|  |  |  | | | | | TE-RegenMed-StemCell feed | | | | | | | | | | | | | | | | | | The California stem cell agency today officially notified the public for the first time about next month's sweeping review of its operations and said the public may attend for one hour at the beginning of two days of meetings.
CIRM's announcement was the first such notice that it has given the public via its Web site about the sessions that begin Oct. 13 at its San Francisco headquarters. The | | | | | | | | | | | | | | | | | | | | | | | | | Perfusion cell seeding on large porous PLA/calcium phosphate composite scaffolds in a perfusion bioreactor system under varying perfusion parameters. J Biomed Mater Res A. 2010 Sep 24; Authors: Koch MA, Vrij EJ, Engel E, Planell JA, Lacroix D A promising approach to bone tissue engineering lies in the use of perfusion bioreactors where cells are seeded and cultured on scaffolds under conditions of enhanced nutrient supply and removal of metabolic products. Fluid flow alterations can stimulate cell activity, making the engineering of tissue more efficient. Most bioreactor systems are used to culture cells on thin scaffold discs. In clinical use, however, bone substitutes of large dimensions are needed. In this study, MG63 osteoblast-like cells were seeded on large porous PLA/glass scaffolds with a custom developed perfusion bioreactor system. Cells were seeded by oscillating perfusion of cell suspension through the scaffolds. Applicable perfusion parameters for successful cell seeding were determined by varying fluid flow velocity and perfusion cycle number. After perfusion, cell seeding, the cell distribution, and cell seeding efficiency were determined. A fluid flow velocity of 5 mm/s had to be exceeded to achieve a uniform cell distribution throughout the scaffold interior. Cell seeding efficiencies of up to 50% were achieved. Results suggested that perfusion cycle number influenced cell seeding efficiency rather than fluid flow velocities. The cell seeding conducted is a promising basis for further long term cell culture studies in large porous scaffolds. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010. PMID: 20872752 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | TGF-β3 immobilized PLGA-gelatin/chondroitin sulfate/hyaluronic acid hybrid scaffold for cartilage regeneration. J Biomed Mater Res A. 2010 Sep 24; Authors: Fan H, Tao H, Wu Y, Hu Y, Yan Y, Luo Z Although most in vitro studies indicate that transforming growth factor β3 (TGF-β3) immobilized scaffold is suitable for cartilage tissue engineering, in vivo studies of implanting immobilized scaffold for chondral defect repair are still lacking. This study is to evaluate the potentials of TGF-β3 immobilized poly-(lactic-co-glycolic acid)-gelatin/chondroitin sulfate/hyaluronic acid (PLGA-GCH) hybrid scaffold for cartilage regeneration. The scaffold was fabricated by incorporating GCH micro-sponges into PLGA frameworks and then crosslinked with TGF-β3 to mimic natural cartilaginous extra cellular matrix (ECM). In vitro study demonstrated that MSCs proliferated vigorously and produced abundant ECM on scaffold. The immunohistochemistry staining and alcian blue staining confirmed the cartilaginous ECM production. The chondrogenic differentiation of MSCs on scaffold was proved by the expression of collagen II gene in mRNA and protein level. Then MSCs/TGF-β3 immobilized scaffolds were implanted in rabbits for chondral defects repair. After eight weeks, histological observation showed that differentiated MSCs were located in lacunae within the metachromatic staining matrix and exhibited typical chondrocyte morphology. Histological grading scores also indicated the congruent cartilage was regenerated. In conclusion, the TGF-β3 immobilized PLGA-GCH hybrid scaffold has great potential in constructing the tissue-engineered cartilage. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010. PMID: 20872747 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Gelatin microspheres crosslinked with genipin for local delivery of growth factors. J Tissue Eng Regen Med. 2010 Oct;4(7):514-523 Authors: Solorio L, Zwolinski C, Lund AW, Farrell MJ, Stegemann JP A main challenge in tissue engineering and regenerative medicine is achieving local and efficient growth factor release to guide cell function. Gelatin is a denatured form of collagen that cells can bind to and degrade through enzymatic action. In this study, gelatin microspheres were used to release bone morphogenetic protein 2 (BMP2). Spherical microparticles with diameters in the range of 2-6 µm were created by an emulsification process and were stabilized by crosslinking with the small molecule genipin. The degree of crosslinking was varied by controlling the incubation time in genipin solution. Loading rate studies, using soy bean trypsin inhibitor as a model protein, showed rapid protein uptake over the first 24 h, followed by a levelling off and then a further increase after approximately 3 days, as the microspheres swelled. Growth factor release studies using microspheres crosslinked to 20%, 50% and 80% of saturation and then loaded with BMP2 showed that higher degrees of crosslinking resulted in higher loading efficiency and slower protein release. After 24 h, the concentration profiles produced by all microsphere formulations were steady and approximately equal. Microspheres incubated with adult human mesenchymal stem cells accumulated preferentially on the cell surface, and degraded over time in culture. BMP2-loaded microspheres caused a three- to eight-fold increase in expression of the bone sialoprotein gene after 14 days in culture, with more crosslinked beads producing a greater effect. These results demonstrate that genipin-crosslinked gelatin microspheres can be used to deliver growth factors locally to cells in order to direct their function. Copyright © 2010 John Wiley & Sons, Ltd. PMID: 20872738 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Two staged laryngeal regeneration using tissue engineering techniques in a canine model. Laryngoscope. 2010 Oct;120(S3):S58 Authors: Kitani Y, Kanemaru SI, Hirano S, Ohno S, Kojima T, Ito J PMID: 20872503 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Engineering of a multi-functional extracellular matrix protein for immobilization to bone mineral hydroxyapatite. Biotechnol Lett. 2010 Sep 25; Authors: Kang W, Kim TI, Yun Y, Kim HW, Jang JH PURPOSE OF WORK: We have developed a strategy of designing multi-functional extracellular matrix proteins for functionalizing bone tissue engineering scaffolds and other biomedical surfaces to achieve improvements in bone grafting, bone repair and bone regeneration. We developed a novel extracellular matrix protein designed to have a cell adhesive RGD sequence derived from fibronectin and active functional unit of osteocalcin (OC) containing Ca(2+)-binding sites for immobilization to mineral component of bone, hydroxyapatite (HA). The fusion protein, designated FN(RGD)/OC, was expressed in Escherichia coli and purified with affinity chromatography using a His-tag. The resultant FN(RGD)/OC fusion protein preferentially bound to HA, promoted cell adhesive activity, and stimulated differentiation of MC3T3-E1 cell. PMID: 20872161 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | [Induction of human dental pulp stem cells of premolars into osteoblast-like cells by mineralizing culture medium.] Shanghai Kou Qiang Yi Xue. 2010 Aug;19(4):398-402 Authors: Zhang XY, Li XT, Zeng XL PURPOSE: To induce human dental pulp stem cells(HDPSCs) of premolars into osteoblast-like cells by mineralizing culture medium. METHODS: HDPSCs of premolars were induced by mineralizing culture medium for 28 days.The activity of ALP was examined by enzyme histochemical staining on the 7th and 28th day. The ability of mineralization of HDPSCs was detected by Alizarin-red staining on the 14th,21st and 28th day. The gene expression of ALP,DSPP,BSP,OCN on day 0,3,5,7,14,21 and 28 was evaluated by RT-PCR. The protein expression