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PubMed2003

Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre.

DNA double-strand break repair (DSBR) is an essential process for preserving genomic integrity in all organisms. To investigate this process at the cellular level, we engineered a system of fluorescently marked DNA double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae to visualize in vivo DSBR in single cells. Using this system, we demonstrate for the first time that Rad52 DNA repair foci and DSBs colocalize. Time-lapse microscopy reveals that the relocalization of Rad52 protein into a focal assembly is a rapid and reversible process. In addition, analysis of DNA damage checkpoint-deficient cells provides direct evidence for coordination between DNA repair and subsequent release from checkpoint arrest. Finally, analyses of cells experiencing multiple DSBs demonstrate that Rad52 foci are centres of DNA repair capable of simultaneously recruiting more than one DSB.

Bacterial Proteins↗

PubMed2006

Transverse aortic constriction leads to accelerated heart failure in mice lacking PPAR-gamma coactivator 1alpha.

Heart failure is accompanied by important defects in metabolism. The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) is a powerful regulator of mitochondrial biology and metabolism. PGC-1alpha and numerous genes regulated by PGC-1alpha are repressed in models of cardiac stress, such as that generated by transverse aortic constriction (TAC). This finding has suggested that PGC-1alpha repression may contribute to the maladaptive response of the heart to chronic hemodynamic loads. We show here that TAC in mice genetically engineered to lack PGC-1alpha leads to accelerated cardiac dysfunction, which is accompanied by signs of significant clinical heart failure. Treating cardiac cells in tissue culture with the catecholamine epinephrine leads to repression of PGC-1alpha and many of its target genes, recapitulating the findings in vivo in response to TAC. Importantly, introduction of ectopic PGC-1alpha can reverse the repression of most of these genes by epinephrine. Together, these data indicate that endogenous PGC-1alpha serves a cardioprotective function and suggest that repression of PGC-1alpha significantly contributes to the development of heart failure. Moreover, the data suggest that elevating PGC-1alpha activity may have therapeutic potential in the treatment of heart failure.

Animals↗

PubMed1996

Long-term functional recovery from age-induced spatial memory impairments by nerve growth factor gene transfer to the rat basal forebrain.

Nerve growth factor (NGF) stimulates functional recovery from cognitive impairments associated with aging, either when administered as a purified protein or by means of gene transfer to the basal forebrain. Because gene transfer procedures need to be tested in long-term experimental paradigms to assess their in vivo efficiency, we have used ex vivo experimental gene therapy to provide local delivery of NGF to the aged rat brain over a period of 2.5 months by transplanting immortalized central nervous system-derived neural stem cells genetically engineered to secrete NGF. By grafting them at two independent locations in the basal forebrain, medial septum and nucleus basalis magnocellularis, we show that functional recovery as assessed in the Morris water maze can be achieved by neurotrophic stimulation of any of these cholinergic cell groups. Moreover, the cholinergic neurons in the grafted regions showed a hypertrophic response resulting in a reversal of the age-associated atrophy seen in the learning-impaired aged control rats. Long-term expression of the transgene lead to an increased NGF tissue content (as determined by NGF-ELISA) in the transplanted regions up to at least 10 weeks after grafting. We conclude that the gene transfer procedure used here is efficient to provide the brain with a long-lasting local supply of exogenous NGF, induces long-term functional recovery of cognitive functions, and that independent trophic stimulation of the medial septum or nucleus basalis magnocellularis has similar consequences at the behavioral level.

Aging↗

PubMed2003

Pyridoxal phosphate inhibits dynamic subunit interchange among serine hydroxymethyltransferase tetramers.

