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1.
PLoS One ; 8(12): e81728, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24339957

RESUMO

BACKGROUND: High proliferative and differentiation capacity renders embryonic stem cells (ESCs) a promising cell source for tissue engineering and cell-based therapies. Harnessing their potential, however, requires well-designed, efficient and reproducible expansion and differentiation protocols as well as avoiding hazardous by-products, such as teratoma formation. Traditional, standard culture methodologies are fragmented and limited in their fed-batch feeding strategies that afford a sub-optimal environment for cellular metabolism. Herein, we investigate the impact of metabolic stress as a result of inefficient feeding utilizing a novel perfusion bioreactor and a mathematical model to achieve bioprocess improvement. METHODOLOGY/PRINCIPAL FINDINGS: To characterize nutritional requirements, the expansion of undifferentiated murine ESCs (mESCs) encapsulated in hydrogels was performed in batch and perfusion cultures using bioreactors. Despite sufficient nutrient and growth factor provision, the accumulation of inhibitory metabolites resulted in the unscheduled differentiation of mESCs and a decline in their cell numbers in the batch cultures. In contrast, perfusion cultures maintained metabolite concentration below toxic levels, resulting in the robust expansion (>16-fold) of high quality 'naïve' mESCs within 4 days. A multi-scale mathematical model describing population segregated growth kinetics, metabolism and the expression of selected pluripotency ('stemness') genes was implemented to maximize information from available experimental data. A global sensitivity analysis (GSA) was employed that identified significant (6/29) model parameters and enabled model validation. Predicting the preferential propagation of undifferentiated ESCs in perfusion culture conditions demonstrates synchrony between theory and experiment. CONCLUSIONS/SIGNIFICANCE: The limitations of batch culture highlight the importance of cellular metabolism in maintaining pluripotency, which necessitates the design of suitable ESC bioprocesses. We propose a novel investigational framework that integrates a novel perfusion culture platform (controlled metabolic conditions) with mathematical modeling (information maximization) to enhance ESC bioprocess productivity and facilitate bioprocess optimization.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Reatores Biológicos , Células-Tronco Embrionárias/citologia , Modelos Biológicos , Perfusão , Animais , Técnicas de Cultura Celular por Lotes/instrumentação , Proliferação de Células , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Camundongos , Células-Tronco Pluripotentes/citologia
2.
Tissue Eng Part C Methods ; 18(4): 263-72, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22047052

RESUMO

Application of stem cells for cell therapy of respiratory diseases is a developing field. We have previously established several protocols for the differentiation of embryonic stem cells (ESC) into alveolar epithelial cells, which require a high degree of operator interference and result in a low yield of target cells. Herein, we have shown that, by provision of a medium conditioned using A549 cells and by integration of classic steps of ESC differentiation into a single step through encapsulation in hydrogels (three-dimensional) and culture in a rotary bioreactor, murine ESC (mESC) could be directed to differentiate into distal respiratory epithelial cells. Type I and II pneumocytes (with a yield of 50% for type II) and Clara cells were demonstrated by the expression of aquaporin 5, surfactant protein C, and Clara cell secretory protein, respectively. We identified target cells as early as day 5 of culture and stably maintained our differentiated cells in vitro for 100 days. Electron microscopy demonstrated microvilli and intracellular lamellar bodies (LB), and fluorescent staining confirmed the active process of exocytosis of these LB in differentiated type II cells. When these cells were decapsulated and cultured in static conditions in flask cultures (two-dimensional), they retained their characteristic type II phenotype and morphology. In conclusion, our protocol offers integrated bioprocessing, shorter time of differentiation, lower cost, no use of growth factors, high reproducibility, and high phenotypic and functional stability, as well as being amenable to automation and being scalable, which would move this field closer to future clinical applications.


Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Alvéolos Pulmonares/citologia , Rotação , Animais , Anticorpos/imunologia , Automação , Agregação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Imobilizadas/citologia , Células Imobilizadas/efeitos dos fármacos , Células Imobilizadas/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/ultraestrutura , Humanos , Camundongos , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Br Med Bull ; 98: 117-26, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21565803

RESUMO

BACKGROUND: Considerable amount of information about the potential of stem cell therapy in regenerative medicine is available today. Scientific meetings and publications in specialized journals enable experts in stem cell science and regenerative medicine to follow worldwide cutting-edge research. However, controversial information plaguing the media and the Internet lead patients to believe that stem cells are the long-awaited panacea even though there are little or no stringent factual data available yet. SOURCES OF DATA: PubMed database systematically searched in the period 4-6 January 2011. AREAS OF AGREEMENT: Stem cell-based therapy is a future of regenerative medicine. AREAS OF CONTROVERSY: Based on unsubstantial claims fueled by media, patients are frequently seeking advice about the risks and prospects of specific therapeutic regimes from their physicians. Reports in specialized journals written in a scientific vocabulary are difficult to evaluate for many primary-care physicians. Hence, physicians are reluctant to provide advice or endorse treatment options for cell-based therapies. AREAS TIMELY FOR FURTHER DEVELOPMENT: We wish to fill the gap and offer physicians suitable guidance. By giving a comprehensive overview of different types of stem cells and their potential in a simple language, here we are introducing a series of articles written by world-renowned experts on regenerative medicine about the current status and prospects of the field from the point of view of the standard level of patient safety and efficacy for the healthcare industry.


Assuntos
Transplante de Células-Tronco/métodos , Células-Tronco Adultas/transplante , Diferenciação Celular , Células-Tronco Fetais/transplante , Humanos , Medicina Regenerativa/métodos , Células-Tronco/citologia
4.
Biomaterials ; 31(12): 3244-52, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20149448

RESUMO

Embryonic stem cells (ESCs) are pluripotent and have the ability to differentiate into mineralising cells in vitro. The use of pluripotent cells in engineered bone substitutes will benefit from the development of bioactive scaffolds which encourage cell differentiation and tissue development. Extracellular matrix (ECM) may be a suitable candidate for use in such scaffolds since it plays an active role in cellular differentiation. Here, we test the hypothesis that tissue-specific ECM influences the differentiation of murine ESCs. We induced murine ESCs to differentiate by embryoid body formation, followed by dissociation and culture on ECM prepared by decellularisation of either osteogenic cell (MC3T3-E1) or non-osteogenic cell (A549) cultures, or on defined collagen type I matrix. We assessed osteogenic differentiation by formation of mineralised tissue and osteogenic gene expression, and found it to be significantly greater on MC3T3-E1 matrices than on any other matrix. The osteogenic effect of MC3T3-E1 matrix was reduced by heat treatment and abolished by trypsin, suggesting a bioactive proteinaceous component. These results demonstrate that decellularised bone-specific ECM promotes the osteogenic differentiation of ESCs. Our results are of fundamental interest and may help in tailoring scaffolds for tissue engineering applications which both incorporate tissue-specific ECM signals and stimulate stem-cell differentiation.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Matriz Extracelular , Osteoblastos/citologia , Células 3T3 , Animais , Adesão Celular , Divisão Celular , Imuno-Histoquímica , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase
5.
Biomaterials ; 30(21): 3542-50, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19339047

RESUMO

Bioactive glasses bond strongly to bone in vivo and their ionic dissolution products have previously been shown to have stimulatory properties on adult and fetal osteoblasts and to induce the differentiation of embryonic stem cells towards the osteoblastic lineage in vitro. In the present study, the effect of 45S5 Bioglass conditioned medium with two different Si concentrations (15 microg/ml (BGCM/15) and 20 microg/ml (BGCM/20)) on human fetal osteoblast growth, differentiation and extracellular matrix production and mineralization was investigated. In the first instance, primary fetal osteoblasts were examined for the osteoblast phenotypic markers alkaline phosphatase (ALP), collagen type I (Col I) and OB Cadherin (Cadherin 11) (OB Cad) as well as for the mesenchymal stem cell markers CD105 and CD166. At passage 0 more than 50% of the population was positive for Col I and ALP, but at passage 2, the proportion of cells expressing ALP increased. In addition at passage 0 more than 50% of the fetal osteoblasts expressed the mesenchymal stem cell surface markers CD105 and CD166. Treatment with BGCM/15 and BGCM/20 in the absence of osteogenic supplements increased the gene expression of the bone extracellular matrix proteins alkaline phosphatase, osteonectin and bone sialoprotein as determined by quantitative real time reverse transcriptase-polymerase chain reaction (rt RT-PCR) analysis. Extracellular matrix production was also enhanced in the absence of osteogenic supplements by the 45S5 Bioglass conditioned medium as demonstrated by ALP enzymatic activity, osteocalcin and Col I protein synthesis. Furthermore, BGCM/15 and BGCM/20 significantly enhanced the formation of mineralized nodules, based on alizarin red histochemical staining, without necessitating the addition of beta-glycerophosphate, l-ascorbate-2-phosphate or dexamethasone (commonly used osteogenic supplements).


