Bio‐electrospraying: A potentially safe technique for delivering progenitor cells

Bio‐electrospraying is fast becoming an attractive tool for in situ cell delivery into scaffolds for tissue engineering applications, with several cell types been successfully electrosprayed. Bone marrow derived mesenchymal progenitor/stem cells (BMSC), which are an important cell source for tissue engineering, have not been explored in detail and the effect of electrospraying on their “stemness” is not known. This study therefore investigates the effects of electrospraying on BMSC viability, proliferation, and multilineage differentiation potential. Electrospraying a BMSC suspension at flow rate of 6 mL/h and voltages of 7.5–15 kV could successfully generate a continuous, stable and linearly directed electrospray of cells. Morphological observation, trypan blue tests and alamar blue based metabolic assays revealed about 88% of these electrosprayed cells were viable, and proliferated at rates similar to native BMSCs. However, at higher voltages, electrospraying became unstable and reduced cell viability, possibly due to electrical or thermal damage to the cells. BMSCs electrosprayed at 7.5 kV also retained their multipotency and could be successfully differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating similar morphology and gene expression levels as induced native BMSCs. These results indicate that bio‐electrospraying could be safely used as a progenitor/stem cell delivery technique for tissue engineering and regenerative medicine applications. Biotechnol. Bioeng. 2010;106: 690–698. © 2010 Wiley Periodicals, Inc.     

种子细胞——组织工程连载之三

种子细胞也是组织工程的核心研究内容,获得足够数量和质量的种子细胞是开展体外组织工程的必要基础。用于组织工程的种子细胞必须具有形成新组织结构的能力,主要来源于自体、同种异体或异种,在具体应用时各有利弊。一些成体干细胞由于不存在伦理争议以及发育分化条件相对简单等优势是重要的种子细胞,包括造血干细胞、骨髓干细胞、神经干细胞、脂肪干细胞、皮肤干细胞。人胚胎干细胞及其组织工程要真正在临床医学中得到应用,还有很长的一段路要走。其他一些细胞也可以作为组织工程种子细胞,包括内皮细胞、上皮细胞、成纤维细胞、骨细胞、成骨细胞、角质细胞、前脂肪细胞、脂肪细胞、肌腱细胞等。这些细胞已分化,分裂能力有限,但仍应用于组织工程。理想的种子细胞具有一定标准。     

骨髓间充质干细胞在组织工程研究中的应用新进展

骨髓间充质干细胞又称为骨髓源性间充质干细胞,是指存在于骨髓基质细胞系统中的一类干细胞,具有高度稳定的体外扩增能力和多向分化潜能等特点。骨髓间充质干细胞因其取材方便,易于分离和培养,以及在适当条件下可诱导分化为皮肤、骨骼、内脏、血液、神经等多种组织细胞的独特优势,目前被广泛应用于药物开发、免疫调节、组织修复、器官重建等多个研究领域。近年来,骨髓间充质干细胞作为种子细胞在组织工程领域有着非常诱人的潜在应用前景。本文就骨髓间充质干细胞在组织工程学研究中应用的最新进展作一综述。     

组蛋白乙酰化对成体干细胞生物学性状的影响

成体干细胞（adult stem cells,ASCs）是指存在于一种已经分化组织中的未分化细胞,它们可以再生修复损伤的组织和器官,是组织工程和细胞治疗的理想细胞。但是ASCs在体外扩增过程中容易发生自主分化和衰老,影响其在临床的广泛应用。组蛋白乙酰化作为表观遗传调节的重要机制,参与细胞分化、衰老及凋亡等众多细胞活动的调控。该文就组蛋白乙酰化对成体干细胞生物学性状的影响进行综述。     

