|  |  |  | | | | | TE-RegenMed-StemCell feed | | | | | | | | | | | | | | | | | | | Selective improvement of tumor necrosis factor capture in a cytokine hemoadsorption device using immobilized anti-tumor necrosis factor. J Biomed Mater Res B Appl Biomater. 2010 Nov 17; Authors: Dileo MV, Fisher JD, Burton BM, Federspiel WJ Sepsis is a harmful hyper-inflammatory state characterized by overproduction of cytokines. Removal of these cytokines using an extracorporeal device is a potential therapy for sepsis. We are developing a cytokine adsorption device (CAD) filled with porous polymer beads which efficiently depletes middle-molecular weight cytokines from a circulating solution. However, removal of one of our targeted cytokines, tumor necrosis factor (TNF), has been significantly lower than other smaller cytokines. We addressed this issue by incorporating anti-TNF antibodies on the outer surface of the beads. We demonstrated that covalent immobilization of anti-TNF increases overall TNF capture from 55% (using unmodified beads) to 69%. Passive adsorption increases TNF capture to over 99%. Beads containing adsorbed anti-TNF showed no significant loss in their ability to remove smaller cytokines, as tested using interleukin-6 (IL-6) and interleukin-10 (IL-10). We also detail a novel method for quantifying surface-bound ligand on a solid substrate. This assay enabled us to rapidly test several methods of antibody immobilization and their appropriate controls using dramatically fewer resources. These new adsorbed anti-TNF beads provide an additional level of control over a device which previously was restricted to nonspecific cytokine adsorption. This combined approach will continue to be optimized as more information becomes available about which cytokines play the most important role in sepsis. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010. PMID: 21086427 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Refined human artificial chromosome vectors for gene therapy and animal transgenesis. Gene Ther. 2010 Nov 18; Authors: Kazuki Y, Hoshiya H, Takiguchi M, Abe S, Iida Y, Osaki M, Katoh M, Hiratsuka M, Shirayoshi Y, Hiramatsu K, Ueno E, Kajitani N, Yoshino T, Kazuki K, Ishihara C, Takehara S, Tsuji S, Ejima F, Toyoda A, Sakaki Y, Larionov V, Kouprina N, Oshimura M Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.Gene Therapy advance online publication, 18 November 2010; doi:10.1038/gt.2010.147. PMID: 21085194 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Cardiogenesis From Human Embryonic Stem Cells. Circ J. 2010 Nov 12; Authors: Mignone JL, Kreutziger KL, Paige SL, Murry CE Over the past decade, the ability to culture and differentiate human embryonic stem cells (ESCs) has offered researchers a novel therapeutic that may, for the first time, repair regions of the damaged heart. Studies of cardiac development in lower organisms have led to identification of the transforming growth factor-β superfamily (eg, activin A and bone morphogenic protein 4) and the Wnt/β-catenin pathway as key inducers of mesoderm and cardiovascular differentiation. These factors act in a context-specific manner (eg, Wnt/β-catenin is required initially to form mesoderm but must be antagonized thereafter to make cardiac muscle). Different lines of ESCs produce different levels of agonists and antagonists for these pathways, but with careful optimization, highly enriched populations of immature cardiomyocytes can be generated. These cardiomyocytes survive transplantation to infarcted hearts of experimental animals, where they create new human myocardial tissue and improve heart function. The grafts generated by cell transplantation have been small, however, leading to an exploration of tissue engineering as an alternate strategy. Engineered tissue generated from preparations of human cardiomyocytes survives poorly after transplantation, most likely because of ischemia. Creation of pre-organized vascular networks in the tissue markedly enhances survival, with human capillaries anastomosed to the host coronary circulation. Thus, pathways controlling formation of the human cardiovascular system are emerging, yielding the building blocks for tissue regeneration that may address the root causes of heart failure. PMID: 21084757 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Drag-reducing polymers diminish near-wall concentration of platelets in microchannel blood flow. Biorheology. 2010 Jan 1;47(3):193-203 Authors: Zhao R, Marhefka JN, Antaki JF, Kameneva MV The accumulation of platelets near the blood vessel wall or artificial surface is an important factor in the cascade of events responsible for coagulation and/or thrombosis. In small blood vessels and flow channels this phenomenon has been attributed to the blood phase separation that creates a red blood cell (RBC)-poor layer near the wall. We hypothesized that blood soluble drag-reducing polymers (DRP), which were previously shown to lessen the near-wall RBC depletion layer in small channels, may consequently reduce the near-wall platelet excess. This study investigated the effects of DRP on the lateral distribution of platelet-sized fluorescent particles (diam. = 2 μm, 2.5 × 108/ml) in a glass square microchannel (width and depth = 100 μm). RBC suspensions in PBS were mixed with particles and driven through the microchannel at flow rates of 6-18 ml/h with and without added DRP (10 ppm of PEO, MW = 4500 kDa). Microscopic flow visualization revealed an elevated concentration of particles in the near-wall region for the control samples at all tested flow rates (between 2.4 ± 0.8 times at 6 ml/h and 3.3 ± 0.3 times at 18 ml/h). The addition of a minute concentration of DRP virtually eliminated the near-wall particle excess, effectively resulting in their even distribution across the channel, suggesting a potentially significant role of DRP in managing and mitigating thrombosis. PMID: 21084744 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Identification of Hematopoietic Stem Cell-Specific miRNAs Enables Gene Therapy of Globoid Cell Leukodystrophy. Sci Transl Med. 2010 Nov 17;2(58):58ra84 Authors: Gentner B, Visigalli I, Hiramatsu H, Lechman E, Ungari S, Giustacchini A, Schira G, Amendola M, Quattrini A, Martino S, Orlacchio A, Dick JE, Biffi A, Naldini L Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal storage disorder caused by mutations in the galactocerebrosidase (GALC) gene. Hematopoietic stem cell (HSC)-based gene therapy is being explored for GLD; however, we found that forced GALC expression was toxic to HSCs and early progenitors, highlighting the need for improved regulation of vector expression. We used a genetic reporter strategy based on lentiviral vectors to detect microRNA activity in hematopoietic cells at single-cell resolution. We report that miR-126 and miR-130a were expressed in HSCs and early progenitors from both mice and humans, but not in differentiated progeny. Moreover, repopulating HSCs could be purified solely on the basis of miRNA expression, providing a new method relevant for human HSC isolation. By incorporating miR-126 target sequences into a GALC-expressing vector, we suppressed GALC