Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney.

With the prevalence of end-stage renal disease rising 8% per annum globally, there is an urgent need for renal regenerative strategies. The kidney is a mesodermal organ that differentiates from the intermediate mesoderm (IM) through the formation of a ureteric bud (UB) and the interaction between this bud and the adjacent IM-derived metanephric mesenchyme (MM). The nephrons arise from a nephron progenitor population derived from the MM (ref. ). The IM itself is derived from the posterior primitive streak. Although the developmental origin of the kidney is well understood, nephron formation in the human kidney is completed before birth. Hence, there is no postnatal stem cell able to replace lost nephrons. In this study, we have successfully directed the differentiation of human embryonic stem cells (hESCs) through posterior primitive streak and IM under fully chemically defined monolayer culture conditions using growth factors used during normal embryogenesis. This differentiation protocol results in the synchronous induction of UB and MM that forms a self-organizing structure, including nephron formation, in vitro. Such hESC-derived components show broad renal potential ex vivo, illustrating the potential for pluripotent-stem-cell-based renal regeneration.

The functional and molecular characterisation of human embryonic stem cell-derived insulin-positive cells compared with adult pancreatic beta cells.

AIMS/HYPOTHESIS: Using a novel directed differentiation protocol, we recently generated up to 25% insulin-producing cells from human embryonic stem cells (hESCs) (insulin(+) cells). At this juncture, it was important to functionally and molecularly characterise these hESC-derived insulin(+) cells and identify key differences and similarities between them and primary beta cells. METHODS: We used a new reporter hESC line with green fluorescent protein (GFP) cDNA targeted to the INS locus by homologous recombination (INS (GFP/w)) and an untargeted hESC line (HES2). INS (GFP/w) allowed efficient identification and purification of GFP-producing (INS:GFP(+)) cells. Insulin(+) cells were examined for key features of adult beta cells using microarray, quantitative PCR, secretion assays, imaging and electrophysiology. RESULTS: Immunofluorescent staining showed complete co-localisation of insulin with GFP; however, cells were often multihormonal, many with granules containing insulin and glucagon. Electrophysiological recordings revealed variable K(ATP) and voltage-gated Ca(2+) channel activity, and reduced glucose-induced cytosolic Ca(2+) uptake. This translated into defective glucose-stimulated insulin secretion but, intriguingly, appropriate glucagon responses. Gene profiling revealed differences in global gene expression between INS:GFP(+) cells and adult human islets; however, INS:GFP(+) cells had remarkably similar expression of endocrine-lineage transcription factors and genes involved in glucose sensing and exocytosis. CONCLUSIONS/INTERPRETATION: INS:GFP(+) cells can be purified from differentiated hESCs, providing a superior source of insulin-producing cells. Genomic analyses revealed that INS:GFP(+) cells collectively resemble immature endocrine cells. However, insulin(+) cells were heterogeneous, a fact that translated into important functional differences within this population. The information gained from this study may now be used to generate new iterations of functioning beta cells that can be purified for transplant.

INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.

AIMS/HYPOTHESIS: We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro. METHODS: Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS⁺ cells. RESULTS: Differentiation of INS(GFP/w) hESCs using published protocols demonstrated that all GFP⁺ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP⁺ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS(GFP/w) hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP⁺ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP⁺ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS⁺ cells. CONCLUSIONS/INTERPRETATION: INS(GFP/w) hESCs are a valuable tool for investigating the nature of early INS⁺ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS⁺ cells from differentiating hESCs.

Expression pattern of the stem cell leukaemia gene in the CNS of the embryonic and adult mouse.

The basic helix-loop-helix (bHLH) transcription factor stem cell leukaemia (SCL) is a 'master regulator' of haematopoiesis, where SCL is pivotal in cell fate determination and differentiation. SCL has also been detected in CNS, where other members of the bHLH-family have been shown to be indispensable for neuronal development; however, no detailed expression pattern of SCL has so far been described. We have generated a map of SCL expression in the embryonic and adult mouse brain based on histochemical analysis of LacZ reporter gene expression in sequential sections of brain tissue derived from SCL-LacZ knockin mice. The expression of LacZ was confirmed to reflect SCL expression by in situ hybridisation. LacZ expression was found in a range of different diencephalic, mesencephalic and metencephalic brain nuclei in adult CNS. Co-localisation of LacZ with the neuronal marker NeuN indicated expression in post-mitotic neurons in adulthood. LacZ expression by neurons was confirmed in tissue culture analysis. The nature of the pretectal, midbrain and hindbrain regions expressing LacZ suggest that SCL in adult CNS is potentially involved in processing of visual, auditory and pain related information. During embryogenesis, LacZ expression was similarly confined to thalamus, midbrain and hindbrain. LacZ staining was also evident in parts of the intermediate and marginal zone of the aqueduct and ventricular zone of the fourth ventricle at E12.5 and E14. These cells may represent progenitor stages of differentiating neural cells. Given the presence of SCL in both the developing brain and in post-mitotic neurons, it seems likely that the function of SCL in neuronal differentiation may differ from its function in maintaining the differentiated state of the mature neuron.

