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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.