RESUMO
Toward developing biologically sound models for the study of heart regeneration and disease, we cultured heart cells on a biodegradable, microfabricated poly(glycerol sebacate) (PGS) scaffold designed with micro-structural features and anisotropic mechanical properties to promote cardiac-like tissue architecture. Using this biomimetic system, we studied individual and combined effects of supplemental insulin-like growth factor-1 (IGF-1) and electrical stimulation (ES). On culture day 8, all tissue constructs could be paced and expressed the cardiac protein troponin-T. IGF-1 reduced apoptosis, promoted cell-to-cell connectivity, and lowered excitation threshold, an index of electrophysiological activity. ES promoted formation of tissue-like bundles oriented in parallel to the electrical field and a more than ten-fold increase in matrix metalloprotease-2 (MMP-2) gene expression. The combination of IGF-1 and ES increased 2D projection length, an index of overall contraction strength, and enhanced expression of the gap junction protein connexin-43 and sarcomere development. This culture environment, designed to combine cardiac-like scaffold architecture and biomechanics with molecular and biophysical signals, enabled functional assembly of engineered heart muscle from dissociated cells and could serve as a template for future studies on the hierarchy of various signaling domains relative to cardiac tissue development.
Assuntos
Materiais Biomiméticos/farmacologia , Coração , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Miocárdio/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Animais Recém-Nascidos , Fenômenos Biomecânicos , Materiais Biomiméticos/química , Células Cultivadas , Estimulação Elétrica/métodos , Coração/efeitos dos fármacos , Coração/fisiologia , Microtecnologia , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual/instrumentaçãoRESUMO
Human mesenchymal stem cells, or multipotent stromal cells (MSCs), are of interest for clinical therapy, in part because of their capacity for proliferation and differentiation. However, results from clinical trials and in vitro models have been variable, possibly because of MSC heterogeneity and a lack of standardization between MSC in vitro expansion protocols. Here we defined changes in MSCs during expansion in vitro. In low-density cultures, MSCs expand through distinct lag, exponential growth, and stationary phases. We assayed cultures of passage 2 human MSCs from three donors at low density (50 cells per cm(2)) at approximately 5% confluence on day 2 and after the cultures had expanded to approximately 70% confluence on day 7. On day 2, genes involved in cell division were upregulated. On day 7, genes for cell development were upregulated. The variations among three donors were less than the variation within the expansion of MSCs from a single donor. The microarray data for selected genes were confirmed by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and FACScan analysis. Approximately 50% of cells at day 2 were in S-phase compared with 10% at day 7. The results demonstrated major differences in early and late stage cultures of MSCs that should be considered in using the cells in experiments and clinical applications.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Células Estromais/citologia , Divisão Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The development of mechanically functional cartilage and bone tissue constructs of clinically relevant size, as well as their integration with native tissues, remains an important challenge for regenerative medicine. The objective of this study was to assess adult human mesenchymal stem cells (MSCs) in large, three-dimensionally woven poly(ε-caprolactone; PCL) scaffolds in proximity to viable bone, both in a nude rat subcutaneous pouch model and under simulated conditions in vitro. In Study I, various scaffold permutations-PCL alone, PCL-bone, "point-of-care" seeded MSC-PCL-bone, and chondrogenically precultured Ch-MSC-PCL-bone constructs-were implanted in a dorsal, ectopic pouch in a nude rat. After 8 weeks, only cells in the Ch-MSC-PCL constructs exhibited both chondrogenic and osteogenic gene expression profiles. Notably, although both tissue profiles were present, constructs that had been chondrogenically precultured prior to implantation showed a loss of glycosaminoglycan (GAG) as well as the presence of mineralization along with the formation of trabecula-like structures. In Study II of the study, the GAG loss and mineralization observed in Study I in vivo were recapitulated in vitro by the presence of either nearby bone or osteogenic culture medium additives but were prevented by a continued presence of chondrogenic medium additives. These data suggest conditions under which adult human stem cells in combination with polymer scaffolds synthesize functional and phenotypically distinct tissues based on the environmental conditions and highlight the potential influence that paracrine factors from adjacent bone may have on MSC fate, once implanted in vivo for chondral or osteochondral repair.