of BSP,OCN was analyzed by Western blotting and immunocytochemistry. RESULTS: ALP was expressed and mineralized nodules were observed after induction of HDPSCs. ALP mRNA was detected since the 3rd day, while no expression of DSPP mRNA. The gene of BSP,OCN was expressed from the 5th day and exhibited increment with time. The expression trend of BSP protein was consistent with BSP mRNA. OCN stained positive since the 5th day. CONCLUSIONS: HDPSCs of premolars could be induced to differentiate into osteoblast-like cells,which suggests that HDPSCs of premolars may be a potential source of cells used for bone-tissue engineering in the future. Supported by National Natural Science Foundation of China(Grant No.30772452). PMID: 20871957 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Controlling the Spatial Organization of Cells and Extracellular Matrix Proteins in Engineered Tissues Using Ultrasound Standing Wave Fields. Ultrasound Med Biol. 2010 Sep 24; Authors: Garvin KA, Hocking DC, Dalecki D Tissue engineering holds great potential for saving the lives of thousands of organ transplant patients who die each year while waiting for donor organs. However, to successfully fabricate tissues and organs in vitro, methodologies that recreate appropriate extracellular microenvironments to promote tissue regeneration are needed. In this study, we have developed an application of ultrasound standing wave field (USWF) technology to the field of tissue engineering. Acoustic radiation forces associated with USWF were used to noninvasively control the spatial distribution of mammalian cells and cell-bound extracellular matrix proteins within three-dimensional (3-D) collagen-based engineered tissues. Cells were suspended in unpolymerized collagen solutions and were exposed to a continuous wave USWF, generated using a 1 MHz source, for 15 min at room temperature. Collagen polymerization occurred during USWF exposure resulting in the formation of 3-D collagen gels with distinct bands of aggregated cells. The density of cell bands was dependent on both the initial cell concentration and the pressure amplitude of the USWF. Importantly, USWF exposure did not decrease cell viability but rather enhanced cell function. Alignment of cells into loosely clustered, planar cell bands significantly increased levels of cell-mediated collagen gel contraction and collagen fiber reorganization compared with sham-exposed samples with a homogeneous cell distribution. Additionally, the extracellular matrix protein, fibronectin, was localized to cell banded areas by binding the protein to the cell surface prior to USWF exposure. By controlling cell and extracellular organization, this application of USWF technology is a promising approach for engineering tissues in vitro. (E-mail: dalecki@bme.rochester.edu). PMID: 20870341 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Efficacy of hESC-MSCs in knitted silk-collagen scaffold for tendon tissue engineering and their roles. Biomaterials. 2010 Sep 25; Authors: Chen JL, Yin Z, Shen WL, Chen X, Heng BC, Zou XH, Ouyang HW Human embryonic stem cells (hESC) and their differentiated progenies are an attractive cell source for transplantation therapy and tissue engineering. Nevertheless, the utility of these cells for tendon tissue engineering has not yet been adequately explored. This study incorporated hESC-derived mesenchymal stem cells (hESC-MSCs) within a knitted silk-collagen sponge scaffold, and assessed the efficacy of this tissue-engineered construct in promoting tendon regeneration. When subjected to mechanical stimulation in vitro, hESC-MSCs exhibited tenocyte-like morphology and positively expressed tendon-related gene markers (e.g. Collagen type I & III, Epha4 and Scleraxis), as well as other mechano-sensory structures and molecules (cilia, integrins and myosin). In ectopic transplantation, the tissue-engineered tendon under in vivo mechanical stimulus displayed more regularly aligned cells and larger collagen fibers. This in turn resulted in enhanced tendon regeneration in situ, as evidenced by better histological scores and superior mechanical performance characteristics. Furthermore, cell labeling and extracellular matrix expression assays demonstrated that the transplanted hESC-MSCs not only contributed directly to tendon regeneration, but also exerted an environment-modifying effect on the implantation site in situ. Hence, tissue-engineered tendon can be successfully fabricated through seeding of hESC-MSCs within a knitted silk-collagen sponge scaffold followed by mechanical stimulation. PMID: 20870282 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Surface Modification with Fibrin/Hyaluronic acid Hydrogel on Solid-Free Form-based Scaffolds Followed by BMP-2 loading to Enhance Bone Regeneration. Bone. 2010 Sep 23; Authors: Kang SW, Kim JS, Park KS, Cha BH, Shim JH, Kim JY, Cho DW, Rhie JW, Lee SH Bone tissue engineering often requires a well-defined scaffold that is highly porous. The multi-head deposition system (MHDS), a form of solid freeform fabrication, has raised great interest as a method for fabricating scaffolds, since it yields a highly porous inter-connective structure without the use of cytotoxic solvents, and permits the diffusion of nutrients and oxygen. However, this method is not suitable for introducing proteins, as it includes a heating process. Hydrogels incorporated with protein coating of the scaffold surface could overcome this limitation of the MHDS. In present study, the surface of a scaffold fabricated using MHDS was coated with a mixture of fibrin and hyaluronic acid (HA) and used as a vehicle for delivery of both bone morphogenetic protein-2 (BMP-2) and adipose-derived stromal cells (ASCs). Fibrin/HA coating of the scaffold significantly enhanced initial cell attachment. Furthermore, the in vitro release of BMP-2 from fibrin/HA-coated scaffolds was sustained for three days and it stimulated the alkaline phosphatase activity of ASCs seeded on scaffold for 10 days more actively and continuously than did the soluble BMP-2 that was added to the culture media, not the scaffold itself. Importantly, the transplantation of undifferentiated ASCs inoculated on BMP-2-loaded, fibrin/HA-coated scaffolds resulted in more improved bone formation and mineralization than did the transplantation of undifferentiated ASCs seeded on uncoated scaffolds or on fibrin/HA-coated scaffolds without BMP-2, but containing BMP-2 in the cell suspension medium. These results show that BMP-2-loaded, fibrin/HA-coated scaffolds fabricated using MHDS may be useful in stimulating bone regeneration from undifferentiated ASCs in vivo. PMID: 20870047 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Self-assembly of three-dimensional DNA nanostructures and potential biological applications. Curr Opin Chem Biol. 2010 Sep 23; Authors: Lo PK, Metera KL, Sleiman HF A current challenge in nanoscience is to achieve controlled organization in three-dimensions, to provide tools for biophysics, molecular sensors, enzymatic cascades, drug delivery, tissue engineering, and device fabrication. DNA displays some of the most predictable and programmable interactions of any molecule, natural or synthetic. As a result, 3D-DNA nanostructures have emerged as promising tools for biology and materials science. In this review, strategies for 3D-DNA assembly are discussed. DNA cages, nanotubes, dendritic networks, and crystals are formed, with deliberate variation of their size, shape, persistence length, and porosities. They can exhibit dynamic character, allowing their selective switching with external stimuli. They can encapsulate and position materials into arbitrarily designed patterns, and show promise for numerous biological and materials applications. PMID: 20869905 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Fluorescent PLLA-nanodiamond composites for bone tissue engineering. Biomaterials. 