Generation Of Different Cell Types Research Articles

Abstract 195: Dose-Dependent Induction of Nitric Oxide Production in Human Induced Pluripotent Stem Cell-Derived Endothelial Cells by Poloxamer 188

Background: Poloxamer 188 (P188), a triblock copolymer, protects against ischemia/reperfusion injury. The precise mechanisms remain incompletely understood. Human induced pluripotent stem cells (hiPSCs) constitute a unique model system, allowing the parallel generation of different cell types from individual patients to study drug-specific effects in human tissue. Here, we aimed to investigate the potential of P188 to induce nitric oxide (NO) production in hiPSC-derived endothelial cells (ECs) and its effect on cell viability during hypoxia-reoxygenation. Methods: hiPSC-ECs were cultured in phenol red-free endothelial Cell Growth Medium-2 and subjected to hypoxia (1% oxygen) at 37°C for 36 hours. At the end of hypoxia, the cell culture medium was supplemented with NO-sensitive fluorescent dye DAF-FM diacetate (1:5000 dilution, final concentration 1 uM, 30 min). Hypoxic medium was removed, and P188 at different concentrations (0.1 nM to 100 nM) was added for 8 hours. DAF fluorescence and cell viability were assessed at the end of reoxygenation. One-Way ANOVA & Dunn's post hoc test was utilized for statistical analysis (* P < 0.05 vs. untreated control group). Results: P188 dose-dependently increased hiPSC-ECs viability (control group: 53±2%, 0.01 nM P188: 59±2%, 100 nM P188: 64±5%). Interestingly, DAF fluorescence increased dose-dependently as well (control group: 182±9%, 0.1 nM P188: 199±11%, 100 nM P188: 264±4%, data expressed as percent of normoxic cells, n=3 / treatment). Conclusion: This suggests that P188 protects hiPSC-ECs with increased NO production, shedding new light on the underlying mechanisms of its cardioprotective effects.

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders.

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

Hematopoiesis: A Layered Organization Across Chordate Species.

The identification of distinct waves of progenitors during development, each corresponding to a specific time, space, and function, provided the basis for the concept of a “layered” organization in development. The concept of a layered hematopoiesis was established by classical embryology studies in birds and amphibians. Recent progress in generating reliable lineage tracing models together with transcriptional and proteomic analyses in single cells revealed that, also in mammals, the hematopoietic system evolves in successive waves of progenitors with distinct properties and fate. During embryogenesis, sequential waves of hematopoietic progenitors emerge at different anatomic sites, generating specific cell types with distinct functions and tissue homing capacities. The first progenitors originate in the yolk sac before the emergence of hematopoietic stem cells, some giving rise to progenies that persist throughout life. Hematopoietic stem cell-derived cells that protect organisms against environmental pathogens follow the same sequential strategy, with subsets of lymphoid cells being only produced during embryonic development. Growing evidence indicates that fetal immune cells contribute to the proper development of the organs they seed and later ensure life-long tissue homeostasis and immune protection. They include macrophages, mast cells, some γδ T cells, B-1 B cells, and innate lymphoid cells, which have “non-redundant” functions, and early perturbations in their development or function affect immunity in the adult. These observations challenged the view that all hematopoietic cells found in the adult result from constant and monotonous production from bone marrow-resident hematopoietic stem cells. In this review, we evaluate evidence for a layered hematopoietic system across species. We discuss mechanisms and selective pressures leading to the temporal generation of different cell types. We elaborate on the consequences of disturbing fetal immune cells on tissue homeostasis and immune development later in life.

A Gene Regulatory Network Balances Neural and Mesoderm Specification during Vertebrate Trunk Development

SummaryTranscriptional networks, regulated by extracellular signals, control cell fate decisions and determine the size and composition of developing tissues. One example is the network controlling bipotent neuromesodermal progenitors (NMPs) that fuel embryo elongation by generating spinal cord and trunk mesoderm tissue. Here, we use single-cell transcriptomics to identify the molecular signature of NMPs and reverse engineer the mechanism that regulates their differentiation. Together with genetic perturbations, this reveals a transcriptional network that integrates opposing retinoic acid (RA) and Wnt signals to determine the rate at which cells enter and exit the NMP state. RA, produced by newly generated mesodermal cells, provides feedback that initiates NMP generation and induces neural differentiation, thereby coordinating the production of neural and mesodermal tissue. Together, the data define a regulatory network architecture that balances the generation of different cell types from bipotential progenitors in order to facilitate orderly axis elongation.