Graphene for improved femtosecond laser based pluripotent stem cell transfection

Pluripotent stem cells are hugely attractive in the tissue engineering research field as they can self‐renew and be selectively differentiated into various cell types. For stem cell and tissue engineering research it is important to develop new, biocompatible scaffold materials and graphene has emerged as a promising material in this area as it does not compromise cell proliferation and accelerates specific cell differentiation. Previous studies have shown a non‐invasive optical technique for mouse embryonic stem (mES) cell differentiation and transfection using femtosecond (fs) laser pulses. To investigate cellular responses to the influence of graphene and laser irradiation, here we present for the first time a study of mES cell fs laser transfection on graphene coated substrates. First we studied the impact of graphene on Chinese Hamster Ovary (CHO‐K1) cell viability and cell cytotoxicity in the absence of laser exposure. These were tested via evaluating the mitochondrial activity through adenosine triphosphates (ATP) luminescence and breakages on the cell plasma membrane assessed using cytosolic lactate dehydrogenase (LDH) screening. Secondly, the effects of fs laser irradiation on cell viability and cytotoxicity at 1064 and 532 nm for cells plated and grown on graphene and pure glass were assessed. Finally, optical transfection of CHO‐K1 and mES cells was performed on graphene coated versus plain glass substrates. Our results show graphene stimulated cell viability whilst triggering a mild release of intracellular LDH. We also observed that compared to pure glass substrates; laser irradiation at 1064 nm on graphene plates was less cytotoxic. Finally, in mES cells efficient optical transfection at 1064 (82%) and 532 (25%) nm was obtained due to the presence of a graphene support as compared to pristine glass. Here we hypothesize an up‐regulation of cell adhesion promoting peptides or laminin‐related receptors of the extracellular matrix (ECM) in cell samples grown and irradiated on graphene substrates. By bringing together advances in optics and nanomaterial sciences we demonstrate pathways for enhancement of pluripotent stem cell biology. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

小鼠胚胎干细胞移植入成体大鼠脑内的区域特异性存活与分化

全能区域非特异性的胚胎干细胞是研究成体不同脑区控制干细胞分化能力的十分有力的工具。胚胎干细胞源性神经前体细胞移植入成体脑后可分化为功能性神经元,但是未分化的胚胎干细胞在成体脑内各个部位的存活、生长与分化的潜能差异尚不清楚。本文旨在探讨成体脑组织对胚胎干细胞的影响及胚胎干细胞在成体脑内的一系列行为。将少量转绿色荧光蛋白未分化的小鼠胚胎干细胞移植入成体大鼠脑内不同部位,分别于移植5、14和28d后处死大鼠,进行形态学观察及免疫组化定性,以了解未分化的小鼠胚胎干细胞在大鼠脑内不同区域的存活、生长与分化。结果发现未分化的小鼠胚胎干细胞可逐步整合入受体组织并向nestin阳性神经前体细胞分化。移植细胞及其后裔在海马生长最为旺盛,而在隔区最差（P〈0．01）;移植细胞分化为神经干细胞的效率也是在海马最高,而在隔区最低（P〈0．01）。提示只有部分脑区适合胚胎干细胞及其后裔生存,并提供促进其分化的有益环境。因此,由于位置特异的微环境因子及环境因素的存在,宿主组织特性对决定中枢神经系统疾病的细胞替代疗法策略是相当重要的。     

INHIBITION OF EHRLICH TUMOR GROWTH IN MICE BY ELECTRIC INTERFERENCE THERAPY (IN VIVO STUDIES)

A study of solid tumor growth retardation by employing extremely low frequency (ELF) electric fields has been carried out. ELF electric fields were generated in tumor tissue in mice by the interference of two high frequency sinusoidal waves with the beat frequency centered at the tumor core. The results indicated a pronounced decrease in tumor growth rate in animals exposed to a 5-Hz interferential frequency for 1 hr daily. The 1 hr/day treatment produced a greater retardation effect than the 1 hr/week treatment. This indicates that treatment duration at the applied field frequency appears to play an important role in tumor growth delay. The dielectric properties of the tumor cells showed higher permittivity and conductivity values than homologous normal tissue. The permittivity of tumor cells treated daily with 5 Hz reaches nearly the same value as control tissue. Moreover, histological studies show that tumor tissues treated daily with the same frequency undergo partial regression and shrinkage of the aggregates of neoplastic cells leaving very little of them. We conclude that this new interferential technique is promising for tumor treatment in which a resonating electric field affects cell-to-cell communication.     