expression in HSCs while maintaining robust expression in mature hematopoietic cells. This approach protected HSCs from GALC toxicity and allowed successful treatment of a mouse GLD model, providing a rationale to explore HSC-based gene therapy for GLD. PMID: 21084719 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Identification and Characterization of Adenovirus Early Region 1B-Associated Protein 5 as a Surface Marker on Undifferentiated Human Embryonic Stem Cells. Stem Cells Dev. 2010 Nov 18; Authors: Choi HS, Kim WT, Kim H, Kim JJ, Ko JY, Lee SW, Jang YJ, Kim SJ, Lee MJ, Jung HS, Kzhyshkowska J, Um SJ, Lee MY, Lee SH, Kim CH, Ryu CJ Pluripotent human embryonic stem cells (hESCs) provide appropriate systems for developmental studies and prospective donor cell sources for regenerative medicine. Identification of surface markers specific to hESCs is a prerequisite for studying hESC biology, and can be used to generate clinical-level donor cell preparations that are free from tumorigenic undifferentiated hESCs. We previously reported the generation of monoclonal antibodies (MAbs) that specifically recognize hESC surface antigens using a decoy immunization strategy. In this study, we show that MAb 57-C11 recognizes a phosphorylated form of adenovirus early region 1B-associated protein 5 (E1B-AP5). E1B-AP5 is a nuclear RNA-binding protein, but we report that 57-C11-reactive E1B-AP5 is expressed on the surface of undifferentiated hESCs. In undifferentiated hESCs, 57-C11-reactive E1B-AP5 is localized to SSEA3-, SSEA4-, TRA-1-60-, TRA-1-81-, OCT4-, SOX2-, and NANOG-positive hESCs. In mixtures of undifferentiated hESCs and hESC-derived neurons, 57-C11 exclusively recognizes undifferentiated hESCs, and not hESC-derived neuronal cells. Furthermore, the expression of 57-C11-reactive E1B-AP5 decreases upon differentiation. Our results demonstrate that 57-C11-reactive E1B-AP5 is a novel surface molecule which is involved in the undifferentiated state of hESCs. As far as we know, this is the first report demonstrating that heterogeneous nuclear RNA-binding protein is expressed on the surface of undifferentiated hESCs. PMID: 21083500 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Rational design of murine secreted alkaline phosphatase for enhanced performance as a reporter gene in mouse gene therapy preclinical studies. Hum Gene Ther. 2010 Nov 17; Authors: Christou C, Parks R Many preclinical gene therapy studies utilize a reporter gene to evaluate vector design and performance in mouse models of human disease. Unfortunately, most commonly used reporter genes are immunogenic in mice, which confounds accurate evaluation of vector function. In previous studies, we showed that the murine secreted alkaline phosphatase (mSEAP) gene functions well as a simple and sensitive reporter gene in mice. In this study, we have used rational design to enhance mSEAP performance. The majority of native mSEAP remains attached to the outer surface of the cell through glycan phosphatidylinositol linkage; removal of the carboxy-terminal tail of mSEAP resulted in a dramatic enhancement in release of the protein into cell culture medium and into mouse plasma in whole animal experiments. We increased the heat stability of mSEAP through mutation of a key residue in the crown domain of the protein (H451E), thus allowing us to reduce endogenous, background AP activity through heat inactivation for enhanced sensitivity. We show that these alterations in mSEAP result in enhanced performance in tissue culture and mouse studies. Taken together, these data illustrate that mSEAP is a sensitive, non-immunogenic reporter for preclinical mouse studies. PMID: 21083426 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Acknowledgements. Regen Med. 2010 Nov;5(6):987 Authors: PMID: 21082896 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Regenerative medicine in Europe: global competition and innovation governance. Regen Med. 2010 Nov;5(6):971-985 Authors: Hogarth S, Salter B Leading European nations with strong biotech sectors, such as the UK and Germany, are investing heavily in regenerative medicine, seeking competitive advantage in this emerging sector. However, in the broader biopharmaceutical sector, the EU is outperformed by the USA on all metrics, reflecting longstanding problems: limited venture capital finance, a fragmented patent system, and relatively weak relations between academia and industry. The current global downturn has exacerbated these difficulties. The crisis comes at a time when the EU is reframing its approach to the governance of innovation and renewing its commitment to the goal of making Europe the leading player in the global knowledge economy. If the EU is to gain a competitive advantage in the regenerative medicine sector then it must coordinate a complex multilevel governance framework that encompasses the EU, member states and regional authorities. This article takes stock of Europe's current competitive position within the global bioeconomy, drawing on a variety of metrics in the three intersecting spheres of innovation governance: science, market and society. These data then provide a platform for reviewing the problems of innovation governance faced by the EU and the strategic choices that have to be confronted in the regenerative medicine sector. PMID: 21082895 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Mining the extracellular matrix for tissue engineering applications. Regen Med. 2010 Nov;5(6):961-970 Authors: Pradhan S, Farach-Carson MC Tissue engineering is a rapidly evolving interdisciplinary field that aims to regenerate new tissue to replace damaged tissues or organs. The extracellular matrix (ECM) of animal tissues is a complex mixture of macromolecules that play an essential instructional role in the development of tissues and organs. Therefore, tissue engineering approaches rely on the need to present the correct cues to cells, to guide them to maintain tissue-specific functions. Recent research efforts have allowed us to mine various sequences and motifs, which play key roles in these guidance functions, from the ECM. Small conserved peptide sequences mined from ECM molecules can mimic some of the biological functions of their large parent molecules. In addition, these peptide sequences can be linked to various biomaterial scaffolds that can provide the cells with mechanical support to ensure appropriate cell growth and aid the formation of the correct tissue structure. The tissue engineering field will continue to benefit from the advent of these mined ECM sequences which have two major advantages over recombinant ECM molecules: material consistency and scalability. PMID: 21082894 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Myc transcription factors: key regulators behind establishment and maintenance of pluripotency. Regen Med. 2010 Nov;5(6):947-959 Authors: Smith K, Dalton S The interplay between transcription factors, epigenetic modifiers, chromatin remodelers and miRNAs form the foundation of a complex regulatory network required for establishment and maintenance of the pluripotent state. Recent work indicates that Myc transcription