Cells Tissues Organs

Publications in this journal

• Article: Paracrine Effects and Heterogeneity of Marrow-Derived Stem/Progenitor Cells: Relevance for the Treatment of Respiratory Diseases.

  Massimo Conese, Annalucia Carbone, Stefano Castellani, Sante Di Gioia
  [show abstract] [hide abstract]
  ABSTRACT: Stem cell-based treatment may represent a hope for the treatment of acute lung injury and pulmonary fibrosis, and other chronic lung diseases, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease (COPD). It is well established in preclinical models that bone marrow-derived stem and progenitor cells exert beneficial effects on inflammation, immune responses and repairing of damage in virtually all lung-borne diseases. While it was initially thought that the positive outcome was due to a direct engraftment of these cells into the lung as endothelial and epithelial cells, paracrine factors are now considered the main mechanism through which stem and progenitor cells exert their therapeutic effect. This knowledge has led to the clinical use of marrow cells in pulmonary hypertension with endothelial progenitor cells (EPCs) and in COPD with mesenchymal stromal (stem) cells (MSCs). Bone marrow-derived stem cells, including hematopoietic stem/progenitor cells, MSCs, EPCs and fibrocytes, encompass a wide array of cell subsets with different capacities of engraftment and injured tissue-regenerating potential. The characterization/isolation of the stem cell subpopulations represents a major challenge to improve the efficacy of transplantation protocols used in regenerative medicine and applied to lung disorders.
  Cells Tissues Organs 05/2013;
• Article: A Two-Step Method of Constructing Mature Cartilage Using Bone Marrow-Derived Mesenchymal Stem Cells.

  Ke Xue, Lin Qi, Guangdong Zhou, Kai Liu
  [show abstract] [hide abstract]
  ABSTRACT: Bone marrow-derived mesenchymal stem cells (BMSCs) are a promising source of stem cells for tissue engineering in cartilage repair. However, construction of cartilage using BMSCs can involve many problems, such as fibrosis, vascularization, the 'hollow' phenomenon and shrinkage, which may be caused by the incomplete differentiation of BMSCs and prevent the clinical application of tissue-engineered cartilage. A novel induction system that facilitates chondrogenesis by swine BMSCs has been developed. In this study, we constructed cartilage using a two-step procedure: first, promoting complete chondrogenic differentiation of BMSCs in 8 weeks, and second, these chondrocytes which differentiated from BMSCs in vitro were provided with a three-dimensional scaffold, which was then implanted subcutaneously. The results indicate that this two-step construction procedure can promote the full chondrogenic differentiation of BMSCs in vitro and the formation of mature ectopic cartilage by BMSCs in vivo.
  Cells Tissues Organs 04/2013;
• Article: Histomorphometric Study and Three-Dimensional Reconstruction of the Osteocyte Lacuno-Canalicular Network One Hour after Applying Tensile and Compressive Forces.

  Carola B Bozal, Luciana M Sánchez, Patricia M Mandalunis, Angela M Ubios
  [show abstract] [hide abstract]
  ABSTRACT: The occurrence of very early morphological changes in the osteocyte lacuno-canalicular network following application of tensile and/or compressive forces remains unknown to date. Thus, the aim of this study was to perform a morphological and morphometric evaluation of the changes in the three-dimensional structure of the lacuno-canalicular network and the osteocyte network of alveolar bone that take place very early after applying tensile and compressive forces in vivo, conducting static histomorphometry on bright-field microscopy and confocal laser scanning microscopy images. Our results showed that both the tensile and compressive forces induced early changes in osteocytes and their lacunae, which manifested as an increase in lacunar volume and changes in lacunar shape and orientation. An increase in canalicular width and a decrease in the width and an increase in the length of cytoplasmic processes were also observed. The morphological changes in the lacuno-canalicular and osteocyte networks that occur in vivo very early after application of tensile and compressive forces would be an indication of an increase in permeability within the system. Thus, both compressive and tensile forces would cause fluid displacement very soon after being applied; the latter would in turn rapidly activate alveolar bone osteocytes, enhancing transmission of the signals to the entire osteocyte network and the effector cells located at the bone surface.
  Cells Tissues Organs 04/2013;
• Article: Delivery of Mesenchymal Stem Cells in Chitosan/Collagen Microbeads for Orthopedic Tissue Repair.