Assuntos
Meios de Cultivo Condicionados/farmacologia , Vidro , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteogênese/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cerâmica , Feminino , Feto/citologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Osteogênese/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Células-Tronco/fisiologia
6.
Biomaterials ; 30(4): 499-507, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18977027

RESUMO

The application of embryonic stem cells (ESCs) in bone tissue engineering and regenerative medicine requires the development of suitable bioprocesses that facilitate the integrated, reproducible, automatable production of clinically-relevant, scaleable, and integrated bioprocesses that generate sufficient cell numbers resulting in the formation of three-dimensional (3D) mineralised, bone tissue-like constructs. Previously, we have reported the enhanced differentiation of undifferentiated mESCs toward the osteogenic lineage in the absence of embryoid body formation. Herein, we present an efficient and integrated 3D bioprocess based on the encapsulation of undifferentiated mESCs within alginate hydrogels and culture in a rotary cell culture microgravity bioreactor. Specifically, for the first 3 days, encapsulated mESCs were cultured in 50% (v/v) HepG2 conditioned medium to generate a cell population with enhanced mesodermal differentiation capability followed by osteogenic differentiation using osteogenic media containing ascorbic acid, beta-glycerophosphate and dexamethasone. 3D mineralised constructs were generated that displayed the morphological, phenotypical, and molecular attributes of the osteogenic lineage, as well mechanical strength and mineralised calcium/phosphate deposition. Consequently, this bioprocess provides an efficient, automatable, scalable and functional culture system for application to bone tissue engineering in the context of macroscopic bone formation.


Assuntos
Alginatos/metabolismo , Reatores Biológicos , Osso e Ossos/metabolismo , Células-Tronco Embrionárias/citologia , Engenharia Tecidual , Ausência de Peso , Animais , Fenômenos Biomecânicos , Osso e Ossos/citologia , Osso e Ossos/ultraestrutura , Calcificação Fisiológica , Proliferação de Células , Forma Celular , Sobrevivência Celular , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/metabolismo , Camundongos , Osteogênese/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
J R Soc Interface ; 6(32): 209-32, 2009 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-19033137

RESUMO

In recent years, the potential of stem cell research for tissue engineering-based therapies and regenerative medicine clinical applications has become well established. In 2006, Chung pioneered the first entire organ transplant using adult stem cells and a scaffold for clinical evaluation. With this a new milestone was achieved, with seven patients with myelomeningocele receiving stem cell-derived bladder transplants resulting in substantial improvements in their quality of life. While a bladder is a relatively simple organ, the breakthrough highlights the incredible benefits that can be gained from the cross-disciplinary nature of tissue engineering and regenerative medicine (TERM) that encompasses stem cell research and stem cell bioprocessing. Unquestionably, the development of bioprocess technologies for the transfer of the current laboratory-based practice of stem cell tissue culture to the clinic as therapeutics necessitates the application of engineering principles and practices to achieve control, reproducibility, automation, validation and safety of the process and the product. The successful translation will require contributions from fundamental research (from developmental biology to the 'omics' technologies and advances in immunology) and from existing industrial practice (biologics), especially on automation, quality assurance and regulation. The timely development, integration and execution of various components will be critical-failures of the past (such as in the commercialization of skin equivalents) on marketing, pricing, production and advertising should not be repeated. This review aims to address the principles required for successful stem cell bioprocessing so that they can be applied deftly to clinical applications.


