A molecular roadmap of definitive erythropoiesis from human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) are being considered for use in understanding haematopoietic disorders and as a potential source of in vitro manufactured red cells. Here, we show that hiPSCs are able to recapitulate various stages of developmental erythropoiesis. We show that primitive erythroblasts arise first, express CD31+ with CD235a+ , embryonic globins and red cell markers, but fail to express the hallmark red cell transcripts of adult erythropoiesis. When hiPSC-derived CD45+ CD235a- haematopoietic progenitors are isolated on day 12 and further differentiated on OP9 stroma, they selectively express CD36+ and CD235a+ , adult erythroid transcripts for transcription factors (e.g., BCL11A, KLF1) and fetal/adult globins (HBG1/2, HBB). Importantly, hiPSC- and cord-derived CD36+ CD235a+ erythroblasts show a striking homology by transcriptome array profiling (only 306 transcripts with a 2Log fold change >1·5- or 2·8-fold). Phenotypic and transcriptome profiling of CD45+ CD117+ CD235a+ pro-erythroblasts and terminally differentiated erythroblasts is also provided, including evidence of a HbF (fetal) to HbA (adult) haemoglobin switch and enucleation, that mirrors their definitive erythroblast cord-derived counterparts. These findings provide a molecular roadmap of developmental erythropoiesis from hiPSC sources at several critical stages, but also helps to inform on their use for clinical applications and modelling human haematopoietic disease.

Distinct gene expression program dynamics during erythropoiesis from human induced pluripotent stem cells compared with adult and cord blood progenitors.

BACKGROUND: Human-induced pluripotent stem cells (hiPSCs) are a potentially invaluable resource for regenerative medicine, including the in vitro manufacture of blood products. HiPSC-derived red blood cells are an attractive therapeutic option in hematology, yet exhibit unexplained proliferation and enucleation defects that presently preclude such applications. We hypothesised that substantial differential regulation of gene expression during erythroid development accounts for these important differences between hiPSC-derived cells and those from adult or cord-blood progenitors. We thus cultured erythroblasts from each source for transcriptomic analysis to investigate differential gene expression underlying these functional defects. RESULTS: Our high resolution transcriptional view of definitive erythropoiesis captures the regulation of genes relevant to cell-cycle control and confers statistical power to deploy novel bioinformatics methods. Whilst the dynamics of erythroid program elaboration from adult and cord blood progenitors were very similar, the emerging erythroid transcriptome in hiPSCs revealed radically different program elaboration compared to adult and cord blood cells. We explored the function of differentially expressed genes in hiPSC-specific clusters defined by our novel tunable clustering algorithms (SMART and Bi-CoPaM). HiPSCs show reduced expression of c-KIT and key erythroid transcription factors SOX6, MYB and BCL11A, strong HBZ-induction, and aberrant expression of genes involved in protein degradation, lysosomal clearance and cell-cycle regulation. CONCLUSIONS: Together, these data suggest that hiPSC-derived cells may be specified to a primitive erythroid fate, and implies that definitive specification may more accurately reflect adult development. We have therefore identified, for the first time, distinct gene expression dynamics during erythroblast differentiation from hiPSCs which may cause reduced proliferation and enucleation of hiPSC-derived erythroid cells. The data suggest several mechanistic defects which may partially explain the observed aberrant erythroid differentiation from hiPSCs.

Human induced pluripotent stem cell derived erythroblasts can undergo definitive erythropoiesis and co-express gamma and beta globins.

Human induced pluripotent stem cells (hiPSCs), like embryonic stem cells, are under intense investigation for novel approaches to model disease and for regenerative therapies. Here, we describe the derivation and characterization of hiPSCs from a variety of sources and show that, irrespective of origin or method of reprogramming, hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144(+) endothelium, CD235a(+) erythrocytes (myeloid lineage) and CD19(+) B lymphocytes (lymphoid lineage). Within the erythroblast lineage, we were able to demonstrate by single cell analysis (flow cytometry), that hiPSC-derived erythroblasts express alpha globin as previously described, and that a sub-population of these erythroblasts also express haemoglobin F (HbF), indicative of fetal definitive erythropoiesis. More notably however, we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner, but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover, the HbA expressing erythroblast population could be greatly enhanced (44·0 ± 6·04%) when a defined serum-free approach was employed to isolate a CD31(+) CD45(+) erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine.

