RESUMO
Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) promote the healing of chronic wounds, and that the exposure of ASCs to hypoxia enhances their wound healing effect. To aid the translation of these findings into clinical use, robust wound models are necessary to explore each aspect of wound healing. The aspect of re-epithelization is often studied in a scratch assay based on transformed keratinocytes. However, there are concerns regarding the validity of this model, since these cell lines differ from normal keratinocytes, both in terms of proliferative capacity and differentiation, and sensitivity to environmental cues. In this study, the main challenge of using primary keratinocytes to examine the effects of ASCs was identified to be their different requirements for calcium in the culture media. We confirmed that a high calcium content led to morphological and cytoskeletal changes in primary keratinocytes, and demonstrated that a low calcium content compromised the growth of ASCs. We found that it is possible to perform the wound healing assay with primary keratinocytes, if the conditioned media from the ASCs is dialyzed to reduce the calcium concentration. Additionally, using this model of re-epithelization, conditioned media from normoxic ASCs was shown to markedly increase the rate of wound closure by primary keratinocytes, and this effect was significantly enhanced with media from the hypoxia-exposed ASCs. These findings, which are in line with the observations from previous in vivo studies, highlight the validity of this modified assay to investigate the wound healing properties of ASCs in vitro.
Assuntos
Tecido Adiposo/citologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Diferenciação Celular , Hipóxia/metabolismo , Queratinócitos/metabolismo , Cicatrização , Cálcio , Técnicas de Cultura de Células , Células Cultivadas , Meios de Cultura/química , HumanosRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0151264.].
RESUMO
Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.
Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Microesferas , Vitronectina/farmacologia , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacosRESUMO
The generation of induced pluripotent stem cells (iPSCs) from somatic cells has enabled the possibility of providing unprecedented access to patient-specific iPSC cells for drug screening, disease modeling, and cell therapy applications. However, a major obstacle to the use of iPSC for therapeutic applications is the potential of genomic modifications caused by insertion of viral transgenes in the cellular genome. A second concern is that reprogramming often requires the use of animal feeder layers and reagents that contain animal origin products, which hinder the generation of clinical-grade iPSCs. Here, we report the generation of iPSCs by an RNA Sendai virus vector that does not integrate into the cells genome, providing transgene-free iPSC line. In addition, reprogramming can be performed in feeder-free condition with StemPro hESC SFM medium and in xeno-free (XF) conditions. Generation of an integrant-free iPSCs generated in xeno-free media should facilitate the safe downstream applications of iPSC-based cell therapies.
RESUMO
Skeletal muscle differentiation and regeneration are regulated by interactions between exogenous hormone- and growth factor-activated signaling cascades and endogenous muscle-specific transcriptional programs. IGF-I and IGF-II can promote muscle differentiation in vitro and can enhance muscle maintenance and repair in vivo. In contrast, members of the TGF-ß superfamily prominently inhibit muscle differentiation and regeneration. In this study, we have evaluated functional interactions between IGF- and TGF-ß-regulated signaling pathways during skeletal muscle differentiation. In the mouse C2 muscle cell line and in human myoblasts in primary culture, addition of TGF-ß1 blocked differentiation in a dose-dependent way, inhibited expression of muscle-specific mRNAs and proteins, and impaired myotube formation. TGF-ß1 also diminished stimulation of IGF-II gene expression in myoblasts, decreased IGF-II secretion, and reduced IGF-I receptor activation. To test the hypothesis that TGF-ß1 prevents muscle differentiation primarily by blocking IGF-II production, we examined effects of IGF analogues on TGF-ß actions in myoblasts. Although both IGF-I and IGF-II restored muscle gene and protein expression, and stimulated myotube formation in the presence of TGF-ß1, they did not reduce TGF-ß1-stimulated signaling, as measured by no decline in phosphorylation of SMA and mothers against decapentaplegic homolog (Smad)3, or in induction of TGF-ß-activated target genes, including a Smad-dependent promoter-reporter plasmid. Our results demonstrate that TGF-ß disrupts an IGF-II-stimulated autocrine amplification cascade that is necessary for muscle differentiation in vitro. Because this inhibitory pathway can be overcome by exogenous IGFs, our observations point toward potential strategies to counteract disorders that reduce muscle mass and strength.
