mosquitoes are the greatest animal threat to human health, causing hundreds of millions of infections and around 1 million deaths each year. All mosquito-borne pathogens must traverse the salivary glands (SGs) to be transmitted to the next host, making this organ an ideal target for interventions. The adult SG develops from precursor cells located in the larval SG duct bud. Characterization of the larval SG has been limited. We sought to better understand larval SG architecture, secretion and gene expression. We developed an optimized method for larval SG staining and surveyed hundreds of larval stage 4 (L4) SGs using fluorescence confocal microscopy. Remarkable variation in SG cell and chromatin organization differed among individuals and across the L4 stage. Lumen formation occurred during L4 stage through secretion likely involving a coincident cellular apical lipid enrichment and extracellular vesicle-like structures. Meta-analysis of microarray data showed that larval SG gene expression is divergent from adult SGs, more similar to larval gastric cecae, but different from other larval gut compartments. This work highlights the variable cell architecture of larval Anopheles gambiae SGs and provides candidate targets for genetic strategies aiming to disrupt SGs and transmission of mosquito-borne pathogens.     

Optimal timing and frequency of bone marrow soup therapy for functional restoration of salivary glands injured by single‐dose or fractionated irradiation

Injections of bone marrow (BM) cell extract, known as ‘BM soup’, were previously reported to mitigate ionizing radiation (IR) injury to salivary glands (SGs). However, the optimal starting time and frequency to maintain BM soup therapeutic efficacy remains unknown. This study tested the optimal starting time and frequency of BM soup injections in mice radiated with either a single dose or a fractionated dose. First, BM soup treatment was started at 1, 3 or 7 weeks post‐IR; positive (non‐IR) and negative (IR) control mice received injections of saline (vehicle control). Second, BM soup‐treated mice received injections at different frequencies (1, 2, 3 and 5 weekly injections). Third, a 'fractionated‐dose radiation' model to injure mouse SGs was developed (5 Gy × 5 days) and compared with the single high dose radiation model. All mice (n = 65) were followed for 16 weeks post‐IR. The results showed that starting injections of BM soup between 1 and 3 weeks mitigated the effect of IR‐induced injury to SGs and improved the restoration of salivary function. Although the therapeutic effect of BM soup lessens after 8 weeks, it can be sustained by increasing the frequency of weekly injections. Moreover, both single‐dose and fractionated‐dose radiation models are efficient and comparable in inducing SG injury and BM soup treatments are effective in restoring salivary function in both radiation models. In conclusion, starting injections of BM soup within 3 weeks post‐radiation, with 5 weekly injections, maintains 90–100% of saliva flow in radiated mice.     

Impact of modified gelatin on valvular microtissues

A significant challenge in the field of tissue engineering is the biofabrication of three‐dimensional (3D) functional tissues with direct applications in organ‐on‐a‐chip systems and future organ engineering. Multicellular valvular microtissues can be used as building blocks for the formation of larger scale valvular macrotissues. Yet, for the controlled biofabrication of 3D macrotissues with predefined complex shapes, directed assembly of microtissues through bioprinting is needed. This study aimed to investigate if modified gelatin is an instructive material for valvular microtissues. Valvular microtissues were encapsulated in modified gelatin hydrogels and cross‐linked in the presence of a photoinitiator (Irgacure 2959 or VA‐086). Hydrogel properties were determined, and valvular interstitial cell functions like phenotype, proliferation, migration, mRNA expression of extracellular matrix (ECM) molecules, ECM deposition, and tissue fusion were characterized by histochemical stainings and RT‐qPCR. Encapsulated microtissues remained viable, produced heart valve‐related ECM components, and remained in a quiescent state. However, encapsulation induced some changes in ECM formation and gene expression. Encapsulated microtissues showed lower remodeling capacity and increased expression levels of Col I/V, elastin, hyaluronan, biglycan, decorin, and Sox9 compared with nonencapsulated microtissues. Furthermore, this study demonstrated that proliferation, migration, and tissue fusion was more pronounced in softer gels. In general, we evidenced that modified gelatin is an instructive material for physiologically relevant valvular microtissues and provided a proof of concept for the formation of larger valvular tissue by assembling microtissues at random in soft gels.     