Cytoplasmic serine hydroxymethyltransferase (cSHMT) is a tetrameric, pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. The enzyme has four active sites and is best described as a dimer of obligate dimers. Each monomeric subunit within the obligate dimer contributes catalytically important amino acid residues to both active sites. To investigate the interchange of subunits among cSHMT tetramers, a dominant-negative human cSHMT enzyme (DNcSHMT) was engineered by making three amino acid substitutions: K257Q, Y82A, and Y83F. Purified recombinant DNcSHMT protein was catalytically inactive and did not bind 5-formyltetrahydrofolate. Coexpression of the cSHMT and DNcSHMT proteins in bacteria resulted in the formation of heterotetramers with a cSHMT/DNcSHMT subunit ratio of 1. Characterization of the cSHMT/DNcSHMT heterotetramers indicates that DNcSHMT and cSHMT monomers randomly associate to form tetramers and that cSHMT/DNcSHMT obligate dimers are catalytically inactive. Incubation of recombinant cSHMT protein with recombinant DNcSHMT protein did not result in the formation of hetero-oligomers, indicating that cSHMT subunits do not exchange once the tetramer is assembled. However, removal of the active site PLP cofactor does permit exchange of obligate dimers among preformed cSHMT and DNcSHMT tetramers, and the formation of heterotetramers from cSHMT and DNcSHMT homodimers does not affect the activity of the cSHMT homodimers. The results of these studies demonstrate that PLP inhibits dimer exchange among cSHMT tetramers and suggests that cellular PLP concentrations may influence the stability of cSHMT protein in vivo.

Binding Sites↗

PubMed2006

Betanodavirus infection in the freshwater model fish medaka (Oryzias latipes).

Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite, positive-sense RNA genomes. As their genomes are the smallest and simplest among viruses, betanodaviruses have been studied in detail as model viruses by using a genetic-engineering system, as has occurred with the insect alphanodaviruses, the other members of the family Nodaviridae. However, studies of virus-host interactions have been limited, as betanodaviruses basically infect marine fish at early developmental stages (larval and juvenile). These fish are only available for a few months of the year and are not suitable for the construction of a reverse-genetics system. To overcome these problems, several freshwater fish species were tested for their susceptibility to betanodaviruses. It was found that adult medaka (Oryzias latipes), a well-known model fish, was susceptible to both Striped jack nervous necrosis virus (the type species of the genus Betanodavirus) and Redspotted grouper nervous necrosis virus (RGNNV), which have different host specificities in marine fish species. Infected medaka exhibited erratic swimming and the viruses were localized specifically in the brain, spinal cord and retina of the infected fish, similar to the pattern of infection in naturally infected marine fish. Moreover, medaka were susceptible to RGNNV at the larval stage. This is the first report of a model virus-model host infection system in fish. This system should facilitate elucidation of the mechanisms underlying RNA virus infections in fish.

Animals↗

PubMed2002

The capsid of infectious bursal disease virus contains several small peptides arising from the maturation process of pVP2.

The capsid proteins VP2 and VP3 of infectious bursal disease virus, a birnavirus, are derived from the processing of a large polyprotein: NH2-pVP2-VP4-VP3-COOH. Although the primary cleavage sites at the pVP2-VP4 and VP4-VP3 junctions have been identified, the proteolytic cascade involved in the processing of this polyprotein is not yet fully understood, particularly the maturation of pVP2. By using different approaches, we showed that the processing of pVP2 (residues 1 to 512) generated VP2 and four small peptides (residues 442 to 487, 488 to 494, 495 to 501, and 502 to 512). We also showed that in addition to VP2, at least three of these peptides (residues 442 to 487, 488 to 494, and 502 to 512) were associated with the viral particles. The importance of the small peptides in the virus cycle was assessed by reverse genetics. Our results showed that the mutants lacking the two smaller peptides were viable, although the virus growth was affected. In contrast, deletions of the domain 442 to 487 or 502 to 512 did not allow virus recovery. Several amino acids of the peptide 502 to 512 appeared essential for virus viability. Substitutions of the P1 and/or P1" position were engineered at each of the cleavage sites (P1-P1": 441-442, 487-488, 494-495, 501-502, and 512-513). Most substitutions at the pVP2-VP4 junction (512-513) and at the final VP2 maturation cleavage site (441-442) were lethal. Mutations of intermediate cleavage sites (487-488, 494-495, and 501-502) led to viable viruses showing different but efficient pVP2 processing. Our data suggested that while peptides 488 to 494 and 495 to 501 play an accessory role, peptides 442 to 487 and 502 to 512 have an unknown but important function within the virus cycle.

Amino Acid Sequence↗

PubMed1999

Binding kinetics of engineered mutants provide insight about the pathway for entering and exiting the intestinal fatty acid binding protein.