Assuntos
Medicina Regenerativa/métodos , Células-Tronco/citologia , Engenharia Tecidual/métodos , Animais , Humanos , Alicerces Teciduais
8.
Stem Cells Dev ; 18(1): 77-91, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-18393639

RESUMO

Visceral endoderm (VE) is an extraembryonic cell layer formed before gastrulation that secretes critical factors involved in embryonic development with inductive effects on embryonic stem cell (ESC) differentiation. We utilized the conditioned media (CM) from the VE-like cell lines, HepG2 and END2, to enhance lineage-specific differentiation of murine ESCs (mESCs) toward the osteogenic lineage. Previously, we have demonstrated that use of the HepG2-CM resulted in the efficient osteogenic/chondrogenic differentiation of mESCs without embryoid body (EB) formation. In this study, we demonstrate, for the first time, the osteogenic-inducing activity of END2-CM, suggesting a potential shared protein profile between HepG2- and END2-CM. To identify the active factors in the CM, proteomic analysis using differential gel electrophoresis coupled with matrix-assisted laser desorption/ionization was performed on the two CM, resulting in six proteins being established to be present in both CM, including ones that may function on the epithelial-mesenchymal transition (EMT). Our results represent the first study on the VE-like cell line secretome and provide information on VE proteins identifying possible candidate proteins to be used for mesoderm-specific osteogenic differentiation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Meios de Cultivo Condicionados , Células-Tronco Embrionárias , Endoderma/metabolismo , Osteogênese/efeitos dos fármacos , Proteoma , Animais , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Linhagem Celular , Condrogênese/fisiologia , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/fisiologia , Endoderma/citologia , Humanos , Camundongos , Osteogênese/fisiologia
9.
Biomaterials ; 29(29): 3946-52, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18639332

RESUMO

The majority of methodologies for maintaining human embryonic stem cell (hESC) pluripotency require the use of human or animal feeder cell layers, the most common being murine embryonic fibroblasts. In this study, we applied a protocol aimed at maintaining hESCs in culture without exposure to animal cells or proteins. hESCs were encapsulated in 1.1% (w/v) calcium alginate hydrogels and grown in basic maintenance medium for a period of up to 260 days. Investigation of the cell aggregates formed within the hydrogels yielded no evidence of the formation of any of the three germ layers, although the hESCs retained their pluripotency and could differentiate when they were subsequently cultured in a conditioned environment. Immunohistochemistry and RT-PCR showed that the hESC aggregates expressed protein and gene markers characteristic of pluripotency including Oct-4, Nanog, SSEA-4, TRA-1-60 and TRA-1-81. At the ultrastructural level, the cells were arranged in closely packed clusters and showed no cytoplasmic organelles, suggesting an undifferentiated state. These data show that it is possible to maintain hESCs in an undifferentiated state, without passaging or embryoid body formation, and without animal contamination.


Assuntos
Técnicas de Cultura de Células/métodos , Técnicas de Cocultura , Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Biomarcadores/metabolismo , Forma Celular , Células-Tronco Embrionárias/citologia , Expressão Gênica , Humanos , Hidrogéis/química , Hidrogéis/metabolismo , Teste de Materiais , Camundongos , Células-Tronco Pluripotentes/citologia
10.
Stem Cells Dev ; 17(5): 963-70, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18564030

RESUMO

Embryonic stem cells (ESCs) posses the ability to self-renew and differentiate into a multitude of lineages, including the osteogenic lineage in vitro. Currently, most approaches have focused on embryonic body (EB)-mediated osteogenic differentiation, which relies on formation of all three germ layers resulting in limited yields and labour-intensive culture processes. Our study aimed at developing an efficient culture strategy resulting in the upregulated in vitro osteogenic differentiation of murine ESCs (mESCs), which completely avoided EB formation. Specifically, mESCs were cultured in HepG2 conditioned medium for 3 days and then directed into osteogenic differentiation for 21 days without prior EB formation. The mineralised bone nodules generated were characterized by Alizarin red S-staining, phenotypic alkaline phosphatase expression, time-course analysis of ALPase activity, the presence of type I collagen and osteopontin, and osteocalcin, cbfa-1/runx-2, and osterix gene expression. Our method of direct osteogenic differentiation of mESCs represents a novel and efficient approach that results in enhanced yields and could have significant applications in bone tissue engineering.