Assuntos
Comunicação Autócrina/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Fator de Crescimento Insulin-Like II/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/biossíntese , Camundongos , Modelos Biológicos , Músculo Esquelético/efeitos dos fármacos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Sus scrofa , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Repulsive guidance molecule c (RGMc or hemojuvelin), a glycosylphosphatidylinositol-linked glycoprotein expressed in liver and striated muscle, plays a central role in systemic iron balance. Inactivating mutations in the RGMc gene cause juvenile hemochromatosis (JH), a rapidly progressing iron storage disorder with severe systemic manifestations. RGMc undergoes complex biosynthetic steps leading to membrane-bound and soluble forms of the protein, including both 50 and 40 kDa single-chain species. RESULTS: We now show that pro-protein convertases (PC) are responsible for conversion of 50 kDa RGMc to a 40 kDa protein with a truncated COOH-terminus. Unlike related molecules RGMa and RGMb, RGMc encodes a conserved PC recognition and cleavage site, and JH-associated RGMc frame-shift mutants undergo COOH-terminal cleavage only if this site is present. A cell-impermeable peptide PC inhibitor blocks the appearance of 40 kDa RGMc in extra-cellular fluid, as does an engineered mutation in the conserved PC recognition sequence, while the PC furin cleaves 50 kDa RGMc in vitro into a 40 kDa molecule with an intact NH2-terminus. Iron loading reduces release of RGMc from the cell membrane, and diminishes accumulation of the 40 kDa species in cell culture medium. CONCLUSION: Our results define a role for PCs in the maturation of RGMc that may have implications for the physiological actions of this critical iron-regulatory protein.
Assuntos
Proteínas Reguladoras de Ferro/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Pró-Proteína Convertases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Furina/metabolismo , Proteínas Ligadas por GPI , Proteína da Hemocromatose , Ferro/metabolismo , Proteínas Reguladoras de Ferro/genética , Proteínas de Membrana/genética , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Peso Molecular , Proteínas Musculares/genética , Mutação , Isoformas de Proteínas/metabolismoRESUMO
Juvenile hemochromatosis is a severe and rapidly progressing hereditary disorder of iron overload, and it is caused primarily by defects in the gene encoding repulsive guidance molecule c/hemojuvelin (RGMc/HJV), a recently identified protein that undergoes a complicated biosynthetic pathway in muscle and liver, leading to cell membrane-linked single-chain and heterodimeric species, and two secreted single-chain isoforms. RGMc modulates expression of the hepatic iron regulatory factor, hepcidin, potentially through effects on signaling by the bone morphogenetic protein (BMP) family of soluble growth factors. To date, little is known about specific pathogenic defects in disease-causing RGMc/HJV proteins. Here we identify functional abnormalities in three juvenile hemochromatosis-linked mutants. Using a combination of approaches, we first show that BMP-2 could interact in biochemical assays with single-chain RGMc species, and also could bind to cell-associated RGMc. Two mouse RGMc amino acid substitution mutants, D165E and G313V (corresponding to human D172E and G320V), also could bind BMP-2, but less effectively than wild-type RGMc, while G92V (human G99V) could not. In contrast, the membrane-spanning protein, neogenin, a receptor for the related molecule, RGMa, preferentially bound membrane-associated heterodimeric RGMc and was able to interact on cells only with wild-type RGMc and G92V. Our results show that different isoforms of RGMc/HJV may play unique physiological roles through defined interactions with distinct signaling proteins and demonstrate that, in some disease-linked RGMc mutants, these interactions are defective.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína Morfogenética Óssea 2 , Linhagem Celular , Membrana Celular/metabolismo , Proteínas Ligadas por GPI , Regulação da Expressão Gênica/fisiologia , Hemocromatose/genética , Proteína da Hemocromatose , Humanos , Proteínas de Membrana/genética , Mutação , Ligação Proteica , Isoformas de ProteínasRESUMO
We describe a simple, robust, and relatively inexpensive non-radioactive in vitro assay for measuring histone acetyl-transferase activity. The assay takes advantage of easy to purify recombinant E. coli-derived fusion proteins containing the NH(2)-terminal tails of histones H3 and H4 linked to epitope-tagged maltose-binding protein (MBP), and immunoblotting with antibodies specific to acetylated H3 and H4. Here we show the specificity and dynamic range of this assay for the histone acetyl-transferases, p300 and PCAF. This assay may be adapted readily for other substrates by simply generating new fusion proteins and for other acetyl-transferases by modifying reaction conditions.