  Limin Wang, Rameshwar R Rao, Jan P Stegemann
  [show abstract] [hide abstract]
  ABSTRACT: Microencapsulation and delivery of stem cells in biomaterials is a promising approach to repairing damaged tissue in a minimally invasive manner. An appropriate biomaterial niche can protect the embedded cells from the challenging environment in the host tissue, while also directing stem cell differentiation toward the desired lineage. In this study, adult human mesenchymal stem cells (MSC) were embedded in hydrogel microbeads consisting of chitosan and type I collagen using an emulsification process. Glyoxal and β-glycerophosphate were used as chemical and physical crosslinkers to initiate copolymerization of the matrix materials. The average size and size distribution of the microbeads could be varied by controlling the emulsification conditions. Spheroidal microbeads ranging in diameter from 82 ± 19 to 290 ± 78 µm were produced. Viability staining showed that MSC survived the encapsulation process (>90% viability) and spread inside the matrix over a period of 9 days in culture. Induced osteogenic differentiation using medium supplements showed that MSC increased gene expression of osterix and osteocalcin over time in culture, and also deposited calcium mineral. Bone sialoprotein and type I collagen gene expression were not affected. Delivery of microbeads through standard needles at practically relevant flow rates did not adversely affect cell viability, and microbeads could also be easily molded into prescribed geometries for delivery. Such protein-based microbeads may have utility in orthopedic tissue regeneration by allowing minimally invasive delivery of progenitor cells in microenvironments that are both protective and instructive.
  Cells Tissues Organs 04/2013;
• Article: Immunohistochemical Localization and Characterization of Putative Mesenchymal Stem Cell Markers in the Retinal Capillary Network of Rodents.

  Dierk Wittig, József Jászai, Denis Corbeil, Richard H W Funk
  [show abstract] [hide abstract]
  ABSTRACT: Perivascular cells of microvascular niches are the prime candidates for being a reservoire of mesenchymal stem cell (MSC)-like cells in many tissues and organs that could serve as a potential source of cells and a target of novel cell-based therapeutic approaches. In the present study, by utilising typical markers of pericytes (neuronal-glial antigen 2, NG2, a chondroitin sulphate proteoglycan) and those of MSCs (CD146 and CD105) and primitive pluripotent cells (sex-determining region Y-box 2, Sox2), the phenotypic traits and the distribution of murine and rat retinal perivascular cells were investigated in situ. Our findings indicate that retinal microvessels of juvenile rodents are highly covered by NG2-positive branching processes of pericytic (perivascular) cells that are less prominent in mature capillary networks of the adult retina. In the adult rodent retinal vascular bed, NG2 labeling is mainly confined to membranes of the cell body resulting in a pearl-chain-like distribution along the vessels. Retinal pericytes, which were identified by their morphology and NG2 expression, simultaneously express CD146. Furthermore, CD146-positive cells located at small arteriole-to-capillary branching points appear more intensely stained than elsewhere. Evidence for a differential expression of the two markers around capillaries that would hint at a clonal heterogeneity among pericytic cells, however, is lacking. In contrast, the expression of CD105 is exclusively restricted to vascular endothelial cells and Sox2 is detected neither in perivascular nor in endothelial cells. In dissociated retinal cultures, however, simultaneous expression of NG2 and CD105 was observed. Collectively, our data indicate that vascular wall resident retinal pericytes share some phenotypic features (i.e. CD146 expression) with archetypal MSCs, which is even more striking in dissociated retinal cultures (i.e. CD105 expression). These findings might have implications for the treatment of retinal pathologies.
  Cells Tissues Organs 04/2013;
• Article: Strain Magnitude-Dependent Calcific Marker Expression in Valvular and Vascular Cells.