2010 Sep 23; Authors: Zhang Q, Mochalin VN, Neitzel I, Knoke IY, Han J, Klug CA, Zhou JG, Lelkes PI, Gogotsi Y Superior mechanical properties, rich surface chemistry, and good biocompatibility of diamond nanoparticles make them attractive in biomaterial applications. A multifunctional fluorescent composite bone scaffold material has been produced utilizing a biodegradable polymer, poly(l-lactic acid) (PLLA), and octadecylamine-functionalized nanodiamond (ND-ODA). The uniform dispersion of nanoparticles in the polymer led to significant increase in hardness and Young's modulus of the composites. Addition of 10%wt of ND-ODA resulted in more than 200% increase in Young's modulus and 800% increase in hardness, bringing the nanocomposite properties close to that of the human cortical bone. Testing of ND-ODA/PLLA as a matrix supporting murine osteoblast (7F2) cell growth for up to 1 week showed that the addition of ND-ODA had no negative effects on cell proliferation. ND-ODA serves as a multifunctional additive providing improved mechanical properties, bright fluorescence, and options for drug loading and delivery via surface modification. Thus ND-ODA/PLLA composites open up numerous avenues for their use as components of bone scaffolds and smart surgical tools such as fixation devices in musculoskeletal tissue engineering and regenerative medicine. Intense fluorescence of ND-ODA/PLLA scaffolds can be used to monitor bone re-growth replacing the implant in vivo. PMID: 20869765 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | PLGA: Poloxamer blend micro- and nanoparticles as controlled release systems for synthetic proangiogenic factors. Eur J Pharm Sci. 2010 Sep 22; Authors: Parajó Y, d'Angelo I, Horváth A, Vantus T, György K, Welle A, Garcia-Fuentes M, Alonso MJ Tissue engineering is one of the most promising research areas in bioregenerative medicine. However, the restoration of biological functionalities by implanting bioartificially-engineered tissues is still highly limited because of their lack of vascular networks. The use of proangiogenic molecules delivered from a controlled release device is a promising strategy to induce tissue vascularization. Indeed, the controlled release system can enhance the therapeutic effect in vivo of many short half-life drugs, while circumventing the need for repeated administrations. In this work, PLGA:poloxamer blend based micro- and nanoparticles have been developed for the sustained delivery of a recently developed synthetic proangiogenic compound: SHA-2-22. Drug-loaded PLGA:poloxamer blend microparticles were prepared by an oil-in-oil solvent extraction/evaporation technique. Drug-loaded PLGA:poloxamer nanoparticles were prepared by a modified solvent diffusion technique. These drug carriers were characterized with regard to their physicochemical properties, morphology, drug encapsulation efficiency and release kinetics in vitro. The results show that by adjusting the formulation conditions, it is possible to obtain PLGA:Poloxamer micro- and nanoparticles with very high drug loadings, and with the capacity to release the active compound in a controlled way for up to one month. In vitro cell assays performed in an endothelial cell model confirmed the bioactivity of SHA-22-2 encapsulated in PLGA:poloxamer microparticles. PMID: 20869438 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Construction of a multi-layer skin substitute: Simultaneous cultivation of keratinocytes and preadipocytes on a dermal template. Burns. 2010 Sep 22; Authors: Keck M, Haluza D, Lumenta DB, Burjak S, Eisenbock B, Kamolz LP, Frey M BACKGROUND: After deep excision of burn eschar down to the muscle fascia patients have a non-reversible loss of the skin and underlying subcutaneous tissue. These patients would benefit from the development of a sufficient epidermal, dermal, and hypodermal tissue-engineered replacement provided by new technologies of tissue engineering. The aim of the present study was to determine whether keratinocytes and preadipocytes grow simultaneously on a bovine-derived collagen-elastin matrix under in vitro conditions in order to obtain a multi-layer skin substitute. METHODS: Human keratinocytes as well as human preadipocytes were seeded onto a collagen-elastin matrix (Matriderm(®)). Human preadipocytes were isolated from human subcutaneous adipose tissue and seeded onto the scaffold directly after isolation. Keratinocytes were isolated from fresh human split-thickness skin harvests and seeded onto the surface of the scaffold after 4 days of proliferation. Twenty one days after seeding all scaffolds were histologically evaluated, using hematoxylin eosin, immunohistochemical staining with collagen IV as well as immunofluorescence labeling with anti-Ki67 antibody and DAPI (4',6-diamidino-2-phenylindole). RESULTS: Simultaneous growth of keratinocytes and preadipocytes could be observed on the collagen-elastin matrix. Keratinocytes adhered well to the surface of the matrix and formed a confluent epidermis-like layer. Preadipocytes adhered well and also penetrated into the deeper layers of the matrix. CONCLUSION: In this study, a collagen-elastin matrix served as a suitable scaffold for simultaneous culturing of preadipocytes and keratinocytes. Preadipocytes showed good penetration into deeper layers of the scaffold, whereas keratinocytes attached only to the uppermost surface of the matrix. This approach towards a multi-layered skin substitute might be a useful asset for future reconstructive surgery. PMID: 20869175 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Adipose-derived stem cells modified genetically in vivo promote reconstruction of bone defects. Cytotherapy. 2010 Oct;12(6):831-840 Authors: Chen Q, Yang Z, Sun S, Huang H, Sun X, Wang Z, Zhang Y, Zhang B Abstract Aims. Bone defects induced by different causes are difficult to replace and repair. We sought to repair bone defects by transplantation of genetically modified adipose-derived stem cells (ADSC) and acellular bone matrix (ACBM). Methods. We constructed the biologic material of ACBM and evaluated its mechanical properties, general biocompatibility and biosafety. ADSC isolated from minipigs were cultured in vitro and then transfected by recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF) plasmids, respectively. Subsequently, the compounds of ACBM/ADSC/rhBMP-2/rhVEGF were used to repair bone defects of the ulna in minipigs. X-ray examination, radionuclide bone imaging and single photon emission computerized tomography (SPECT) were employed to monitor the therapeutic effects 2, 4, 8 and 12 weeks after operation. Histologic experiments were carried out 12 weeks after operation. Results. ACBM had no or weak antigenicity and the natural mechanical properties of ACBM were preserved. In vitro, ADSC transfected by rhBMP-2 and rhVEGF, respectively, could release rhBMP-2 or rhVEGF for at least 4 weeks. The X-ray, radionuclide bone imaging and SPECT examinations indicated that the compound of ACBM/ADSC/rhBMP-2/rhVEGF had better treatment effects on bone defects compared with the controls. Conclusions. Scaffolds, seed cells and bioactive factors are key points in tissue engineering. This research indicates that ACBM is a good biologic material for tissue repair, and ACBM/ADSC/rhBMP-2/rhVEGF can accelerate bone formation significantly. PMID: 20868218 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Autologous Valve Replacement - CD133<sup>+</sup> Stem Cell-plus-Fibrin Composite based Sprayed Cell Seeding for Intra-Operative
 Heart Valve Tissue Engineering. Tissue Eng Part C Methods. 2010 Sep 24; Authors: Kaminski A, Klopsch C, Mark P, Yerebakan C, Donndorf P, Gaebel R, Eisert F, Hasken S, Kreitz S, Glass A, Jockenhoevel S, Ma N, Kundt G, Liebold A, Steinhoff G Objective:
 The development of biological valve prostheses with lifetime native-like performance and optimal host engraftment is an ultimate goal of heart valve tissue-engineering. We describe a new concept for autologous graft coating based on a CD133<sup>+</sup>-stem-cells-plus-fibrin-complex (SC+F) processed from bone marrow and peripheral blood of a single patient. 