Assuntos
Bioensaio/métodos , Histonas/química , Immunoblotting/métodos , Fatores de Transcrição de p300-CBP/análise , Acetilação , Animais , Mapeamento de Epitopos , Marcação por Isótopo , Camundongos , Camundongos Endogâmicos C3HRESUMO
The recently discovered repulsive guidance molecule c (RGMc or hemojuvelin) gene encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein that is expressed in striated muscle and in liver. Mutations in this gene have been linked to the severe iron storage disease, juvenile hemochromatosis, although the mechanisms of action of RGMc in iron metabolism are unknown. As a first step toward understanding the molecular physiology of this protein, we studied its biosynthesis, processing and maturation. Production of RGMc occurs as an early and sustained event during skeletal muscle differentiation in culture and is secondary to RGMc gene activation. As assessed by pulse-chase studies and cell-surface labeling experiments, two classes of GPI-anchored and glycosylated RGMc molecules are targeted to the membrane and undergo distinct fates. Full-length RGMc is released from the cell surface and accumulates in extracellular fluid, where its half-life exceeds 24 hours. By contrast, the predominant membrane-associated isoform, a disulfide-linked heterodimer composed of N- and C-terminal fragments, is not found in the extracellular fluid, and is short-lived, as it disappears from the cell surface with a half-life of <3 hours after interruption of protein synthesis. A natural disease-associated RGMc mutant, with valine substituted for glycine at residue 320 (313 in mouse RGMc), does not undergo processing to generate the heterodimeric membrane-linked isoform of RGMc, and is found on the cell surface only as larger protein species. Our results define a series of biosynthetic steps leading to the normal production of different RGMc isoforms in cells, and provide a framework for understanding the biochemical basis of defects in the maturation of RGMc in juvenile hemochromatosis.
Assuntos
Diferenciação Celular , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Músculo Esquelético/metabolismo , Adenoviridae , Animais , Células Cultivadas , Líquido Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas Ligadas por GPI , Glicosilação , Glicosilfosfatidilinositóis/metabolismo , Proteína da Hemocromatose , Homeostase , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/fisiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Mutação , Mioblastos/citologia , Mioblastos/metabolismo , CoelhosRESUMO
Normal skeletal muscle development requires the proper orchestration of genetic programs by myogenic regulatory factors (MRFs). The actions of the MRF protein MyoD are enhanced by the transcriptional coactivators p300 and the p300/CBP-associated factor (PCAF). We previously described C2 skeletal myoblasts lacking expression of insulin-like growth factor-II (IGF-II) that underwent progressive apoptotic death when incubated in differentiation-promoting medium. Viability of these cells was sustained by addition of IGF analogs or unrelated peptide growth factors. We now show that p300 or PCAF maintains myoblast viability as effectively as added growth factors through mechanisms requiring the acetyltransferase activity of PCAF but not of p300. The actions of p300 to promote cell survival were not secondary to increased expression of known MyoD targets, as evidenced by results of gene microarray experiments, but rather appeared to be mediated by induction of other genes, including fibroblast growth factor-1 (FGF-1). Conditioned culture medium from cells expressing p300 increased myoblast viability, and this was blocked by pharmacological inhibition of FGF receptors. Our results define a role for p300 in promoting cell survival, which is independent of its acetyltransferase activity and acts at least in part through FGF-1.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/fisiologia , Proteína p300 Associada a E1A/fisiologia , Histona Acetiltransferases/fisiologia , Mioblastos/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Fator 1 de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica , Humanos , Camundongos , Mioblastos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Transcrição de p300-CBPRESUMO
Peptide growth factors regulate cell fate by activating distinct signal transduction pathways that ultimately influence gene expression. Insulin-like growth factors (IGFs) play central roles in controlling somatic growth and participate in skeletal muscle development and regeneration. In cultured muscle cells, IGF action is critical both for maintaining viability during the transition from proliferating to differentiating myoblasts and for facilitating differentiation. By contrast, platelet-derived growth factor (PDGF) can sustain cell survival but inhibits differentiation. Here we examine the genetic programs that accompany IGF and PDGF action in myoblasts. Through analysis of high-density oligonucleotide arrays containing approximately 36,000 mouse probe sets, we identify 90 transcripts differentially induced by IGF-I, including 28 muscle-specific genes and 33 previously unannotated mRNAs, and 55 transcripts specifically stimulated by PDGF, including 14 unknowns. Detailed study of one IGF-induced mRNA shows that it encodes a protein related to a recently characterized repulsive guidance molecule postulated to regulate neuronal targeting during development. Our results demonstrate the power of transcriptional profiling for gene discovery and provide opportunities for investigating new proteins potentially involved in different aspects of growth factor action in muscle.
