Contrasting effects of IRS-1 versus IRS-2 gene disruption on carbohydrate and lipid metabolism in vivo

To examine the impact of homozygous genetic disruption of insulin receptor substrate (IRS)-1 (IRS-1(-/-)) or IRS-2 (IRS-2(-/-)) on basal and insulin-stimulated carbohydrate and lipid metabolism in vivo, we infused 18-h fasted mice (wild-type (WT), IRS-1(-/-), and IRS-2(-/-)) with [3-(3)H]glucose and [(2)H(5)]glycerol and assessed rates of glucose and glycerol turnover under basal (0-90 min) and hyperinsulinemic-euglycemic clamp (90-210 min; 5 mm glucose, and 5 milliunits of insulin.kg(-)(1).min(-)(1)) conditions. Both IRS-1(-)(/-) and IRS-2(-)(/-) mice were insulin-resistant as reflected by markedly impaired insulin-stimulated whole-body glucose utilization compared with WT mice. Insulin resistance in the IRS-1(-)(/-) mice could be ascribed mainly to decreased insulin-stimulated peripheral glucose metabolism. In contrast, IRS-2(-)(/-) mice displayed multiple defects in insulin-mediated carbohydrate metabolism as reflected by (i) decreased peripheral glucose utilization, (ii) decreased suppression of endogenous glucose production, and (iii) decreased hepatic glycogen synthesis. Additionally, IRS-2(-)(/-) mice also showed marked insulin resistance in adipose tissue as reflected by reduced suppression of plasma free fatty acid concentrations and glycerol turnover during the hyperinsulinemic-euglycemic clamp. These data suggest important tissue-specific roles for IRS-1 and IRS-2 in mediating the effect of insulin on carbohydrate and lipid metabolism in vivo in mice. IRS-1 appears to have its major role in muscle, whereas IRS-2 appears to impact on liver, muscle, and adipose tissue.     

Bioluminescence reporter gene imaging characterize human embryonic stem cell-derived teratoma formation

Human embryonic stem (hES) cells have a potential use for the repair and regeneration of injured tissues. However, teratoma formation can be a major obstacle for hES-mediated cell therapy. Therefore, tracking the fate and function of transplanted hES cells with noninvasive imaging could be valuable for a better understanding of the biology and physiology of teratoma formation. In this study, hES cells were stably transduced with a double fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. Following bioluminescence imaging and histology, we demonstrated that engraftment of hES cells was followed by dramatically increasing signaling and led to teratoma formation confirmed by histology. Studies of the angiogenic processes within teratomas revealed that their vasculatures were derived from both differentiated hES cells and host. Moreover, FACS analysis showed that teratoma cells derived from hES cells expressed high levels of CD56 and SSEA-4, and the subcultured SSEA-4(+) cells showed a similar cell surface marker expression pattern when compared to undifferentiated hES cells. We report here for the first time that SSEA-4(+) cells derived from teratoma exhibited multipotency, retained their differentiation ability in vivo as confirmed by their differentiation into representative three germ layers.     

Electrophysiological properties of embryonic stem cell-derived neurons

In vitro generation of functional neurons from embryonic stem (ES) cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP) and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP(+) neurons in culture display functional neuronal properties even at early stages of differentiation.     

Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids

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Differential lipolytic regulation in human embryonic stem cell-derived adipocytes

Objective: Human embryonic stem cells (hESCs) have raised great hopes for future clinical applications. Several groups have succeeded in differentiating hESCs into adipocytes, as determined by morphology, mRNA expression, and protein secretion. However, determination of lipolytic response, the most important characteristic of adipocytes, has not been performed. This work was intended to study adipogenic conversion of hESCs by functional assessment of differentiation. Research Methods and Procedures: Single undifferentiated colonies were allowed to transform into embryonic bodies. mRNA expression for a set of adipocyte‐specific genes and leptin/adiponectin secretion and lipolysis were assessed at different time‐points after differentiation. Results: In contrast to primary human adipocytes, hESC‐derived adipocytes showed a very small response to classical β‐adrenergic agonists, although they expressed the major genes in the lipolytic cascade. In contrast, there was a significant lipolytic response to atrial natriuretic peptide. Discussion: Although hESC‐derived adipocytes seem to be morphologically and expressionally similar to mature adipocytes, there are important functional differences that could depend on their early developmental origin. We conclude that, in contrast to mature adipocytes, hESC‐derived adipocytes display a differential response to atrial natriuretic peptide and catecholamines.     