 Methods: 
 CD133<sup>+</sup>-SC (1x10<sup>6</sup> cells/ml) from human bone marrow and autologous fibrin (20mg/ml) were administered simultaneously via spray administration using the novel Vivostat Co-Delivery System. During static cultivation, SC+F performance was monitored for 20 days after delivery and compared to controls. For dynamic testing SC+F-composite was sprayed on a decellularized porcine pulmonary valve and transferred to a bioreactor under pulsatile flow conditions for 7 days.
 Results:
 Static cultivation of SC+F-composite induced significant improvements in stem cell proliferation as compared to controls. For dynamic testing, microscopic analyses on a smooth engineered heart valve surface detected homogenous distribution of stem cells. Ultrasonic analysis revealed native-like valve performance. Applied CD133<sup>+</sup> stem cells differentiated into endothelial-like cells positive for CD31 and VEGFR2 and engrafted the valve. However, occasional delamination was observed. 
 Conclusion:
 SC+F serves as an excellent autologous matrix for intra-operative tissue-engineering of valve prostheses promising optimal in-vivo integration. However, stability remains an issue.
 PMID: 20868207 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Promising Efficacy of E. coli rhBMP-2 in Collagen Sponge for Ectopic and Orthotopic Bone formation and Comparison with Mammalian Cell rhBMP-2. Tissue Eng Part A. 2010 Sep 24; Authors: Kim IS, Lee EN, Cho TH, Song YM, Hwang SJ, Oh JH, Park EK, Koo TY, Seo YK Non-glycosylated recombinant human bone morphogenetic protein (rhBMP)-2 prepared in E. coli (E. coli rhBMP-2) has recently been considered as an alternative to mammalian cell rhBMP-2. However, its clinical use is still limited due to lack of evidence for osteogenic activity comparable with that of mammalian cell rhBMP-2 via micro-computed tomography (μCT)-based analysis. Therefore, this study aimed to evaluate the ability of E. coli rhBMP-2 in absorbable collagen sponge (ACS) to form ectopic and orthotopic bone and to compare it to that of mammalian rhBMP-2. In vitro investigation was performed to study osteoblast differentiation of human mesenchymal stromal cells (hMSCs). Both types of rhBMP-2 enhanced proliferation, alkaline phosphatase activity, and matrix mineralization of hMSCs at similar levels. Similar tendencies were observed in µCT analysis, which determined bone volume, fractional bone volume, trabecular thickness, trabecular separation, bone mineral density and other characteristics. Histology from an in vivo osteoinductivity test and from a rat calvarial defect model demonstrated a dose-dependent increase in local bone formation. The E. coli rhBMP-2 group (5 μg) not only induced complete regeneration of an 8 mm critical-sized defect at four weeks, but also led to new bone with the same bone mineral density as normal bone at eight weeks, with the same efficiency as that of mammalian cell rhBMP-2 (5 μg). These uniformly favorable results provide evidence that the osteogenic activity of E. coli rhBMP-2 is not inferior to that of mammalian cell rhBMP-2 despite its low solubility and lack of gylcosylation. These results suggest that the application of E. coli rhBMP-2 in ACS may be a promising equivalent to mammalian cell rhBMP-2 in bone tissue engineering. PMID: 20868206 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Implantation of Cardiac Progenitor Cells using Self-assembling Peptide Improves Cardiac Function after Myocardial Infarction. J Mol Cell Cardiol. 2010 Sep 23; Authors: Tokunaga M, Liu ML, Nagai T, Iwanaga K, Matsuura K, Takahashi T, Kanda M, Kondo N, Wang P, Naito AT, Komuro I Implantation of various types of cells into the heart has been reported to be effective for heart failure, however, it is unknown what kinds of cells are most suitable for myocardial repair. To examine which types of cells are most effective, we injected cells-Puramatrix(TM) (PM) complex into the border area and overlaid the cell-PM patch on the myocardial infarction (MI) area. We compared cardiac morphology and function at 2 weeks after transplantation. Among clonal stem cell antigen-1 positive cardiac progenitors with PM (cSca-1/PM), bone marrow mononuclear cells with PM (BM/PM), skeletal myoblasts with PM (SM/PM), adipose tissue-derived mesenchymal cells with PM (AMC/PM), PM alone (PM), and non-treated MI group (MI), infarct area of cSca-1/PM was smaller than that of BM/PM, SM/PM, PM and MI. cSca-1/PM and AMC/PM attenuated ventricular enlargement and restored cardiac function in comparison with MI. Capillary density in the infarct area of cSca-1/PM was higher than that of other five groups. The percentage of TUNEL positive cardiomyocytes in the infarct area of cSca-1/PM was lower than that of MI and PM. cSca-1 secreted VEGF and some of them differentiated into cardiomyocytes and vascular smooth muscle cells. These results suggest that transplantation of cSca-1/PM most effectively prevents cardiac remodeling and dysfunction through angiogenesis, inhibition of apoptosis and myocardial regeneration. PMID: 20869968 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Early combined treatment with sildenafil and adipose-derived mesenchymal stem cells preserves heart function in rat dilated cardiomyopathy. J Transl Med. 2010 Sep 26;8(1):88 Authors: Lin YC, Leu S, Sun CK, Yen CH, Kao YH, Chang LT, Tsai TH, Chua S, Fu M, Ko SF, Wu CJ, Lee FY, Yip HK ABSTRACT: BBackground: We investigated whether early combined autologous adipose-derived mesenchymal stem cell (ADMSC) and sildenafil therapy offer an additive benefit in preserving heart function in rat dilated cardiomyopathy (DCM). METHODS: Adult Lewis rats (n=8 per group) were divided into group 1 (normal control), group 2 (saline-treated DCM rats), group 3 [2.0x106 ADMSC implanted into left ventricular (LV) myocardium of DCM rats], group 4 (DCM rats with sildenafil 30 mg/kg/day, orally), and group 5 (DCM rats with combined ADMSC-sildenafil). Treatment was started 1 week after DCM induction and the rats were sacrificed on day 90. RESULTS: The results showed that mitochondrial protein expressions of connexin43 and cytochrome-C were lowest in group 2, and lower in groups 3 and 4 than in group 5 (p<0.002). Conversely, oxidative index was highest in group 2, and also higher in groups 3 and 4 than in group 5 (p<0.0003). The mRNA expressions of interleukin (IL)-10, Gro/IL-8, endothelial nitric oxide synthase, and Bcl-2 were lowest in group 2, and lower in groups 3 and 4 compared with group 5 (p<0.0001). The mRNA expressions of matrix metalloproteinase-9, Bax, caspase 3, and stromal-cell derived factor-1alpha were highest in group 2, and higher in groups 3 and 4 than in group 5 (p<0.0004). Apoptosis and fibrosis in LV myocardium were most prominent in group 2 and higher in groups 3 and 4 than in group 5, whereas angiogenesis and LV ejection fraction were lowest in group 2 and lower in groups 3 and 4 than in group 5 (p<0.003). Conclusion: Early combined ADMSC/sildenafil is superior to either treatment alone in preserving LV function. PMID: 20868517 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Constrained watershed method to infer morphology of mammalian cells in microscopic images. Cytometry