  Zannatul Ferdous, Hanjoong Jo, Robert M Nerem
  [show abstract] [hide abstract]
  ABSTRACT: Aortic valve disease and atherosclerosis tend to coexist in most patients with cardiovascular disease; however, the causes and mechanisms of disease development in heart valves are still not clearly understood. To understand the contributions of the magnitude of cyclic strain (5% hypotension, 10% physiological, and 15% hypertension) in calcification, we used a model system of tissue-engineered collagen gels containing human aortic smooth muscle cells and human aortic valvular interstitial cells, both isolated from noncalcific heart transplant tissue. The compacted collagen gels were cultured in osteogenic media for 3 weeks in a custom-designed bioreactor and all assessments were performed at the end of the culture period. The major finding of this study is that bone morphogenic protein (BMP)-2 and BMP-4 and transforming growth factor-β1 mRNA expression significantly changed in response to the magnitude of applied strain in valvular cells, while the lowest expression was observed for the representative physiological strain. On the other hand, mRNA expression in vascular cells did not vary in response to the magnitude of strain. Regarding BMP-2 and BMP-4 protein expression determined by immunostaining, trends were similar to mRNA expression in vascular and valvular cells, where only valvular cells showed a varied protein expression depending on the magnitude of the strain applied. Our results suggest that cellular differences exist between vascular and valvular cells in their response to altered levels of cyclic strain during calcification. Copyright © 2013 S. Karger AG, Basel.
  Cells Tissues Organs 03/2013;
• Article: DiI Labeling of Human Adipose-Derived Stem Cells: Evaluation of DNA Damage, Toxicity and Functional Impairment.

  K Froelich, G Steussloff, K Schmidt, M Ramos Tirado, A Technau, A Scherzed, S Hackenberg, A Radeloff, R Hagen, N Kleinsasser
  [show abstract] [hide abstract]
  ABSTRACT: Introduction: Adipose tissue-derived stem cells (ASCs) have become the primary focus of tissue engineering research. To understand their functions and behavior in in vitro and in vivo models, it is mandatory to track the implanted cells and distinguish them from the resident or host cells. A common labeling method is the use of fluorescent dyes, e.g. the lipophilic carbocyanine dye, DiI. This study aimed to analyze potential DNA damage, toxicity and impairment of the functional properties of human ASCs after labeling with DiI. Methods: Cytotoxicity was measured using the MTT assay and DNA damage was determined by means of the comet assay. Potential apoptotic effects were determined using the annexin V-propidium iodide test. Differentiation potential was evaluated by trilineage differentiation procedures in labeled and unlabeled ASCs. Proliferation as well as migration capability was analyzed, and the duration and stability of DiI labeling in ASCs during in vitro expansion was observed over a period of 35 days. Results: DiI labeling did not cause genotoxic effects 15, or 30 min or 24 h after the labeling procedure, and there were no cytotoxic effects until 72 h afterwards. No impairment of proliferation or migration capability or differentiation potential could be determined. However, after 35 days, only 37% of labeled cells could be detected using the fluorescence microscope, which indicates a decrease in staining stability during in vitro expansion. Conclusion: DiI is a convenient method for ASCs labeling which causes no toxic effects and does not impair the proliferation, migration or differentiation potential of ASCs after the labeling procedure.
  Cells Tissues Organs 03/2013;
• Article: Alteration of the Fibrocartilaginous Nature of Scaffoldless Constructs Formed from Leporine Meniscus Cells and Chondrocytes through Manipulation of Culture and Processing Conditions.