Enhanced tissue remodelling efficacy of adipose‐derived mesenchymal stem cells using injectable matrices in radiation‐damaged salivary gland model

The present study was conducted to introduce the use of a delivery carrier for local transplantation of human adipose tissue‐derived mesenchymal stem cells (AdMSCs) into the salivary gland (SG) and analyse its ability to enhance radioprotection of AdMSCs against irradiation (IR)‐induced damage. An injectable porcine small intestinal submucosa (SIS) matrix was used as a cell delivery carrier, and human AdMSCs were contained within SIS hydrogel (AdMSC/SIS). After local injection into SGs of mice following local IR, morphological and functional changes were evaluated in the sham, vehicle [phosphate‐buffered saline (PBS)], SIS, AdMSC and AdMSC/SIS groups. Local transplantation of AdMSC resulted in less fibrosis, regardless of the use of a carrier, but the AdMSC/SIS group showed more mucin‐producing acini relative to those in the PBS group. Functional restoration of salivation capacity and salivary protein synthesis was achieved in AdMSC and AdMSC/SIS groups, with a greater tendency being observed in the AdMSC/SIS group. AdMSC treatment resulted in tissue remodelling with a greater number of salivary epithelial cells (AQP‐5), SG progenitor cells (c‐Kit), endothelial cells (CD31) and myoepithelial cells (α‐SMA), among which endothelial and myoepithelial cells significantly increased in the AdMSC/SIS group relative to the AdMSC group. AdMSC treatment alleviated IR‐induced cell death, and the anti‐apoptotic and anti‐oxidative effects of AdMSC were enhanced in the AdMSC/SIS group relative to the AdMSC group. These results suggest local transplantation of AdMSC improves tissue remodelling following radiation damage in SG tissue, and that use of a carrier enhances the protective effects of AdMSC‐mediated cellular protection against IR via paracrine secretion. Copyright © 2016 John Wiley & Sons, Ltd.     

Cell extracts from spleen and adipose tissues restore function to irradiation‐injured salivary glands

A cell extract from whole bone marrow (BM), which we named “BM Soup,” has the property to restore saliva secretion to irradiation (IR)‐injured salivary glands (SGs). However, BM cell harvesting remains an invasive procedure for the donor. The main objective of this study was to test the therapeutic effect of “Cell Soups” obtained from alternate tissues, such as adipose‐derived stromal cells (ADSCs) and spleen cells to repair SGs. BM Soup, Spleen Soup, ADSC Soup, or saline (vehicle control) was injected intravenously into mice with IR‐injured SGs (13Gy). Results demonstrated that all three cell soups restored 65–70% of saliva secretion, protected acinar cells, blood vessels, and parasympathetic nerves, and increased cell proliferation. Although protein array assays identified more angiogenesis‐related growth factors in ADSC Soup, the length of its therapeutic efficiency on saliva flow was less than that of the BM Soup and Spleen Soup. Another objective of this study was to compare “Fresh” versus “Cryopreserved (?80 °C)” BM Soup. It was found that the therapeutic effect of 12‐month “Cryopreserved BM Soup” was comparable to that of “Fresh BM Soup” on the functional restoration of IR‐injured SGs. In conclusion, both Spleen Soup and ADSC Soup can be used to mitigate IR‐damaged SGs.     