To better understand the mechanism by which fatty acids bind to and dissociate from the binding cavities of fatty acid binding proteins (FABPs), we constructed 31 single amino acid mutants of the intestinal FABP (I-FABP) and determined the rate constants for binding and dissociation, primarily for long-chain fatty acids (FA). FA dissociation from these proteins was measured both by the ADIFAB method and by the change in tryptophan fluorescence of the FABPs. Rate constants for binding (kon) were calculated from the rate constants for dissociation (koff) and the equilibrium binding affinities. Amino acid substitutions were made at locations within the binding cavity, in the region of the gap between the betaD- and betaE-strands, and within the "portal" region of the protein. The koff values for the mutant proteins ranged from about 20-fold slower to 4-fold faster than the wild-type (WT) protein. Values for kon were as much as 20-fold slower than the WT protein, but in no case was kon significantly faster than the WT. Mutants with slower and faster koff values were generally those involving sites within the binding cavity and, relative to the WT protein, revealed higher and lower affinities, respectively. Reduced rates of binding were generally, but not exclusively, associated with sites within the portal region. For example, for F68A which is located closer to the opposite end of the protein from the portal region, the kon is more than 10-fold slower than WT. Even for these distal sites, however, the evidence is consistent with reductions in kon being due to alterations of the portal region. Binding affinities and rate constants measured as a function of ionic strength also suggest that the FA initially binds, through an electrostatic interaction, to Arg-56 on the surface of the protein, before inserting into the binding cavity. Thus, the results of this study are consistent with FA binding to I-FABP involving an initial interaction with Arg-56 followed by insertion of the FA, through the portal region, into the binding cavity and with a reversal of these steps for the dissociation reaction.

2-Naphthylamine↗

PubMed2006

Biomimetic porous scaffolds made from poly(L-lactide)-g-chondroitin sulfate blend with poly(L-lactide) for cartilage tissue engineering.

A novel biodegradable graft copolymer chondroitin sulfate-grafted poly(L-lactide) (CS-PLLA) was synthesized. The graft copolymer was blended with PLLA to form biomimetic porous scaffolds. Natural CS was introduced into the polyester matrix to promote the proliferation of cells. Three-dimensional spongelike scaffolds were fabricated by a combination of salt leaching and solvent casting methods. The morphology of the scaffolds was observed with scanning electron microscopy with an average pore size between 50 and 250 microm, and its porosity was high (>85%). Compression analysis indicated that the mechanical properties of the scaffold were adequate to support the proliferation of cells. The hydrophilicity increased with an increase in the copolymer content in the blend, as determined by measuring the contact angle. Hematoxylin and eosin, Masson, and Safranin-O staining showed that cells formed a chondro tissue gradually. Histological results revealed that abundant cartilaginous matrixes surrounded spherical chondrocytes in the center of the explants. Chondrocytes cultured in this extracellular-matrix-like scaffold maintained a round morphology phenotype, characterized by a significant quantity of extracellular matrixes of sulfated glycosaminoglycans and collagens. Additionally, phenotypic gene expression (reverse transcriptase-polymerase chain reaction) indicated that chondrocytes expressed transcripts that encoded type II collagen and aggrecan and generated sulfated glycosaminoglycans.

Aggrecans↗

PubMed1998

Recombinant production and purification of novel antisense antimicrobial peptide in Escherichia coli.

A fusion protein was genetically engineered that contains an antimicrobial peptide, designated P2, at its carboxy terminus and bovine prochymosin at its amino terminus. Bovine prochymosin was chosen as the fusion partner because of its complete insolubility in Escherichia coli, a property utilized to protect the cells from the toxic effects of the antimicrobial peptide. This fusion protein was purified by centrifugation as an insoluble inclusion body. A methionine linker between prochymosin and the P2 peptide enabled P2 to be released by digestion with cyanogen bromide. Cation exchange HPLC followed by reversed-phase HPLC were used to purify the P2 peptide. The recombinant P2 peptide's molecular mass was confirmed by mass spectrometry to within 0.1% of the theoretical value (2480.9 Da), and the antimicrobial activity of the purified recombinant P2 against E. coli D31 was determined to be identical to that of the chemically synthesized peptide (minimal inhibitory concentration of 5 mg/mL). Although the yield of the fusion protein after expression by the cells was high (16% of the total cell protein), the percentage recovery of the P2 peptide in the inclusion bodies was relatively low, which appears to be due to losses in the cyanogen bromide digestion step.