  Daniel J Huey, Kyriacos A Athanasiou
  [show abstract] [hide abstract]
  ABSTRACT: Articular cartilage and the menisci of the knee joint lack intrinsic repair capacity; thus, injuries to these tissues result in eventual osteoarthrotic changes to the joint. Tissue engineering offers the potential to replace damaged cartilage and mitigate long-term debilitating changes to the joint. In an attempt to enhance the ability of adult articular chondrocytes (ACs) and meniscus cells (MCs) to produce robust scaffoldless neocartilage, the effects of passage number, cryopreservation, and redifferentiation prior to construct formation were studied. By increasing passage number, smaller donor biopsies could be used to generate sufficient cells for tissue engineering and, in this study, no detrimental effects were observed when employing passage-4 versus passage-3 cells. Cryopreservation of cells would enable the generation of a cell bank thus reducing lead time and enhancing consistency of cell-based therapies. Interestingly, cryopreservation was shown to enhance the biomechanical properties of the resultant self-assembled constructs. With regard to redifferentiation prior to construct formation, aggregate redifferentiation was shown to enhance the biochemical and biomechanical properties of self-assembled constructs. By increasing passaging number, cryopreserving cells, and applying aggregate redifferentiation prior to neotissue formation, the utility of ACs and MCs in tissue engineering can be enhanced.
  Cells Tissues Organs 02/2013;
• Article: Cholinergic Modulation of Epithelial Integrity in the Proximal Colon of Pigs.

  Szilvia Lesko, Ignaz Wessler, Gotthold Gäbel, Carola Petto, Helga Pfannkuche
  [show abstract] [hide abstract]
  ABSTRACT: Background: Within the gut, acetylcholine (ACh) is synthesised by enteric neurons, as well as by 'non-neuronal' epithelial cells. In studies of non-intestinal epithelia, ACh was involved in the generation of an intact epithelial barrier. In the present study, primary cultured porcine colonocytes were used to determine whether treatment with exogenous ACh or expression of endogenous epithelium-derived ACh may modulate epithelial tightness in the gastrointestinal tract. Methods: Piglet colonocytes were cultured on filter membranes for 8 days. The tightness of the growing epithelial cell layer was evaluated by measuring transepithelial electrical resistance (TEER). To determine whether ACh modulates the tightness of the cell layer, cells were treated with cholinergic, muscarinic and/or nicotinic agonists and antagonists. Choline acetyltransferase (ChAT), cholinergic receptors and ACh were determined by immunohistochemistry, RT-PCR and HPLC, respectively. Results: Application of the cholinergic agonist carbachol (10 µm) and the muscarinic agonist oxotremorine (10 µM) resulted in significantly higher TEER values compared to controls. The effect was completely inhibited by the muscarinic antagonist atropine. Application of atropine alone (without any agonist) led to significantly lower TEER values compared to controls. Synthesis of ACh by epithelial cells was proven by detection of muscarinic and nicotinic receptor mRNAs, immunohistochemical detection of ChAT and detection of ACh by HPLC. Conclusion: ACh is strongly involved in the regulation of epithelial tightness in the proximal colon of pigs via muscarinic pathways. Non-neuronal ACh seems to be of particular importance for epithelial cells forming a tight barrier.
  Cells Tissues Organs 02/2013;
• Article: Differentiation Patterns of Embryonic Stem Cells in Two- versus Three-Dimensional Culture.

  Emma T Pineda, Robert M Nerem, Tabassum Ahsan
  [show abstract] [hide abstract]
  ABSTRACT: Pluripotent stem cells are attractive candidates as a cell source for regenerative medicine and tissue engineering therapies. Current methods of differentiation result in low yields and impure populations of target phenotypes, with attempts for improved efficiency often comparing protocols that vary multiple parameters. This basic science study focused on a single variable to understand the effects of two-dimensional (2D) versus three-dimensional (3D) culture on directed differentiation. We compared mouse embryonic stem cells (ESCs) differentiated on collagen type I-coated surfaces (SLIDEs), embedded in collagen type I gels (GELs), and in suspension as embryoid bodies (EBs). For a systematic analysis in these studies, key parameters were kept identical to allow for direct comparison across culture configurations. We determined that all three configurations supported differentiation of ESCs and that the kinetics of differentiation differed greatly for cells cultured in 2D versus 3D. SLIDE cultures induced overall differentiation more quickly than 3D configurations, with earlier expression of cytoskeletal and extracellular matrix proteins. For 3D culture as GELs or EBs, cells clustered similarly, formed complex structures and promoted differentiation towards cardiovascular phenotypes. GEL culture, however, also allowed for contraction of the collagen matrix. For differentiation towards fibroblasts and smooth muscle cells which actively remodel their environment, GEL culture may be particularly beneficial. Overall, this study determined the effects of dimensionality on differentiation and helps in the rational design of protocols to generate phenotypes needed for tissue engineering and regenerative medicine.
  Cells Tissues Organs 02/2013;
• Article: Expression of Intermediate Filaments in the Balbiani Body and Ovarian Follicular Wall of the Japanese Quail (Coturnix japonica).