factors are essential elements of this regulatory system. However, despite numerous studies, aspects of how Myc controls self-renewal and pluripotency remain obscure. This article reviews evidence supporting the placement of Myc as a central regulator of the pluripotent state and discusses possible mechanisms of action. PMID: 21082893 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Mesenchymal stem cells for the treatment of neurodegenerative disease. Regen Med. 2010 Nov;5(6):933-946 Authors: Joyce N, Annett G, Wirthlin L, Olson S, Bauer G, Nolta JA Mesenchymal stem cells/marrow stromal cells (MSCs) present a promising tool for cell therapy, and are currently being tested in US FDA-approved clinical trials for myocardial infarction, stroke, meniscus injury, limb ischemia, graft-versus-host disease and autoimmune disorders. They have been extensively tested and proven effective in preclinical studies for these and many other disorders. There is currently a great deal of interest in the use of MSCs to treat neurodegenerative diseases, in particular for those that are fatal and difficult to treat, such as Huntington's disease and amyotrophic lateral sclerosis. Proposed regenerative approaches to neurological diseases using MSCs include cell therapies in which cells are delivered via intracerebral or intrathecal injection. Upon transplantation into the brain, MSCs promote endogenous neuronal growth, decrease apoptosis, reduce levels of free radicals, encourage synaptic connection from damaged neurons and regulate inflammation, primarily through paracrine actions. MSCs transplanted into the brain have been demonstrated to promote functional recovery by producing trophic factors that induce survival and regeneration of host neurons. Therapies will capitalize on the innate trophic support from MSCs or on augmented growth factor support, such as delivering brain-derived neurotrophic factor or glial-derived neurotrophic factor into the brain to support injured neurons, using genetically engineered MSCs as the delivery vehicles. Clinical trials for MSC injection into the CNS to treat traumatic brain injury and stroke are currently ongoing. The current data in support of applying MSC-based cellular therapies to the treatment of neurodegenerative disorders are discussed. PMID: 21082892 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Skeletal myoblasts for cardiac repair. Regen Med. 2010 Nov;5(6):919-932 Authors: Durrani S, Konoplyannikov M, Ashraf M, Haider KH Stem cells provide an alternative curative intervention for the infarcted heart by compensating for the cardiomyocyte loss subsequent to myocardial injury. The presence of resident stem and progenitor cell populations in the heart, and nuclear reprogramming of somatic cells with genetic induction of pluripotency markers are the emerging new developments in stem cell-based regenerative medicine. However, until safety and feasibility of these cells are established by extensive experimentation in in vitro and in vivo experimental models, skeletal muscle-derived myoblasts, and bone marrow cells remain the most well-studied donor cell types for myocardial regeneration and repair. This article provides a critical review of skeletal myoblasts as donor cells for transplantation in the light of published experimental and clinical data, and indepth discussion of the advantages and disadvantages of skeletal myoblast-based therapeutic intervention for augmentation of myocardial function in the infarcted heart. Furthermore, strategies to overcome the problems of arrhythmogenicity and failure of the transplanted skeletal myoblasts to integrate with the host cardiomyocytes are discussed. PMID: 21082891 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Graft and host interactions following transplantation of neural stem cells to organotypic striatal cultures. Regen Med. 2010 Nov;5(6):901-917 Authors: Jäderstad LM, Jäderstad J, Herlenius E Aims: To investigate neural stem cell (NSC) interactions with striatal tissue following engraftment and the effects of growth factors. Materials & methods: Organotypic striatal slice cultures established from neonatal rats were used as an ex vivo model system. Survival, integration and differentiation of grafted NSCs from the previously generated C17.2 clone and host tissue response were investigated weekly for 28 days in vitro. To direct grafted cells towards a neuronal lineage, the role of growth factor supplementation and serum-free culturing conditions was studied using neural stem cells overexpressing neurotrophin-3 and Neurobasal™/B27 culture medium. Results: Following engraftment, NSCs gradually integrated morphologically and formed a part of the host 3D cytoarchitecture. Compared with nongrafted cultures, NSC engraftment increased the overall survival of the organotypic cultures by 39%, and reduced the host cell necrosis by more than 80% (from 2.1 ± 0.5% to 0.3 ± 0.1%), the host cell apoptosis by more than 60% (from 1.4 ± 0.4% to 0.5 ± 0.1%) and the reactions to mechanical trauma by 30% (estimated by nestin and glial fibrillary acidic protein immunohistochemistry) 7 days after engraftment. Elevated neurotrophin-3 production in NSCs and serum-free culturing conditions directed grafted NSCs towards a neuronal lineage as indicated by increased Tuj1 and Map2ab expression. However, this did not alter the survival of organotypic cultures. Conclusions: NSC engraftment was associated with rescue of imperiled host cells and reduction of host cell gliosis. These NSC effects were not related to the addition of growth factors, suggesting that other factors are involved in the supportive effects of the host following NSC engraftment. PMID: 21082890 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | An efficient method for generation of neural-like cells from adult human bone marrow-derived mesenchymal stem cells. Regen Med. 2010 Nov;5(6):891-900 Authors: Alexanian AR Background: Stem cell-based therapies to repair and replace lost neural cells are a highly promising treatment for CNS diseases. Bone marrow (BM)-derived mesenchymal stem cells (MSCs) have great potential as therapeutic agents against neurological maladies, since they have the ability to differentiate into neural phenotypes and can be readily isolated and expanded for autotransplantation with no risk of rejection. In our previous studies, we demonstrated that neural cells could be efficiently generated from mouse BM-derived MSCs by exposing cells to epigenetic modifiers and a neural environment. The main idea of this approach was the reactivation of pluripotency-associated genes in MSCs before exposing them to neural-inducing factors. Aim: In this study, we used a similar approach to efficiently generate neural cells from human BM-derived MSCs. Method: Neural induction was achieved by exposing cells simultaneously to inhibitors of DNA methylation and histone deacetylation, and pharmacological agents that increase cAMP levels. Results: The expression of pluripotency and neural markers was confirmed with immunocytochemistry, western blot and real-time PCR. ELISA studies showed that these neurally induced-human MSCs cells released the