Chemicals as the Sole Transformers of Cell Fate.

Forced expression of lineage-specific transcription factors in somatic cells can result in the generation of different cell types in a process named direct reprogramming, bypassing the pluripotent state. However, the introduction of transgenes limits the therapeutic applications of the produced cells. Numerous small-molecules have been introduced in the field of stem cell biology capable of governing self-renewal, reprogramming, transdifferentiation and regeneration. These chemical compounds are versatile tools for cell fate conversion toward desired outcomes. Cell fate conversion using small-molecules alone (chemical reprogramming) has superiority over arduous traditional genetic techniques in several aspects. For instance, rapid, transient, and reversible effects in activation and inhibition of functions of specific proteins are of the profits of small-molecules. They are cost-effective, have a long half-life, diversity on structure and function, and allow for temporal and flexible regulation of signaling pathways. Additionally, their effects could be adjusted by fine-tuning concentrations and combinations of different small-molecules. Therefore, chemicals are powerful tools in cell fate conversion and study of stem cell and chemical biology in vitro and in vivo. Moreover, transgene-free and chemical-only transdifferentiation approaches provide alternative strategies for the generation of various cell types, disease modeling, drug screening, and regenerative medicine. The current review gives an overview of the recent findings concerning transdifferentiation by only small-molecules without the use of transgenes.

Zinc finger transcription factor Casz1 expression is regulated by homeodomain transcription factor Prrxl1 in embryonic spinal dorsal horn late-born excitatory interneurons.

The transcription factor Casz1 is required for proper assembly of vertebrate vasculature and heart morphogenesis as well as for temporal control of Drosophila neuroblasts and mouse retina progenitors in the generation of different cell types. Although Casz1 function in the mammalian nervous system remains largely unexplored, Casz1 is expressed in several regions of this system. Here we provide a detailed spatiotemporal characterization of Casz1 expression along mouse dorsal root ganglion (DRG) and dorsal spinal cord development by immunochemistry. In the DRG, Casz1 is broadly expressed in sensory neurons since they are born until perinatal age. In the dorsal spinal cord, Casz1 displays a more dynamic pattern being first expressed in dorsal interneuron 1 (dI1) progenitors and their derived neurons and then in a large subset of embryonic dorsal late-born excitatory (dILB) neurons that narrows gradually to become restricted perinatally to the inner portion. Strikingly, expression analyses using Prrxl1-knockout mice revealed that Prrxl1, a key transcription factor in the differentiation of dILB neurons, is a positive regulator of Casz1 expression in the embryonic dorsal spinal cord but not in the DRG. By performing chromatin immunoprecipitation in the dorsal spinal cord, we identified two Prrxl1-bound regions within Casz1 introns, suggesting that Prrxl1 directly regulates Casz1 transcription. Our work reveals that Casz1 lies downstream of Prrxl1 in the differentiation pathway of a large subset of dILB neurons and provides a framework for further studies of Casz1 in assembly of the DRG-spinal circuit.

Bone marrow-derived cells migrate to the liver and contribute to the generation of different cell types in chronic Schistosoma mansoni infection

The main pathogenic event caused by Schistosoma mansoni infection is characterized by a granulomatous inflammatory reaction around parasite eggs and fibrosis in the liver. We have previously shown that transplantation of bone marrow cells (BMC) promotes a reduction in liver fibrosis in chronically S. mansoni-infected mice. Here we investigated the presence and phenotype of bone marrow-derived cells in livers of S. mansoni-infected mice. During the chronic phase of infection, C57BL/6 mice had an increased number of circulating mesenchymal stem cells and endothelial progenitor cells in the peripheral blood when compared to uninfected controls. In order to investigate the fate of BMC in the liver, we generated bone marrow chimeric mice by transplanting BMC from transgenic green fluorescent protein (GFP) mice into lethally irradiated wild-type C57BL/6 mice. S. mansoni-infected chimeric mice did not demonstrate increased mortality and developed similar liver histopathological features, when compared to wild-type S. mansoni-infected mice. GFP+ bone marrow-derived cells were found in the liver parenchyma, particularly in periportal regions. CD45+GFP+ cells were found in the granulomas. Flow cytometry analysis of digested liver tissue characterized GFP+ cells as lymphocytes, myeloid cells and stem cells. GFP+ cells were also found in areas of collagen deposition, although rare GFP+ cells expressed the myofibroblast cell marker α-SMA. Additionally GFP+ endothelial cells (co-stained with von Willebrand factor) were frequently observed, while BMC-derived hepatocytes (GFP+ albumin+ cells) were sparsely found in the liver of chimeric mice chronically infected with S. mansoni. In conclusion, BMC are recruited to the liver during chronic experimental infection with S. mansoni and contribute to the generation of different cell types involved, not only in disease pathogenesis, but possibly in liver regeneration and repair.