Assuntos
Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Osteogênese , Fosfatase Alcalina/metabolismo , Animais , Antraquinonas/metabolismo , Calcificação Fisiológica , Diferenciação Celular , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Meios de Cultivo Condicionados , Células-Tronco Embrionárias/enzimologia , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Osteogênese/genética , Osteopontina/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
Stem Cells Dev ; 17(5): 971-8, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18564031

RESUMO

Current approaches on the chondrogenic differentiation of embryonic stem cells (ESCs) involve embryoid body (EB) formation, resulting in a fragmented process where control of differentiation, integration, and scalability are difficult to achieve, thus hampering any potential application to cartilage tissue engineering and regenerative medicine. Our study aimed at developing a simplified two-step process which avoids EB formation and results in enhanced chondrogenic differentiation of murine ESCs. Specifically, mESCs were cultured in HepG2 conditioned medium for 3 days and then directed into chondrogenic differentiation for 15 days without prior EB formation. Analysis of chondrogenic differentiation demonstrated well-developed Alcian blue-stained cartilage nodules, production of sulfated glycosaminoglycan and collagen matrix, the presence of structured type II collagen and sox-9 molecules, as well as distinct gene expression of type II collagen, aggrecan, link-protein, scleraxis and sox-9 transcripts. To our knowledge, this represents one of the first reports demonstrating the enhanced derivation of chondrogenic cells from mESCs without EB formation in a simplified and easily integrateable and scalable bioprocess with potential applications in cartilage tissue engineering.


Assuntos
Condrogênese , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Agrecanas/genética , Agrecanas/metabolismo , Azul Alciano , Animais , Cartilagem/citologia , Cartilagem/embriologia , Linhagem Celular Tumoral , Forma Celular , Condrogênese/genética , Colágeno/genética , Colágeno/metabolismo , Meios de Cultivo Condicionados , Células-Tronco Embrionárias/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Glicosaminoglicanos/biossíntese , Humanos , Imuno-Histoquímica , Camundongos , Proteoglicanas/genética , Proteoglicanas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo
12.
Pediatr Res ; 63(5): 461-6, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18427288

RESUMO

Regenerative Medicine is a new, multidisciplinary field that combines expertise in biology, chemistry, engineering, materials, and medicine, to find solutions to some of the most challenging medical problems faced by humankind. Regenerative Medicine has the potential to impact the whole spectrum of health care, such as heart disease, emphysema, and diabetes. Regenerative Medicine employs various combinations of specially grown cells, tissues, and laboratory-made compounds to replace or amplify the body's natural healing process. The impact of Regenerative Medicine to the health care industry is likely to be comparable with that of antibiotics, vaccines and lately, monoclonal antibodies have had in clinical care. Regenerative Medicine is growing and maturing steadily; however, many challenges lie ahead. These include best cell source, most appropriate biomaterials, and reliable ways of expanding the cells and growing them in a three-dimensional environment (stem cell bioprocessing). This concise review deals with current achievements in the field, challenges that lie ahead and potential ways of having robust and reliable "off the shelf" cellular products.


Assuntos
Técnicas de Cultura de Células/tendências , Medicina Regenerativa/tendências , Células-Tronco , Materiais Biocompatíveis , Reatores Biológicos , Técnicas de Cultura de Células/métodos , Humanos , Medicina Regenerativa/métodos , Pesquisa/tendências , Engenharia Tecidual/tendências
13.
Reprod Sci ; 15(1): 91-6, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18212359