A. 2010 Sep 24; Authors: Kachouie NN, Fieguth P, Gamble D, Jervis E, Ezziane Z, Khademhosseini A Precise information about the size, shape, temporal dynamics, and spatial distribution of cells is beneficial for the understanding of cell behavior and may play a key role in drug development, regenerative medicine, and disease research. The traditional method of manual observation and measurement of cells from microscopic images is tedious, expensive, and time consuming. Thus, automated methods are in high demand, especially given the increasing quantity of cell data being collected. In this article, an automated method to measure cell morphology from microscopic images is proposed to outline the boundaries of individual hematopoietic stem cells (HSCs). The proposed method outlines the cell regions using a constrained watershed method which is derived as an inverse problem. The experimental results generated by applying the proposed method to different HSC image sequences showed robust performance to detect and segment individual and dividing cells. The performance of the proposed method for individual cell segmentation for single frame high-resolution images was more than 97%, and decreased slightly to 90% for low-resolution multiframe stitched images. © 2010 International Society for Advancement of Cytometry. PMID: 20872884 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | An Essential and Evolutionarily Conserved Role of Protein Arginine Methyltransferase 1 for Adult Intestinal Stem Cells During Postembryonic Development. Stem Cells. 2010 Sep 24; Authors: Matsuda H, Shi YB Organ-specific adult stem cells are critical for the homeostasis of adult organs and organ repair and regeneration. Unfortunately, it has been difficult to investigate the origins of these stem cells and the mechanisms of their development, especially in mammals. Intestinal remodeling during frog metamorphosis offers a unique opportunity for such studies. During the transition from an herbivorous tadpole to a carnivorous frog, the intestine is completely remodeled as the larval epithelial cells undergo apoptotic degeneration and are replaced by adult epithelial cells developed de novo. The entire metamorphic process is under the control of thyroid hormone, making it possible to control the development of the adult intestinal stem cells. We show here that the thyroid hormone receptor-coactivator PRMT1 (protein arginine methyltransferase 1) is upregulated in a small number of larval epithelial cells and that these cells dedifferentiate to become the adult stem cells. More importantly, transgenic overexpression of PRMT1 leads to increased adult stem cells in the intestine and conversely knocking down the expression of endogenous PRMT1 reduces the adult stem cell population. In addition, PRMT1 expression pattern during zebrafish and mouse development suggests that PRMT1 may play an evolutionally conserved role in the development of adult intestinal stem cells throughout vertebrates. These findings are not only important for the understanding of organ-specific adult stem cell development but also have important implications in regenerative medicine of the digestive tract. PMID: 20872846 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Gelatin microspheres crosslinked with genipin for local delivery of growth factors. J Tissue Eng Regen Med. 2010 Oct;4(7):514-523 Authors: Solorio L, Zwolinski C, Lund AW, Farrell MJ, Stegemann JP A main challenge in tissue engineering and regenerative medicine is achieving local and efficient growth factor release to guide cell function. Gelatin is a denatured form of collagen that cells can bind to and degrade through enzymatic action. In this study, gelatin microspheres were used to release bone morphogenetic protein 2 (BMP2). Spherical microparticles with diameters in the range of 2-6 µm were created by an emulsification process and were stabilized by crosslinking with the small molecule genipin. The degree of crosslinking was varied by controlling the incubation time in genipin solution. Loading rate studies, using soy bean trypsin inhibitor as a model protein, showed rapid protein uptake over the first 24 h, followed by a levelling off and then a further increase after approximately 3 days, as the microspheres swelled. Growth factor release studies using microspheres crosslinked to 20%, 50% and 80% of saturation and then loaded with BMP2 showed that higher degrees of crosslinking resulted in higher loading efficiency and slower protein release. After 24 h, the concentration profiles produced by all microsphere formulations were steady and approximately equal. Microspheres incubated with adult human mesenchymal stem cells accumulated preferentially on the cell surface, and degraded over time in culture. BMP2-loaded microspheres caused a three- to eight-fold increase in expression of the bone sialoprotein gene after 14 days in culture, with more crosslinked beads producing a greater effect. These results demonstrate that genipin-crosslinked gelatin microspheres can be used to deliver growth factors locally to cells in order to direct their function. Copyright © 2010 John Wiley & Sons, Ltd. PMID: 20872738 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Expansion of human mesenchymal stem cells in a fixed-bed bioreactor system based on non-porous glass carrier - Part A: Inoculation, cultivation, and cell harvest procedures. Int J Artif Organs. 2010 Aug;33(8):512-525 Authors: Weber C, Freimark D, Pörtner R, Pino-Grace P, Pohl S, Wallrapp C, Geigle P, Czermak P Human mesenchymal stem cells (hMSC) are a promising cell source for several applications of regenerative medicine. The cells employed are either autologous or allogenic; by using stem cell lines in particular, allogenic cells enable the production of therapeutic cell implants or tissue engineered implants in stock. For these purposes, the generally small initial cell number has to be increased; this requires the use of bioreactors, which offer controlled expansion of the hMSC under GMP-conform conditions. In this study, divided into part A and B, a fixed bed bioreactor system based on non-porous borosilicate glass spheres for the expansion of hMSC, demonstrated with the model cell line hMSCTERT, is introduced. The system offers convenient automation of the inoculation, cultivation, and harvesting procedures. Furthermore, the bioreactor has a simple design which favors its manufacturing as a disposable unit. Part A is focused on the inoculation, cultivation, and harvesting procedures. Cultivations were performed in lab scales up to a bed volume of 300 cm3. The study showed that the fixed bed system, based on 2-mm borosilicate glass spheres, as well as the inoculation, cultivation, and harvesting procedures are suitable for the expansion of hMSC with high yield and vitality. PMID: 20872346 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Risk factors and prevention of upper gastrointestinal hemorrhage after a coronary artery bypass grafting operation. Surg Today. 