Human induced pluripotent stem cells are capable of B-cell lymphopoiesis.

Induced pluripotent stem (iPS) cells offer a unique potential for understanding the molecular basis of disease and development. Here we have generated several human iPS cell lines, and we describe their pluripotent phenotype and ability to differentiate into erythroid cells, monocytes, and endothelial cells. More significantly, however, when these iPS cells were differentiated under conditions that promote lympho-hematopoiesis from human embryonic stem cells, we observed the formation of pre-B cells. These cells were CD45(+)CD19(+)CD10(+) and were positive for transcripts Pax5, IL7αR, λ-like, and VpreB receptor. Although they were negative for surface IgM and CD5 expression, iPS-derived CD45(+)CD19(+) cells also exhibited multiple genomic D-J(H) rearrangements, which supports a pre-B-cell identity. We therefore have been able to demonstrate, for the first time, that human iPS cells are able to undergo hematopoiesis that contributes to the B-cell lymphoid lineage.

The RNA binding protein Larp1 regulates cell division, apoptosis and cell migration.

The RNA binding protein Larp1 was originally shown to be involved in spermatogenesis, embryogenesis and cell-cycle progression in Drosophila. Our data show that mammalian Larp1 is found in a complex with poly A binding protein and eukaryote initiation factor 4E and is associated with 60S and 80S ribosomal subunits. A reduction in Larp1 expression by siRNA inhibits global protein synthesis rates and results in mitotic arrest and delayed cell migration. Consistent with these data we show that Larp1 protein is present at the leading edge of migrating cells and interacts directly with cytoskeletal components. Taken together, these data suggest a role for Larp1 in facilitating the synthesis of proteins required for cellular remodelling and migration.

Neonatal respiratory consequences from water birth.

AIM: Differentiating features were sought for respiratory distress after water birth versus air birth in term low-risk babies. Clinical and X-ray features were to be assessed to determine if the disease processes could be differentiated. METHODS: Review of case records and X-rays over a 7-year period for all admitted babies with respiratory distress after water birth and a similar group of babies with respiratory distress after air birth. RESULTS: There were 14 water birth babies and 24 air birth babies in the study. The water birth babies showed greater acidosis, greater requirement for ventilation, greater requirement for nitric oxide treatment and greater time to establish feeding. The X-rays could not be reliably allocated to the correct group, but the water birth X-rays were judged to have more severe changes than the air birth babies. CONCLUSION: In low-risk babies with respiratory distress, water birth is associated with a greater level of respiratory morbidity than seen after air birth.

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi.

BACKGROUND: Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages. RESULTS: We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages. CONCLUSION: Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.

Inhibition of protein kinase C delta protects rat INS-1 cells against interleukin-1beta and streptozotocin-induced apoptosis.

Exposure of pancreatic beta-cells to cytokines, such as interleukin-1beta (IL-1beta), is thought to contribute to the beta-cell apoptosis that underlies the onset of type 1 diabetes. One important event triggered by IL-1beta is induction of nitric oxide synthase (iNOS), an enzyme that catalyzes intracellular generation of the cytotoxic free radical NO. We recently described a novel requirement for the protein kinase C (PKC) isozyme PKCdelta in this process. Our current aim, therefore, was to assess whether PKCdelta also plays a role in beta-cell apoptosis. As assessed by either annexin V staining or DNA fragmentation, IL-1beta caused INS-1 cells to undergo apoptosis. This was completely blocked by adenoviral overexpression of a dominant-negative, kinase-dead (KD) PKCdelta mutant. The corresponding PKCalpha virus was without effect. However, apoptosis caused by the cytotoxic agent streptozotocin (STZ), which acts independent of iNOS, was also inhibited by overexpression of PKCdeltaKD. STZ was additionally shown to activate the proteolytic enzyme caspase-3, a key biochemical effector of end-stage apoptosis. Moreover, STZ caused a caspase-dependent cleavage of PKCdelta, thereby releasing a COOH-terminal fragment corresponding to the kinase catalytic domain. Thus, proteolytic activation of PKCdelta seems to be important in the distal apoptotic pathway induced by STZ. That IL-1beta also activated caspase-3 and promoted PKCdelta cleavage suggests that this distal pathway also contributes in the apoptotic response to the cytokine. These data therefore support a dual role for PKCdelta in IL-1beta-mediated cell death: it is required for efficient NO generation through regulation of iNOS levels but also contributes to apoptotic pathways downstream of caspase activation.

