Vimentin expression is retained in erythroid cells differentiated from human iPSC and ESC and indicates dysregulation in these cells early in differentiation

Background: Pluripotent stem cells are attractive progenitor cells for the generation of erythroid cells in vitro as have expansive proliferative potential. However, although embryonic (ESC) and induced pluripotent (iPSC) stem cells can be induced to undergo erythroid differentiation, the majority of cells fail to enucleate and the molecular basis of this defect is unknown. One protein that has been associated with the initial phase of erythroid cell enucleation is the intermediate filament vimentin, with loss of vimentin potentially required for the process to proceed. Methods: In this study, we used our established erythroid culture system along with western blot, PCR and interegation of comparative proteomic data sets to analyse the temporal expression profile of vimentin in erythroid cells differentiated from adult peripheral blood stem cells, iPSC and ESC throughout erythropoiesis. Confocal microscopy was also used to examine the intracellular localisation of vimentin. Results: We show that expression of vimentin is turned off early during normal adult erythroid cell differentiation, with vimentin protein lost by the polychromatic erythroblast stage, just prior to enucleation. In contrast, in erythroid cells differentiated from iPSC and ESC, expression of vimentin persists, with high levels of both mRNA and protein even in orthochromatic erythroblasts. In the vimentin-positive iPSC orthochromatic erythroblasts, F-actin was localized around the cell periphery; however, in those rare cells captured undergoing enucleation, vimentin was absent and F-actin was re-localized to the enucleosome as found in normal adult orthrochromatic erythroblasts. Conclusion: As both embryonic and adult erythroid cells loose vimentin and enucleate, retention of vimentin by iPSC and ESC erythroid cells indicates an intrinsic defect. By analogy with avian erythrocytes which naturally retain vimentin and remain nucleated, retention in iPSC- and ESC-derived erythroid cells may impede enucleation. Our data also provide the first evidence that dysregulation of processes in these cells occurs from the early stages of differentiation, facilitating targeting of future studies

High efficiency serum free feeder free erythroid differentiation of human pluripotent stem cells using small molecules

This article describes a good manufacturing practice (GMP)-compatible, feeder-free and serum-free method to produce large numbers of erythroid cells from human pluripotent stem cells (hPSCs), either embryonic or induced. This multistep protocol combines cytokines and small molecules to mimic and surpass the early stages of development. It produces, without any selection or sorting step, a population of cells in which 91.8% ± 5.4% express CD34 at day 7, 98.6% ± 1.3% express CD43 at day 10, and 99.1% ± 0.95% of cells are CD235a positive by day 31 of the differentiation process. Moreover, this differentiation protocol supports extensive expansion, with a single hPSC producing up to 150 hematopoietic progenitor cells by day 10 and 50,000–200,000 erythroid cells by day 31. The erythroid cells produced exhibit a definitive fetal hematopoietic type, with 90%–95% fetal globin and variable proportion of embryonic and adult globin at the protein level. The presence of small molecules during the differentiation protocol has quantitative and qualitative effects; it increases the proportion of adult globin and decreases the proportion of embryonic globin. Given its level of definition, this system provides a powerful tool for investigation of the mechanisms governing early hematopoiesis and erythropoiesis, including globin switching and enucleation. The early stages of the differentiation protocol could also serve as a starting point for the production of endothelial cells and other hematopoietic cells, or to investigate the production of long-term reconstituting hematopoietic stem cells from hPSCs

In cardiac myocytes, cAMP elevation triggers the down-regulation of transcripts and promoter activity for cyclic AMP phosphodiesterase-4A10 (PDE4A10)

Transcripts for the PDE4A10 cyclic AMP phosphodiesterase isoform are present in a wide variety of rat tissues including the heart. Sequence comparisons between the putative human and mouse promoters revealed a number of conserved regions including both an Sp1 and a CREB-binding site. The putative mouse PDE4A10 promoter was amplified from genomic DNA and sub-cloned into a luciferase reporter vector for investigation of activity in neonatal cardiac myocytes. Transfection with this construct identified a high level of luciferase expression in neonatal cardiac myocytes. Surprisingly, this activity was down-regulated by elevation of intracellular cAMP through a process involving PKA, but not EPAC, signalling. Such inhibition of the rodent PDE4A10 promoter activity in response to elevated cAMP levels is in contrast to the PDE4 promoters so far described. Site-directed mutagenesis revealed that the Sp1 binding site at promoter position - 348 to - 336 is responsible for the basal constitutive expression of murine PDE4A10. The conserved CREB-binding motif at position - 370 to - 363 also contributes to basal promoter activity but does not in itself confer cAMP inhibition upon the PDE4A10 promoter. EMSA analysis confirmed the authenticity of CREB and Sp1 binding sites. The transcriptional start site was identified to be an adenine residue at position - 55 in the mouse PDE4A10 promoter. We present evidence that this novel down-regulation of PDE4A10 is mediated by the transcription factor ICER in a PKA dependent manner. The pool of cAMP in cardiac myocytes that down-regulates PDE4A10 is regulated b

1H NMR structural and functional characterisation of a cAMP-specific phosphodiesterase-4D5 (PDE4D5) N-terminal region peptide that disrupts PDE4D5 interaction with the signalling scaffold proteins, βarrestin and RACK1