2010 Oct;40(10):931-935 Authors: Fan H, Zheng Z, Feng W, Wang W, Song Y, Lin Y, Hu S PURPOSE: Upper gastrointestinal (GI) hemorrhage is a serious complication of coronary artery bypass grafting (CABG). The aim of this study was to retrospectively investigate the risk factors and prevention of upper GI bleeding after CABG. METHODS: This study followed 6316 coronary patients who underwent CABG from 1998 to 2005. The perioperative parameters were recorded. Data from patients who experienced major gastrointestinal complications were analyzed retrospectively by univariate and multivariate analyses. RESULTS: The rate of upper GI bleeding was 0.3%. The overall mortality for patients complicated by upper GI bleeding was 47.6%. The risk factors for upper GI bleeding were age (odds ratio [OR] = 3.18, 95% confidence interval [CI] = 1.73-5.87, P < 0.01), extracorporeal circulation time (OR = 1.30, 95% CI = 1.11-1.52, P < 0.01) and the prophylactic use of omeprazole (OR = 0.19, 95% CI = 0.04-0.89, P < 0.05). The long-term mortality was significantly different between the upper GI bleeding group and the controls (P < 0.01). CONCLUSION: Advanced age and extracorporeal circulation time were risk factors for upper GI bleeding after CABG, and the prophylactic use of omeprazole decreased the rate of upper GI bleeding. PMID: 20872195 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | The effect of source animal age upon extracellular matrix scaffold properties. Biomaterials. 2010 Sep 24; Authors: Tottey S, Johnson SA, Crapo PM, Reing JE, Zhang L, Jiang H, Medberry CJ, Reines B, Badylak SF Biologic scaffold materials composed of mammalian extracellular matrix (ECM) are commonly used for the repair and reconstruction of injured tissues. An important, but unexplored variable of biologic scaffolds is the age of the animal from which the ECM is prepared. The objective of the present study was to compare the structural, mechanical, and compositional properties of small intestinal submucosa (SIS)-ECM harvested from pigs that differed only in age. Degradation product bioactivity of these ECM materials was also examined. Results showed that there are distinct differences in each of these variables among the various age source ECM scaffolds. The strength and growth factors content of ECM from 3-week-old animals is less than that of ECM harvested from 12, 26 or >52-week-old animals. The elastic modulus of SIS-ECM for 3 week and >52-week-old source was less than that of the 12 and 26 week source. Degradation products from all age source ECMs were chemotactic for perivascular stem cells, with the 12 week source the most potent, while the oldest source caused the greatest increase in proliferation. In summary, distinct differences exist in the mechanical, structural, and biologic properties of SIS-ECM harvested from different aged animals. PMID: 20870285 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Fluorescent PLLA-nanodiamond composites for bone tissue engineering. Biomaterials. 2010 Sep 23; Authors: Zhang Q, Mochalin VN, Neitzel I, Knoke IY, Han J, Klug CA, Zhou JG, Lelkes PI, Gogotsi Y Superior mechanical properties, rich surface chemistry, and good biocompatibility of diamond nanoparticles make them attractive in biomaterial applications. A multifunctional fluorescent composite bone scaffold material has been produced utilizing a biodegradable polymer, poly(l-lactic acid) (PLLA), and octadecylamine-functionalized nanodiamond (ND-ODA). The uniform dispersion of nanoparticles in the polymer led to significant increase in hardness and Young's modulus of the composites. Addition of 10%wt of ND-ODA resulted in more than 200% increase in Young's modulus and 800% increase in hardness, bringing the nanocomposite properties close to that of the human cortical bone. Testing of ND-ODA/PLLA as a matrix supporting murine osteoblast (7F2) cell growth for up to 1 week showed that the addition of ND-ODA had no negative effects on cell proliferation. ND-ODA serves as a multifunctional additive providing improved mechanical properties, bright fluorescence, and options for drug loading and delivery via surface modification. Thus ND-ODA/PLLA composites open up numerous avenues for their use as components of bone scaffolds and smart surgical tools such as fixation devices in musculoskeletal tissue engineering and regenerative medicine. Intense fluorescence of ND-ODA/PLLA scaffolds can be used to monitor bone re-growth replacing the implant in vivo. PMID: 20869765 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Selective Isolation and Differentiation of a Stromal Population of Human Embryonic Stem Cells with Osteogenic Potential. Bone. 2010 Sep 22; Authors: Harkness LM, Mahmood A, Ditzel N, Abdallah BM, Nygaard JV, Kassem M The derivation of osteogenic cells from human embryonic stem cells (hESC) has been hampered by the absence of easy and reproducible protocols. hESC grown in feeder-free conditions, often show a sub population of fibroblast-like, stromal cells growing between the colonies. Thus, we examined the possibility that these cells represent a population of stromal (mesenchymal) stem cells (hESC-stromal). Two in house derived hES cell lines (Odense3 and KMEB3) as well as an externally derived cell line (Hues8) were transitioned to feeder-free conditions. A sub population of fibroblast-like cells established between the hESC colonies were isolated by selective adherence to hyaluronic acid-coated plates (100μg/ml) and were characterized using a combination of FACS analysis and staining. The cells were CD44(+), CD29(+), CD73(+), CD166(+), CD146(+), and CD105(+); and, Oct4(-), CD34(-), CD45(-) and CXCR4(-). When cultured in osteogenic differentiation media, up regulation of osteoblastic lineage markers (DLX5, MSX2, RUNX2, SPARC, ALP, COL1a1, BGLAP, IBSP, DCN, LOX-L4) and production of in vitro mineralized matrix was detected. hESC-stromal cells loaded on a carrier and implanted either subcutaneously or in a critical size calvarial defect in immune deficient mice for 10 weeks, resulted in new bone formation and partial repair of the calvarial defect. In conclusion, hESC-stromal can be isolated from hESC cultures and represent a good source for obtaining cells with osteogenic differentiation potential suitable for regenerative medicine protocols. PMID: 20869473 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Acknowledgements. Regen Med. 2010 Sep;5(5):837 Authors: PMID: 20868337 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Advanced Tissue Sciences Inc.: learning from the past, a case study for regenerative medicine. Regen Med. 2010 Sep;5(5):823-835 Authors: Pangarkar N, Pharoah M, Nigam A, Hutmacher DW, Champ S On 31st March 2003 Advanced Tissue Sciences (ATS) was liquidated, with the effect that in excess of US$300 million of stakeholder financing was destroyed. Although successful in the development of breakthrough technologies in the regenerative medicine arena and the building of a substantial portfolio of patents, the company never made a profit. In this case study, ATS’ business strategy, market and competitive environment will be discussed in the context of the company’s historical development. A number of important lessons from this case are discussed. From a management perspective the most critical lesson is the importance of effective financial planning and management of costs, and in particular R&D costs, including the significant costs