Electron microscopic study of mouse embryonic stem cell-derived cardiomyocytes

Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs’ development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs’ outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences.     

Characterization of human embryonic stem cell-derived hematopoietic progenitor phenotype

Differentiation of human embryonic stem cells (hESCs) into hematopoietic lineages using various methods has been reported. However, the phenotype that precisely defines the hematopoietic progenitor compartment with clonogenic activities has yet to be determined. Here, we measured and characterized progenitor function of subfractions of cells prospectively isolated from human embryoid bodies (hEBs) during hematopoietic differentiation basing on surface markers CD45, CD34, CD43, and CD38. We report that hematopoietic progenitors predominantly resided in the CD45⁺ subset. CD43⁺ cells lacking CD45 expression were largely devoid of progenitor activity. However, progenitor activity and multipotentiality was more enriched in CD45⁺ cells co-expressing CD43. CD45⁺ subset co-expressing CD34 but lacking CD38 expression (CD45⁺CD34⁺CD38^-) were further enriched for CFU capacity compared to the CD45⁺CD34⁺CD38⁺ subset. Our study demonstrates a role of CD43 in enriching hematopoietic progenitors derived from hEBs and reveals a hierarchical organization of hESC-derived hematopoietic progenitor compartments defined by phenotypic markers.     

Microcarrier expansion of mouse embryonic stem cell-derived neural stem cells in stirred bioreactors

Neural stem cells (NSCs) are self-renewing multipotent cells, able to differentiate into the phenotypes present in the central nervous system. Applications of NSCs may include toxicology, fundamental research, or cell therapies. The culture of floating cell clusters, called neurospheres, is widely used for the propagation of NSC populations in vitro but shows several limitations, which may be circumvented by expansion under adherent conditions. In particular, the derivation of distinct populations of NSCs from embryonic stem cells capable of long-term culture under adherent conditions without losing differentiation potential was recently described. However, the expansion of these cells in agitated bioreactors has not been addressed until now and was the aim of this study. Selected microcarriers were tested under dynamic conditions in spinner flasks. Superior performance was observed with polystyrene beads coated with a recombinant peptide containing the Arg-Gly-Asp (RGD) motif (Pronectin F). After optimization of the culture, a 35-fold increase in cell number was achieved after 6 days. High cellular viability and multipotency were maintained throughout the culture. The study presented here may be the basis for the development of larger scale bioprocesses for expansion of these and other populations of adherent NSCs, either from mouse or human origin.     

Detecting de novo insulin synthesis in embryonic stem cell-derived populations

Several studies in recent years have described protocols, both genetic- and culture-based, that induce the differentiation of embryonic stem (ES) cells towards a pancreatic beta-cell type. The success of previous protocols in generating insulin-producing beta-cells has been questioned due in part to uncertainty regarding cell lineage but also due to the controversy regarding the source of any insulin detected in these cells. In an attempt to address the latter, we designed a novel assay that can identify de novo insulin synthesis. The method is based on metabolic labeling combined with a modified radio-immunoassay and will routinely detect less than 5 pg/microl of de novo insulin synthesis in lysates from the insulinoma cell line MIN6. This assay failed to detect any newly translated insulin in an ES cell-derived population generated using an adapted version of a previously published, 5-stage differentiation protocol. In combination with other techniques, including immunofluorescent staining and western blot analysis to detect and quantify C-peptide, we conclude that the majority of the insulin found in these differentiated ES cell cultures is medium-derived.     

