Amniotic membrane as novel scaffold for human iPSC-derived cardiomyogenesis.

Recent approaches of using decellularized organ matrices for cardiac tissue engineering prompted us to culture human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) on the human amniotic membrane (hAM). Since hAM has been used lately to patch diseased hearts in patients and has shown anti-inflammatory and anti-fibrotic benefits, it qualifies as a cardiac compatible and clinically relevant heart tissue scaffold. The aim of this study was to test the ability of the hAM to support attachment, differentiation, and maturation of hiPSC-derived CMs in vitro. hAMs were prepared from term placenta. An in-house generated hiPSC line was used for CM derivation. hiPSC-derived cardiac progenitors were cultured on the surface of cryopreserved hAMs and in the presence of cytokines promoting cardiac differentiation. CMs grown on hAM and popular basement membrane matrix (BMM) Matrigel™ were compared for the following aspects of cardiac development: the morphology of cardiomyocytes with respect to shape and cellular alignments, levels of cardiac-related gene transcript expression, functionality in terms of spontaneous calcium fluxes and mitochondrial densities and distributions. hAM is biocompatible with hiPSC-derived CMs. hAM increased cardiac transcription regulator and myofibril protein transcript expression, accelerated intracellular calcium transients, and enhanced cellular mitochondrial complexity of its cardiomyocytes in comparison to cardiomyocytes differentiated on Matrigel™. Our data suggests that hAM supports differentiation and improves cardiomyogenesis in comparison to Matrigel™. hAMs are natural, easily and largely available. The method of preparing hAM cardiac sheets described here is simple with potential for clinical transplantation. Graphical abstract A An outline of the differentiation protocol with stage-specific growth factors and culture media used. B Cell fates from pluripotent stem cells to cardiomyocytes during differentiation on the amniotic membrane. C-FPhotomicrographs of cells at various stages of differentiation. Scale bars represent 100 µm.

Generation of an induced pluripotent stem cell line from chorionic villi of a Turner syndrome spontaneous abortion.

A major cause of spontaneous abortions is chromosomal abnormality of foetal cells. We report the generation of an induced pluripotent stem cell line from the fibroblasts isolated from chorionic villi of an early spontaneously aborted foetus with Turner syndrome. The Turner syndrome villus induced pluripotent stem cell line is transgene free, retains the original XO karyotype, expresses pluripotency markers and undergoes trilineage differentiation. This pluripotent stem cell model of Turner syndrome should serve as a tool to study the developmental abnormalities of foetus and placenta that lead to early embryo lethality and profound symptoms like infertility in 45 XO survivors.

Transplantation of hippocampal cell lines restore spatial learning in rats with ventral subicular lesions.

We have demonstrated in our previous studies that ventral subicular lesion induces neurodegeneration of the hippocampus and produces cognitive impairment in rats. In the present study, the efficacy of transplanted green fluorescent protein (GFP)-labeled hippocampal cell line (H3-GFP) cells in establishing functional recovery in ventral subicular lesioned rats has been evaluated. The survival of H3-GFP transplants and their ability to express trophic factors in vivo were also investigated. Adult male Wistar rats were subjected to selective lesioning of ventral subiculum and were transplanted with H3-GFP cells into the cornu ammonis 1 (CA1) hippocampus. The transplants settled mainly in the dentate gyrus and expressed neurotrophic factors, brain-derived neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF). The ventral subicular lesioned (VSL) rats with H3-GFP transplants showed enhanced expression of BDNF in the hippocampus and performed well in eight-arm radial maze and Morris water maze tasks. The VSL rats without hippocampal transplants continued to show cognitive impairment in task learning. The present study demonstrated the H3-GFP transplants mediated recovery of cognitive functions in VSL rats. Our study supports the notion of graft meditated host regeneration and functional recovery through trophic support, although these mechanisms require further investigation.

Cloning and functional expression of alternative spliced variants of the rho1 gamma-aminobutyrate receptor.