PKC alpha is activated but not required during glucose-induced insulin secretion from rat pancreatic islets.

The role of protein kinase C (PKC) in glucose-stimulated insulin secretion (GSIS) is controversial. Using recombinant adenoviruses for overexpression of PKC alpha and PKC delta, in both wild-type (WT) and kinase-dead (KD) forms, we here demonstrate that activation of these two PKCs is neither necessary nor sufficient for GSIS from batch-incubated, rat pancreatic islets. In contrast, responses to the pharmacologic activator 12-O-tetradecanoylphorbol-13-acetate (TPA) were reciprocally modulated by overexpression of the PKC alpha WT or PKC alpha KD but not the corresponding PKC delta adenoviruses. The kinetics of the secretory response to glucose (monitored by perifusion) were not altered in either cultured islets overexpressing PKC alpha KD or freshly isolated islets stimulated in the presence of the conventional PKC (cPKC) inhibitor Go6976. However, the latter did inhibit the secretory response to TPA. Using phosphorylation state-specific antisera for consensus PKC phosphorylation sites, we also showed that (compared with TPA) glucose causes only a modest and transient functional activation of PKC (maximal at 2-5 min). However, glucose did promote a prolonged (15 min) phosphorylation of PKC substrates in the presence of the phosphatase inhibitor okadaic acid. Overall, the results demonstrate that glucose does stimulate PKC alpha in pancreatic islets but that this makes little overall contribution to GSIS.

Human induced pluripotent stem cell-derived B lymphocytes express sIgM and can be generated via a hemogenic endothelium intermediate.

The differentiation of human pluripotent stem cells to the B-cell lymphoid lineage has important clinical applications that include in vitro modeling of developmental lymphogenesis in health and disease. Here, we first demonstrate the capacity of human induced pluripotent stem cells (hiPSCs) to differentiate into CD144(+)CD73(-)CD43/CD235a(-) cells, characterized as hemogenic endothelium, and show that this population is capable of differentiating to CD10(+)CD19(+) B lymphocytes. We also demonstrate that B lymphocytes generated from hiPSCs are able to undergo full VDJ rearrangement and express surface IgM (sIgM(+)), thus representing an immature B-cell subset. Efficiency of sIgM expression on the hiPSC-derived B lymphocytes (∼ 5% of CD19(+) cells) was comparable with B lymphocytes generated from human umbilical cord blood (UCB) hematopoietic progenitor cells. Importantly, when assessed by global transcriptional profiling, hiPSC-derived B-cells show a very high level of similarity when compared with their UCB-derived counterparts, such that from more than 47,000 different transcripts, only 45 were significantly different (with a criteria adjusted P value P<0.05, log FC >1.5 or 2.8-fold). This represents a unique in vitro model to delineate critical events during lymphogeneisis in development and lymphoid diseases such as acute lymphocytic leukemia.

Qualitative and quantitative comparison of the proteome of erythroid cells differentiated from human iPSCs and adult erythroid cells by multiplex TMT labelling and nanoLC-MS/MS.