Fetal and adult liver stem cells for liver regeneration and tissue engineering

For the development of innovative cell-based liver directed therapies, e.g. liver tissue engineering, the use of stem cells might be very attractive to overcome the limitation of donor liver tissue. Liver specific differentiation of embryonic, fetal or adult stem cells is currently under investigation. Different types of fetal liver (stem) cells during development were identified, and their advantageous growth potential and bipotential differentiation capacity were shown. However, ethical and legal issues have to be addressed before using fetal cells. Use of adult stem cells is clinically established, e.g. transplantation of hematopoietic stem cells. Other bone marrow derived liver stem cells might be mesenchymal stem cells (MSC). However, the transdifferentiation potential is still in question due to the observation of cellular fusion in several in vivo experiments. In vitro experiments revealed a crucial role of the environment (e.g. growth factors and extracellular matrix) for specific differentiation of stem cells. Co-cultured liver cells also seemed to be important for hepatic gene expression of MSC. For successful liver cell transplantation, a novel approach of tissue engineering by orthotopic transplantation of gel-immobilized cells could be promising, providing optimal environment for the injected cells. Moreover, an orthotopic tissue engineering approach using bipotential stem cells could lead to a repopulation of the recipients liver with healthy liver and biliary cells, thus providing both hepatic functions and biliary excretion. Future studies have to investigate, which stem cell and environmental conditions would be most suitable for the use of stem cells for liver regeneration or tissue engineering approaches.     

The effect of amino density on the attachment, migration, and differentiation of rat neural stem cells In Vitro

Artificial extracellular matrices play important roles in the regulation of stem cell behavior. To generate materials for tissue engineering, active functional groups, such as amino, carboxyl, and hydroxyl, are often introduced to change the properties of the biomaterial surface. In this study, we chemically modified coverslips to create surfaces with different amino densities and investigated the adhesion, migration, and differentiation of neural stem cells (NSCs) under serum-free culture conditions. We observed that a higher amino density significantly promoted NSCs attachment, enhanced neuronal differentiation and promoted excitatory synapse formation in vitro. These results indicate that the amino density significantly affected the biological behavior of NSCs. Thus, the density and impact of functional groups in extracellular matrices should be considered in the research and development of materials for tissue engineering.     

A novel method for culturing neural stem cells

The standard culture method for neural stem cells cannot prevent the attachment of neurospheres, which eventually result in differentiation. This study developed a new method for long-term neural stem cell cultivation. In the antiattachment group, neural stem cells were cultured in flasks coated with 1.5% agarose gel. As a control, cells were cultured in plastic flasks. The 5-bromine-deoxyuridine incorporation assay was used to determine the S-phase labeling index of both groups. The methyl thiazolyl tetrazolium (MTT) colorimetric assay was used to determine the total cell vitality. After a 3-mo culture, the spontaneous differentiation of stem cells was studied using immunocytochemistry for neuroepithelial stem cell protein. We found that neural stem cells grew rapidly in the antiattachment flasks. There was no statistically significant difference between the two groups in terms of the S-phase labeling index or MTT assay. When cultured for 3 mo in vitro, many more cells differentiated in the control than in the antiattachment group (32.05 vs. 0.64%, P < 0.01). Moreover, the neural stem cells in the antiattachment group remained multipotent. Therefore, flasks coated with agarose gel are suitable for long-term neural stem cell culture.     

干细胞的蛋白质组研究

干细胞研究和蛋白质组研究同属于21世纪生命科学的热点领域。将蛋白质组学技术应用于干细胞的研究,能够为了解干细胞提供蛋白质水平的信息,揭示干细胞的增殖、定向分化和迁移的机制,为人们更好地将干细胞技术应用于组织工程、基因治疗及药物开发等领域奠定基础。     

Synovium-derived stem cell-based chondrogenesis

Synovium is considered a candidate source of cells for cartilage tissue engineering. Compared with mesenchymal stem cells (MSCs) from other sources, synovium-derived stem cells (SDSCs) have a higher capacity for chondrogenic differentiation. Our objective was to define cocktails of growth factors that support the growth and chondrogenic differentiation of SDSCs in chemically defined medium. We established a fast and highly selective technique of negative isolation of SDSC populations. The individual and combined effects of three growth factors-transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (FGF-2)-were evaluated in serum-free pellet cultures of SDSCs for the chondrogenesis of SDSCs using histology, biochemical analysis, and real-time RT-PCR. In vitro studies identified TGF-beta1 as the key factor for both the growth and chondrogenesis of SDSCs. The highest rates of SDSC growth were observed with the synergistic interaction of all three factors. With respect to chondrogenic differentiation of SDSCs, the interaction of TGF-beta1 and IGF-I applied simultaneously was superior to the sequential application of these two factors or any other combination of growth factors studied. Based on these findings, we propose a two-step protocol for the derivation of chondrogenic SDSCs: a cocktail of TGF-beta1, IGF-I, and FGF-2 is applied first to induce cell growth followed by a cocktail of TGF-beta1 and IGF-I applied to induce chondrogenesis.     