The rho1 gamma-aminobutyrate receptor (GABArho1) is expressed predominantly in the retina and forms homomeric GABA-gated Cl- channels that are clearly different from the multisubunit GABAA receptors. In contrast to these, GABArho1 receptors desensitize very little and are not blocked by bicuculline. In addition to GABArho1, two new variants were identified in human retina cDNA libraries. Cloning and sequence analysis showed that both variants contain large deletions in the putative extracellular domain of the receptor. These deletions extend from a common 5' site to different 3' sites. The cDNA with the largest deletion, named GABArho1Delta450, contains a complete ORF identical to that of GABArho1 but missing 450 nt. This cDNA encodes a protein of 323 aa, identical to the GABArho1, but has a deletion of 150 aa in the amino-terminal extracellular domain. GABArho1Delta450 mRNA injected into Xenopus oocytes did not produce functional GABA receptors. The second GABArho1 variant (GABArho1Delta51) contains a 51-nt deletion. In Xenopus oocytes, GABArho1Delta51 led to the expression of GABA receptors that had the essential GABArho1 characteristics of low desensitization and bicuculline resistance. Therefore, alternative splicing increases the coding potential of this gene family expressed in the human retina, but the functional diversity created by the alternative spliced forms is still not understood.

Multiple transcripts encode the 5-HT1F receptor in rodent brain.

The mouse serotonin 1F (5-HT1F) receptor is encoded by at least three transcripts in mouse brain. These transcripts are expressed predominantly in cortex and hippocampus. Similar transcripts are seen in Northern analysis of rat brain mRNA. 5' RACE showed a predominant transcription start site around 350 bp upstream of the translational start present in mouse cDNA. Our results suggest that the heterogeneity seen in transcript size is due to differences in the 3' untranslated region, which could play a critical role in mRNA targeting and localization. The mouse 5-HT1F genomic clone shows the coding region to be intronless and an intron splice junction is seen in the 5' untranslated region which is conserved in both rat and mouse.

Efficient coupling of 5-HT1a receptors to the phospholipase C pathway in Xenopus oocytes.

To investigate the receptor-channel coupling pathway, the coding region of the 5-HT1a receptor was subcloned into two plasmid vectors pSP64(polyA+) and pSP64T. Compared to the original 5-HT1a receptor construct G-21, both new constructs increased greatly the expression of functional 5-HT1a receptors in Xenopus oocytes, which developed large inward current responses to 5-HT. These responses were dose-dependent (EC50 approximately 150 nM), and could be elicited also by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The 5-HT1a receptor mediated current had an oscillatory time course, and a reversal potential close to the equilibrium potential for Cl- (ca. -25 mV). Moreover, during and for some minutes following the application of 5-HT, these oocytes acquired the property of generating a transient inward current when their membrane was hyperpolarized. These features are characteristic of responses mediated by other receptors (e.g. muscarinic, angiotensin, serum receptors, etc.) that are known to couple to the endogenous PLC/PI second messenger pathway in Xenopus oocytes. In particular, the 5-HT1a receptor mediated current was very similar to the current induced by 5-HT-stimulation of heterogenic 5-HT2c receptors. Our results show further that the 5-HT1a receptor couples to the endogenous PLC/PI pathway much less efficiently than the 5-HT2c receptor. These results demonstrate clearly that the human 5-HT1a receptor can couple efficiently to the Xenopus oocyte endogenous PLC/PI pathway, and provide additional evidence for cell-specific signal transduction.

Purification and properties of the F sex factor TraD protein, an inner membrane conjugal transfer protein.

Using a traD overexpression plasmid, we purified the F sex factor TraD protein in milligram quantities. The purified protein has an apparent molecular weight of 82,000 and an amino acid composition rich in acidic residues. Using specific antibodies, TraD was localized to the inner membrane of F+ cells under conditions where it is produced in physiologically normal amounts. Furthermore, the protein was soluble only in the presence of detergents, but there is evidence that the carboxyl terminus is water-soluble. The purified protein shows pH-sensitive binding to DNA cellulose columns.