Cloning, expression analysis, and chromosomal localization of murine and human homologues of a Xenopus mix gene.

We report the cloning and chromosomal localization of murine and human Mix genes, members of a subclass of paired-like homeobox genes of which the Xenopus laevis Mix.1 gene is the founding member. The murine Mix gene was mapped to the distal region of chromosome 1 and the human region to the syntenic region 1q41-42. Northern analysis and RT-PCR of murine adult and embryonic tissues demonstrated that Mix expression was restricted to the early embryo. Whole-mount in situ hybridization revealed patchy but symmetrical Mix expression in visceral endoderm of embryonic day (E)5.5 embryos. In slightly older embryos, the expression was skewed to one side of the embryo and by E6.5, at the onset of gastrulation, expression was seen in the epiblast, visceral endoderm, nascent mesoderm, and the primitive streak. This expression pattern was maintained in mid- and late-streak embryos. In early bud-stage embryos, expression was strongest in the proximal two thirds of the streak, extending to the base of the allantois. By the headfold-stage, expression was confined to the remnant of the primitive streak in the caudal region of the embryo and, after E8.0, in the caudal notochord and tail bud mesoderm. Mix transcripts were no longer detectable after embryonic day 9.5.

Leishmania major proteophosphoglycan is expressed by amastigotes and has an immunomodulatory effect on macrophage function.

Proteophosphoglycan (PPG) is a newly described mucin-like glycoprotein found on the surface of Leishmania major promastigotes and secreted in the culture supernatant. We show here that antigenically similar PPGs are present in several Leishmania species. PPG could also be detected on the surface of amastigotes and in small, parasite-free vesicles in infected macrophages. Because of the similarity of its carbohydrate chains to lipophosphoglycan, a parasite receptor for host macrophages, PPG was tested for binding to macrophages. PPG bound to macrophages and was internalized in a time-dependent manner. PPG inhibited the production of tumor necrosis factor-alpha and synergized with interferon-gamma to stimulate the production of nitric oxide by macrophages. PPG may contribute to the binding of Leishmania to host cells and may play a role in modulating the biology of the infected macrophage at the early stage of infection.

SCL expression in the mouse embryo detected with a targeted lacZ reporter gene demonstrates its localization to hematopoietic, vascular, and neural tissues.

The helix-loop-helix transcription factor SCL (TAL1) is indispensable for blood cell formation in the mouse embryo. We have explored the localization and developmental potential of cells fated to express SCL during murine development using SCL-lacZ mutant mice in which the Escherichia coli lacZ reporter gene was 'knocked in' to the SCL locus. In addition to the hematopoietic defect associated with SCL deficiency, the yolk sac blood vessels in SCL(lacZ/lacZ) embryos formed an abnormal primary vascular plexus, which failed to undergo subsequent remodeling and formation of large branching vessels. Intraembryonic vasculogenesis in precirculation SCL(lacZ/lacZ) embryos appeared normal but, in embryos older than embryonic day (E) 8.5 to E9, absolute anemia leading to severe hypoxia precluded an accurate assessment of further vascular development. In heterozygous SCL(lacZ/w) embryos, lacZ was expressed in the central nervous system, vascular endothelia, and primitive and definitive hematopoietic cells in the blood, aortic wall, and fetal liver. Culture of fetal liver cells sorted for high and low levels of beta galactosidase activity from SCL(lacZ/w) heterozygous embryos indicated that there was a correlation between the level of SCL expression and the frequency of hematopoietic progenitor cells.

Suppressors of cytokine signaling (SOCS): negative regulators of signal transduction.