Matrix‐directed differentiation of human adipose‐derived mesenchymal stem cells to dermal‐like fibroblasts that produce extracellular matrix

Commercially available skin substitutes lack essential non‐immune cells for adequate tissue regeneration of non‐healing wounds. A tissue‐engineered, patient‐specific, dermal substitute could be an attractive option for regenerating chronic wounds, for which adipose‐derived mesenchymal stem cells (ADMSCs) could become an autologous source. However, ADMSCs are multipotent in nature and may differentiate into adipocytes, osteocytes and chondrocytes in vitro, and may develop into undesirable tissues upon transplantation. Therefore, ADMSCs committed to the fibroblast lineage could be a better option for in vitro or in vivo skin tissue engineering. The objective of this study was to standardize in vitro culture conditions for ADMSCs differentiation into dermal‐like fibroblasts which can synthesize extracellular matrix (ECM) proteins. Biomimetic matrix composite, deposited on tissue culture polystyrene (TCPS), and differentiation medium (DM), supplemented with fibroblast‐conditioned medium and growth factors, were used as a fibroblast‐specific niche (FSN) for cell culture. For controls, ADMSCs were cultured on bare TCPS with either DM or basal medium (BM). Culture of ADMSCs on FSN upregulated the expression of differentiation markers such as fibroblast‐specific protein‐1 (FSP‐1) and a panel of ECM molecules specific to the dermis, such as fibrillin‐1, collagen I, collagen IV and elastin. Immunostaining showed the deposition of dermal‐specific ECM, which was significantly higher in FSN compared to control. Fibroblasts derived from ADMSCs can synthesize elastin, which is an added advantage for successful skin tissue engineering as compared to fibroblasts from skin biopsy. To obtain rapid differentiation of ADMSCs to dermal‐like fibroblasts for regenerative medicine, a matrix‐directed differentiation strategy may be employed. Copyright © 2014 John Wiley & Sons, Ltd.     

Prevention of irradiation-induced salivary hypofunction by microvessel protection in mouse salivary glands.

Treatment of most head and neck cancers includes radiotherapy. Salivary glands (SGs) in the irradiation (IR) field are irreversibly damaged resulting in severe hyposalivation. We evaluated the importance of SG endothelial cells to this IR-induced injury, and whether serotype 5 adenoviral (Ad5) vector-mediated transfer of basic fibroblast growth factor (AdbFGF) or vascular endothelial growth factor (AdVEGF) complementary DNAs would afford radioprotection. Four hours after IR, microvessel density (MVD) in SGs decreased by approximately 45%. However, if mice were pre-treated with either AdVEGF or AdbFGF 48 hours before IR the loss in MVD was significantly reduced. An irrelevant vector, AdLacZ, encoding Escherichia coli beta-galactosidase, was without effect. After 8 weeks, IR reduced salivary flow by approximately 65% in untreated mice. Mice pre-treated (using 5 x 10(9) particles/gland 48 hours prior to IR) with AdLacZ exhibited a reduction in salivary flow similar to untreated mice receiving IR. However, irradiated mice pre-treated with AdbFGF or AdVEGF showed a significant improvement in their salivary flow, to approximately 70% (P < 0.01) and 80% (P < 0.01), respectively, compared to non-irradiated control mice. These results are consistent with the notion that injury to the adjacent microvasculature may play an important role in SG radiation damage. Furthermore, our results suggest that a local transient treatment directed at protecting SG endothelial cells may be beneficial for patients undergoing IR for head and neck cancer.     

A magnetic three‐dimensional levitated primary cell culture system for the development of secretory salivary gland‐like organoids

Salivary gland (SG) hypofunction and oral dryness can be induced by radiotherapy for head and neck cancers or autoimmune disorders. These are common clinical conditions that involve loss of saliva‐secreting epithelial cells. Several oral complications arise with SG hypofunction that interfere with routine daily activities such as chewing, swallowing, and speaking. Hence, there is a need for replacing these saliva‐secreting cells. Recently, researchers have proposed to repair SG hypofunction via various cell‐based approaches in three‐dimensional (3D) scaffold‐based systems. However, majority of the scaffolds used cannot be translated clinically due to the presence of non‐human‐based substrates. Herein, saliva‐secreting organoids/mini‐glands were developed using a new scaffold/substrate‐free culture system named magnetic 3D levitation (M3DL), which assembles and levitates magnetized primary SG‐derived cells (SGDCs), allowing them to produce their own extracellular matrices. Primary SGDCs were assembled in M3DL to generate SG‐like organoids in well‐established SG epithelial differentiation conditions for 7 days. After such culture time, these organoids consistently presented uniform spheres with greater cell viability and pro‐mitotic cells, when compared with conventional salisphere cultures. Additionally, organoids formed by M3DL expressed SG‐specific markers from different cellular compartments: acinar epithelial including adherens junctions (NKCC1, cholinergic muscarinic receptor type 3, E‐cadherin, and EpCAM); ductal epithelial and myoepithelial (cytokeratin 14 and α‐smooth muscle actin); and neuronal (β3‐tubulin and vesicular acetylcholine transferase). Lastly, intracellular calcium and α‐amylase activity assays showed functional organoids with SG‐specific secretory activity upon cholinergic stimulation. Thus, the functional organoid produced herein indicate that this M3DL system can be a promising tool to generate SG‐like mini‐glands for SG secretory repair.     