Assuntos
Perfilação da Expressão Gênica , Fator de Crescimento Insulin-Like I/fisiologia , Mioblastos Esqueléticos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Crescimento Derivado de Plaquetas/fisiologia , Animais , Sequência de Bases , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Dados de Sequência Molecular , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Células Musculares/fisiologia , Músculo Esquelético/crescimento & desenvolvimento , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologiaRESUMO
Specific gene ablation by RNA inference (RNAi) involves the binding of short interfering RNA (siRNA), 21 to 22 nucleotides long, to complementary mRNA sequences, leading to sequence-specific posttranslational gene silencing, thus providing a powerful tool for studying gene function with potential therapeutic applications. Here we describe the development of a two-vector adenovirus system for efficient, tightly controlled hairpin siRNA expression (shRNA). Regulated expression of the shRNA is conferred within an adenoviral vector by a modified RNA polymerase III promoter containing a Tet operator element adjacent to the transcription start site. In the presence of the tetracycline repressor protein (TetR), encoded in a second adenovirus, shRNA expression is repressed. Addition of tetracycline abolishes TetR binding, allowing shRNA transcription to proceed, and leading to reduced mRNA and protein expression. Here we establish the efficacy of this system by delivering siRNA targeted against the transcriptional coactivator p300. Our results show tetracycline-mediated inhibition of p300 mRNA and protein accumulation in the presence of both viruses, but no effect in the absence of antibiotic. Regulated adenoviral shRNA vectors offer the advantages of being able to infect a wide array of replicating and nonreplicating cells and of allowing temporal control of gene silencing.
Assuntos
Adenoviridae/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA/genética , Linhagem Celular , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Células HeLa , Humanos , Imuno-Histoquímica , Cinética , Regiões Promotoras Genéticas , RNA/metabolismo , RNA Polimerase III/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/química , Proteínas Repressoras/metabolismo , Tetraciclina/farmacologiaRESUMO
It has been shown that p300 binds to MDM2 and leads to down-regulation of the p53 function. However, it remains unclear whether the acetylase activity of p300 is necessary for regulating MDM2 stability. In this study, we address this issue. First, p300 did not acetylate MDM2 in solution and in cells. Second, overexpression of p300 in cells increased the level of the MDM2 protein but not its mRNA. Similarly, the acetylase-defective p300 AT2 mutant stabilized the MDM2 protein as well. Consistently, the deacetylase inhibitor, trichostatin A, did not significantly affect the half-life of the endogenous MDM2 protein, whereas p300 enhanced the half-life of MDM2. Finally, both wild type and acetylase-defective mutant p300 proteins associated with MDM2 in nuclear body-like structures where MDM2 might be protected from proteasomal degradation. Thus, these results suggest that p300 appears to stabilize MDM2 by retaining this protein in a specific nuclear structure rather than by acetylating it.
Assuntos
Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/fisiologia , Acetilação , Adenoviridae/genética , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Cicloeximida/farmacologia , Proteína p300 Associada a E1A , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Microscopia de Fluorescência , Mutação , Proteínas Proto-Oncogênicas c-mdm2 , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
The major human AP-endonuclease 1 (APE1) is a multifunctional protein that plays a central role in the repair of damaged DNA by acting as a dual-function nuclease in the base excision repair pathway. This enzyme was also independently identified as a redox activator of AP-1 DNA-binding activity and has subsequently been shown to activate a variety of transcription factors via a redox mechanism. In a third distinct role, APE1 was identified as a component of a trans-acting complex that acts as a repressor by binding to the negative calcium responsive elements (nCaRE)-A and nCaRE-B, which were first discovered in the promoter of the human parathyroid gene and later in the APE1 promoter itself. Here we show that the nuclear protein complex which binds to the nCaRE-B2 of the hAPE1 gene contains APE1 itself and the heterogeneous nuclear ribonucleoprotein L (hnRNP-L). The interaction between the APE1 and hnRNP-L proteins does not require the presence of nCaRE-B2. Our results support the possibility that the APE1 gene is down-regulated by its own product, which would be the first such example of the regulation of a DNA repair enzyme, and identify a novel function of hnRNP-L in transcriptional regulation.