Assuntos
Diferenciação Celular , Condrogênese , Células-Tronco Mesenquimais/citologia , Osteogênese , Alicerces Teciduais/química , Adulto , Animais , Bovinos , Diferenciação Celular/genética , Condrogênese/genética , Feminino , Regulação da Expressão Gênica , Humanos , Hipertrofia , Implantes Experimentais , Osteogênese/genética , Poliésteres/química , Ratos Nus , Microtomografia por Raio-XRESUMO
Tough scaffolds comprised of aligned and conductive fibers are promising for peripheral nerve regeneration due to their unique mechanical and electrical properties. Several studies have confirmed that electrical stimulation can control the axonal extension in vitro. However, the stimulatory effects of scaffold architecture and electrical stimulation have not yet been investigated in detail. Here, we assessed a comparison between aligned and random fibers made of graphene (Gr) embedded sodium alginate (SA) polyvinyl alcohol (PVA) (Gr-AP scaffolds) for peripheral nerve engineering. The effects of applied electrical stimulation and orientation of the fabricated fibers on the in vitro attachment, alignment, and proliferation of PC12 cells (a rat neuronal cell line) were investigated. The results revealed that the aligned fibrous Gr-AP scaffolds closely mimicked the anisotropic structure of the native sciatic nerve. Aligned fibrous Gr-AP scaffolds significantly improved mechanical properties as well as cell-scaffold integration compared to random fibrous scaffolds. In addition, electrical stimulation significantly improved PC12 cell proliferation. In summary, our findings revealed that aligned fibrous Gr-AP scaffolds offered superior mechanical characteristics and structural properties that enhanced neural cell-substrate interactions, resulting in a promising construct for nerve tissue regeneration.
RESUMO
In the stem-cell niche, the extracellular matrix (ECM) serves as a structural support that additionally provides stem cells with signals that contribute to the regulation of stem-cell function, via reciprocal interactions between cells and components of the ECM. Recently, cell-derived ECMs have emerged as in vitro cell culture substrates to better recapitulate the native stem-cell microenvironment outside the body. Significant changes in cell number, morphology and function have been observed when mesenchymal stem cells (MSC) were cultured on ECM substrates as compared to standard tissue-culture polystyrene (TCPS). As select ECM components are known to regulate specific stem-cell functions, a robust characterization of cell-derived ECM proteomic composition is critical to better comprehend the role of the ECM in directing cellular processes. Here, we characterized and compared the protein composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts from different donors, employing quantitative proteomic methods. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential survival and gene-expression profiles among the cell types and as compared to TCPS, indicating that the cell-derived ECMs influence each cell type in a different manner. This general approach to understanding the protein composition of different tissue-specific and cell-derived ECM will inform the rational design of defined systems and biomaterials that recapitulate critical ECM signals for stem-cell culture and tissue engineering.
Assuntos
Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteômica/métodos , Medula Óssea/metabolismo , Proliferação de Células , Sobrevivência Celular , Matriz Extracelular/ultraestrutura , Humanos , Recém-Nascido , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/ultraestrutura , Doadores de Tecidos , Transcriptoma/genéticaRESUMO
Cellular microarrays have become extremely useful in expediting the investigation of large libraries of (bio)materials for both in vitro and in vivo biomedical applications. An exceedingly simple strategy is developed for the fabrication of non-cell-adhesive substrates supporting the immobilization of diverse (bio)material features, including both monomeric and polymeric adhesion molecules (e.g., RGD and polylysine), hydrogels, and polymers.