Liver extracellular matrix promotes BM‐MSCs hepatic differentiation and reversal of liver fibrosis through activation of integrin pathway

In cell‐based therapies for liver injuries, the clinical outcomes are closely related to the surrounding microenvironment of the transplanted bone marrow mesenchymal stem cells (BM‐MSCs). However, whether liver‐specific ECM (L‐ECM), as one of major microenvironment signals, could regulate the therapeutic effect of BM‐MSCs through changing their biological characteristics is unclear. This study aimed to investigate the hepatogenicity and underlying mechanism of L‐ECM as well as its potential regulative role in the MSC‐based liver recovery. L‐ECM was prepared by homogenization of decellularized whole porcine liver. After three‐dimensional culture with or without the presence of L‐ECM, BM‐MSCs expressed hepatocyte‐specific genes and proteins in an L‐ECM concentration‐dependent manner. Further analysis showed that L‐ECM could activate specific types of integrins (ITGs) as well as their downstream signalling pathways. When the cell/ECM interaction was enhanced by incorporating BM‐MSCs with Mn²⁺, ITGs were activated and the hepatogenic capacity of L‐ECM was improved. The regeneration of rat livers from either acute or chronic fibrosis could also be accelerated after transplantation of Mn²⁺‐treated BM‐MSCs. L‐ECM therefore promotes hepatic differentiation of BM‐MSCs via the ITG pathway and plays a therapeutically beneficial role for stem cell‐based liver regeneration. Copyright © 2016 John Wiley & Sons, Ltd.     

Conditioned medium as a strategy for human stem cells chondrogenic differentiation

Paracrine signalling from chondrocytes has been reported to increase the synthesis and expression of cartilage extracellular matrix (ECM) by stem cells. The use of conditioned medium obtained from chondrocytes for stimulating stem cells chondrogenic differentiation may be a very interesting alternative for moving into the clinical application of these cells, as chondrocytes could be partially replaced by stem cells for this type of application. In the present study we aimed to achieve chondrogenic differentiation of two different sources of stem cells using conditioned medium, without adding growth factors. We tested both human bone marrow‐derived mesenchymal stem cells (hBSMCs) and human Wharton's jelly‐derived stem cells (hWJSCs). Conditioned medium obtained from a culture of human articular chondrocytes was used to feed the cells during the experiment. Cultures were performed in previously produced three‐dimensional (3D) scaffolds, composed of a blend of 50:50 chitosan:poly(butylene succinate). Both types of stem cells were able to undergo chondrogenic differentiation without the addition of growth factors. Cultures using hWJSCs showed significantly higher GAGs accumulation and expression of cartilage‐related genes (aggrecan, Sox9 and collagen type II) when compared to hBMSCs cultures. Conditioned medium obtained from articular chondrocytes induced the chondrogenic differentiation of MSCs and ECM formation. Obtained results showed that this new strategy is very interesting and should be further explored for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Fabrication of viable centimeter‐sized 3D tissue constructs with microchannel conduits for improved tissue properties through assembly of cell‐laden microbeads