  Daniela Rodler, Fred Sinowatz
  [show abstract] [hide abstract]
  ABSTRACT: In the present study, we examined the distribution of 6 groups of intermediate filaments (IFs; cytokeratins, CKs, vimentin, synemin, desmin, glial fibrillary acidic protein and lamins) in oocytes and follicular walls of the Japanese quail (Coturnix japonica) during their development using immunohistochemical and ultrastructural techniques. A distinctly vimentin- and synemin-positive Balbiani body, which is a transient accumulation of organelles (mitochondria, Golgi complex and endoplasmic reticulum) that occurs in the oocytes of all vertebrates including birds, could be detected in the oocytes of primordial and early pre-vitellogenic follicles. In larger pre-vitellogenic follicles, the Balbiani body has dispersed and the positivity of the granulosa cells appeared to concentrate in the basal portion of their cytoplasm. Our ultrastructural data demonstrated that the matrix of the Balbiani body consists of fine IFs, which may play a role in the formation and dispersion of the Balbiani body. Of the CKs studied (panCK, CK5, CK7, CK8, CK14, CK15, CK18 and CK19), only CK5 showed a slight positive staining in both the theca externa and the Balbiani bodies of pre-vitellogenic oocytes. In conclusion, our data, which describe the changes in avian IF protein expression during folliculogenesis, suggest that the functions of the IFs (vimentin and synemin) of oocytes and follicular walls are not primarily mechanical but may be involved in the transient tethering of mitochondria in the area of the Balbiani body and in the gain of endocrine competence during the differentiation of granulosa cells.
  Cells Tissues Organs 02/2013;
• Article: Ultrastructure of Interstitial Cells in Subserosa of Human Colon.

  Jüri Johannes Rumessen, Jean-Marie Vanderwinden, Alastair Hansen, Thomas Horn
  [show abstract] [hide abstract]
  ABSTRACT: We studied the ultrastructure of interstitial cells in the subserosal/adventitial layer in human colon. An interstitial cell type with an ultrastructure intermediate between fibroblast-like cells (FLC) and interstitial cells of Cajal was identified (IC-SS). IC-SS had thin and flattened branching processes, most densely arranged close to the longitudinal muscle cells. Caveolae, bundles of intermediate filaments and membrane-associated dense bands, often with a patchy basal lamina, were characteristic. Secretory organelles (granular endoplasmic reticulum, smooth endoplasmic reticulum, Golgi, coated vesicles) were prominent. The IC-SS ultrastructure was different from that of FLC in the longitudinal layer, which had no caveolae and fewer intermediate filaments. Peg-and-socket junctions between IC-SS and between IC-SS and muscle cells were present, and IC-SS processes had close, selective appositions to muscle cells. Gap junctions were not observed. Small nerve bundles were abundant, but close contacts (<100 nm) between IC-SS or muscle cells and nerves were inconspicuous. Close appositions between IC-SS and mast cells were present; close relations to macrophages were not observed. The myoid features of IC-SS are thus more pronounced in comparison with FLC of other locations in the gastrointestinal muscle. The organization and ultrastructure may suggest a regulatory nature of IC-SS on the colonic muscle layers.
  Cells Tissues Organs 02/2013;
• Article: Characterization of Vitamin C-Induced Cell Sheets Formed from Primary and Immortalized Human Corneal Stromal Cells for Tissue Engineering Applications.