Role of Notch Signaling in Hematopoietic Stem Cell Function

Abstract SCI-15The Notch pathway controls the generation of different cell types in most tissues including blood, and dysregulation of this pathway is strongly associated with oncogenic processes. In many systems, Notch is also required for the maintenance of the stem cell populations. However, in the adult hematopoietic system this link between Notch and stemness has not been established. Instead, work of several groups, including ours, has clearly demonstrated that Notch has a prominent role in the generation of hematopoietic stem cells (HSC) during embryonic development. Although the first wave of blood cells appears in the mouse embryo around day 7.5 of development and is independent of Notch function, embryonic HSC are formed around day 10 of development from endothelial-like progenitors that reside in the embryonic aorta surrounded by the gonad and mesonephros, also called AGM region. By analyzing different Notch pathway mutant mouse embryos, we have demonstrated the involvement of the Jagged1-Notch1-GATA2 axis in this event. However, the formal demonstration that Notch regulates the GATA2 gene during HSC generation is still lacking. We have now found that GATA2 is a direct Notch target in vivo during embryonic HSC generation. However, whereas Notch positively activates GATA2 transcription in the HSC precursors, it simultaneously activates hes1 transcription, which acts a repressor of the same GATA2 gene. This finding directly implicates hes1 in the regulation of HSC development although further studies using loss-of-function mutant embryos are still needed. Altogether, our results indicate that both Notch and hes1 are required to finely regulate the levels, distribution, and likely the timing of GATA2 expression through an incoherent feed-forward loop. In parallel, we have identified other downstream targets of Notch in the AGM region by ChIP-on-chip and expression microarray analysis that we are currently characterizing. Disclosures:No relevant conflicts of interest to declare.

Genome Medicine: stem cells, genomics and translational research

Genome Medicine: stem cells, genomics and translational research

Modularity map of the network of human cell differentiation

Cell differentiation in multicellular organisms is a complex process whose mechanism can be understood by a reductionist approach, in which the individual processes that control the generation of different cell types are identified. Alternatively, a large-scale approach in search of different organizational features of the growth stages promises to reveal its modular global structure with the goal of discovering previously unknown relations between cell types. Here, we sort and analyze a large set of scattered data to construct the network of human cell differentiation (NHCD) based on cell types (nodes) and differentiation steps (links) from the fertilized egg to a developed human. We discover a dynamical law of critical branching that reveals a self-similar regularity in the modular organization of the network, and allows us to observe the network at different scales. The emerging picture clearly identifies clusters of cell types following a hierarchical organization, ranging from sub-modules to super-modules of specialized tissues and organs on varying scales. This discovery will allow one to treat the development of a particular cell function in the context of the complex network of human development as a whole. Our results point to an integrated large-scale view of the network of cell types systematically revealing ties between previously unrelated domains in organ functions.