Bottom‐up approaches have emerged as a new philosophy in tissue engineering, enabling precise control over tissue morphogenesis at the cellular level. We previously prepared large bone‐like tissues using cell‐laden microbeads (microtissues) by following a modular approach to ensure cell viability. However, a long‐term culture of such avascular macroscopic tissues (macrotissues) has not been evaluated. In the present study, microtissues were fabricated by cultivating human fibroblasts on Cytopore‐2 microbeads in spinner flasks for 16 days. We then examined the long‐term perfusion culture for macrotissues. Specifically, following assembly in a perfusion chamber for 15 days, cell death was found to be prominent at a depth of 500 µm from the surface of macrotissues towards the interior, suggesting that there was a new mass transfer limit leading to cell death instead of tissue maturation. Subsequently, we developed a strategy by incorporating microchannel structures in centimeter‐sized tissue constructs to promote mass transport. By installing glass rods (1 mm diameter, 1 mm wall‐to‐wall spacing) in the perfusion chamber, stable microchannel architectures were introduced during the microtissue assembly process. Based on live/dead assay and scanning electron microscopy (SEM), these channelled macrotissues (length × diameter, 1.6 × 2.0 cm) demonstrated high cell viability and compact packing of microbeads. Comparative biochemical analysis further suggested a more homogeneous spatial distribution of cells and extracellular matrix (ECM) in the channelled macrotissues than in solid ones. Viable 3D large tissues can therefore be prepared by assembling cell‐laden microbeads in conjunction with microchannel carving, meeting clinical needs in tissue repair. Copyright © 2012 John Wiley & Sons, Ltd.     

Small leucine‐rich proteoglycans (SLRPs): characteristics and function in the intervertebral disc

The intervertebral disc (IVD) is responsible for normal spinal motion and load distribution. However, degeneration may occur due to age‐ and non‐age‐related processes and is primarily characterized by a reduction in the number of chondrocyte‐like cells and abnormal extracellular matrix (ECM) structure in the nucleus pulposus. Although IVD progenitor cells have been identified, the local microenvironment components regulating the behaviour of these progenitor cell populations remain unknown. Small leucine‐rich proteoglycans (SLRPs) are bioactive components of the ECM associated with fibrillogenesis, cellular growth and apoptosis and tissue remodelling. SLRPs support the survival of IVD progenitor cells under hypoxic conditions via the activation of specific hypoxia‐inducible factors. Additionally, SLRPs deficiency (biglycan) in knockout mice is sufficient to accelerate the IVD degenerative process. These data suggest that SLRPs play an important role in the homeostasis of IVD. Given their specific properties and physiological functions, we propose a role of SLRPs in IVD degeneration and potential application in its regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Nanoparticle-mediated Gene Silencing Confers Radioprotection to Salivary Glands In Vivo

Radiation treatment of head and neck cancers causes irreversible damage of the salivary glands (SG). Here, we introduce a preclinical mouse model for small-interfering RNA (siRNA)-based gene silencing to provide protection of SG from radiation-induced apoptosis. Novel, pH-responsive nanoparticles complexed with siRNAs were introduced into mouse submandibular glands (SMG) by retroductal injection to modulate gene expression in vivo. To validate this approach, we first targeted Nkcc1, an ion transporter that is essential for saliva secretion. Nkcc1 siRNA delivery resulted in efficient knockdown, as quantified at the mRNA and the protein levels, and the functional result of Nkcc1 knockdown phenocopied the severe decrease in saliva secretion, characteristic of the systemic Nkcc1 gene knockout. To establish a strategy to prevent apoptotic cell loss due to radiation damage, siRNAs targeting the proapoptotic Pkcδ gene were administered into SMG before ionizing radiation. Knockdown of Pkcδ not only reduced the number of apoptotic cells during the acute phase of radiation damage, but also markedly improved saliva secretion at 3 months in irradiated animals, indicating that this treatment confers protection from hyposalivation. These results demonstrate that nanoparticle delivery of siRNAs targeting a proapoptotic gene is a localized, nonviral, and effective means of conferring radioprotection to the SGs.     

Human hematopoietic stem cell adherence to cytokines and matrix molecules.