Assuntos
Materiais Biocompatíveis/farmacologia , Células-Tronco Mesenquimais/citologia , Impressão/métodos , Animais , Adesão Celular/efeitos dos fármacos , Células HeLa , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Água/químicaRESUMO
UNLABELLED: We assayed gene expressions during adipogenesis of human MSCs. Microarray assays demonstrated time-dependent increases in expression of 67 genes, including 2 genes for transcription factors that were not previously shown to be expressed during adipogenesis. INTRODUCTION Increased numbers of bone marrow adipocytes have been observed in patients with osteoporosis and aplastic anemia, but the pathological mechanisms remain unknown. Recently, microarray assays for mRNAs were used to follow adipogenic differentiation of the preadipocytic cell line, 3T3-L1, but adipogenic differentiation has not been examined in primary cells from bone marrow. Here we defined the sequence of gene expression during the adipogenesis ex vivo of human cells from bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). MATERIALS AND METHODS: MSCs were plated at extremely low densities to generate single-cell derived colonies, and adipogenic differentiation of the colonies assayed by accumulation of fat vacuoles, time-lapse photomicroscopy, microarrays, and reverse transcriptase-polymerase chain reaction (RT-PCR) assays. RESULTS AND CONCLUSIONS About 30% of the colonies differentiated to adipocytes in 14 days and about 60% in 21 days. Cell proliferation was inhibited by approximately 50% in adipogenic medium. The differentiation occurred primarily at the center of the colonies, and a few adipocytes that formed near the periphery migrated toward the centers. RT-PCR assays demonstrated that the differentiation was accompanied by increases in a series of genes previously shown to increase with adipogenic differentiation: peroxisome proliferator activated receptor gamma, CCAAT enhancer-binding protein alpha, acylCoA synthetase, lipoprotein lipase, and fatty acid binding protein 4. We also followed differentiation with microarray assays. Sixty-seven genes increased more than 10-fold at day 1 and 20-fold at day 7, 14, or 21. Many of the genes identified were previously shown to be expressed during adipocytic differentiation. However, others, such as zinc finger E-box binding protein and zinc finger protein 145, were not. This study should serve as a basis for future study to clarify the mechanisms of adipocyte differentiation of MSCs.
Assuntos
Tecido Adiposo/citologia , Células da Medula Óssea/citologia , Células Estromais/citologia , Adulto , Sequência de Bases , Diferenciação Celular , Primers do DNA , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Tissue engineered heart valves (TEHVs) that can grow and remodel have the potential to serve as permanent replacements of the current non-viable prosthetic valves particularly for pediatric patients. A major challenge in designing functional TEHVs is to mimic both structural and anisotropic mechanical characteristics of the native valve leaflets. To establish a more biomimetic model of TEHV, we fabricated tri-layered scaffolds by combining electrospinning and microfabrication techniques. These constructs were fabricated by assembling microfabricated poly(glycerol sebacate) (PGS) and fibrous PGS/poly(caprolactone) (PCL) electrospun sheets to develop elastic scaffolds with tunable anisotropic mechanical properties similar to the mechanical characteristics of the native heart valves. The engineered scaffolds supported the growth of valvular interstitial cells (VICs) and mesenchymal stem cells (MSCs) within the 3D structure and promoted the deposition of heart valve extracellular matrix (ECM). MSCs were also organized and aligned along the anisotropic axes of the engineered tri-layered scaffolds. In addition, the fabricated constructs opened and closed properly in an ex vivo model of porcine heart valve leaflet tissue replacement. The engineered tri-layered scaffolds have the potential for successful translation towards TEHV replacements.
Assuntos
Próteses Valvulares Cardíacas , Polímeros/farmacologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Anisotropia , Valva Aórtica/efeitos dos fármacos , Valva Aórtica/fisiologia , Elastômeros , Humanos , Técnicas In Vitro , Teste de Materiais , Microscopia Eletrônica de Varredura , Valva Pulmonar/efeitos dos fármacos , Valva Pulmonar/fisiologia , OvinosRESUMO
Tissue engineered heart valves (TEHV) can be useful in the repair of congenital or acquired valvular diseases due to their potential for growth and remodeling. The development of biomimetic scaffolds is a major challenge in heart valve tissue engineering. One of the most important structural characteristics of mature heart valve leaflets is their intrinsic anisotropy, which is derived from the microstructure of aligned collagen fibers in the extracellular matrix (ECM). In the present study, a directional electrospinning technique is used to fabricate fibrous poly(glycerol sebacate):poly(caprolactone) (PGS:PCL) scaffolds containing aligned fibers, which resemble native heart valve leaflet ECM networks. In addition, the anisotropic mechanical characteristics of fabricated scaffolds are tuned by changing the ratio of PGS:PCL to mimic the native heart valve's mechanical properties. Primary human valvular interstitial cells (VICs) attach and align along the anisotropic axes of all PGS:PCL scaffolds with various mechanical properties. The cells are also biochemically active in producing heart-valve-associated collagen, vimentin, and smooth muscle actin as determined by gene expression. The fibrous PGS:PCL scaffolds seeded with human VICs mimick the structure and mechanical properties of native valve leaflet tissues and would potentially be suitable for the replacement of heart valves in diverse patient populations.