GABAergic interneurons and the development of network oscillations

Event Abstract Back to Event GABAergic interneurons and the development of network oscillations Thomas Voigt1* 1 Otto-von-Guericke University, Institute of Physiology, Germany Neocortical neurons dissociated from embryonic tissue and grown in a standardized environment form synaptically interconnected and electrically active networks. Depending on the age of dissociation, the major developmental steps (cell proliferation, cell migration, generation of different cell types, synaptogenesis and emergence of spontaneous activity) occur in the culture dish within a schedule comparable to the development in the intact cortex. With certain preparation and cultivation techniques raising neuronal networks with defined cellular content is possible. Our cell culture experiments reveal a close interdependence between functional network development and the maturation of GABAergic neurons and synapses. GABAergic interneurons with large axonal arbors advance the initiation of large scale synchronous network activity, which is similar to the cortical early network oscillations (cENOs) observed in cortical slices 1-3. The emergent network activity in turn promotes growth and axonal maturation of the pioneer GABAergic neurons, a process modulated by thyroid hormones 4. Early synchronous network activity also supports the activation of immature glutamatergic synapses but restrains the formation of long range axonal connections 5. Specially the integration of late-born GABAergic interneurons depends on the developing network activity, which promotes their survival and limits their migration 6,7. Apart from the initiation of the early network activity, GABAergic signaling contributes to the regression and decorrelation of early oscillations 8. This developmental process is promoted by the expression of the potassium-chloride transporter KCC2 and the gradual change of the chloride equilibrium potential, which shifts GABA action from depolarizing to hyperpolarizing. The decorrelation of network activity requires the presence of GABAergic interneurons and can be induced by blocking the sodium-potassium-chloride transporter NKCC1 with bumetanide.

Wnt signaling determines ventral spinal cord cell fates in a time-dependent manner

The identity of distinct cell types in the ventral neural tube is generally believed to be specified by sonic hedgehog (Shh) in a concentration-dependent manner. However, recent studies have questioned whether Shh is the sole signaling molecule determining ventral neuronal cell fates. Here we provide evidence that canonical Wnt signaling is involved in the generation of different cell types in the ventral spinal cord. We show that Wnt signaling is active in the mouse ventral spinal cord at the time when ventral cell types are specified. Furthermore, using an approach that stabilizes beta-catenin protein in small patches of ventral spinal cord cells at different stages, we show that Wnt signaling activates different subsets of target genes depending on the time when Wnt signaling is amplified. Moreover, disruption of Wnt signaling results in the expansion of ventrally located progenitors. Finally, we show genetically that Wnt signaling interacts with Hh signaling at least in part through regulating the transcription of Gli3. Our results reveal a novel mechanism by which ventral patterning is achieved through a coordination of Wnt and Shh signaling.

Putting a Notch in Our Understanding of Nuclear Migration

The nuclei of progenitor cells in developing neural epithelia change their position during the cell cycle through a process called interkinetic nuclear migration. Del Bene et al. (2008) report that defects in the machinery controlling this process lead to altered exposure to Notch signals and systemic effects on neurogenesis in the retina.

Regulation of Stem Cell Differentiation by Histone Methyltransferases and Demethylases

The generation of different cell types from stem cells containing identical genetic information and their organization into tissues and organs during development is a highly complex process that requires defined transcriptional programs. Maintenance of such programs is epigenetically regulated and the factors involved in these processes are often essential for development. The activities required for cell-fate decisions are frequently deregulated in human tumors, and the elucidation of the molecular mechanisms that regulate these processes is therefore important for understanding both developmental processes and tumorigenesis.

Hematopoietic cytokine receptor signaling

Hematopoiesis is the cumulative result of intricately regulated signaling pathways that are mediated by cytokines and their receptors. Proper culmination of these diverse pathways forms the basis for an orderly generation of different cell types. Recent studies conducted over the past 10-15 years have revealed that hematopoietic cytokine receptor signaling is largely mediated by a family of tyrosine kinases termed Janus kinases (JAKs) and their downstream transcription factors termed STATs (signal transducers and activators of transcription). Aberration in these pathways, such as that caused by the recently identified JAK2V617F mutation, is an underlying cause for diseases such as leukemias and other myeloproliferative disorders. This recent discovery, when coupled with the fact that STATs are activated by oncoproteins such as BCR-ABL, underscores the importance of the JAK-STAT pathway in both normal cellular development and disease states.

Pancreatic Development and Disease

Pancreatic Development and Disease

Cytokinesis Monitoring during Development: Rapid Pole-to-Pole Shuttling of a Signaling Protein by Localized Kinase and Phosphatase in Caulobacter

For successful generation of different cell types by asymmetric cell division, cell differentiation should be initiated only after completion of division. Here, we describe a control mechanism by which Caulobacter couples the initiation of a developmental program to the completion of cytokinesis. Genetic evidence indicates that localization of the signaling protein DivK at the flagellated pole prevents premature initiation of development. Photobleaching and FRET experiments show that polar localization of DivK is dynamic with rapid pole-to-pole shuttling of diffusible DivK generated by the localized activities of PleC phosphatase and DivJ kinase at opposite poles. This shuttling is interrupted upon completion of cytokinesis by the segregation of PleC and DivJ to different daughter cells, resulting in disruption of DivK localization at the flagellated pole and subsequent initiation of development in the flagellated progeny. Thus, dynamic polar localization of a diffusible protein provides a control mechanism that monitors cytokinesis to regulate development.