SOCS-1 was originally identified as an inhibitor of interleukin-6 signal transduction and is a member of a family of proteins (SOCS-1 to SOCS-7 and CIS) that contain an SH2 domain and a conserved carboxyl-terminal SOCS box motif. Mutation studies have established that critical contributions from both the amino-terminal and SH2 domains are essential for SOCS-1 and SOCS-3 to inhibit cytokine signaling. Inhibition of cytokine-dependent activation of STAT3 occurred in cells expressing either SOCS-1 or SOCS-3, but unlike SOCS-1, SOCS-3 did not directly interact with or inhibit the activity of JAK kinases. Although the conserved SOCS box motif appeared to be dispensable for SOCS-1 and SOCS-3 action when overexpressed, this domain interacts with elongin proteins and may be important in regulating protein turnover. In gene knockout studies, SOCS-1(-/-) mice were born but failed to thrive and died within 3 weeks of age with fatty degeneration of the liver and hemopoietic infiltration of several organs. The thymus in SOCS-1(-/-) mice was small, the animals were lymphopenic, and deficiencies in B lymphocytes were evident within hemopoietic organs. We propose that the absence of SOCS-1 in these mice prevents lymphocytes and liver cells from appropriately controlling signals from cytokines with cytotoxic side effects.

Aberrant hematopoiesis in mice with inactivation of the gene encoding SOCS-1.

Mice with homozygous inactivation of the gene encoding the suppressor of cytokine signaling-1 (SOCS-1) protein die within 21 days of birth with low body weight, fatty degeneration and necrosis of the liver, infiltration of the lung, pancreas, heart and skin by macrophages and granulocytes and a profound depletion of T- and B-lymphocytes. In the present study, SOCS-1 -/- mice were found to have a moderate neutrophilia, and reduced platelet and hematocrit levels. Replacement of the SOCS-1 gene by a lac-Z reporter gene allowed documentation by FACS sorting that at least a proportion of granulocyte-macrophage progenitor cells transcribe SOCS-1. Most hematopoietic progenitor cell frequencies were normal in -/- marrow as were the size and cellular content of colonies formed by -/- progenitor cells in response to various stimulating factors. However, there was an increased frequency of macrophage progenitor cells in -/- mice and, abnormally, one quarter of all progenitor cells were located in the liver. Progenitor cells from -/- mice were hyper-responsive to stimulation by GM-CSF but not by M-CSF or Multi-CSF (IL-3). Progenitor cells from -/- mice were also hypersensitive to inhibition by interferon-gamma (IFN-gamma), the degree of inhibition varying markedly with the stimulating factor used. The suppressive effects of IFN-gamma therefore appear to involve interactions with particular growth factor-initiated signals in -/- cells--interactions that are strongly modulated by the action of the SOCS-1 protein.

Expression of BCR - ABL in M1 myeloid leukemia cells induces differentiation without arresting proliferation.

The mechanism leading to the expanding population of maturing myeloid cells which characterises chronic myeloid leukemia (CML) remains obscure. Because of its ability to mimic the proliferative and cell survival functions of hematopoietic growth factors, we hypothesized that the oncogene activated in CML, BCR-ABL, might also influence differentiation. To test this hypothesis, we examined the effects of expressing BCR-ABL on the myeloid differentiation of murine M1 leukemic cells, which cease dividing and differentiate into macrophages in the presence of the cytokines leukemia inhibitory factor (LIF) or interleukin (IL)-6. We found that BCR-ABL induced macrophage differentiation in M1 cells, accompanied by increased expression of macrophage cell surface markers and the acquisition of phagocytic ability. interestingly, clones of M1 cells which expressed BCR-ABL remained in cell cycle and were refractory to the growth inhibition and apoptosis induced by IL-6 or LIF in parental M1 cells. These cells also expressed inappropriately high levels of c-MYC mRNA for their degree of differentiation, which may have been important in maintaining cellular proliferation. These data suggest that BCR-ABL can stimulate both differentiation and proliferation and that these characteristics may contribute to the phenotype observed in CML.

Apoptosis and beta-cell destruction in pancreatic islets of NOD mice with spontaneous and cyclophosphamide-accelerated diabetes.

Autoimmune-mediated destruction of pancreatic islet beta cells leads to insulin-dependent diabetes in non-obese diabetic (NOD) mice. Although both direct cytotoxic T cell- and indirect cytokine-, nitric oxide- or free radical-mediated mechanisms induce beta-cell apoptosis in vitro, beta-cell death in vivo in spontaneous autoimmune diabetes is not well-characterized. Furthermore, whether beta cells die gradually, or rapidly in the late pre-clinical stage, is a question of current interest. To investigate beta-cell death in vivo, we measured the frequency and intra-islet localisation of apoptosis, defined as DNA strand breaks by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) technique, during spontaneous and cyclophosphamide-accelerated diabetes in NOD mice. In spontaneous diabetes, the frequency of apoptosis in islets correlated with the progression of beta-cell destruction with age. Although apoptosis was detected at low frequency within the reduced insulin-positive islet area of pre-diabetic mice at 90 days of age, it was rarely co-localised to beta cells. After acceleration of beta-cell destruction with cyclophosphamide, the frequency of apoptosis reached maximum at 12 days, at which time 3.2 % of apoptotic cells were beta cells. Apoptosis was most frequent in the insulin-negative islet area comprised of mononuclear cell infiltrate and was localized to CD8+ T cells. The rarity of detectable apoptotic beta cells in spontaneous pre-diabetic mice with pronounced insulitis and reduced insulin-positive islet areas most likely reflects the rapid clearance of apoptotic beta cells. Our findings are more consistent with gradual destruction of non-renewable beta-cells in spontaneous diabetes, than with their rapid, accelerated destruction (as after cyclophosphamide) in the late pre-clinical stage.

Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1.

SOCS-1, a member of the suppressor of cytokine signaling (SOCS) family, was identified in a genetic screen for inhibitors of interleukin 6 signal transduction. SOCS-1 transcription is induced by cytokines, and the protein binds and inhibits Janus kinases and reduces cytokine-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 and the gp130 component of the interleukin 6 receptor. Thus, SOCS-1 forms part of a feedback loop that modulates signal transduction from cytokine receptors. To examine the role of SOCS-1 in vivo, we have used gene targeting to generate mice lacking this protein. SOCS-1(-/-) mice exhibited stunted growth and died before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. In addition, the thymus of SOCS-1(-/-) mice was reduced markedly in size, and there was a progressive loss of maturing B lymphocytes in the bone marrow, spleen, and peripheral blood. Thus, SOCS-1 is required for in vivo regulation of multiple cell types and is indispensable for normal postnatal growth and survival.

The hemangioblast--an elusive cell captured in culture.

The close temporal and spatial association between blood and endothelial cell development during embryogenesis was first documented almost 100 years ago. In recent years, gene expression studies have further strengthened this link. Now, using cultures of mouse embryonic stem cells, a common progenitor cell that gives rise to both blood cells and vascular endothelial cells, has been identified. The existence of the hemangioblast has been proved and experiments addressing its unique properties can begin.

Beta-cell apoptosis in an accelerated model of autoimmune diabetes.

BACKGROUND: The non-obese diabetic (NOD) mouse is a model of human type 1 diabetes in which autoreactive T cells mediate destruction of pancreatic islet beta cells. Although known to be triggered by cytotoxic T cells, apoptosis has not been unequivocally localized to beta cells in spontaneously diabetic NOD mice. We created a model of accelerated beta-cell destruction mediated by T cells from spontaneously diabetic NOD mice to facilitate the direct detection of apoptosis in beta cells. MATERIALS AND METHODS: NOD.scid (severe combined immunodeficiency) mice were crossed with bm1 mice transgenically expressing the costimulatory molecule B7-1 (CD80) in their beta cells, to generate B7-1 NOD.scid mice. Apoptosis in islet cells was measured as DNA strand breakage by the TdT-mediated-dUTP-nick end labeling (TUNEL) technique. RESULTS: Adoptive transfer of splenocytes from spontaneously diabetic NOD mice into B7-1 NOD.scid mice caused diabetes in recipients within 12-16 days. Mononuclear cell infiltration and apoptosis were significantly greater in the islets of B7-1 NOD.scid mice than in nontransgenic NOD.scid mice. Dual immunolabeling for TUNEL and either B-7 or insulin, or the T cell markers CD4 and CD8, and colocalization by confocal microscopy clearly demonstrated apoptosis in beta cells as well in a relatively larger number of infiltrating T cells. The clearance time of apoptotic beta cells was estimated to be less than 6 min. CONCLUSIONS: B7-1 transgenic beta cells undergo apoptosis during their accelerated destruction in response to NOD mouse effector T cells. Rapid clearance implies that beta cells undergoing apoptosis would be detected only rarely during more protracted disease in spontaneously diabetic NOD mice.

Characterization of hematopoietic progenitor cells that express the transcription factor SCL, using a lacZ "knock-in" strategy.