Stem cell mechanobiology

Stem cells are undifferentiated cells that are capable of proliferation, self‐maintenance and differentiation towards specific cell phenotypes. These processes are controlled by a variety of cues including physicochemical factors associated with the specific mechanical environment in which the cells reside. The control of stem cell biology through mechanical factors remains poorly understood and is the focus of the developing field of mechanobiology. This review provides an insight into the current knowledge of the role of mechanical forces in the induction of differentiation of stem cells. While the details associated with individual studies are complex and typically associated with the stem cell type studied and model system adopted, certain key themes emerge. First, the differentiation process affects the mechanical properties of the cells and of specific subcellular components. Secondly, that stem cells are able to detect and respond to alterations in the stiffness of their surrounding microenvironment via induction of lineage‐specific differentiation. Finally, the application of external mechanical forces to stem cells, transduced through a variety of mechanisms, can initiate and drive differentiation processes. The coalescence of these three key concepts permit the introduction of a new theory for the maintenance of stem cells and alternatively their differentiation via the concept of a stem cell ‘mechano‐niche’, defined as a specific combination of cell mechanical properties, extracellular matrix stiffness and external mechanical cues conducive to the maintenance of the stem cell population. J. Cell. Biochem. 112: 1–9, 2011. © 2010 Wiley‐Liss, Inc.     

Cytoskeletal forces produced by extremely low‐frequency electric fields acting on extracellular glycoproteins

The physical mechanism by which cells transduce an applied electric field is not well understood. This article establishes for the first time a direct, quantitative model that links the field to cytoskeletal forces. In a previous article, applied electric fields of physiological strength were shown to produce significant mechanical torques at the cellular level. In this article, the corresponding forces exerted on the cytoskeleton are computed and found to be comparable in magnitude to mechanical forces known to produce physiological effects. In addition to the electrical force, the viscous drag force exerted by the surrounding medium and the restoring force exerted by the neighboring structures are considered in the analysis. For an applied electric field of 10 V/m, the force transmitted to the CD44 receptor of a hyaluronan chain in cartilage is about 1 pN at 10 Hz and 7 pN at 1 Hz. For an applied electric field of 100 V/m, the force transmitted to the cytoskeleton at one focus of the glycocalyx is about 0.5 pN at 10 Hz and 1.3 pN at 1 Hz. Mechanical forces of similar magnitude have been observed to produce physiological effects. Hence, this electromechanical transduction process is a plausible mechanism for the production of physiological effects by such electric fields. Bioelectromagnetics 31:77–84, 2010. © 2009 Wiley‐Liss, Inc.     

A novel method to reconstruct epithelial tissue using high-purity keratinocyte lineage cells induced from human embryonic stem cells

The treatment of oral mucosa defect such as autologous oral mucosa caused by resection of oral mucosa carcinoma is still not ideal in clinical practice. However, Tissue engineering gives us the possibility to solve this problem. As we all know, Human embryonic stem cells (hESCs) have the ability to give rise to various cell types. We can take advantage of the totipotency of human embryonic stem cells to acquire keratinocytes. Directing the epithelial differentiation of hESCs can provide seed cells for the construction of epithelium tissue by tissue engineering. But, how to get high purity keratinocytes by induced stem cells then Applied to tissue engineering mucosa is an important challenge. We described a novel method to directly induce hESCs to differentiate into keratinocytes. Retinoic acid, ascorbic acid, and bone morphogenetic protein induced hESCs to differentiate into cells that highly expressed cytokeratin (CK)14. Our findings suggest that the retinoic acid, ascorbic acid and bone morphogenetic proteins induced hESCs to form high purity keratinocyte cell populations. In addition, we found that the highly pure keratinocyte populations reconstructed artificial tissue resembling epithelial tissue when inoculated in vitro on a biological scaffold.     