JAKs, STATs and Src kinases in hematopoiesis.

Hematopoiesis is the cumulative result of intricately regulated signal transduction cascades that are mediated by cytokines and their cognate receptors. Proper culmination of these diverse signaling pathways forms the basis for an orderly generation of different cell types and aberrations in these pathways is an underlying cause for diseases such as leukemias and other myeloproliferative and lymphoproliferative disorders. Over the past decade, downstream signal transduction events initiated upon cytokine/growth factor stimulation have been a major focus of basic and applied biomedical research. As a result, several key concepts have emerged allowing a better understanding of the complex signaling processes. A group of transcription factors, termed signal transducers and activators of transcription (STATs) appear to orchestrate the downstream events propagated by cytokine/growth factor interactions with their cognate receptors. Similarly, cytoplasmic Janus protein tyrosine kinases (JAKs) and Src family of kinases seem to play a critical role in diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. Accumulating evidence suggests that STAT protein activation may be mediated by members of both JAK and Src family members following cytokine/growth factor stimulation. In addition, JAK kinases appear to be essential for the phosphorylation of the cytokine receptors which results in the creation of docking sites on the receptors for binding of SH2-containing proteins such as STATs, Src-kinases and other signaling intermediates. Cell and tissue-specificity of cytokine action appears to be determined by the nature of signal transduction pathways activated by cytokine/receptor interactions. The integration of these diverse signaling cues from active JAK kinases, members of the Src-family kinases and STAT proteins, leads to cell proliferation, cell survival and differentiation, the end-point of the cytokine/growth factor stimulus.

Notch Regulates RNA Translation

The asymmetric cell divisions that occur during the development of certain neuronal systems account for the generation of different cell types from a common precursor cell. Such is true during mechanosensory bristle development in Drosophila where the first asymmetric cell division of a sensory organ precursor cell gives rise to a neuronal and nonneuronal precursor cell. Inhibition of Notch signaling by the intracellular protein Numb accounts for generation of the neuronal cell, but it is not clear how Notch signaling regulates formation of the nonneuronal cell. Okabe et al. report that Notch somehow inhibits the activity of Musashi (MSI), an RNA-binding protein. MSI was found to bind to the 3' untranslated region of an mRNA that encodes Tramtrack69 (TTK69), a transcriptional repressor implicated in determining nonneuronal cell fate. MSI association with TTK69 mRNA inhibited translation in a reporter assay. Hence, MSI-mediated repression of TTK69 translation may account for neuronal cell fate. M. Okabe, T. Imai, M. Kurusu, Y. Hiromi, H. Okano, Translational repression determines a neuronal potential in Drosophila asymmetric cell division. Nature 411 , 94-98 (2001). [Online Journal]

Notch Regulates RNA Translation

The asymmetric cell divisions that occur during the development of certain neuronal systems account for the generation of different cell types from a common precursor cell. Such is true during mechanosensory bristle development in Drosophila where the first asymmetric cell division of a sensory organ precursor cell gives rise to a neuronal and nonneuronal precursor cell. Inhibition of Notch signaling by the intracellular protein Numb accounts for generation of the neuronal cell, but it is not clear how Notch signaling regulates formation of the nonneuronal cell. Okabe et al. report that Notch somehow inhibits the activity of Musashi (MSI), an RNA-binding protein. MSI was found to bind to the 3' untranslated region of an mRNA that encodes Tramtrack69 (TTK69), a transcriptional repressor implicated in determining nonneuronal cell fate. MSI association with TTK69 mRNA inhibited translation in a reporter assay. Hence, MSI-mediated repression of TTK69 translation may account for neuronal cell fate.M. Okabe, T. Imai, M. Kurusu, Y. Hiromi, H. Okano, Translational repression determines a neuronal potential in Drosophila asymmetric cell division. Nature 411, 94-98 (2001). [Online Journal]