RESUMO

The authors previously demonstrated nerve trunks and autonomic ganglia of the hypogastric plexus within the uterosacral ligament (USL) and the cardinal ligaments. The nerve content of these ligaments is greatest closer to the pelvic sidewalls and diminishes toward the insertion of the ligaments into the uterus, with the greater nerve content in the USL. Here the authors determine whether the nerve content of the superficial and deep portion of the USLs, where they are divided at a radical hysterectomy, differ. Biopsies were taken from the right and left superficial and deep USL in 6 patients during radical hysterectomy for early-stage cervical cancer. Indirect immunofluorescence was performed using primary antibodies to (1) the panneuronal marker PGP 9.5, (2) the parasympathetic marker vasoactive intestinal peptide, (3) the sympathetic markers tyrosine hydroxylase and neuropeptide-Y, (4) the sensory and nociceptive nerve marker substance P, and (5) the sensory and sensory-motor nerve marker calcitonin gene-related peptide. The percentage area of immunoreactivity (PAI) was determined using a computer-assisted image analyzer as an objective measure of nerve content. There was a lower nerve content in the superficial USL compared with the deep USL. The PAI of the deep USL was greater than that of the superficial USL for all the nerve markers (P < .05). The PAI was greatest for sympathetic and sensory/nociceptive nerve markers. There were relatively more sympathetic nerve fibers than parasympathetic nerve fibers in the deep USL. These data provide further indirect evidence that pelvic dysfunction following radical hysterectomy is associated with division of the deep portion of the USL.


Assuntos
Vias Autônomas/lesões , Plexo Hipogástrico/lesões , Histerectomia/efeitos adversos , Ligamentos/inervação , Ligamentos/cirurgia , Complicações Pós-Operatórias/etiologia , Vias Autônomas/metabolismo , Vias Autônomas/patologia , Biomarcadores/metabolismo , Biópsia , Feminino , Humanos , Plexo Hipogástrico/metabolismo , Plexo Hipogástrico/patologia , Histerectomia/métodos , Ligamentos/patologia , Neurotransmissores/metabolismo , Útero/cirurgia
14.
Tissue Eng ; 13(12): 2957-70, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17988191

RESUMO

Embryonic stem cell (ESC) culture is fragmented and laborious and involves operator decisions. Most protocols consist of 3 individual steps: maintenance, embryoid body (EB) formation, and differentiation. Integration will assist automation, ultimately aiding scale-up to clinically relevant numbers. These problems were addressed by encapsulating undifferentiated murine ESCs (mESCs) in 1.1% (w/v) low-viscosity alginic acid, 0.1% (v/v) porcine gelatin hydrogel beads (d = 2.3 mm). Six hundred beads containing 10,000 mESCs per bead were cultured in a 50-mL high-aspect-ratio vessel bioreactor. Bioreactor cultures were rotated at 17.5 revolutions per min, cultured in maintenance medium containing leukemia inhibitory factor for 3 days, replaced with EB formation medium for 5 days followed by osteogenic medium containing L-ascorbate-2-phosphate (50 microg/mL), beta-glycerophosphate (10 mM), and dexamethasone (1 microM) for an additional 21 days. After 29 days, 84 times as many cells per bead were observed and mineralized matrix was formed within the alginate beads. Osteogenesis was confirmed using von Kossa, Alizarin Red S staining, alkaline phosphatase activity, immunocytochemistry for osteocalcin, OB-cadherin, collagen type I, reverse transcriptase polymerase chain reaction, microcomputed tomography (micro-computed tomography) and Fourier transform infrared spectroscopic imaging. This simplified, integrated, and potentially scaleable methodology could enable the production of 3-demensional mineralized tissue from ESCs for potential clinical applications.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteogênese/fisiologia , Engenharia Tecidual/métodos , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células , Camundongos
15.
Biomaterials ; 28(9): 1653-63, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17175022

RESUMO

Sol-gel derived bioactive glasses of the 70S30C (70mol% SiO2, 30mol% CaO) composition have been foamed to produce 3D bioactive scaffolds with hierarchical interconnected pore morphologies similar to trabecular bone. The aim of this study was to investigate primary human osteoblast response to porous bioactive glass scaffolds. The scaffolds supported osteoblast growth and induced differentiation, within the 3-week culture period, as depicted by enhanced ALPase enzymatic activity, without the addition of supplementary factors such as ascorbic acid, beta-glycerophosphate and dexamethasone. This is the first time this has been observed on a bioactive glass that does not contain phosphate. Deposition of extracellular matrix was also confirmed by enhanced production of the extracellular matrix protein collagen type I. SEM showed indications of mineralized bone nodule formation without the addition of growth factors. The 70S30C bioactive glass scaffolds therefore fulfil many of the criteria for an ideal scaffold for bone tissue engineering applications.