Amino Acid Sequence↗

PubMedDate not supplied

Injectable liver: a novel approach using fibrin gel as a matrix for culture and intrahepatic transplantation of hepatocytes.

Cell transplantation and tissue engineering with liver cells are currently under investigation as experimental therapies for certain liver diseases. In this study we evaluated a fibrin-based gel matrix as carrier for hepatocytes in culture. Furthermore, a novel technique for direct intrahepatic injection of fibrin gel-immobilized hepatocytes was developed and evaluated in a rat model. Hepatocytes were harvested from rats. Fibrin matrix was generated with modified fibrin sealant. Cells, in medium containing epidermal growth factor and insulin, were seeded in a drop of fibrin matrix onto plastic culture dishes. Cell numbers were assessed by DNA content. Hepatocyte differentiation was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistology (IH) for cytokeratin (CK)-18 and albumin. PKH26-labeled fibrin gel-immobilized hepatocytes were transplanted into liver by direct injection underneath the capsule. Fluorescence microscopy of explanted liver was performed to identify PKH26+ donor cells. Neotissue was characterized by IH for the markers CK-18, ED1, and desmin. Culture in a fibrin matrix allowed stable cell numbers and three-dimensional neotissue formation. RT-PCR and IH showed preservation of liver-specific markers CK-18 and albumin in vitro. Transplanted cells were identified by fluorescence microscopy after 2 and 7 days. CK-18 and desmin staining showed integration of hepatocytes and hepatic stellate cells into the host liver. Fibrin matrix is an appropriate environment for hepatocytes in culture. Direct intrahepatic injection of fibrin gel-immobilized hepatocytes is technically feasible. We conclude that fibrin gel immobilization is an attractive tool for the development of tissue engineering-based liver support systems.

Animals↗

PubMed2005

Three-dimensional composites manufactured with human mesenchymal cambial layer precursor cells as an alternative for sinus floor augmentation: an in vitro study.

Bone tissue engineering is a promising approach for treatment of defective and lost bone in the maxillofacial region. Creating functional tissue for load bearing bone reconstruction using biocompatible and biodegradable scaffolds seeded with living cells is of crucial importance. The aim of our study was to compare the effects of poly-lactic-co-glycolic acid (PLGA) and hydroxyapatite (HA) ceramic granulae on growth, differentiation, mineralization and gene expression of mandibular mesenchymal cambial layer precursor cells (MCLPCs) cultured onto tissue engineered three-dimensional (3-D) composites in vitro. These 3-D composites were cultivated in a rotating cultivation system under osteogenic differentiation conditions for a maximum period of 21 days. After 6 and 21 days, histological examination was performed; scanning electron microscopy (SEM), alkaline phosphatase (ALP) activity and levels of DNA were investigated. Expression of bone-specific genes osteocalcin, osteonectin, osteopontin, ALP, core binding factor alpha 1 and collagen type I were investigated by using a reverse transcription-polymerase chain reaction (RT-PCR) method. After 6 and 21 days of incubation an initiation of mineralization and the presence of newly formed bone at the surface of the composites were shown after evaluation of ALP activity, DNA content, SEM and histological staining. Expression of bone-specific genes confirmed the bone-like character of these composites and different effects of PLGA or HA granulae on the osteogenic differentiation of human MCLPCs in vitro. The results of this study support the concept that substrate signals significantly influence MCLPCs growth, differentiation, mineralization and gene expression in vitro, and that the use of these cells in the manufacturing of 3-D cell/HA composites is a promising approach for load bearing bone reconstruction in the maxillofacial region in vivo.

Alkaline Phosphatase↗

PubMed2002

Nanoscale phenomena in synthetic functional oxide heterostructures.

This paper reviews nanoscale phenomena such as polarization relaxation dynamics and piezoelectric characterization in model ferroelectric thin films and nanostructures using voltage-modulated scanning force microscopy. Using this technique we show the three-dimensional reconstruction of the polarization vector in lead zirconate titanate (PZT) thin films. Second, the time-dependent relaxation of remanent polarization in epitaxial PZT ferroelectric thin films, containing a uniform two-dimensional grid of 90 degrees domains (c-axis in the plane of the film), has been investigated extensively. The 90 degrees domain walls preferentially nucleate the 180 degrees reverse domains during relaxation. Relaxation occurs through the nucleation and growth of reverse 180 degrees domains, which subsequently coalesce and consume the entire region as a function of relaxation time. In addition we also present results on investigation of the relaxation phenomenon on a very local scale, where pinning and bowing of domain walls has been observed. We also show how this technique is used for obtaining quantitative information on piezoelectric constants and by engineering special structures, and how we realize ultrahigh values of piezoconstants. Last, we also show direct hysteresis measurements on nanoscale capacitors, where there is no observable loss of polarization in capacitors as small as 0.16 microm2 in area.