  Gesa Maria Grobe, Stephan Reichl
  [show abstract] [hide abstract]
  ABSTRACT: The purpose of this study was to compare the ability of primary human corneal stromal cells (HuFib cells) and SV40-immortalized human corneal keratocytes (HCK cells) to synthesize their own extracellular matrix induced by vitamin C supplementation. Therefore, the amount of collagen secreted and resulting biomechanical properties based on the culture duration were assessed. Cells were cultivated for several weeks with or without vitamin C. The amount of collagen secreted by the cells was quantified based on the culture duration. Cell viability was simultaneously determined via the MTT assay. Collagen secretion was increased as a result of vitamin C supplementation. The effect was stronger in primary cells. In addition, vitamin C supplementation had a positive effect on HuFib cell viability. Vitamin C supplementation induced the formation of detachable cell sheets in both primary and immortalized cells. The biomechanical properties of the sheets were evaluated using a static material testing machine, and the ultrastructure of the cell sheets was examined using scanning electron microscopy. The cell sheets formed from HuFib cells had a higher percentage of light transmission between 400 and 800 nm and were superior in terms of E-modulus and ultimate strength testing. Indirect immunofluorescence and Western blot confirmed the presence of collagen type I in the HuFib and HCK cell cultures. Stimulating secretion of the extracellular matrix in corneal stromal cells is a promising approach for corneal stroma reconstruction for tissue engineering applications.
  Cells Tissues Organs 02/2013;
• Article: Embryonic Stem Cells Facilitate the Isolation of Persistent Clonal Cardiovascular Progenitor Cell Lines and Leukemia Inhibitor Factor Maintains Their Self-Renewal and Myocardial Differentiation Potential in vitro.

  Julia Hoebaus, Philipp Heher, Teresa Gottschamel, Matthias Scheinast, Harmen Auner, Diana Walder, Marc Wiedner, Jasmin Taubenschmid, Maximilian Miksch, Thomas Sauer, Martina Schultheis, Alexey Kuzmenkin, Christian Seiser, Juergen Hescheler, Georg Weitzer
  [show abstract] [hide abstract]
  ABSTRACT: Compelling evidence for the existence of somatic stem cells in the heart of different mammalian species has been provided by numerous groups; however, so far it has not been possible to maintain these cells as self-renewing and phenotypically stable clonal cell lines in vitro. Thus, we sought to identify a surrogate stem cell niche for the isolation and persistent maintenance of stable clonal cardiovascular progenitor cell lines, enabling us to study the mechanism of self-renewal and differentiation in these cells. Using postnatal murine hearts with a selectable marker as the stem cell source and embryonic stem cells and leukemia inhibitory factor (LIF)-secreting fibroblasts as a surrogate niche, we succeeded in the isolation of stable clonal cardiovascular progenitor cell lines. These cell lines self-renew in an LIF-dependent manner. They express both stemness transcription factors Oct4, Sox2, and Nanog and early myocardial transcription factors Nkx2.5, GATA4, and Isl-1 at the same time. Upon LIF deprivation, they exclusively differentiate to functional cardiomyocytes and endothelial and smooth muscle cells, suggesting that these cells are mesodermal intermediates already committed to the cardiogenic lineage. Cardiovascular progenitor cell lines can be maintained for at least 149 passages over 7 years without phenotypic changes, in the presence of LIF-secreting fibroblasts. Isolation of wild-type cardiovascular progenitor cell lines from adolescent and old mice has finally demonstrated the general feasibility of this strategy for the isolation of phenotypically stable somatic stem cell lines.
  Cells Tissues Organs 01/2013;
• Article: The Effect of Testosterone on the Formation of Brain Structures.