Biochemical characterization of Escherichia coli DNA helicase I.

The gene product of F tral is a bifunctional protein which nicks and unwinds the F plasmid during conjugal DNA transfer. Further biochemical characterization of the Tral protein reveals that it has a second, much lower, Km for ATP hydrolysis, in addition to that previously identified. Measurement of the single-stranded DNA-stimulated ATPase rate indicates that there is co-operative interaction between the enzyme monomers for maximal activity. Furthermore, 18O-exchange experiments indicate that Tral protein hydrolyses ATP with, at most, a low-level reversal of the hydrolytic step during each turnover.

Actions of dopamine and dopaminergic drugs on cloned serotonin receptors expressed in Xenopus oocytes.

Using electrophysiological techniques, we studied interactions of dopamine and selected dopaminergic drugs with serotonin (5-hydroxytryptamine; 5-HT) receptors expressed in Xenopus oocytes by RNAs transcribed from cloned cDNAs. Oocytes showing strong expression of 5-HT1c and 5-HT2 receptors became weakly responsive to the neurotransmitter dopamine, which, like 5-HT, elicited Cl- currents through activation of the phosphatidylinositol/Ca2+ messenger pathway. The two types of 5-HT receptors showed similar sensitivity to dopamine; threshold responses were activated at concentrations as low as 1 microM. However, maximum dopamine responses were only 5-20% of maximum responses activated by 5-HT. The dopamine D1 receptor antagonist SCH 23390 was a potent agonist on 5-HT1c and 5-HT2 receptors. SCH 23390 elicited currents at concentrations as low as 1 nM, but maximum responses were again only 5-20% of those activated by 5-HT. Fenoldopam, a dopamine D1 receptor agonist, also interacted with 5-HT1c and 5-HT2 receptors, eliciting threshold responses between 10 and 20 nM. Our experiments raise the possibility that low micromolar concentrations of dopamine can cause weak activation and concomitant desensitization of serotoninergic systems in vivo and demonstrate that benzazepines can interact with 5-HT receptors at nanomolar concentrations.

Receptors of the serotonin 1C subtype expressed from cloned DNA mediate the closing of K+ membrane channels encoded by brain mRNA.

The modulation of K+ channels by serotonin (5-HT) receptors was studied by coinjecting Xenopus oocytes with mRNA transcribed in vitro from a cloned 5-HT 1C subtype (5-HT1C) receptor gene, together with size-fractionated mRNA isolated from rat cerebral cortex that expresses K+ channels. After intracellular loading with EGTA to block Ca2(+)-dependent chloride currents, these oocytes responded to 5-HT with an inward current associated with a decrease in membrane conductance. Membrane current responses were small or absent in oocytes injected with either mRNA alone. We conclude that 5-HT1C receptors are able to cause the closing of a class of K+ channels expressed by cortex mRNA in a Ca2(+)-independent manner. The coupling between the receptors and channels appears to be mediated by the inositol phospholipid second messenger pathway, since activation of this pathway by application of serum evoked a similar closing current.

Serotonin receptors expressed in Xenopus oocytes by mRNA from brain mediate a closing of K+ membrane channels.

Membrane currents evoked by serotonin (5-HT) were studied in Xenopus oocytes injected with rat brain mRNA. Intracellular EGTA was used to abolish the Ca2(+)-dependent oscillatory Cl- current to 5-HT, revealing an underlying smooth inward current. This was associated with a decreased membrane conductance, was antagonized by Ba2+ and Zn2+ (but not TEA), and probably arises through a closing of K+ channels. Half-maximal responses were obtained with 30 nM 5-HT, while 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) was ineffective. Furthermore, methysergide, mianserin and lysergic acid antagonized the K(+)-closing response to 5-HT, consistent with it being mediated through 5-HT1C receptors. The largest K(+)-closing responses were induced by a size fraction of mRNA which also induced a large K+ conductance, suggesting that the response requires expression of both receptors and K+ channels. The K(+)-closing response induced in the oocyte resembles the M- and S-type currents described in, respectively, mammalian and invertebrate neurons.