Assuntos
Decanoatos/química , Glicerol/análogos & derivados , Valvas Cardíacas/citologia , Poliésteres/química , Polímeros/química , Actinas/metabolismo , Animais , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Módulo de Elasticidade , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Glicerol/química , Valvas Cardíacas/metabolismo , Humanos , Suínos , Resistência à Tração , Engenharia Tecidual , Alicerces Teciduais , Vimentina/metabolismoRESUMO
A biodegradable microvessel scaffold comprised of distinct parenchymal and vascular compartments separated by a permeable membrane interface was conceptualized, fabricated, cellularized, and implanted. The device was designed with perfusable microfluidic channels on the order of 100 µm to mimic small blood vessels, and high interfacial area to an adjacent parenchymal space to enable transport between the compartments. Poly(glycerol sebacate) (PGS) elastomer was used to construct the microvessel framework, and various assembly methods were evaluated to ensure robust mechanical integrity. In vitro studies demonstrated the differentiation of human skeletal muscle cells cultured in the parenchymal space, a 90% reduction in muscle cell viability due to trans-membrane transport of a myotoxic drug from the perfusate, and microvessel seeding with human endothelial cells. In vivo studies of scaffolds implanted subcutaneously and intraperitoneally, without or with exogenous cells, into nude rats demonstrated biodegradation of the membrane interface and host blood cell infiltration of the microvessels. This modular, implantable scaffold could serve as a basis for building tissue constructs of increasing scale and clinical relevance.
Assuntos
Decanoatos/química , Glicerol/análogos & derivados , Polímeros/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Glicerol/química , Humanos , Microscopia Eletrônica de Varredura , Músculo Esquelético/citologia , RatosRESUMO
Multi-layered poly(glycerol-sebacate) (PGS) scaffolds with controlled pore microarchitectures were fabricated, combined with heart cells, and cultured with perfusion to engineer contractile cardiac muscle constructs. First, one-layered (1L) scaffolds with accordion-like honeycomb shaped pores and elastomeric mechanical properties were fabricated by laser microablation of PGS membranes. Second, two-layered (2L) scaffolds with fully interconnected three dimensional pore networks were fabricated by oxygen plasma treatment of 1L scaffolds followed by stacking with off-set laminae to produce a tightly bonded composite. Third, heart cells were cultured on scaffolds with or without interstitial perfusion for 7 days. The laser-microablated PGS scaffolds exhibited ultimate tensile strength and strain-to-failure higher than normal adult rat left ventricular myocardium, and effective stiffnesses ranging from 220 to 290 kPa. The 7-day constructs contracted in response to electrical field stimulation. Excitation thresholds were unaffected by scaffold scale up from 1L to 2L. The 2L constructs exhibited reduced apoptosis, increased expression of connexin-43 (Cx-43) and matrix metalloprotease-2 (MMP-2) genes, and increased Cx-43 and cardiac troponin-I proteins when cultured with perfusion as compared to static controls. Together, these findings suggest that multi-layered, microfabricated PGS scaffolds may be applicable to myocardial repair applications requiring mechanical support, cell delivery and active implant contractility.