Gene targeting experiments have demonstrated that the transcription factor SCL is essential for primitive and definitive hematopoiesis in the mouse. To study the functional properties of hematopoietic cells expressing SCL, we have generated mutant mice (SCLlacZ/w) in which the Escherichia coli lacZ reporter gene has been "knocked in" to the SCL locus, thereby linking beta-galactosidase expression to transcription from the SCL promoter. Bone marrow cells from heterozygous SCLlacZ/w mice were sorted into fractions expressing high, intermediate and low levels of beta-galactosidase (designated lacZhigh, lacZint, and lacZneg). Cells that were lacZhigh or lacZint were enriched for day 12 spleen colony-forming units and myeloid and erythroid colony-forming cells (CFCs). These fractions included >99% of the erythroid and >90% of the myeloid CFCs. Culture of sorted bone marrow populations on stromal cells secreting interleukin-7 or in fetal thymic organ cultures showed that B and T lymphoid progenitors were also present in the lacZhigh and lacZint fractions. These data provide a functional correlation between SCL expression and colony-forming ability in immature hematopoietic cells. Our data also suggested that expression of SCL was transient and confined to hematopoietic stem and/or progenitor cells, because the differentiated progeny of most lineages (except the erythroid) were beta-galactosidase-negative.

BCR-ABL activates pathways mediating cytokine independence and protection against apoptosis in murine hematopoietic cells in a dose-dependent manner.

The hallmark of chronic myeloid leukemia (CML) is the chimeric tyrosine kinase oncogene bcr-abl. Since expression of bcr-abl mRNA frequently increases with disease progression and a duplication of the Philadelphia chromosome (harbouring the bcr-abl hybrid locus) represents the most frequent karyotypic abnormality in acute phase CML, we hypothesized that the level of BCR-ABL protein may affect the disease phenotype. Therefore, the biological effects of high and low levels of BCR-ABL expression were compared in growth factor-dependent and -independent myeloid and lymphoid cell lines. Our results demonstrated that low levels of BCR - ABL were sufficient to render these cell lines growth factor independent and tumorigenic, but higher levels were mandatory for additional protection against apoptotic stimuli. The provision of growth factor or an activated ras oncogene did not afford the same degree of protection as high levels of BCR-ABL and there were qualitative differences between the survival signals mediated by BCR-ABL and Bcl-2. These results have enabled us to establish a dose-dependent hierarchy of BCR-ABL induced biological effects, thus distinguishing the activation of pathways mediating protection from cytokine withdrawal from those protecting against other apoptotic stimuli.

Transcriptional regulation of vav, a gene expressed throughout the hematopoietic compartment.

The vav gene is expressed in all hematopoietic but few other cell types. To explore its unusual compartment-wide regulation, we cloned the murine gene, sequenced its promoter region, identified DNase I hypersensitive (HS) sites in the chromatin, and tested their promoter activity with a beta-galactosidase (beta-gal) reporter gene in cell lines and transgenic mice. Whereas fibroblasts had no HS sites, a myeloid and an erythroid cell line contained five, located 0.2 kb (HS1), 1.9 kb (HS2), and 3.6 kb (HS3) upstream from the transcription start and 0.6 kb (HS4) and 10 kb (HS5) downstream. A vav DNA fragment including HS1 promoted beta-gal expression in a myeloid but not a fibroblast line. Expression in leukocytes of transgenic mice also required HS2 and HS5. Only hematopoietic organs contained beta-gal, but virtually all beta-gal+ cells were B or T lymphocytes. Expression was always variegated (mosaic), and the proportion of beta-gal+ cells declined with lymphoid maturation and animal age. Thus, these vav regulatory elements promoted hematopoietic-specific expression in vivo, at least in lymphocytes, but the transgene was sporadically silenced. Maintaining pan-hematopoietic expression may require additional vav elements or an alternative reporter.

Transcription of the SCL gene in erythroid and CD34 positive primitive myeloid cells is controlled by a complex network of lineage-restricted chromatin-dependent and chromatin-independent regulatory elements.

The SCL gene (also known as TAL-1) encodes a basic helix-loop-helix transcription factor that is essential for the development of all haematopoietic lineages, and ectopic expression of which results in T cell leukaemia. SCL is expressed in normal pluripotent haematopoietic stem cells and its expression is maintained during differentiation along erythroid, mast and megakaryocytic lineages, but is extinguished following commitment to other cell types. The mechanisms responsible for this pattern of expression are poorly understood, but are likely to illuminate the molecular basis for stem cell development and lineage commitment. We have identified multiple lineage-restricted DNase I hypersensitive sites in a 45 kb region spanning the murine SCL locus. Committed erythroid cells and CD34 positive primitive myeloid cells exhibited both shared and unique DNase I hypersensitive sites whereas none were found in T cells. The function of each hypersensitive site was studied using both transient and stable reporter assays in erythroid, primitive myeloid and T cells. Multiple positive and negative regulatory elements were characterised and found to display lineage-specificity, promoter-specificity and/or chromatin-dependence. These results represent the first description of key components of a complex network of regulatory elements controlling SCL expression during haematopoiesis.