Induced pluripotent stem cells (iPSC) are an attractive progenitor source for the generation of in vitro blood products. However, before iPSC-derived erythroid cells can be considered for therapeutic use their similarity to adult erythroid cells must be confirmed. We have analysed the proteome of erythroid cells differentiated from the iPSC fibroblast derived line (C19) and showed they express hallmark RBC proteins, including all those of the ankyrin and 4.1R complex. We next compared the proteome of erythroid cells differentiated from three iPSC lines (C19, OCE1, OPM2) with that of adult and cord blood progenitors. Of the 1989 proteins quantified <3% differed in level by 2-fold or more between the different iPSC-derived erythroid cells. When compared to adult cells, 11% of proteins differed in level by 2-fold or more, falling to 1.9% if a 5-fold threshold was imposed to accommodate slight inter-cell line erythropoietic developmental variation. Notably, the level of >30 hallmark erythroid proteins was consistent between the iPSC lines and adult cells. In addition, a sub-population (10-15%) of iPSC erythroid cells in each of the iPSC lines completed enucleation. Aberrant expression of some cytoskeleton proteins may contribute to the failure of the majority of the cells to enucleate since we detected some alterations in cytoskeletal protein abundance. In conclusion, the proteome of erythroid cells differentiated from iPSC lines is very similar to that of normal adult erythroid cells, but further work to improve the induction of erythroid cells in existing iPSC lines or to generate novel erythroid cell lines is required before iPSC-derived red cells can be considered suitable for transfusion therapy.

Efficient differentiation of human induced pluripotent stem cells generates cardiac cells that provide protection following myocardial infarction in the rat.

Induced pluripotent stem (iPS) cells are being used increasingly to complement their embryonic counterparts to understand and develop the therapeutic potential of pluripotent cells. Our objectives were to identify an efficient cardiac differentiation protocol for human iPS cells as monolayers, and demonstrate that the resulting cardiac progenitors could provide a therapeutic benefit in a rodent model of myocardial infarction. Herein, we describe a 14-day protocol for efficient cardiac differentiation of human iPS cells as a monolayer, which routinely yielded a mixed population in which over 50% were cardiomyocytes, endothelium, or smooth muscle cells. When differentiating, cardiac progenitors from day 6 of this protocol were injected into the peri-infarct region of the rat heart; after coronary artery ligation and reperfusion, we were able to show that human iPS cell-derived cardiac progenitor cells engrafted, differentiated into cardiomyocytes and smooth muscle, and persisted for at least 10 weeks postinfarct. Hearts injected with iPS-derived cells showed a nonsignificant trend toward protection from decline in function after myocardial infarction, as assessed by magnetic resonance imaging at 10 weeks, such that the ejection fraction at 10 weeks in iPS treated hearts was 62%±4%, compared to that of control infarcted hearts at 45%±9% (P<0.2). In conclusion, we demonstrated efficient cardiac differentiation of human iPS cells that gave rise to progenitors that were retained within the infarcted rat heart, and reduced remodeling of the heart after ischemic damage.

The impact of proliferative potential of umbilical cord-derived endothelial progenitor cells and hypoxia on vascular tubule formation in vitro.

Revascularization of the damaged tissue is pivotal to tissue repair. Here, by bringing together two in vitro model systems, we have been able to examine (1) the ability of human umbilical vein endothelial cells (HUVEC) containing a complete hierarchy of endothelial progenitors derived from the human umbilical cord to generate vascular tubules within a human stromal niche in vitro and (2) the effects of exposure to low oxygen tensions on endothelial progenitor cell proliferation and tubule formation in vitro. Our results demonstrate that high proliferative potential endothelial colony forming cells (HPP-ECFC) from cultured HUVEC preferentially contribute to vascular tubule formation in vitro and that these progenitor cells are concentrated in the CD34(lo/-) fraction. HUVEC were initially resistant when exposed to hypoxia (1.5% O(2)) for short periods (1-2 days), but sustained chronic hypoxia (4-14 days) inhibited their ability to proliferate. This was reflected by a loss in their ability to form tubules in cocultures of human dermal fibroblasts (hDFs). In contrast, an acute exposure to low oxygen tensions (1.5% O(2) for 24 h) followed by reoxygenation did not adversely affect the capacity of these cells to both proliferate and form vascular tubules in vitro.These studies therefore provide a model system to study the influences of the microenvironmental niche and modification of this niche on vascular tubule formation in vitro from HPP-ECFC.

The diverse roles of protein kinase C in pancreatic beta-cell function.