Assuntos
Calcificação Fisiológica/fisiologia , Cerâmica/química , Matriz Extracelular/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteogênese/fisiologia , Engenharia Tecidual/métodos , Técnicas de Cultura de Células/métodos , Células Cultivadas , Matriz Extracelular/ultraestrutura , Humanos , Fosfatos/química , Propriedades de Superfície
16.
Methods Enzymol ; 418: 333-49, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17141045

RESUMO

Repair or regeneration of defective lung epithelium would be of great therapeutic potential. Cellular sources for such repair have long been searched for within the lung, but the identification and characterization of stem or progenitor cells have been hampered by the complexity and cellular heterogeneity of the organ. In recent years, various pulmonary cells have been identified that meet the criteria for stem cells but it remains to be seen how far manipulation of these tissue-specific cell pools can upregulate epithelial repair. The initial excitement that greeted the results of animal experiments showing cells of bone marrow origin in murine lung has been tempered by more recent data suggesting that the cells do not repair pulmonary epithelium. However, there are reports of engraftment of bone marrow-derived cells in human lung, albeit at a low level, so the administration of cell therapy via the circulation, for repair and/or gene delivery, needs further investigation. The potential of human embryonic stem cells to generate any cell, tissue, or organ on demand for tissue repair or replacement is promising to revolutionize the treatment of human disease. Although some headway has been made into making pulmonary epithelium from these stem cells, human embryonic stem cell technology is still in its infancy and many technical, safety, and ethical hurdles must be cleared before clinical trials can begin. This chapter focuses on the potential role of stem cells in future approaches to lung repair and regeneration.


Assuntos
Pulmão/citologia , Pulmão/fisiologia , Mucosa Respiratória/citologia , Mucosa Respiratória/fisiologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Técnicas de Cocultura/métodos , Meios de Cultura , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Humanos , Mesoderma/citologia , Regeneração , Mucosa Respiratória/transplante , Células-Tronco/citologia , Células-Tronco/fisiologia
17.
J Mater Sci Mater Med ; 17(11): 1029-35, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17122915

RESUMO

Professors Larry Hench and Julia Polak formed the Tissue Engineering and Regenerative Medicine Centre (TERM) at Imperial College London to foster collaborations between biologists and materials scientists. Early work at the center elucidated the biomolecular interactions between primary human osteoblasts and 45S5 Bioglass . As research efforts expanded, the team discovered that the dissolution products of both 45S5 Bioglass and 58S sol-gel bioactive glasses had osteoblastic stimulatory properties. To address the shortage of appropriate cells for bone tissue engineering applications, TERM scientists also demonstrated the differentiation of embryonic stem (ES) cells to osteoblasts when treated with the dissolution products of bioactive glasses. They also found that the soluble factors ascorbic acid, beta -glycerophosphate, and dexamethasone preferentially differentiated ES cells to osteoblasts, and their combination with the dissolution products of bioactive glasses stimulated differentiation even further. Taken together, these results demonstrate the suitability of bioactive glasses as scaffolds for bone tissue engineering as they not only provide an osteoconductive and osteoproductive substrate, but also actively stimulate cells to express appropriate osteoblastic phenotypes. Professor Hench's vision to pioneer regenerative medicine research continues with the aim of developing novel therapeutics to treat musculoskeletal disability.


Assuntos
Substitutos Ósseos/história , Engenharia Tecidual/história , Engenharia Tecidual/tendências , Animais , História do Século XX , História do Século XXI , Humanos
18.
Tissue Eng ; 12(7): 2025-30, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16889530

RESUMO

Embryonic stem cells (ESCs) are being investigated increasingly for their potential as a cell source for tissue engineering. Antibiotics are regularly used in ESC culture media to control contamination, although they can be cytotoxic and interfere with protein synthesis. Our aim was to examine the effects of the frequently used antibiotics gentamicin and combined penicillin and streptomycin on ESC culture using differentiation of murine ESC into type II pneumocytes as a model. Antibiotics reduced the expression of the specific marker for type II pneumocytes, SPC mRNA, by up to 60%. We also identified an adverse effect on the growth rate of differentiating embryoid bodies, causing a significant ( p < 0.05) reduction of up to 40%, and an increase in population doubling time of up to 48%. No contamination was seen in any of the cultures. Our findings suggest that the routine use of antibiotics in ESC culture should be avoided as it may reduce the efficiency of the culture system.