associated with clinical trials. In addition, a clear strategic focus is extremely important due to the significant resources required in the development of a new therapy. From an investor’s perspective the lessons to be gathered from the ATS case are related to the risk involved in investing in the field of regenerative medicine. This case indicates that both professional and private investors did not fully question the validity of ATS’ business strategy and financial forecasts. A clear and focused strategy based on long-term investor commitment is essential for the successful commercialization of regenerative medicine. PMID: 20868336 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Hydrogels in regenerative medicine: towards understanding structure–function relationships. Regen Med. 2010 Sep;5(5):809-821 Authors: Mather ML, Tomlins PE Hydrogels are playing an increasing role in regenerative medicine owing to their growing functional sophistication. This is being underpinned by advances in hydrogel synthesis, particularly through molecular and genetic engineering, which provide greater control of hydrogel structure and hence the emergence of hydrogels with new functionalities. In order to exploit this capability it is necessary to fully understand the relationship between hydrogel structure and function. This article will investigate the nature of hydrogel-structure relationships by: highlighting the key attributes of hydrogels that modulate their function, discussing the link between these attributes and hydrogel behavior, and identifying possible measurement strategies to elucidate them. PMID: 20868335 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Stimulating progress in regenerative medicine: improving the cloning and recovery of cryopreserved human pluripotent stem cells with ROCK inhibitors. Regen Med. 2010 Sep;5(5):799-807 Authors: Rizzino A Until recently, culturing human pluripotent stem cells was hampered by three prominent technical problems: a high degree of unwanted cellular stress when the cells are passaged, unacceptably low cloning efficiency and poor recovery of cryopreserved stocks. This review discusses recent developments that address these problems. A major focus of the review is the use of p160 Rho-associated coiled-coil kinase inhibitors for improving both the cloning efficiency and the recovery of cryopreserved human embryonic stem cells and human induced pluripotent stem cells. An underlying theme of this review is that the three problems have a common cause: separation of human pluripotent stem cells from one another increases cellular stress, which greatly decreases their viability unless special steps are taken. PMID: 20868334 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Understanding graft-induced dyskinesia. Regen Med. 2010 Sep;5(5):787-797 Authors: Lane E, Smith G The transplantation of dopaminergic cells for the treatment of symptoms of Parkinson’s disease has several hurdles to overcome before it can be considered a successful therapeutic approach. One issue is the development of abnormal involuntary movements in the absence of L-3,4-dihydroxyphenylalanine following the transplantation of fetal ventral mesencephalon identified in three different clinical trials. Hypotheses as to the cause of these movements include: the composition of the graft, size of the graft, L-3,4-dihydroxyphenylalanine exposure and L-3,4-dihydroxyphenylalanine-induced dyskinesia prior to transplantation and inflammatory responses in and around the graft. We evaluate the clinical evidence supporting these hypotheses and the preclinical models upon which experiments are being based to resolve them. PMID: 20868333 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Blood supply of the graft after cellular cardiomyoplasty. Regen Med. 2010 Sep;5(5):777-786 Authors: Reffelmann T, Kloner RA Cellular cardiomyoplasty is under extensive investigation as a potential therapeutic strategy after myocardial infarction, in congestive heart failure and chronic ischemic heart disease. Various cell sources and techniques for transplantation have been studied in animal models of cardiac disease. The initial goal of replacing myocardial scar tissue by vital myocardial cells, integrated into the host, simultaneously beating and contributing to systolic force, has not yet been accomplished. However, most experimental models provided evidence for enhanced vascularization after cell transplantation. In some investigations, neovascularization was also shown to be accompanied by increased myocardial perfusion. Mechanisms by which vascularization occurs have not been fully elucidated: either the transplanted cells provide an angiogenic stimulus, involving various paracrine or hormone-like factors, which induces the formation of a new vasculature or, depending on the source of transplanted cells, the cells incorporate into the vascular network after proliferation and differentiation. This review summarizes research that specifically studied the occurrence, magnitude and mechanisms of enhanced myocardial blood supply after cellular cardiomyoplasty. PMID: 20868332 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Cardiac regeneration using human embryonic stem cells: producing cells for future therapy. Regen Med. 2010 Sep;5(5):763-775 Authors: Wong SS, Bernstein HS Directed differentiation of human embryonic stem cells (hESCs) has generated much interest in the field of regenerative medicine. Because of their ability to differentiate into any cell type in the body, hESCs offer a novel therapeutic paradigm for myocardial repair by furnishing a supply of cardiomyocytes (CMs) that would ultimately restore normal myocardial function when delivered to the damaged heart. Spontaneous CM differentiation of hESCs is an inefficient process that yields very low numbers of CMs. In addition, it is not clear that fully differentiated CMs provide the benefits sought from cell transplantation. The need for new methods of directed differentiation of hESCs into functional CMs and cardiac progenitors has led to an explosion of research utilizing chemical, genetic, epigenetic and lineage selection strategies to direct cardiac differentiation and enrich populations of cardiac cells for therapeutic use. Here, we review these approaches and highlight their increasingly important roles in stem cell biology and cardiac regenerative medicine. PMID: 20868331 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Similarly derived and cultured hESC lines show variation in their developmental potential towards neuronal cells in long-term culture. Regen Med. 2010 Sep;5(5):749-762 Authors: Lappalainen RS, Salom X00e4 Ki M, Yl X00e4-Outinen L, Heikkil X00e4 TJ, Hyttinen JA, Pihlajam X00e4 Ki H, Suuronen R, Skottman H, Narkilahti S Background: Human embryonic stem cells (hESCs) can differentiate into any human cell type, including CNS cells, and thus have high potential in regenerative medicine. Several protocols exist for neuronal differentiation of hESCs, which do not necessarily work for all hESC lines. Materials & methods: We tested the differentiation capacity of four similarly derived and cultured hESC lines (HS181, HS360, HS362 and HS401) in suspension culture in