Assuntos
Miocárdio/citologia , Alicerces Teciduais/química , Animais , Animais Recém-Nascidos , Células Cultivadas , Eletrofisiologia , Teste de Materiais , Microscopia Eletrônica de Varredura , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Reação em Cadeia da Polimerase , Ratos , Engenharia Tecidual/métodosRESUMO
Polymer scaffolds that direct elongation and orientation of cultured cells can enable tissue engineered muscle to act as a mechanically functional unit. We combined micromolding and microablation technologies to create muscle tissue engineering scaffolds from the biodegradable elastomer poly(glycerol sebacate). These scaffolds exhibited well defined surface patterns and pores and robust elastomeric tensile mechanical properties. Cultured C2C12 muscle cells penetrated the pores to form spatially controlled engineered tissues. Scanning electron and confocal microscopy revealed muscle cell orientation in a preferential direction, parallel to micromolded gratings and long axes of microablated anisotropic pores, with significant individual and interactive effects of gratings and pore design.
Assuntos
Elastômeros/síntese química , Microtecnologia/métodos , Miocárdio/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Linhagem Celular , Forma Celular , Decanoatos/síntese química , Decanoatos/química , Módulo de Elasticidade , Elastômeros/química , Glicerol/análogos & derivados , Glicerol/síntese química , Glicerol/química , Membranas Artificiais , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Mioblastos/citologia , Polímeros/síntese química , Polímeros/química , Resistência à TraçãoRESUMO
There has been considerable interest in developing new therapies with adult multipotent progenitor stromal cells or mesenchymal stem cells (MSCs) in organ replacement and repair. To be effectively seeded into scaffolds for therapy, large numbers of cells are needed, but concerns remain regarding their chromatin stability in long-term culture. We therefore expanded four donors of human MSCs (hMSCs) from bone marrow aspirates with a protocol that maintains the cells at low density. MSCs initially proliferated at average doubling times of 24 h and then gradually reached senescence after 8-15 passages (33-55 population doublings) without evidence of immortalization. Comparative genomic hybridization assays of two preparations revealed no abnormalities through 33 population doublings. One preparation had a small amplification of unknown significance in chromosome 7 (7q21:11) after 55 population doublings. Microarray assays demonstrated progressive changes in the transcriptome of the cells. However, the transcriptomes clustered more closely over time within a single passage, rather than with passage number, indicating a partial reversibility of the patterns of gene expression. One of the largest changes was a decrease in mRNA for Sox11, a transcription factor previously identified in neural progenitor cells. Knockdown of Sox11 with siRNA decreased the proliferation and osteogenic differentiation potential of hMSCs. The results suggested that assays for Sox11 may provide a biomarker for early progenitor hMSCs.
Assuntos
Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Fatores de Transcrição SOXC/metabolismo , Adulto , Contagem de Células , Diferenciação Celular/genética , Proliferação de Células , Forma Celular , Senescência Celular/genética , Dano ao DNA , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXC/genética , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Quantitative assays for human DNA and mRNA were used to examine the paradox that intravenously (i.v.) infused human multipotent stromal cells (hMSCs) can enhance tissue repair without significant engraftment. After 2 x 10(6) hMSCs were i.v. infused into mice, most of the cells were trapped as emboli in lung. The cells in lung disappeared with a half-life of about 24 hr, but <1000 cells appeared in six other tissues. The hMSCs in lung upregulated expression of multiple genes, with a large increase in the anti-inflammatory protein TSG-6. After myocardial infarction, i.v. hMSCs, but not hMSCs transduced with TSG-6 siRNA, decreased inflammatory responses, reduced infarct size, and improved cardiac function. I.v. administration of recombinant TSG-6 also reduced inflammatory responses and reduced infarct size. The results suggest that improvements in animal models and patients after i.v. infusions of MSCs are at least in part explained by activation of MSCs to secrete TSG-6.