Members of the serine/threonine PKC (protein kinase C) family perform diverse functions in multiple cell types. All members of the family are activated in signalling cascades triggered by occupation of cell surface receptors, but the cPKC (conventional PKC) and nPKC (novel PKC) isoforms are also responsive to fatty acid metabolites. PKC isoforms are involved in various aspects of pancreatic beta-cell function, including cell proliferation, differentiation and death, as well as regulation of secretion in response to glucose and muscarinic receptor agonists. Recently, the nPKC isoform, PKCepsilon, has also been implicated in the loss of insulin secretory responsiveness that underpins the development of Type 2 diabetes.

Inhibitors of Polo-like kinase reveal roles in spindle-pole maintenance.

Polo-like kinases (Plks) have several functions in mitotic progression and are upregulated in many tumor types. Small-molecule Plk inhibitors would be valuable as tools for studying Plk biology and for developing antitumor agents. Guided by homology modeling of the Plk1 kinase domain, we have discovered a chemical series that shows potent and selective Plk1 inhibition. The effects of one such optimized benzthiazole N-oxide, cyclapolin 1 (1), on purified centrosomes indicate that Plks are required to generate MPM2 epitopes, recruit gamma-tubulin and enable nucleation of microtubules. The compound can also promote loss of centrosome integrity and microtubule nucleating ability apparently through increased accessibility of protein phosphatases. We show that treatment of living S2 cells with cyclapolin 1 leads to collapsed spindles, in contrast to the metaphase-arrested bipolar spindles observed after RNAi. This different response to protein depletion and protein inhibition may have significance in the development of antitumor agents.

Overexpression of acyl-CoA synthetase-1 increases lipid deposition in hepatic (HepG2) cells and rodent liver in vivo.

Accumulation of intracellular lipid in obesity is associated with metabolic disease in many tissues including liver. Storage of fatty acid as triglyceride (TG) requires the activation of fatty acids to long-chain acyl-CoAs (LC-CoA) by the enzyme acyl-CoA synthetase (ACSL). There are five known isoforms of ACSL (ACSL1, -3, -4, -5, -6), which vary in their tissue specificity and affinity for fatty acid substrates. To investigate the role of ACSL1 in the regulation of lipid metabolism, we used adenoviral-mediated gene transfer to overexpress ACSL1 in the human hepatoma cell-line HepG2 and in liver of rodents. Infection of HepG2 cells with the adenoviral construct AdACSL1 increased ACSL activity >10-fold compared with controls after 24 h. HepG2 cells overexpressing ACSL1 had a 40% higher triglyceride (TG) content (93 +/- 3 vs. 67 +/- 2 nmol/mg protein in controls, P < 0.05) after 24-h exposure to 1 mM oleate. Furthermore, ACSL1 overexpression produced a 60% increase in cellular LCA-CoA content (160 +/- 6 vs. 100 +/- 6 nmol/g protein in controls, P < 0.05) and increased [(14)C]oleate incorporation into TG without significantly altering fatty acid oxidation. In mice, AdACSL1 administration increased ACSL1 mRNA and protein more than fivefold over controls at 4 days postinfection. ACSL1 overexpression caused a twofold increase in TG content in mouse liver (39 +/- 4 vs. 20 +/- 2 mumol/g wet wt in controls, P < 0.05), and overexpression in rat liver increased [1-(14)C]palmitate clearance into liver TG. These in vitro and in vivo results suggest a pivotal role for ACSL1 in regulating TG synthesis in liver.

Directing pluripotent cell differentiation using "diced RNA" in transient transfection.

Embryonic stem (ES) and embryonic carcinoma (EC) cells are pluripotent and have the capacity to differentiate into many cell types. The ability to direct their differentiation should have considerable practical applications. Here, we first report the use of diced short interfering RNAi against Oct4 in a transient approach, to direct differentiation of ES towards the trophectoderm lineage. We then apply this approach to downregulate Smad4 in mouse P19 EC cells. We have found that this leads to an increase in the levels of Pax6 (a neuroectoderm marker), reduction in the levels of Brachyury (a mesoderm marker), and a 3-fold increase in the number of betaIII tubulin-positive colonies when these cells were allowed to differentiate. This indicates a redirection of cell fate towards the neuroectoderm lineage. Thus, transient RNAi could provide a valuable tool to direct pluripotent cells along specific pathways of differentiation while circumventing permanent genetic changes.