neurotrophic factors glial cell-derived neurotrophic factor and brain-derived neurotrophic factor. Conclusion: Human MSCs that are neurally modified with this methodology could be a useful source of cells for CNS repair and regeneration. PMID: 21082889 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Simulation of an in vitro niche environment that preserves conjunctival progenitor cells. Regen Med. 2010 Nov;5(6):877-889 Authors: Schrader S, Notara M, Tuft SJ, Beaconsfield M, Geerling G, Daniels JT Aim: To evaluate a serum-free system where mitotically active subconjunctival fibroblasts were co-cultured with conjunctival epithelial cells to mimic a niche environment for conjunctival progenitor cells. Methods: Human conjunctival epithelial cells were expanded in vitro and evaluated for their colony-forming efficiency and clonal ability. The cells were then transferred to a serum-free co-culture system and cultured in the presence of mitotically active subconjunctival fibroblasts (human conjunctival epithelial cells and human bulbar subconjunctival fibroblasts [HCEC-HCF]). Cells were evaluated by Ki67 staining, total colony-forming efficiency and the number of colonies with a surface area of more than 10 mm(2). The expression of putative progenitor cell markers p63α, ABCG2 and CK15, and the presence of MUC5AC- and periodic acid-Schiff-positive cells was compared with standard culture conditions (HCEC-3T3). Results: Conjunctival epithelial cells cultured under HCEC-HCF and HCEC-3T3 conditions demonstrated strong immunoreactivity to p63α and ABCG2. Co-localization of CK15 and p63α revealed a subpopulation of CK15-positive cells under HCEC-3T3 conditions compared with only a few CK15-positive cells found under HCEC-HCF conditions. MUC5AC- and periodic acid-Schiff-positive cells were much more common under HCEC-3T3 conditions than under HCEC-HCF conditions. These results were confirmed by reverse transcription-PCR. Cells in HCEC-HCF conditions demonstrated a significantly higher total colony-forming efficiency and a significantly higher percentage of colonies with holoclone-like morphology. Conclusions: The simulation of a niche environment in vitro by co-culturing mitotically active subconjunctival fibroblasts with conjunctival epithelial cells supports the maintenance of conjunctival cells with progenitor cell characteristics and therefore might be a useful tool to expand conjunctival epithelial progenitor cells in vitro for clinical use. PMID: 21082888 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Regenerative medicine in Brazil: small but innovative. Regen Med. 2010 Nov;5(6):863-876 Authors: McMahon DS, Singer PA, Daar AS, Thorsteinsdóttir H Aims: Although Brazil has received attention for conducting one of the world's largest stem cell clinical trials for heart disease, little has been published regarding Brazil's regenerative medicine (RM) sector. Here we present a comprehensive case study of RM in Brazil, including analysis of the current activity, the main motivations for engaging in RM and the remaining challenges to development in this field. Methods: Our case study is primarily based on semi-structured interviews with experts on RM in Brazil, including researchers, policymakers, clinicians, representatives of firms and regulators. Results: Driven by domestic health needs and strategic government support, Brazil is producing innovative RM research, particularly for clinical research in cardiology, orthopedics, diabetes and neurology. We describe the main RM research currently taking place in Brazil, as well as some of the economic, regulatory and policy events that have created a favorable environment for RM development. Brazilian RM researchers need to overcome several formidable challenges to research: research funding is inconsistent, importation of materials is costly and slow, and weak linkages between universities, hospitals and industry impede translational research. Conclusions: Although Brazil's contribution to the RM sector is small, its niche emphasis on clinical applications may become of global importance, particularly if Brazil manages to address the challenges currently impinging on RM innovation. PMID: 21082887 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Interview. Regen Med. 2010 Nov;5(6):857-861 Authors: Barker R Dr Roger A Barker is a University Reader in Clinical Neuroscience and Honorary Consultant in Neurology at the Addenbrooke's Hospital. He trained at Oxford and in London and has been in his current position for 10 years, having completed an MRC Clinician Scientist Fellowship prior to this. His main interests are in the neurodegenerative disorders of the nervous system, in particular Parkinson's disease and Huntington's disease. Dr Barker combines basic research investigating cell therapies to treat these conditions, with clinically based work on defining the natural history and heterogeneity of both diseases. He is a member of the CURE PD Research Advisory Panel and the MRC Stem Cell Liaison Committee. He is a member of the Regenerative Medicine editorial board and co-editor-in-chief of the journals ACNR and the Journal of Neurology. PMID: 21082886 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Research Highlights. Regen Med. 2010 Nov;5(6):853-856 Authors: Bates D PMID: 21082885 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Industry Update: Latest developments in stem cell research and regenerative medicine. Regen Med. 2010 Nov;5(6):847-851 Authors: Ilic D PMID: 21082884 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Tissue engineering for clinical applications. Biotechnol J. 2010 Nov 17; Authors: Bhatia SK Tissue engineering is increasingly being recognized as a beneficial means for lessening the global disease burden. One strategy of tissue engineering is to replace lost tissues or organs with polymeric scaffolds that contain specialized populations of living cells, with the goal of regenerating tissues to restore normal function. Typical constructs for tissue engineering employ biocompatible and degradable polymers, along with organ-specific and tissue-specific cells. Once implanted, the construct guides the growth and development of new tissues; the polymer scaffold degrades away to be replaced by healthy functioning tissue. The ideal biomaterial for tissue engineering not only defends against disease and supports weakened tissues or organs, it also provides the elements required for healing and repair, stimulates the body's intrinsic immunological and regenerative capacities, and seamlessly interacts with the living body. Tissue engineering has been investigated for virtually every organ system in the human body. This review describes the potential of tissue engineering to alleviate disease, as well as the latest advances in tissue regeneration. The discussion focuses on three specific clinical applications of tissue engineering: cardiac tissue regeneration for treatment of heart failure; nerve regeneration for treatment of stroke; and lung regeneration for treatment of chronic obstructive pulmonary disease. PMID: 21086454 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Controllable dual-release of dexamethasone and bovine serum albumin from PLGA/β-tricalcium phosphate composite scaffolds. J Biomed Mater Res B Appl Biomater. 