Assuntos
Moléculas de Adesão Celular/metabolismo , Pulmão/metabolismo , Células-Tronco Multipotentes/transplante , Infarto do Miocárdio/terapia , Animais , Ensaios de Migração Celular , Perfilação da Expressão Gênica , Coração/fisiopatologia , Humanos , Mediadores da Inflamação/metabolismo , Infusões Intravenosas , Pulmão/citologia , Camundongos , Células-Tronco Multipotentes/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Embolia Pulmonar/metabolismo , Células Estromais/metabolismoAssuntos
Materiais Biocompatíveis/química , Ácido Gálico/química , Hidrogéis/química , Melaninas/química , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Técnicas de Química Sintética , Células HeLa , Humanos , Hidrogéis/síntese química , Hidrogéis/farmacologiaRESUMO
We developed a strategy for use of microarray data to rapidly identify new downstream targets of transcription factors known to drive differentiation by following the time courses of gene expression as a relatively homogeneous population of stem/progenitor cells are differentiated to multiple phenotypes. Microarray assays were used to follow the differentiation of human marrow stromal cells (MSCs) into chondrocytes or adipocytes in three different experimental conditions. The steps of the analysis were the following: (a) hierarchical clustering was used to define groups of similarly behaving genes in each experiment, (b) candidates for new downstream targets of transcription factors that drive differentiation were then identified as genes that were consistently co-expressed with known downstream target genes of the transcription factors, and (c) the list of candidate new target genes was refined by identifying genes whose signal intensities showed a highly significant linear regression with the signal intensities of the known targets in all the data sets. Analysis of the data identified multiple new candidates for downstream targets for SOX9, SOX5, CCAAT/enhancer binding protein (C/EBP)-alpha, and peroxisome proliferator-activated receptor (PPAR)-gamma. To validate the analysis, we demonstrated that PPAR-gamma protein specifically bound to the promoters of four new targets identified in the analyses. The same multistep analysis can be used to identify new downstream targets of transcription factors in other systems. Also, the same analysis should make it possible to use MSCs from bone marrow to define new mutations that alter chondogenesis or adipogenesis in patients with a variety of syndromes.
Assuntos
Células da Medula Óssea/fisiologia , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Adipogenia/fisiologia , Células da Medula Óssea/citologia , Células Cultivadas , Condrogênese/fisiologia , Perfilação da Expressão Gênica/métodos , Humanos , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células-Tronco/citologia , Células Estromais/citologia , Células Estromais/fisiologiaRESUMO
The human adult stem cells from bone marrow stroma referred to as mesenchymal stem cells or marrow stromal cells (MSCs) are of interest because they are easily isolated and expanded and are capable of multipotential differentiation. Here, we examined the ability of recombinant human bone morphogenetic protein (BMP)-2, -4, and -6 to enhance in vitro cartilage formation of MSCs. Human MSCs were isolated from bone marrow taken from normal adult donors. The cells were pelleted and cultured for 21 days in chondrogenic medium containing transforming growth factor beta3 and dexamethasone with or without BMP-2, -4, or -6. All the BMPs tested increased chondrogenic differentiation as assayed by immunohistochemistry and by the size and weight of the cartilage synthesized. However, BMP-2 was the most effective. Microarray analyses of approximately 12,000 genes and reverse transcription-polymerase chain reaction assays established that the critical genes for cartilage synthesis were expressed in the expected time sequence in response to BMP-2. The tissue engineering of autologous cartilage derived from MSCs in vitro for transplantation will be a future alternative for patients with cartilage injuries. To obtain large amounts of cartilage rich in proteoglycans, the use of BMP-2 is recommended, instead of BMP-4 or -6.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Cartilagem/crescimento & desenvolvimento , Células-Tronco/efeitos dos fármacos , Células Estromais/citologia , Fator de Crescimento Transformador beta/farmacologia , Adulto , Células da Medula Óssea/citologia , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteína Morfogenética Óssea 6 , Proteínas Morfogenéticas Ósseas/genética , Cartilagem/citologia , Cartilagem/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Meios de Cultura/química , Dexametasona/farmacologia , Humanos , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Células-Tronco/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta3RESUMO
One approach to resolving the complexities of chondrogenesis is to examine simplified systems in vitro. We analyzed cartilage differentiation by human adult stem cells from bone marrow stroma. Marrow stromal cells were cultured as micromass pellets for 21 days in serum-free medium containing transforming growth factor (TGF)-beta3, dexamethasone, and bone morphogenetic protein (BMP)-6. Assays for pulse-labeled [3H]DNA and for total DNA indicated that there was little proliferation and a progressive loss of cells in the pellets. There were continuous increases in mRNAs for cartilage matrix (proteoglycans and COL2, -9, -10, and -11), receptors [fibroblast growth factor 2 (FGFR2) and parathyroid hormone-related peptide receptor (PTHrP-R)], and transcription factors (SOX5, -6, and -9) as demonstrated by histochemical and microarray assays. Reverse transcription-PCR assays for 11 mRNAs confirmed the microarray data. SOX4, vascular endothelial growth factor (VEGF), and matrix metalloproteinase 14 (MMP14) increased at day 1 and decreased thereafter, suggesting roles early in chondrogenesis. Also, forkhead, CD10, and MMP13 increased up to day 7 and decreased thereafter, suggesting roles in an intermediate stage of chondrogenesis. In addition, two collagens (COL3A1 and COL16A1), a signaling molecule (WNT11), a homeobox homolog (BAPX1), a receptor (IL-1R1), an IGFs modulator (IGFBP5), and a mettaloproteinase (MMP16) increased progressively up to about day 14, suggesting roles later in chondrogenesis. Our results indicate that the simplicity of the system makes it possible to define in detail the cellular and molecular events during chondrogenesis.