Crystallization↗

PubMed2006

Antisense Smo under the control of the PTCH1 promoter delivered by an adenoviral vector inhibits the growth of human pancreatic cancer.

Hedgehog (Hh) signaling pathway is crucial in growth and patterning during embryonic development. Recent data have shown an association of its activation with cancer formation and maintenance. A ligand-dependent activation, where Hh components (SHH, PTCH1, Smo and GLi1) are aberrantly expressed with PTCH1 being a negative feedback regulator, is a newly identified mechanism for pancreatic carcinogenesis. In this study, we developed a cell-specific cytotoxic model for the treatment of human pancreatic cancer (HPC) in which expression of antisense Smo (SAS) was under the control of the PTCH1 promoter (ptch/p) delivered by an adenoviral vector (Ad-ptch/p-SAS). We observed that the cell-specific cytotoxicity in HPC cells depended on the expressions of inherent PTCH1, Smo and GLi1 in the target cells in which the Hh pathway was presumed to be activated. Fluorescence-activated cell sorting analysis indicated that the cell death was apoptosis. Western blot showed that Smo protein in the infected cells significantly decreased. Furthermore, an in vivo experiment demonstrated that such Hh activity-cell-specific cytotoxicity was achieved by daily intratumoral injection of Ad-ptch/p-SAS (10(9) plaque-forming unit) for 5 days. Our study suggests that targeting at the Hh signaling pathway may be an effective novel gene therapeutic strategy alone or in combination with other agents for the treatment of pancreatic cancer.

Adenoviridae↗

PubMed1996

The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product.

Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL). Immunization of mice with a carcinogen-induced colorectal tumor, CT26, engineered to secrete granulocyte/macrophage colony-stimulating factor, routinely generated both short-term and long-term CTL lines that not only lysed the parental tumor in vitro, but also cured mice of established tumor following adoptive transfer in vivo. When either short-term or long-term CTL lines were used to screen peptides isolated from CT26, one reverse-phase high performance liquid chromatography peptide fraction consistently sensitized a surrogate target for specific lysis. The bioactivity remained localized within one fraction following multiple purification procedures, indicating that virtually all of the CT26-specific CTL recognized a single peptide. This result contrasts with other tumor systems, where multiple bioactive peptide fractions have been detected. The bioactive peptide was identified as a nonmutated nonamer derived from the envelope protein (gp70) of an endogenous ecotropic murine leukemia provirus. Adoptive transfer with CTL lines specific for this antigen demonstrated that this epitope represents a potent tumor rejection antigen. The selective expression of this antigen in multiple non-viral-induced tumors provides evidence for a unique class of shared immunodominant tumor associated antigens as targets for antitumor immunity.

Animals↗

PubMed2004

Gene transfer of cocaine hydrolase suppresses cardiovascular responses to cocaine in rats.

We previously found that injection of a cocaine hydrolase (CocE) engineered from human butyrylcholinesterase will transiently accelerate cocaine metabolism in rats while reducing physiological and behavioral responses. To investigate more extended therapeutic effects, CocE cDNA was incorporated into a replication-incompetent type-5 adenoviral vector with a cytomegalovirus promoter. In rats dosed with this agent (2.2 x 10(9) plaque-forming units), the time course of expression was characterized by reverse transcription polymerase chain reaction for CocE mRNA and by radiometric assay for enzyme activity. Liver and plasma showed comparable expression, beginning 2 days after vector administration and peaking between 5 and 7 days. Plasma CocE content was up to 100 mU/ml, with total cocaine hydrolyzing activity 3000-fold greater than in "empty vector" or untreated controls. This level of expression approximated that found immediately after i.v. injection of purified hydrolase, 3 mg/kg, a dose that shortened cocaine halflife and blunted cardiovascular effects. Sucrose density gradient analysis showed that 96% of the circulating CocE activity was associated with tetrameric enzyme forms, expected to be stable in vivo. Consistent with this expectation, CocE from vector-treated rats showed a plasma t(1/2) of 33 h when reinjected into naive rats. Transduction of another mutant butyrylcholinesterase, Applied Molecular Evolution mutant 359 (AME(359)), caused plasma cocaine hydrolase activity to rise 50,000-fold. At the point of peak AME(359) expression, cocaine was cleared from the blood too rapidly for accurate measurement, and pressor responses to the injection of drug were greatly impaired.