2010 Nov 17; Authors: Yang Y, Tang G, Zhang H, Zhao Y, Yuan X, Wang M, Yuan X Localized dual-drug delivery from biodegradable scaffolds is an important strategy in tissue engineering. In this study, porous poly(L-lactide-co-glycolide) (PLGA)/β-tricalcium phosphate scaffolds containing both dexamethasone (Dex) and bovine serum albumin (BSA) were prepared by incorporating Dex-loaded and BSA-loaded microspheres into the scaffolds. PLGA microspheres containing Dex or BSA were prepared by spray-drying and double emulsion/solvent evaporation, respectively. In vitro release studies indicated that microspheres prepared from PLGA in 3:1 molar ratio of L-lactide/glycolide and 89.5 kDa relative molecular mass showed prolonged release profiles compared with those prepared from PLGA in 1:1 L-lactide/glycolide molar ratio and 30.5 kDa relative molecular mass. Additionally, introduction of poly(ethylene glycol) in the PLGA chain could improve the encapsulation efficiency and reduce the release rate. Based on the above results, controllable dual-release of Dex and BSA with relatively higher or lower release rate was achieved by incorporating Dex-loaded and BSA-loaded microspheres with different release profiles into the PLGA/β-tricalcium phosphate scaffolds. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010. PMID: 21086430 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Skingineering I: engineering porcine dermo-epidermal skin analogues for autologous transplantation in a large animal model. Pediatr Surg Int. 2010 Nov 18; Authors: Braziulis E, Biedermann T, Hartmann-Fritsch F, Schiestl C, Pontiggia L, Böttcher-Haberzeth S, Reichmann E, Meuli M BACKGROUND: Extended full thickness skin defects still represent a considerable therapeutic challenge as ideal strategies for definitive autologous coverage are still not available. Tissue engineering of whole skin represents an equally attractive and ambitious novel approach. We have recently shown that laboratory-grown human skin analogues with near normal skin anatomy can be successfully transplanted on immuno-incompetent rats. The goal of the present study was to engineer autologous porcine skin grafts for transplantation in a large animal model (pig study = intended preclinical study). MATERIALS AND METHODS: Skin biopsies were taken from the pig's abdomen. Epidermal keratinocytes and dermal fibroblasts were isolated and then expanded on culture dishes. Subsequently, highly concentrated collagen hydrogels and collagen/fibrin hydrogels respectively, both containing dermal fibroblasts, were prepared. Fibroblast survival, proliferation, and morphology were monitored using fluorescent labelling and laser scanning confocal microscopy. Finally, keratinocytes were seeded onto this dermal construct and allowed to proliferate. The resulting in vitro generated porcine skin substitutes were analysed by H&E staining and immunofluorescence. RESULTS: Dermal fibroblast proliferation and survival in pure collagen hydrogels was poor. Also, the cells were mainly round-shaped and they did not develop 3D-networks. In collagen/fibrin hydrogels, dermal fibroblast survival was significantly higher. The cells proliferated well, were spindle-shaped, and formed 3D-networks. When these latter dermal constructs were seeded with keratinocytes, a multilayered and partly stratified epidermis readily developed. CONCLUSION: This study provides compelling evidence that pig cell-derived skin analogues with near normal skin anatomy can be engineered in vitro. These tissue-engineered skin substitutes are needed to develop a large animal model to establish standardized autologous transplantation procedures for those studies that must be conducted before "skingineering" can eventually be clinically applied. PMID: 21085972 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Cardiogenesis From Human Embryonic Stem Cells. Circ J. 2010 Nov 12; Authors: Mignone JL, Kreutziger KL, Paige SL, Murry CE Over the past decade, the ability to culture and differentiate human embryonic stem cells (ESCs) has offered researchers a novel therapeutic that may, for the first time, repair regions of the damaged heart. Studies of cardiac development in lower organisms have led to identification of the transforming growth factor-β superfamily (eg, activin A and bone morphogenic protein 4) and the Wnt/β-catenin pathway as key inducers of mesoderm and cardiovascular differentiation. These factors act in a context-specific manner (eg, Wnt/β-catenin is required initially to form mesoderm but must be antagonized thereafter to make cardiac muscle). Different lines of ESCs produce different levels of agonists and antagonists for these pathways, but with careful optimization, highly enriched populations of immature cardiomyocytes can be generated. These cardiomyocytes survive transplantation to infarcted hearts of experimental animals, where they create new human myocardial tissue and improve heart function. The grafts generated by cell transplantation have been small, however, leading to an exploration of tissue engineering as an alternate strategy. Engineered tissue generated from preparations of human cardiomyocytes survives poorly after transplantation, most likely because of ischemia. Creation of pre-organized vascular networks in the tissue markedly enhances survival, with human capillaries anastomosed to the host coronary circulation. Thus, pathways controlling formation of the human cardiovascular system are emerging, yielding the building blocks for tissue regeneration that may address the root causes of heart failure. PMID: 21084757 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Efficacy of engineered FVIII-producing skeletal muscle enhanced by growth factor-releasing co-axial electrospun fibers. Biomaterials. 2010 Nov 15; Authors: Liao IC, Leong KW Co-axial electrospun fibers can offer both topographical and biochemical cues for tissue engineering applications. In this study, we demonstrate the sustained treatment of hemophilia through a non-viral, tissue engineering approach facilitated by growth factor-releasing co-axial electrospun fibers. FVIII-producing skeletal myotubes were first engineered on aligned electrospun fibers in vitro, followed by implantation in hemophilic mice with or without a layer of core-shell electrospun fibers designed to provide sustained delivery of angiogenic or lymphangiogenic growth factors, which serves to stimulate the lymphatic or vascular systems to enhance the FVIII transport from the implant site into systemic circulation. Upon subcutaneous implantation into hemophilic mice, the construct seamlessly integrated with the host tissue within one month, and specifically induced either vascular or lymphatic network infiltration in accordance with the growth factors released from the electrospun fibers. Engineered constructs that induced angiogenesis resulted in sustained elevation of plasma FVIII and significantly reduced blood coagulation time for at least 2-months. Biomaterials-assisted functional tissue engineering