Assuntos
Células da Medula Óssea/fisiologia , Cartilagem/fisiologia , Diferenciação Celular , Células-Tronco/fisiologia , Adulto , Colágeno/genética , Fatores de Crescimento Endotelial/genética , Perfilação da Expressão Gênica , Humanos , Linfocinas/genética , Metaloendopeptidases/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/fisiologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
There is considerable interest in the biology and therapeutic potential of adult stem cells from bone marrow stroma, variously referred to as mesenchymal stem cells or marrow stromal cells (MSCs). Human MSCs can expand rapidly in culture, but the rate of expansion and the yields of multipotential progenitors are inversely related to the plating density and incubation time of each passage. We have defined conditions for optimizing the yields of cultures enriched for early progenitors. Also, we developed a simple method for assessing the quality of the cultures by phase-contrast microscopy and image analysis or by forward light scatter in a flow cytometer. The cells expanded most rapidly on day 4 after plating, with a minimum average doubling time of about 10 hours for cells initially plated at 10 or 50 cells/cm(2). After plating the cells at 1 to 1000 cells/cm(2), the cultures underwent a time-dependent transition from early progenitors, defined as thin, spindle-shaped cells (RS-1A), to wider, spindle-shaped cells (RS-1B), and to still wider, spindle-shaped cells (RS-1C). Assays for adipogenesis demonstrated that the adipogenic potential of cultures was directly related to their ability to generate single-cell-derived colonies and their enrichment for RS-1A cells. In contrast, cultures enriched for RS-1B cells showed the greatest potential to differentiate into cartilage in a serum-free system. The results indicate that, when preparing cultures of human MSCs, it is necessary to compromise between conditions that provide the highest overall yields and those that provide the highest content of early progenitor cells.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Hematopoéticas/citologia , Células Estromais/citologia , Adipócitos/citologia , Adulto , Contagem de Células , Diferenciação Celular , Divisão Celular , Condrócitos/citologia , Feminino , Humanos , Masculino , Células-Tronco Multipotentes/citologia , Transplante de Células-TroncoRESUMO
For reasons that are not apparent, it has been difficult to isolate and expand the adult stem cells referred to as mesenchymal stem cells or marrow stromal cells (MSCs) from murine bone marrow. We developed a protocol that provides rapidly expanding MSCs from 5 strains of inbred mice. The MSCs obtained from 5 different strains of mice were similar to human and rat MSCs in that they expanded more rapidly if plated at very low density, formed single-cell-derived colonies, and readily differentiated into either adipocytes, chondrocytes, or mineralizing cells. However, the cells from the 5 strains differed in their media requirements for optimal growth, rates of propagation, and presence of the surface epitopes CD34, stem cell antigen-1 (Sca-1), and vascular cell adhesion molecule 1 (VCAM-1). The protocol should make it possible to undertake a large number of experiments with MSCs in transgenic mice that have previously not been possible. The differences among MSCs from different strains may explain some of the conflicting data recently published on the engraftment of mouse MSCs or other bone marrow cells into nonhematopoietic tissues.