Mouse embryonic stem cell-derived feeder cells support the growth of their own mouse embryonic stem cells

Feeder cells are usually used in culturing embryonic stem cells (ESCs) to maintain their undifferentiated and pluripotent status. To test whether mouse embryonic stem cells (mESCs) may be a source of feeder cells to support their own growth, 48 fibroblast-like cell lines were isolated from the same mouse embryoid bodies (mEBs) at three phases (10th day, 15th day, 20th day), and five of them, mostly derived from 15th day mEBs, were capable of maintaining mESCs in an undifferentiated and pluripotent state over 10 passages, even up to passage 20. mESCs cultured on the feeder system derived from these five cell lines expressed alkaline phosphatase and specific mESCs markers, including SSEA-1, Oct-4, Nanog, and formed mEBs in vitro and teratomas in vivo. These results suggest that mEB-derived fibroblasts (mEB-dFs) could serve as feeder cells that could sustain the undifferentiated growth and pluripotency of their own mESCs in culture. This study not only provides a novel feeder system for mESCs culture, avoiding a lot of disadvantages of commonly used mouse embryonic fibroblasts as feeder cells, but also indicates that fibroblast-like cells derived from mESCs take on different functions. Investigating the molecular mechanisms of these different functional fibroblast-like cells to act on mESCs will contribute to the understanding of the mechanisms of mESCs self-renewal.     

Heparan sulfate proteoglycan mediates shear stress-induced endothelial gene expression in mouse embryonic stem cell-derived endothelial cells

It has been shown that shear stress plays a critical role in promoting endothelial cell (EC) differentiation from embryonic stem cell (ESC)-derived ECs. However, the underlying mechanisms mediating shear stress effects in this process have yet to be investigated. It has been reported that the glycocalyx component heparan sulfate proteoglycan (HSPG) mediates shear stress mechanotransduction in mature EC. In this study, we investigated whether cell surface HSPG plays a role in shear stress modulation of EC phenotype. ESC-derived EC were subjected to shear stress (5 dyn/cm(2)) for 8 h with or without heparinase III (Hep III) that digests heparan sulfate. Immunostaining showed that ESC-derived EC surfaces contain abundant HSPG, which could be cleaved by Hep III. We observed that shear stress significantly increased the expression of vascular EC-specific marker genes (vWF, VE-cadherin, PECAM-1). The effect of shear stress on expression of tight junction protein genes (ZO-1, OCLD, CLD5) was also evaluated. Shear stress increased the expression of ZO-1 and CLD5, while it did not alter the expression of OCLD. Shear stress increased expression of vasodilatory genes (eNOS, COX-2), while it decreased the expression of the vasoconstrictive gene ET1. After reduction of HSPG with Hep III, the shear stress-induced expression of vWF, VE-cadherin, ZO-1, eNOS, and COX-2, were abolished, suggesting that shear stress-induced expression of these genes depends on HSPG. These findings indicate for the first time that HSPG is a mechanosensor mediating shear stress-induced EC differentiation from ESC-derived EC cells.     

Contrasting effects of ENU induced embryonic lethal mutations of the quaking gene.

Multiple alleles of the quaking (qk) gene have a variety of phenotypes ranging in severity from early embryonic death to viable dysmyelination. A previous study identified a candidate gene, QKI, that contains an RNA-binding domain and encodes at least three protein isoforms (QKI-5, -6 and -7). We have determined the genomic structure of QKI, identifying an additional alternative end in cDNAs. Further we have examined the exons and splice sites for mutations in the lethal alleles qkl-1, qkkt1, qkk2, and qkkt3. The mutation in qkl-1 creates a splice site in the terminal exon of the QKI-6 isoform. Missense mutations in the KH domain and the QUA1 domains in qkk2 and qkkt3, respectively, indicate that these domains are of critical functional importance. Although homozygotes for each ENU induced allele die as embryos, their phenotypes as viable compound heterozygotes with qkv differ. Compound heterozygous qkv animals carrying qkkt1, qkk2, and qkkt3 all exhibit a permanent quaking phenotype similar to that of qkv/qkv animals, whereas qkv/qkl-1 animals exhibit only a transient quaking phenotype. The qkl-1 mutation eliminates the QKI-5 isoform, showing that this isoform plays a crucial role in embryonic survival. The transient quaking phenotype observed in qkv/qkl-1 mice indicates that the QKI-6 and QKI-7 isoforms function primarily during myelination, but that QKI-5 may have a concentration-dependent role in early myelination. This mutational analysis demonstrates the power of series of alleles to examine the function of complex loci and suggests that additional mutant alleles of quaking could reveal additional functions of this complex gene.