  Barbora Filová, Daniela Ostatníková, Peter Celec, Július Hodosy
  [show abstract] [hide abstract]
  ABSTRACT: It has been confirmed in several studies that testosterone can significantly affect brain development. Following metabolism of this hormone by 5α-reductase to dihydrotestosterone, testosterone may act via androgen receptors, or after conversion by aromatase to estradiol, it may act via estrogen receptors. The parts of the brain which are changed under the influence of sex hormones are known as sexually dimorphic nuclei, especially in the preoptic area of the hypothalamus. Nevertheless, evidence suggests that testosterone also influences the structure of the hippocampus, specifically CA1 and CA3 areas of the hippocampus, as well as the amygdala. These brain areas are designed to convert information from short-term into long-term memory. In this review, we summarize the effects of testosterone on the organization of brain structures with respect to spatial cognitive abilities in small rodents.
  Cells Tissues Organs 01/2013;
• Article: Genetic Modification of Primate Amniotic Fluid-Derived Stem Cells Produces Pancreatic Progenitor Cells in vitro.

  Yu Zhou, David L Mack, J Koudy Williams, Sayed-Hadi Mirmalek-Sani, Emily Moorefield, So-Young Chun, Jun Wang, Diego Lorenzetti, Mark Furth, Anthony Atala, Shay Soker
  [show abstract] [hide abstract]
  ABSTRACT: Insulin therapy for type 1 diabetes does not prevent serious long-term complications including vascular disease, neuropathy, retinopathy and renal failure. Stem cells, including amniotic fluid-derived stem (AFS) cells - highly expansive, multipotent and nontumorigenic cells - could serve as an appropriate stem cell source for β-cell differentiation. In the current study we tested whether nonhuman primate (nhp)AFS cells ectopically expressing key pancreatic transcription factors were capable of differentiating into a β-cell-like cell phenotype in vitro. nhpAFS cells were obtained from Cynomolgus monkey amniotic fluid by immunomagnetic selection for a CD117 (c-kit)-positive population. RT-PCR for endodermal and pancreatic lineage-specific markers was performed on AFS cells after adenovirally transduced expression of PDX1, NGN3 and MAFA. Expression of MAFA was sufficient to induce insulin mRNA expression in nhpAFS cell lines, whereas a combination of MAFA, PDX1 and NGN3 further induced insulin expression, and also induced the expression of other important endocrine cell genes such as glucagon, NEUROD1, NKX2.2, ISL1 and PCSK2. Higher induction of these and other important pancreatic genes was achieved by growing the triply infected AFS cells in media supplemented with a combination of B27, betacellulin and nicotinamide, as well as culturing the cells on extracellular matrix-coated plates. The expression of pancreatic genes such as NEUROD1, glucagon and insulin progressively decreased with the decline of adenovirally expressed PDX1, NGN3 and MAFA. Together, these experiments suggest that forced expression of pancreatic transcription factors in primate AFS cells induces them towards the pancreatic lineage.
  Cells Tissues Organs 01/2013;
• Article: Simultaneous Visualization of Myosin Heavy Chain Isoforms in Single Muscle Sections.

  Samo Ribarič, Vita Cebašek
  [show abstract] [hide abstract]
  ABSTRACT: We developed a staining protocol that enables simultaneous visualization of myosin heavy chain (MHC) pure and hybrid muscle fiber types in rat skeletal muscle. Up to eight different muscle fiber types can be visualized in a single section of the rat extensor digitorum longus muscle, which contains all four adult MHC isoforms and shows plasticity during the denervation-reinnervation process. Triple immunofluorescent staining of MHC-1, MHC-2a and MHC-2b with primary antibodies BA-D5 (isotype IgG2b), SC-71 (isotype IgG1) and BF-F3 (isotype IgM) and with three fluorophore-labeled isotype-specific secondary antibodies displays different muscle fiber types in a merged image of red, green and blue channels, each in its own color. Immunoperoxidase staining with primary antibody 6H1 directed against MHC-2x can be additionally applied on the same tissue section to facilitate the identification of muscle fibers containing MHC-2x. Triple staining can also be used in combination with other staining procedures to derive more information about the number of capillaries or the oxidative potential of muscle fiber types. Simultaneous visualization of multiple fiber types in a single merged image enables economical use of muscle samples and provides simple and rapid identification of all fiber types that are present in rat limb muscles.
  Cells Tissues Organs 01/2013;
• Article: Analysis of Differentiation Potentials and Gene Expression Profiles of Mesenchymal Stem Cells Derived from Periodontal Ligament and Wharton's Jelly of the Umbilical Cord.