The hematopoietic microenvironment is a complex structure in which stem cells, progenitor cells, stromal cells, growth factors, and extracellular matrix (ECM) molecules each interact to direct the coordinate regulation of blood cell development. While much is known concerning the individual components of this microenvironment, little is understood of the interactions among these various components or, in particular, the nature of those interactions responsible for the regional localization of specific developmental signals. We hypothesized that cytokines act together with ECM molecules to anchor stem cells within the microenvironment, thus modulating their function. In order to analyze matrix-cytokine-stem cell interactions, we developed an ECM model system in which purified stem cell populations and plastic-immobilized individual proteins are used to assess the role of various matrix molecules and/or cytokines in human hematopoietic cell development. Analysis of these interactions revealed that a single ECM protein, thrombospondin, in conjunction with a single cytokine (e.g., c-kit ligand), constitutes a developmental signal that synergistically modulates hematopoietic stem cell function.     

Advances in tissue engineering through stem cell‐based co‐culture

Stem cells are the future in tissue engineering and regeneration. In a co‐culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co‐culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co‐culture systems are discussed. Direct and indirect co‐culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell–cell contact, cell–extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co‐culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co‐culture use. Copyright © 2014 John Wiley & Sons, Ltd.     

Stem cells display a donor dependent response to escalating levels of growth factor release from extracellular matrix‐derived scaffolds

Numerous growth factor delivery systems have been developed for tissue engineering. However, little is known about how the dose of a specific protein will influence tissue regeneration, or how different patients will respond to altered levels of growth factor presentation. The objective of the present study was to assess stem cell chondrogenesis within extracellular‐matrix (ECM)‐derived scaffolds loaded with escalating levels of transforming growth factor (TGF)‐β3. It was also sought to determine if stem cells display a donor‐dependent response to different doses of TGF‐β3, from low (5 ng) to high (200 ng), released from such scaffolds. It was found that ECM‐derived scaffolds possess the capacity to bind and release increasing amounts of TGF‐β3, with between 60% and 75% of this growth factor released into the media over the first 12 days of culture. After seeding these scaffolds with human infrapatellar fat pad‐derived stem cells (FPSCs), it was found that cartilage‐specific ECM accumulation was greatest for the higher levels of growth factor loading. Importantly, soak‐loading cartilage ECM‐derived scaffolds with high levels of TGF‐β3 always resulted in at least comparable levels of chondrogenesis to controls where this growth factor was continuously added to the culture media. Similar results were observed for FPSCs from all donors, although the absolute level of secreted matrix did vary from donor to donor. Therefore, while no single growth factor release profile will be optimal for all patients, the results of this study suggest that the combination of a highly porous cartilage ECM‐derived scaffold coupled with appropriate levels of TGF‐β3 can consistently drive chondrogenesis of adult stem cells. Copyright © 2016 John Wiley & Sons, Ltd.     

Cell culture of differentiated human salivary epithelial cells in a serum‐free and scalable suspension system: The salivary functional units model

Saliva aids in digestion, lubrication, and protection of the oral cavity against dental caries and oropharyngeal infections. Reduced salivary secretion, below an adequate level to sustain normal oral functions, is unfortunately experienced by head and neck cancer patients treated with radiotherapy and by patients with Sjögren's syndrome. No disease‐modifying therapies exist to date to address salivary gland hypofunction (xerostomia, dry mouth) because pharmacotherapies are limited by the need for residual secretory acinar cells, which are lost at the time of diagnosis, whereas novel platforms such as cell therapies are yet immature for clinical applications. Autologous salivary gland primary cells have clinical utility as personalized cell therapies, if they could be cultured to a therapeutically useful mass while maintaining their in vivo phenotype. Here, we devised a serum‐free scalable suspension culture system that grows partially digested human salivary tissue filtrates composing of acinar and ductal cells attached to their native extracellular matrix components while retaining their 3D in vivo spatial organization; we have coined these salivary spheroids as salivary functional units (SFU). The proposed SFU culture system was sub‐optimal, but we have found that the cells could still survive and grow into larger salivary spheroids through cell proliferation and aggregation for 5 to 10 days within the oxygen diffusion rates in vitro. In summary, by using a less disruptive cell isolation procedure as the starting point for primary cell culture of human salivary epithelial cells, we demonstrated that aggregates of cells remained proliferative and continued to express acinar and ductal cell‐specific markers.     