was shown in this study to offer protein replacement therapy for a genetic disorder such as hemophilia. PMID: 21084118 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Genipin-Cross-linked Electrospun Collagen Fibers. J Biomater Sci Polym Ed. 2010 Nov 17; Authors: Mekhail M, Wong KK, Padavan DT, Wu Y, O'Gorman DB, Wan W The fabrication of a fibrous collagen scaffold using electrospinning is desirable for tissue-engineering applications. Previously, electrospun collagen fibers were shown to be unstable in aqueous environments and, therefore, cross-linking is essential to stabilize these fibers. In this study genipin, a significantly less cytotoxic cross-linking agent compared to glutaraldehyde, was used to cross-link electrospun collagen fibers. The significance of this research lies in the use of four alcohol/water solvent systems to carry out the cross-linking reaction to maintain fibrous morphology during cross-linking. The four cross-linking conditions established were: (1) ethanol, 5% water and 3 days, (2) ethanol, 3% water and 5 days, (3) ethanol, 5% water and 5 days, and (4) isopropanol, 5% water and 5 days at a genipin concentration of 0.03 M. Results illustrated that genipin-cross-linking was effective in maintaining collagen fiber integrity in aqueous and cell culture media environments for up to 7 days. In addition, it was shown that fiber swelling could be controlled by using different cross-linking conditions. Swelling of cross-linked fibers immersed in Dulbecco's modified eagle medium for 7 days ranged from 0 to 59±4%. The cross-linked fibers were analyzed using scanning electron microscopy, Fourier transform infrared spectroscopy and ninhydrin assay. Finally, studies using primary human fibroblasts indicated good cell adhesion to these scaffolds. Overall, our data suggest that these stabilized fibrous collagen scaffolds provide a promising environment for tissue-regeneration applications. PMID: 21083975 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Bone Marrow Combined With Dental Bud Cells Promotes Tooth Regeneration in Miniature Pig Model. Artif Organs. 2010 Nov 18; Authors: Kuo TF, Lin HC, Yang KC, Lin FH, Chen MH, Wu CC, Chang HH Growth factors and morphogens secreted by bone marrow mesenchymal stem cells (BMSCs) of bone marrow fluid may promote tooth regeneration. Accordingly, a tissue engineering approach was utilized to develop an economical strategy for obtaining the growth factors and morphogens from BMSCs. Unerupted second molar tooth buds harvested from miniature pigs were cultured in vitro to obtain dental bud cells (DBCs). Bone marrow fluid, which contains BMSCs, was collected from the porcine mandible before operation. DBCs suspended in bone marrow fluid were seeded into a gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer scaffold (GCHT scaffold). The DBCs/bone marrow fluid/GCHT scaffold was autografted into the original alveolar sockets of the pigs. Radiographic and histological examinations were applied to identify the structure of regenerated tooth at 40 weeks postimplantation. The present results showed that one pig developed a complete tooth with crown, root, pulp, enamel, dentin, odontoblast, cementum, blood vessel, and periodontal ligament in indiscriminate shape. Three animals had an unerupted tooth that expressed dentin matrix protein-1, vascular endothelial growth factor, and osteopontin; and two other pigs also had dental-like structure with dentin tubules. This study reveals that DBCs adding bone marrow fluid and a suitable scaffold can promote the tooth regeneration in autogenic cell transplantation. PMID: 21083830 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Hydrogel-Electrospun Mesh Composites for Coronary Artery Bypass Grafts. Tissue Eng Part C Methods. 2010 Nov 18; Authors: McMahon RE, Qu XP, Bashur C, Goldstein A, Hahn M The aim of the current study was to investigate the potential of hydrogel-electrospun mesh hybrid scaffolds as coronary artery bypass grafts (CABGs). The circumferential mechanical properties of blood vessels modulate a broad range of phenomena, including vessel stress and mass transport, which, in turn, have a critical impact on cardiovascular function. Thus, CABGs should mimic key features of the nonlinear stress-strain behavior characteristic of coronary arteries. In native arteries, this "J-shaped" circumferential stress-strain curve arises primarily from initial load transfer to "low stiffness" elastic fibers followed by progressive recruitment and tensing of "higher stiffness" arterial collagen fibers. This nonlinear mechanical response is difficult to mimic with a single-component scaffold while simultaneously meeting the suture retention strength and tensile strength requirements of an implantable graft. For instance, although electrospun scaffolds have a number of advantages for arterial tissue engineering, including relatively high tensile strengths, tubular mesh constructs formed by conventional electrospinning methods do not generally display biphasic stress-strain curves. In the present work, we demonstrate that a multi-component scaffold comprised of polyurethane electrospun mesh layers (intended to be mimetic of arterial collagen fiber loading) bonded together by a fibrin hydrogel matrix (intended to be mimetic of arterial elastic fiber response) results in a composite construct which retains the high tensile strength and suture retention strength of electrospun mesh but which displays a "J-shaped" mechanical response similar to that of native coronary artery. Moreover, we show that these hybrid constructs support cell infiltration and extracellular matrix accumulation following 12 day exposure to cyclic distension. PMID: 21083438 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Microcarriers and Their Potential in Tissue Regeneration. Tissue Eng Part B Rev. 2010 Nov 18; Authors: Martin Y, Eldardiri M, Lawrence-Watt DJ, Sharpe JR Microcarriers are a versatile tool with applications across a wide range of disciplines within tissue engineering. Large numbers of cells of appropriate phenotypes are required in engineering the many different tissues of the body, and microcarriers facilitate not only the expansion of many cell types but also the investigation of cell behaviour in vitro. Microcarriers can also be used to directly deliver cells in vivo to repair and regenerate tissues. This review summarises and discusses the use of microcarriers in diverse applications of tissue repair, including bone, cartilage, skin, vascular, CNS, adipose tissue and liver repair. It also considers how microcarriers can be used to bulk-culture and deliver stem cells for tissue regeneration. Microcarriers thus have multidisciplinary use and advances in their use are of benefit to the entire tissue engineering field. PMID: 21083436 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Dormant Phase and Multinuclear Cells: Two Key Phenomena in Early Culture of Murine Bone Marrow Mesenchymal Stem Cells. Stem Cells Dev. 2010 Nov 17; Authors: Ahmadbeigi N, Soleimani M, Gheisari Y, Vasei M, Amanpour S, Bagherizadeh I, Shariati SA, Azadmanesh K, Amini S, Shafiee A, Arabkari V, Nardi NB Background: Special features of mesenchymal stem cells (MSC) have made them a popular tool in cell therapy and tissue engineering. Although mouse animal models and murine MSC are common tools in this field, our understanding of the effect of in vitro expansion on the behavior of these cells is poor and controversial. In addition, in comparison to human, isolation of MSC from mouse has been reported to be more difficult and some unexplained features such as heterogeneity and slow growth rate in the culture of these cells have been observed. Methods: Here we followed mouse bone marrow MSC for more than 1 year after isolation and examined the effect of expansion on changes in morphology, growth kinetics, plasticity and chromosomal structure during in vitro culture. Results: Shortly after isolation, the growth rate of the cells decreased until they stopped dividing and entered a dormant state. In this state the size of the cells increased and they became multinuclear. These large multinuclear cells then gave origin to small mononuclear cells which after a while resumed proliferation and could be expanded immortally. The immortal cells had diminished plasticity, were aneuploid but could not form tumors in nude mice. Discussion: These results suggest that mouse bone marrow MSC bear several modifications when expanded in vitro and therefore, the interpretation of the data obtained with these cells should be done more cautiously. PMID: 21083430 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Mining the extracellular matrix for tissue engineering applications. Regen Med. 2010 Nov;5(6):961-970 Authors: Pradhan S, Farach-Carson MC Tissue engineering is a rapidly evolving interdisciplinary field that aims to regenerate new tissue to replace damaged tissues or organs. The extracellular matrix (ECM) of animal tissues is a complex mixture of macromolecules that play an essential instructional role in the development of tissues and organs. Therefore, tissue engineering approaches rely on the need to present the correct cues to cells, to guide them to maintain tissue-specific functions. Recent research efforts have allowed us to mine various sequences and motifs, which play key roles in these guidance functions, from the ECM. Small conserved peptide sequences mined from ECM molecules can mimic some of the biological functions of their large parent molecules. In addition, these peptide sequences can be linked to various biomaterial scaffolds that can provide the cells with mechanical support to ensure appropriate cell growth and aid the formation of the correct tissue structure. The tissue engineering field will continue to benefit from the advent of these mined ECM sequences which have two major advantages over recombinant ECM molecules: material consistency and scalability. PMID: 21082894 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | A Bilayer Construct Controls Adipose Derived Stem Cell Differentiation into Endothelial Cells and Pericytes without Growth Factor Stimulation. Tissue Eng Part A. 2010 Nov 17; Authors: Suggs L, Natesan S, Zhang G, Baer DG, Walters TJ, Christy RJ This work describes the differentiation of ASC in a composite hydrogel for use as a vascularized dermal matrix. Our intent is that such a construct could be utilized following large surface area burn wounds that require extensive skin grafting and that are limited by the availability of uninjured sites. In order to develop engineered skin replacement constructs, we are pursuing the use of adipose-derived mesenchymal stem cells (ASC). We have established that a PEGylated fibrin gel can provide a suitable environment for the proliferation of ASC over a 7 day time-course. Furthermore, we have demonstrated that PEGylated fibrin can be used to control ASC differentiation towards vascular cell types including cells characteristic of both endothelial cells and pericytes. Gene analysis revealed strong upregulation of endothelial markers, CD31 and vWF, up to day 11 in culture with corresponding evidence of protein expression demonstrated by immunocytochemical staining. ASC were not only shown to express endothelial cell phenotype, but a subset of the ASC expressed pericyte markers. The NG2 gene was upregulated over 11 days with corresponding evidence for the cell surface marker. PDGFRβ gene expression decreased as the multipotent ASC differentiated up to day 7. Increased receptor expression at day 11 was likely due to the enhanced pericyte gene expression profile including increased NG2 expression. We have also demonstrated that when cells are loaded onto chitosan microspheres and sandwiched between the PEGylated fibrin gel and a type I collagen gel, the cells can migrate and proliferate within the two different gel types. The matrix composition dictates the lineage specification and is not driven by soluble factors. Utilizing an insoluble bilayer matrix to direct ASC differentiation will allow for the development of both vasculature as well as dermal connective tissue from a single population of ASC. This work underscores the importance of the extracellular matrix in controlling stem cell phenotype. It is our goal to develop layered composites as wound healing dressings or vascularized dermal equivalents that are not limited by nutrient diffusion. PMID: 21083419 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Cell Sheet Technology for Tissue Engineering: The Self-Assembly Approach Using Adipose-Derived Stromal Cells. Methods Mol Biol. 2011;702:429-441 Authors: Labbé B, Marceau-Fortier G, Fradette J In the past years, adipose tissue has spurred a wide interest, not only as a source of adult multipotent stem cells but also as a highly eligible tissue for reconstructive surgery procedures. Tissue engineering is one field of regenerative medicine progressing at great strides in part due to its important use of adipose-derived stem/stromal cells (ASCs). The development of diversified technologies combining ASCs with various biomaterials has lead to the reconstruction of numerous types of tissue-engineered substitutes such as bone, cartilage, and adipose tissues from rodent, porcine, or human ASCs. We have recently achieved the reconstruction of connective and adipose tissues composed entirely of cultured human ASCs and their secreted endogenous extracellular matrix components by a methodology known as the self-assembly approach of tissue engineering. The latter is based on the stimulation of ASCs to secrete and assemble matrix components in culture, leading to the production of cell sheets that can be manipulated and further assembled into thicker multilayer tissues. In this chapter, protocols to generate both reconstructed connective and adipocyte-containing tissues using the self-assembly approach are described in detail. The methods include amplification and cell banking of human ASCs, as well as culture protocols for the production of individual stromal and adipose sheets, which are the building blocks for the reconstruction of multilayered human connective and adipose tissues, respectively. PMID: 21082420 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Soft Tissue Reconstruction. Methods Mol Biol. 2011;702:395-400 