  Shi Yu, Jie Long, Jinhua Yu, Juan Du, Ping Ma, Yushi Ma, Dongmei Yang, Zhipeng Fan
  [show abstract] [hide abstract]
  ABSTRACT: PDLSC-mediated periodontal tissue regeneration is considered a promising method for periodontitis treatment, but the supply of PDLSCs is limited. As a potential alternative, WJCMSCs are readily available; however, there is a lack of evidence proving that WJCMSCs are suitable for periodontal tissue regeneration. In this study, we investigated the characteristics of WJCMSCs and PDLSCs. We found the osteo-/dentinogenic, adipogenic and chondrogenic differentiation potentials of PDLSCs were more powerful than those of WJCMSCs. Microarray analysis discovered that 903 genes were significantly down-regulated and 726 genes up-regulated in WJCMSCs compared with PDLSCs. Based on the microarray data, we found that several genes may be associated with MSCs characteristics. Further bioinformatic analysis identified that TGF-β and WNT signaling pathways, and several genes, including STAT5B and ITGA4, may play key roles in MSCs. Our results indicate that the differentiation potentials of WJCMSCs are far less than those of PDLSCs, and that unmodified WJCMSCs may not be good seeding cells for periodontal tissue regeneration. Our results also help to elucidate the differentiation mechanisms in MSCs and to find the key factors to prompt WJCMSC-mediated periodontal tissue regeneration.
  Cells Tissues Organs 12/2012;
• Article: Stromal Cell-Derived Factor-1 Enhances Wound Healing through Recruiting Bone Marrow-Derived Mesenchymal Stem Cells to the Wound Area and Promoting Neovascularization.

  Xiang Xu, Fangqiang Zhu, Meng Zhang, Dengfen Zeng, Donglin Luo, Guodong Liu, Wenhui Cui, Shali Wang, Wei Guo, Wei Xing, Huaping Liang, Lei Li, Xiaobing Fu, Jianxin Jiang, Hong Huang
  [show abstract] [hide abstract]
  ABSTRACT: Stromal cell-derived factor-1 (SDF-1) is a potent chemokine for bone marrow-derived stromal stem cells (BMSCs) that express CXCR4, the receptor for SDF-1. SDF-1 is considered to play an important role in the trafficking of BMSCs. We investigated the contribution of SDF-1 to the recruitment of BMSCs to the wound area and its promotion of wound repair and neovascularization. BMSCs were pretreated with or without anti-CXCR4 blocking antibody and combined with CM-DiI label, and injected via the tail vein into mice with full-thickness skin wounds on the dorsum. Simultaneously, anti-SDF-1 antibody was injected into local wounds in another group of mice. The results show that blockade of CXCR4 on either infused BMSCs or SDF-1 in the host wounds (1) dramatically impaired the number of infused BMSCs being recruited to the injured tissue, (2) reduced the expression of growth factors involved in the repair of injured tissue such as vascular endothelial growth factor, basic fibroblast growth factor and transforming growth factor beta 1, (3) decreased the resultant neovascularization, and (4) retarded wound healing. Taken together, the findings indicate that the SDF-1/CXCR4 signal pathway facilitates wound healing through augmenting BMSC recruitment to wound tissues, responsive secretion of growth factors by BMSCs and neovascularization in the wound area.
  Cells Tissues Organs 11/2012;