relatively simple neural differentiation medium for up to 20 weeks. Results: All the hESC lines differentiated into neuronal cells, but in a line-dependent manner. Using our method, the HS181- and HS360-derived neurospheres differentiated in vitro into pure neuronal cell populations within 6 weeks, whereas HS362 and HS401 reached their peak of differentiation in 12 weeks, but never produced pure neuronal cell populations using the present method. The withdrawal of FGF from suspension culture increased the in vitro differentiation potential. The hESC-derived neurospheres formed functional neuronal networks when replated on a microelectrode array and responded as expected to pharmacologic modulation. Conclusion: Simple neurosphere culture is a suitable method for producing hESC-derived neuronal cells that can form functional neuronal networks from a number of hESC lines. The variation in the differentiation potential of hESC lines into neuronal cells must be carefully considered by those comparing various differentiation methods and designing transplantation therapies for neuronal disorders. PMID: 20868330 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Skeletal muscle fibrosis: the effect of stromal-derived factor-1α-loaded collagen scaffolds. Regen Med. 2010 Sep;5(5):737-747 Authors: Grefte S, Kuijpers-Jagtman AM, Torensma R, Von den Hoff JW Aim: To develop a model for muscle fibrosis based on full-thickness muscle defects, and to evaluate the effects of implanted stromal-derived factor (SDF)-1α-loaded collagen scaffolds. Methods: Full-thickness defects 2 mm in diameter were made in the musculus soleus of 48 rats and either left alone or filled with SDF-1α-loaded collagen scaffolds. At 3, 10, 28 and 56 days postsurgery, muscles were analyzed for collagen deposition, satellite cells, myofibroblasts and macrophages. Results: A significant amount of collagen-rich fibrotic tissue was formed, which persisted over time. Increased numbers of satellite cells were present around, but not within, the wounds. Satellite cells were further upregulated in regenerating tissue when SDF-1α-loaded collagen scaffolds were implanted. The scaffolds also attracted macrophages, but collagen deposition and myofibroblast numbers were not affected. Conclusion: Persistent muscle fibrosis is induced by full-thickness defects 2 mm in diameter. SDF-1α-loaded collagen scaffolds accelerated muscle regeneration around the wounds, but did not reduce muscle fibrosis. PMID: 20868329 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Novel 3D culture system with similarities to the human heart for studies of the cardiac stem cell niche. Regen Med. 2010 Sep;5(5):725-736 Authors: Jonsson M, Henriksson HB, Hagman M, Kajic K, Lindahl A, Jeppsson A, Berggren HX, Asp J Aims: The aim of this study was to develop a 3D culture system with similarities to the human heart, which was suitable for studies of adult cardiac stem or progenitor cells. Materials & methods: Dissociated cells from human cardiac biopsies were placed in high-density pellet cultures and cultured for up to 6 weeks. Gene and protein expressions, analyzed by quantitative real-time PCR and immunohistochemistry, and morphology were studied in early and late pellets. Results: Cells cultured in the 3D model showed similarities to human cardiac tissue. Moreover, markers for cardiac stem and progenitor cells were also detected after 6 weeks of culture, in addition to markers for signaling pathways active in stem cell niche regulation. Conclusions: The described 3D culture model could be a valuable tool when studying the influence of different compounds on proliferation and differentiation processes in cardiac stem or progenitor cells in cardiac regenerative research. PMID: 20868328 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Shear stress induces osteogenic differentiation of human mesenchymal stem cells. Regen Med. 2010 Sep;5(5):713-724 Authors: Yourek G, McCormick SM, Mao JJ, Reilly GC Aim: To determine whether fluid flow-induced shear stress affects the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) into osteogenic cells. Materials & methods: hMSCs cultured with or without osteogenic differentiation medium were exposed to fluid flow-induced shear stress and analyzed for alkaline phosphatase activity and expression of osteogenic genes. Results: Immediately following shear stress, alkaline phosphatase activity in osteogenic medium was significantly increased. At days 4 and 8 of culture the mRNA expression of bone morphogenetic protein-2 and osteopontin was significantly higher in hMSCs subjected to shear stress than those cultured in static conditions. However, hMSCs cultured in osteogenic differentiation medium were less responsive in gene expression of alkaline phosphatase and bone morphogenetic protein-2. Conclusion: These data demonstrate that shear stress stimulates hMSCs towards an osteoblastic phenotype in the absence of chemical induction, suggesting that certain mechanical stresses may serve as an alternative to chemical stimulation of stem cell differentiation. PMID: 20868327 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med. 2010 Sep;5(5):701-711 Authors: Strassburg S, Richardson SM, Freemont AJ, Hoyland JA Aims: While mesenchymal stem cell (MSC)-based therapies for repair of the degenerate intervertebral disc (IVD) have been proposed, the interaction of MSCs with cells of the degenerate IVD has not been fully investigated. Therefore, it is unclear whether implanted MSCs would differentiate into nucleus pulposus (NP) cells and/or stimulate endogenous NP cells. Here, we investigate the differences in interaction between human MSCs and NP cells from both nondegenerate and degenerate discs during in vitro co-culture with direct cell–cell contact. Materials & methods : Human bone marrow-derived MSCs (labeled with CFDA) were co-cultured with direct cell–cell contact in monolayer with NP cells obtained from nondegenerate or degenerate human NP tissue from lumbar IVDs at 50:50 ratios for 7 days. Differentiation of MSCs and changes of matrix-associated genes in NP cells were assessed by quantitative real-time PCR. Results: MSCs differentiated to an NP-like phenotype following direct co-culture with both nondegenerate and degenerate NP, as shown by a significant upregulation of SOX9, type VI collagen, aggrecan and versican gene expression together with a simultaneous upregulation of CDMP-1, TGF-β1, IGF-1 and CTGF. Direct co-culture of normal NP cells with MSCs had no effect on the phenotype of normal NP cells, while co-culture with degenerate NP cells resulted in enhanced matrix gene expression in degenerate NP cells, accompanied by increases in both TGF-β and CDMP-1 gene expression. Conclusion: Importantly for MSC-based therapies for repair of the degenerate IVD, these data suggest that cellular interactions between MSCs and degenerate NP cells may both stimulate MSC differentiation to an NP-like phenotype and also stimulate the endogenous NP cell population to regain a nondegenerate phenotype and consequently enhance matrix synthesis for self-repair. PMID: 20868326 [PubMed - as supplied by publisher] | | | | | | | | | |  | |  | |  |
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