Authors: Rubin JP, Marra KG The potential of adipose-derived stem cells (ASCs) in clinical applications of soft tissue regeneration is immense. This chapter discusses the isolation and characterization of human ASCs, expansion in vitro, and relevant in vivo models for adipose tissue engineering. PMID: 21082417 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Preservation Protocols for Human Adipose Tissue-Derived Adult Stem Cells. Methods Mol Biol. 2011;702:369-394 Authors: Devireddy R, Thirumala S The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. There are three primary storage techniques, freezing (cryopreservation), drying (anhydrobiosis), and freeze drying (lyophilization), each with its own advantages and disadvantages. Cryopreservation has shown the most promise but is a fairly complex process, necessitating the use of chemicals called cryoprotective agents (CPAs), freezing equipment, and obviously, storage in liquid nitrogen. Preservation by desiccation is an alternative that attempts to reproduce a naturally occurring preservative technique, namely, the phenomenon of anhydrobiosis and requires the use of high (and possibly, toxic) concentration of CPAs as well as disaccharides (sugars). Lyophilization works by first cryopreserving (freezing) the material and then desiccating (drying) it by the process of sublimation or the conversion of ice (solid) to water vapor (gas phase). The purpose of this chapter is to present a general overview of these storage techniques and the optimal protocols/results obtained in our laboratory for long-term storage of adult stem cells using freezing storage and drying storage. PMID: 21082416 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Adipogenic Differentiation of Human Adipose-Derived Stem Cells on 3D Silk Scaffolds. Methods Mol Biol. 2011;702:319-330 Authors: Choi JH, Bellas E, Vunjak-Novakovic G, Kaplan DL Current treatment modalities for soft tissue defects due to various pathologies and trauma include autologous grafting and the use of commercially available fillers. However, these treatment methods are associated with a number of limitations, such as donor site morbidity and volume loss over time. As such, improved therapeutic options are needed. Tissue engineering techniques offer novel solutions to these problems through development of bioactive tissue constructs that can regenerate adipose tissue with an appropriate structure and function. The recent advances in the derivation and characterization of hASCs have led to numerous studies of soft tissue reconstruction. In this chapter, we discuss methods in which our laboratory has used hASCs and silk scaffolds for adipose tissue engineering. The use of naturally occurring and clinically acceptable materials such as silk protein for tissue-engineering applications poses advantages with respect to biocompatibility and mechanical and biological properties. PMID: 21082412 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Osteogenic Differentiation Strategies for Adipose-Derived Mesenchymal Stem Cells. Methods Mol Biol. 2011;702:233-248 Authors: Kroeze RJ, Knippenberg M, Helder MN Adipose stem cell preparations, either obtained as a freshly isolated so-called stromal vascular fraction (SVF) or as cells cultured to homogeneity and then referred to as adipose stem cells (ASCs), have found widespread use in a broad variety of studies on tissue engineering and regenerative medicine applications, including bone repair.For newcomers within the field, but also for established research laboratories having up to 10 years of expertise in this research area, it may be convenient to strive for, and use consensus protocols (1) for studying the osteogenic differentiation potential of ASC preparations in vitro, and (2) for osteogenic induction regimes for in vivo implementation. To assist in achieving this goal, this chapter describes various step-by-step osteogenic differentiation protocols for adipose-derived stem cell populations (SVF as well as ASCs) currently applied within our laboratory, with particular emphasis on protocols aimed at intra-operative use. The protocols describe the use of inducing compounds, including the bone morphogenetic proteins (BMPs), 1,25-dihydroxyvitamin-D3, and polyamines, as well as methods and parameters for evaluating the level of differentiation achieved.We would appreciate receiving feedback on the protocols described; this will facilitate the development of consensus protocols, which in turn will allow better comparison of data sets generated by different research groups. This continuing standardization, which might be reported on at international meetings like those of IFATS ( http://www.IFATS.org ), might be of benefit for the whole ASC research community. PMID: 21082406 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Three-Dimensional Culture Systems to Induce Chondrogenesis of Adipose-Derived Stem Cells. Methods Mol Biol. 2011;702:201-217 Authors: Estes BT, Guilak F Stem cells can easily be harvested from adipose tissue in large numbers for use in tissue-engineering approaches for cartilage repair or regeneration. In this chapter, we describe in vitro tissue-engineering models that we have used in our laboratory for the chondrogenic induction of adipose-derived stem cells (ASC). In addition to the proper growth factor environment, chondrogenesis requires cells to be maintained in a rounded morphology in three-dimensional (3D) culture, and thus properties of the biomaterial scaffold also play a critical role in ASC differentiation. Histologic and immunohistologic methods for assessing chondrogenesis are also presented. In general, 10-12 weeks are required to assess ASC chondrogenesis in these model systems. PMID: 21082404 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Isolation of Human Adipose-Derived Stem Cells from Lipoaspirates. Methods Mol Biol. 2011;702:17-27 Authors: Yu G, Floyd ZE, Wu X, Halvorsen YD, Gimble JM Adipose tissue is as an abundant and accessible source of stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Here, we describe methods from our own laboratory and the literature for the isolation and expansion of adipose-derived stem cells (ASCs). We present a large-scale procedure suitable for processing >100-ml volumes of lipoaspirate tissue specimens by collagenase digestion and a related procedure suitable for processing adipose tissue aspirates without digestion. PMID: 21082391 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Isolation and Culture of Rhesus Adipose-Derived Stem Cells. Methods Mol Biol. 2011;702:3-16 Authors: Gagliardi C, Bunnell BA Adipose tissue is as an abundant and accessible source of stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Rhesus monkeys are physiologically and phylogenetically similar to humans, and they and their cells are valuable for biomedical research and evaluation of preclinical therapies. Here, we describe methods for the isolation, culture, and differentiation of rhesus adipose-derived stem cells (rASCs). PMID: 21082390 [PubMed - as supplied by publisher] | | | | | | | | | |  | |  | |  |
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