Engineered heart tissue enables study of residual undifferentiated embryonic stem cell activity in a cardiac environment

Embryonic stem cell (ESC) derivatives are a promising cell source for cardiac cell therapy. Mechanistic studies upon cell injection in conventional animal models are limited by inefficient delivery and poor cell survival. As an alternative, we have used an engineered heart tissue (EHT) based on neonatal rat cardiomyocytes (CMs) cultivated with electrical field stimulation as an in vitro model to study cell injection. We injected (0.001, 0.01, and 0.1 million) and tracked (by qPCR and histology) undifferentiated yellow‐fluorescent protein transgenic mouse ESCs and Flk1 + /PDGFRα+ cardiac progenitor (CPs) cells, to investigate the effect of the cardiac environment on cell differentiation, as well as to test whether our in vitro model system could recapitulate the formation of teratoma‐like structures commonly observed upon in vivo ESC injection. By 8 days post‐injection, ESCs were spatially segregated from the cardiac cell population; however, ESC injection increased survival of CMs. The presence of ESCs blocked electrical conduction through the tissue, resulting in a 46% increase in the excitation threshold. Expression of mouse cardiac troponin I, was markedly increased in CP injected constructs compared to ESC injected constructs at all time points and cell doses tested. As early as 2 weeks, epithelial and ganglion‐like structures were observed in ESC injected constructs. By 4 weeks of ESC injection, teratoma‐like structures containing neural, epithelial, and connective tissue were observed in the constructs. Non‐cardiac structures were observed in the CP injected constructs only after extended culture (4 weeks) and only at high cell doses, suggesting that these cells require further enrichment or differentiation prior to transplantation. Our data indicate that the cardiac environment of host tissue and electrical field stimulation did not preferentially guide the differentiation of ESCs towards the cardiac lineage. In the same environment, injection of CP resulted in a more robust cardiac differentiation than injection of ESC. Our data demonstrate that the model‐system developed herein can be used to study the functional effects of candidate stem cells on the host myocardium, as well as to measure the residual activity of undifferentiated cells present in the mixture. Biotechnol. Bioeng. 2011; 108:704–719. © 2010 Wiley Periodicals, Inc.     

Differentiation profile of brain tumor stem cells： a comparative study with neural stem cells

Understanding of the differentiation profile of brain tumor stem cells （BTSCs）, the key ones among tumor cell population, through comparison with neural stem cells （NSCs） would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Here, we cultured and purified BTSCs from surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property. As expected, NSCs differentiated into mature neural phenotypes. In the same differentiation condition, however, BTSCs exhibited distinguished differences. Morphologically, cells grew flattened and attached for the first week, but gradually aggregated and reformed floating tumor sphere thereafter. During the corresponding period, the expression rate of undifferentiated cell marker CD 133 and nestin in BTSCs kept decreasing, but 1 week later, they regained ascending tendency. Interestingly, the differentiated cell markers GFAP and β-tubulinlII showed an expression change inverse to that of undifferentiated cell markers. Taken together, BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma.     

A comprehensive overview on utilizing electromagnetic fields in bone regenerative medicine

Stem cells are one of the most important sources to develope a new strategy for repairing bone lesions through tissue engineering. Osteogenic differentiation of stem cells can be affected by various factors such as biological, chemical, physiological, and physical ones. The application of ELF-EMFs has been the subject of many research in bone tissue engineering and evidence suggests that this exogenous physical stimulus can promote osteogenic differentiation in several types of cells. The purpose of this paper is to review the current knowledge on the effects of EMFs on stem cells in bone tissue engineering studies. We recapitulated and analyzed 39 articles that were focused on the application of EMFs for bone tissue engineering purposes. We tabulated scattered information from these articles for easy use and tried to provide an overview of conducted research and identify the knowledge gaps in the field.     

Three‐dimensional cell culture microarray for high‐throughput studies of stem cell fate

We have developed a novel three‐dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high‐throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox‐1, whose levels were also measured in situ using a GFP reporter system. In addition, the high‐throughput capacity of the platform was tested using a dual‐slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor‐4 (FGF‐4) on the pluripotency of mouse ES cells. This high‐throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses. Biotechnol. Bioeng. 2010; 106: 106–118. © 2010 Wiley Periodicals, Inc.