Morphological transformation of hBMSC from 2D monolayer to 3D microtissue on low‐crystallinity SF‐PCL patch with promotion of cardiomyogenesis

The effects of the stiffness of substrates on the cell behaviours of human bone marrow‐derived mesenchymal stem cells (hBMSC) have been investigated, but the effects of the secondary structures of proteins in the substrates on the morphological transformation and differentiation of hBMSC have yet been elucidated. To investigate these issues, silk fibroin‐poly(ε‐caprolactone) SP cardiac patches of poly(ε‐caprolactone; P), on which is grafted by silk fibroin (SF) with various β‐sheet contents (or crystallinity) to provide various degrees of stiffness, were produced to examine the in vitro behaviours of hBMSC during proliferation, and cardiomyogenesis on the SP patches. β‐sheet contents of SF from 20% to 44% (SP20 to SP44, respectively) were induced on patches, which were examined by attenuated total reflection Fourier‐transform infrared (ATR‐FTIR) spectroscopy, and analysed using the Fourier self‐deconvolution method. The stiffness of the SP patches, quantified by their Young's moduli and elasticities, increased with the crystallinity of the SF. During 3 days of proliferation, hBMSC migrated and morphologically transformed into 3D microtissues with diameters of approximately 150–200 μm on low‐stiffness SP20 and SP30 patches, whereas 2D monolayers were observed on the SP37 and SP44 patches. The 3D microtissues/patch yielded more extensive in vitro cardiomyogenesis of hBMSC than the 2D cell monolayer with significantly higher expressions of all examined cardiac‐specific proteins after induction by 5‐aza. Notably, in vivo subcutaneously growing 3D microtissues on SP20 patches and a 2D monolayer on SP44 patches were preliminarily demonstrated in a rat model. Morphological transformations of hBMSC from a 2D monolayer to a 3D microtissue by low‐stiffness SP cardiac patches, promoting cardiomyogenesis, provide a new opportunity for cardiac tissue engineering.     

人胚胎干细胞在无血清mTeSR1培养基中维持培养和向内皮细胞的诱导分化

背景：传统的人胚胎干细胞培养扩增方法中应用含动物血清培养基,并依赖饲养层细胞培养,这种培养方法显著制约了干细胞的体外培养规模;另外异源动物血清成分介入,使病原污染及免疫排斥的概率显著增加。目的：明确应用无血清培养基mTeSR1对人胚胎干细胞进行长期体外培养的可行性,并建立诱导人胚胎干细胞分化为血管内皮细胞的相关技术平台。方法：采用无血清培养基mTeSR1以非饲养层细胞依赖的方式体外培养、扩增人胚胎干细胞株H9。经过40余次体外传代后,于倒置显微镜下观察其生长形态,并利用免疫荧光染色方法评估其细胞表型。此外,应用条件培养基诱导H9细胞株向内皮细胞方向分化。利用免疫荧光染色技术,定量RT-PCR以及低密度脂蛋白摄取实验对该胚胎干细胞源内皮细胞的表型及功能进行评价、分析。结果与结论：mTeSR1培养基能够支持H9细胞株在体外以非饲养层依赖的方式进行长期扩增,同时维持其未分化的干细胞潜能。添加血管内皮细胞的条件培养基能够定向诱导H9细胞向内皮细胞方向分化。该胚胎干细胞源内皮细胞不但表达内皮细胞的标志基因（kdr,pecam）和标记蛋白CD31,而且还能够摄取低密度脂蛋白,形成类似微血管结构。提示实验中所提供的培养及诱导分化体系能够支持胚胎干细胞的增殖与分化行为。