Production of polyclonal antisera to choline acetyltransferase using a fusion protein produced by a cDNA clone.

A fusion protein containing a Drosophila choline acetyltransferase (ChAT) cDNA insert was purified from a lambda gtll lysate of Escherichia coli. The cDNA insert, which contained a 728-amino acid coding region for ChAT, was used for immunizing rabbits. Three different antisera were produced that could recognize native Drosophila ChAT with low titer. In addition, all three antisera stained enzyme polypeptides using the Western blot technique at high titers. The antisera recognized ChAT polypeptides with molecular masses of 67 and 54 kilodaltons in Western blots of partially purified enzyme; these polypeptides had previously been identified using monoclonal anti-ChAT antibodies and are the major components of completely purified enzyme. It was surprising that when these antisera were used to stain Western blots of Drosophila head homogenates, the major immunoreactive band had a molecular mass of 75 kilodaltons. The relationship of this 75-kilodalton polypeptide to ChAT activity was investigated by fractionating fresh fly head homogenates using rapid HPLC gel filtration chromatography. Analysis of column fractions for enzyme activity and immunoreactive polypeptides indicated that the 75- and 67-kilodalton polypeptides can be resolved and are both enzymatically active. In addition, a correlation was observed between the relative immunostaining intensities of both the 75- and 67-kilodalton bands and ChAT activity when supernatants from fresh fly head homogenates were autolyzed at 37 degrees C. Our results indicate that ChAT is present in fresh Drosophila heads primarily as an active enzyme with a molecular mass of 75 kilodaltons.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA transfer occurs during a cell surface contact stage of F sex factor-mediated bacterial conjugation.

Donor bacteria containing JCFL39, a temperature-sensitive traD mutant of the F sex factor, were used at the nonpermissive temperature to accumulate stable mating pairs with recipient cells. At this stage in conjugation, extracellular F pili were removed by treatment with 0.01% sodium dodecyl sulfate. Upon then shifting to the permissive temperature for JCFL39, transfer of the F plasmid was observed. The mating pairs that were accumulated with JCFL39 at the nonpermissive temperature were readily observed by electron microscopy in wall-to-wall contact with the recipient bacteria. These results demonstrate that the traD product, which is known to be required in transferring DNA to a recipient bacterium, acts after the stage at which extracellular F pili are required. In addition, we concluded that DNA transfer takes place while donor and recipient cells are in surface contact and not necessarily through an extended F pilus as envisioned in some models of bacterial conjugation.

Escherichia coli DNA polymerase I. Construction of a polA plasmid for amplification and an improved purification scheme.

Previous attempts to clone the Escherichia coli polA+ gene onto a high copy number plasmid were unsuccessful. The apparent lethality of unregulated overproduction of DNA polymerase I can be eliminated by cutting at a BglII site 100 nucleotides upstream from the ATG start codon of the polA gene. This permitted the construction of plasmid pMP5 which contains both the coding sequence for DNA polymerase I and the lambda pL promoter for conditional control of polA gene expression. BglII cutting only damages but does not eliminate the polA promoter activity; the BglII site thus lies within the polA promoter region. Leakiness of the damaged polA promoter results in overproduction of DNA polymerase I even under conditions where pL is fully repressed. This overproduction is inhibitory of cell growth, as reflected in both growth rate and in the frequency of appearance of mutant plasmids which are nonproducers of DNA polymerase I. Transformation of plasmid pMP5 into E. coli N4830 yields strain ATL100 which under inducing conditions provides 138-fold amplification of DNA polymerase I. Optimization of growth and expression conditions are presented together with an optimized rapid polymerase purification scheme. In addition to providing a convenient source for preparation of DNA polymerase I, this work serves as the basis for a future detailed molecular genetic analysis of the polA gene product.