The unique 88 amino acid N-terminal region of cAMP-specific phosphodiesterase-4D5 (PDE4D5) contains overlapping binding sites conferring interaction with the signaling scaffold proteins, βarrestin and RACK1. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, encompasses the entire N-terminal RACK1 Interaction Domain (RAID1) together with a portion of the βarrestin binding site. 1H NMR and CD analyses indicate that this region has propensity to form a helical structure. The leucine-rich hydrophobic grouping essential for RACK1 interaction forms a discrete hydrophobic ridge located along a single face of an amphipathic α-helix with Arg34 and Asn36, which also play important roles in RACK1 binding. The Asn22/Pro23/Trp24/Asn26 grouping, essential for RACK1 interaction, was located at the N-terminal head of the amphipathic helix that contained the hydrophobic ridge. RAID1 is thus provided by a distinct amphipathic helical structure. We suggest that the binding of PDE4D5 to the WD-repeat protein, RACK1, may occur in a manner akin to the helix–helix interaction shown for Gγ binding to the WD-repeat protein, Gβ. A more extensive section of the PDE4D5 N-terminal sequence (Thr11-Ala85) is involved in βarrestin binding. Several residues within the RAID1 helix contribute to this interaction however. We show here that these residues form a focused band around the centre of the RAID1 helix, generating a hydrophobic patch (from Leu29, Val30 and Leu33) flanked by polar/charged residues (Asn26, Glu27, Asp28, Arg34). The interaction with βarrestin exploits a greater circumference on the RAID1 helix, and involves two residues (Glu27, Asp28) that do not contribute to RACK1 binding. In contrast, the interaction of RACK1 with RAID1 is extended over a greater length of the helix and includes Leu37/Leu38, which do not contribute to βarrestin binding. A membrane-permeable, stearoylated Val12-Ser49 38-mer peptide disrupted the interaction of both βarrestin and RACK1 with endogenous PDE4D5 in HEKB2 cells, whilst a cognate peptide with a Glu27Ala substitution selectively failed to disrupt PDE4D5/RACK1 interaction. The stearoylated Val12-Ser49 38-mer peptide enhanced the isoprenaline-stimulated PKA phosphorylation of the β2-adrenergic receptors (β2AR) and its activation of ERK, whilst the Glu27Ala peptide was ineffective in both these regards

Some lessons from history: Morphomes in diachrony

What follows informally recounts some ‘lessons I have learned’ from several years spent exploring and describing in close detail the history of the inflectional morphology of the Romance verb.1 This programme of research, which has considered the entire range of Romance languages and dialects, and in as much historical depth and comparative breadth as possible, has been concerned in particular to identify, and describe the development of, a number of phenomena for which it can be argued that they are clear-cut,2 unambiguous cases of ‘morphomes’ in the sense of Aronoff; phenomena, that is, for which extramorphological (phonological or morphosyntactic) synchronic conditioning can be ruled out. Reproduction of all the relevant arguments would require far more space than I have here, and they have been published elsewhere. This is not to say, of course, that these arguments are selfevidently correct or that all morphologists would necessarily be persuaded by them (far from it), but I am interested here in standing back and developing some general reflections on what I believe the unusual comparative breadth and historical depth offered by the descriptive literature3 on Romance languages has to offer for the study of morphomes. The principal lessons that have emerged from the diachronic observation of Romance verb morphology seem to me to be the following: i. Diachrony can provide evidence for the psychological reality of putative morphomes. ii. Diachrony can be used as a diagnostic of the synchronically morphomic nature of some pattern of alternation. iii. Typological comparison can serve to falsify the putatively morphomic status of some pattern of alternation iv. Speakers do not especially prefer ‘non-morphomic’ over ‘morphomic’ patterns. v. An alternation pattern can be morphomic even when it appears to be phonologically conditioned. Each of these findings will be explained and illustrated at least in outline4 in the following sections. In conclusion, I shall briefly present some thoughts on why morphomes persist diachronically, which I shall relate to the observation that all morphomes seem to originate in allomorphy affecting lexical roots

Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells

Human embryonic stem cells (hESCs) have enormous potential for use in pharmaceutical development and therapeutics, however to realise this potential there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an established inhibitor of adenosine deaminase (ADA) and cyclic nucleotide phosphodiesterase- 2 (PDE2). hESCs maintained in feeder-free conditions with EHNA for more than 10 passages showed no reduction in hESC associated markers including NANOG, POU5F1 and SSEA 4 compared to cells maintained in feeder-free conditions containing basic fibroblast growth factor (bFGF). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but upon removing EHNA hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure activity relationship analysis found the differentiation blocking properties of EHNA to reside in a pharmacophore comprised of a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large scale or cGMP compliant processes

Scanning peptide array analyses identify overlapping binding sites for the signalling scaffold proteins, β-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5

The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and β-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and β-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK1 and β-arrestin interact at overlapping sites within the unique N-terminal region of PDE4D5 and at distinct sites within the conserved PDE4 catalytic domain. Screening scanning alanine substitution peptide arrays, coupled with mutagenesis and truncation studies, allowed definition of RACK1 and β-arrestin interaction sites. Modelled on the PDE4D catalytic domain, these form distinct well-defined surface-exposed patches on helices-15–16, for RACK1, and helix-17 for β-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased β-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the β(2)AR (β(2)-adrenergic receptor) upon isoproterenol challenge approx. 4-fold and severely attenuated (approx. 4–5 fold) both isoproterenol-stimulated PKA (protein kinase A) phosphorylation of the β(2)AR and activation of ERK (extracellular-signal-regulated kinase). The ability of a catalytically inactive form of PDE4D5 to exert a dominant negative effect in amplifying isoproterenol-stimulated ERK activation was ablated by a mutation that blocked the interaction of PDE4D5 with β-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and β-arrestin compete to sequester distinct ‘pools’ of PDE4D5. In this fashion, alterations in the level of RACK1 expression may act to modulate signal transduction mediated by the β(2)AR