Animals↗

PubMed1993

Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1.

We have identified the tRNAs which are incorporated into both wild-type human immunodeficiency virus type 1 strain IIIB (HIV-1IIIB) produced in COS-7 cells transfected with HIV-1 proviral DNA and mutant, noninfectious HIV-1Lai particles produced in a genetically engineered Vero cell line. The mutant proviral DNA contains nucleotides 678 to 8944; i.e., both long terminal repeats and the primer binding site are absent. As analyzed by two-dimensional polyacrylamide gel electrophoresis, both mutant and wild-type HIV-1 contain four major-abundance tRNA species, which include tRNA(1,2Lys), tRNA(3Lys) (the putative primer for HIV-1 reverse transcriptase) and tRNA(Ile). Identification was accomplished by comparing the electrophoretic mobilities and RNase T1 digests with those of tRNA(3Lys) and tRNA(1,2Lys) purified from human placenta and comparing the partial nucleotide sequence at the 3' end of each viral tRNA species with published tRNA sequences. Thus, the absence of the primer binding site in the mutant virus does not affect tRNA(Lys) incorporation into HIV-1. However, only the wild-type virus contains tRNA(3Lys) tightly associated with the viral RNA genome. The identification of the tightly associated tRNA as tRNA(3Lys) is based upon an electrophoretic mobility identical to that of tRNA(3Lys) and the ability of this RNA to hybridize with a tRNA(3Lys)-specific DNA probe. In addition to the four wild-type tRNA species, the mutant HIV-1-like particle contains two tRNA(His) species and three tRNA-sized species that we have been unable to identify. Their absence in wild-type virus makes it unlikely that they are required for viral infectivity.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre.

Site-specific DNA recombinases from bacteriophage and yeasts have been developed as novel tools for genome engineering both in prokaryotes and eukaryotes. The 38kDa Cre protein efficiently produces both inter- and intramolecular recombination between specific 34bp sites called loxP. We report here the in vivo use of Cre recombinase to manipulate the genome of the protozoan parasite Toxoplasma gondii. Cre catalyzes the precise removal of transgenes from T. gondii genome when flanked by two directly repeated loxP sites. The efficiency of excision has been determined using LacZ as reporter and indicates that it can easily be applied to the removal of undesired sequences such as selectable marker genes and to the determination of gene essentiality. We have also shown that the reversibility of the recombination reaction catalyzed by Cre offers the possibility to target site-specific integration of a loxP-containing vector in a chromosomally placed loxP target in the parasite. In mammalian systems, the Cre recombinase can be regulated by hormone and is used for inducible gene targeting. In T. gondii, fusions between Cre recombinase and the hormone-binding domain of steroids are constitutively active, hampering the utilization of this mode of post-translational regulation as inducible gene expression system.

Animals↗

PubMed2006

Derivation of distal airway epithelium from human embryonic stem cells.

The pluripotency of embryonic stem cells (ESC) is offering new opportunities in tissue engineering and cell therapy. We have shown previously that alveolar epithelial cells, specifically type II pneumocytes, can be derived from murine ESC and hypothesized that a similar protocol could be used successfully on human ESC. Undifferentiated human ESC were induced to form embryoid bodies that were transferred into adherent culture conditions and grown in a medium designed for the maintenance of mature small airway epithelium. On inverted microscopy, the generated cells showed the cobblestone-like morphology of epithelium. The presence of surfactant protein C, a specific marker of type II pneumocytes, and its corresponding RNA were demonstrated by immunostaining and reverse transcription polymerase chain reaction, respectively. Electron microscopy revealed frequent cells with the typical ultrastructure of type II pneumocytes. This study provides evidence for in vitro induction of the differentiation from human ESC of alveolar type II cells, which have the potential for therapeutic use or construction of an in vitro model of human lung.

Animals↗