Assuntos
Antibacterianos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Células-Tronco/metabolismo , Animais , Antígenos de Diferenciação/biossíntese , Linhagem Celular , Embrião de Mamíferos/citologia , Camundongos , Células-Tronco/citologia
19.
Tissue Eng ; 12(6): 1381-92, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16846337

RESUMO

Despite the considerable progress made in directing embryonic stem cell (ESC) differentiation to therapeutically useful lineages, several issues remain to be resolved before ESCs can be used for cell therapy: 1) increasing the efficiency of specific lineage generation, and 2) developing time- and cost-effective culture systems for controlling ESC differentiation. Our study aimed to develop efficient methods to enhance mesodermal differentiation and thereby upregulate osteogenic differentiation of ESCs. Specifically, murine ESCs (mESCs) were cultured in the presence of 50% conditioned medium (CM) from the human hepatocarcinoma cell line HepG2, which resulted in enhanced mesoderm formation during embryoid body (EB) formation in the CM-treated mESCs (CM-mESCs). By varying the length of EB culture time, we achieved the selective control and stimulation of osteogenic differentiation and suppression of cardiogenic differentiation. Hence, reducing the EB culture of the CM-mESCs to 1 day resulted in 5-10-fold enhancement of osteogenic differentiation, as determined by bone nodule formation, higher alkaline phosphatase activity, the presence of well-organized osteoblast-cadherin in the bone nodules, and increased cbfa-1/runx2 gene expression. In contrast, increasing the EB culture of the CM-mESCs to 5 days resulted in three- to four-fold enhanced cardiogenic differentiation. These findings for development of highly efficient culture systems and protocols for mESC differentiation into osteogenic lineage that are time- and cost-effective can be used in skeletal tissue engineering applications.


Assuntos
Diferenciação Celular/fisiologia , Embrião de Mamíferos/citologia , Osteoblastos/citologia , Osteogênese/fisiologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Animais , Linhagem Celular Tumoral , Linhagem da Célula/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Embrião de Mamíferos/fisiologia , Humanos , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos
20.
Tissue Eng ; 12(6): 1687-97, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16846363

RESUMO

We have previously induced differentiation of embryonic stem cells (ESC) to specific phenotypes by manipulating the culture conditions, including the use of indirect co-culture. In this study, we hypothesized that co-culture with primary chondrocytes can induce human embryonic stem cells (hESC) to differentiate towards the chondrocyte lineage. Co-cultures of hESC and chondrocytes were established using well inserts, with control comprising hESC grown alone or with fibroblasts. After 28 days, after removal of the chondrocyte inserts, hESC differentiation was assessed, by morphology, immunocytochemistry, and reverse transcription polymerase chain reaction. hESC, co-cultured or grown alone, were also implanted into SCID mice on a poly-D, L-lactide scaffold, harvested 35 days later and assessed in the same way. hESC co-cultured with chondrocytes formed colonies and secreted extracellular matrix containing glycosaminoglycans (GAG). Quantitative assay showed increased synthesis of sulfated GAG in co-culture as compared with control hESC grown alone for the same period (p < 0.0001). In addition, co-cultured hESC expressed Sox 9 and collagen type II, unlike control hESC. Co-culture with fibroblasts did not induce chondrogenic differentiation. The implanted constructs with co-cultured hESC contained significantly more type II collagen (p < 0.01), type I collagen (p < 0.05), total collagen (p < 0.01), and GAG (p < 0.01) than those with hESC grown alone. Thus, we show for the first time differentiation of hESC to chondrocytes. Our results confirm the potential of the culture micro-environment to influence ESC differentiation and could provide the basis for future generation of chondrogenic cells for use in tissue repair and increase our understanding of the mechanisms that direct differentiation.


Assuntos
Diferenciação Celular/fisiologia , Condrócitos/citologia , Condrogênese/fisiologia , Células-Tronco/citologia , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Humanos
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