Pompe disease results in a Golgi-based glycosylation deficit in human induced pluripotent stem cell-derived cardiomyocytes.

Infantile-onset Pompe disease is an autosomal recessive disorder caused by the complete loss of lysosomal glycogen-hydrolyzing enzyme acid α-glucosidase (GAA) activity, which results in lysosomal glycogen accumulation and prominent cardiac and skeletal muscle pathology. The mechanism by which loss of GAA activity causes cardiomyopathy is poorly understood. We reprogrammed fibroblasts from patients with infantile-onset Pompe disease to generate induced pluripotent stem (iPS) cells that were differentiated to cardiomyocytes (iPSC-CM). Pompe iPSC-CMs had undetectable GAA activity and pathognomonic glycogen-filled lysosomes. Nonetheless, Pompe and control iPSC-CMs exhibited comparable contractile properties in engineered cardiac tissue. Impaired autophagy has been implicated in Pompe skeletal muscle; however, control and Pompe iPSC-CMs had comparable clearance rates of LC3-II-detected autophagosomes. Unexpectedly, the lysosome-associated membrane proteins, LAMP1 and LAMP2, from Pompe iPSC-CMs demonstrated higher electrophoretic mobility compared with control iPSC-CMs. Brefeldin A induced disruption of the Golgi in control iPSC-CMs reproduced the higher mobility forms of the LAMPs, suggesting that Pompe iPSC-CMs produce LAMPs lacking appropriate glycosylation. Isoelectric focusing studies revealed that LAMP2 has a more alkaline pI in Pompe compared with control iPSC-CMs due largely to hyposialylation. MALDI-TOF-MS analysis of N-linked glycans demonstrated reduced diversity of multiantennary structures and the major presence of a trimannose complex glycan precursor in Pompe iPSC-CMs. These data suggest that Pompe cardiomyopathy has a glycan processing abnormality and thus shares features with hypertrophic cardiomyopathies observed in the congenital disorders of glycosylation.

Signals from the surface modulate differentiation of human pluripotent stem cells through glycosaminoglycans and integrins.

The fate decisions of human pluripotent stem (hPS) cells are governed by soluble and insoluble signals from the microenvironment. Many hPS cell differentiation protocols use Matrigel, a complex and undefined substrate that engages multiple adhesion and signaling receptors. Using defined surfaces programmed to engage specific cell-surface ligands (i.e., glycosaminoglycans and integrins), the contribution of specific matrix signals can be dissected. For ectoderm and motor neuron differentiation, peptide-modified surfaces that can engage both glycosaminoglycans and integrins are effective. In contrast, surfaces that interact selectively with glycosaminoglycans are superior to Matrigel in promoting hPS cell differentiation to definitive endoderm and mesoderm. The modular surfaces were used to elucidate the signaling pathways underlying these differences. Matrigel promotes integrin signaling, which in turn inhibits mesendoderm differentiation. The data indicate that integrin-activating surfaces stimulate Akt signaling via integrin-linked kinase (ILK), which is antagonistic to endoderm differentiation. The ability to attribute cellular responses to specific interactions between the cell and the substrate offers new opportunities for revealing and controlling the pathways governing cell fate.

Stem cells and their derivatives: a renaissance in cardiovascular translational research.

Moving from the bench to the bedside is an expensive and arduous journey with a high risk of failure. One roadblock on the path of translational medicine is the paucity of predictive in vitro models available during preclinical drug development. The ability of human embryonic stem (ES) and induced pluripotent stem (iPS) cells to generate virtually any tissue of the body, in vitro, makes these cells an obvious choice for use in drug discovery and translational medicine. Technological advancements in the production of stem cells and their differentiation into relevant cell types, such as cardiomyocytes, has permitted the utility of these cells in the translational medicine setting. In particular, the derivation and differentiation of patient-specific iPS cells will facilitate an understanding of basic disease etiology, enable better drug efficacy and safety screens, and ultimately lead to personalized patient therapies. This review will focus on recent advancements in the derivation and differentiation of human ES and iPS cells into cardiomyocytes and their uses in safety testing and modeling human disease.

Functional cardiomyocytes derived from human induced pluripotent stem cells.

Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapeutic applications, but the ability of human iPS cells to differentiate into functional cardiomyocytes has not yet been demonstrated. The aim of this study was to characterize the cardiac differentiation potential of human iPS cells generated using OCT4, SOX2, NANOG, and LIN28 transgenes compared to human embryonic stem (ES) cells. The iPS and ES cells were differentiated using the embryoid body (EB) method. The time course of developing contracting EBs was comparable for the iPS and ES cell lines, although the absolute percentages of contracting EBs differed. RT-PCR analyses of iPS and ES cell-derived cardiomyocytes demonstrated similar cardiac gene expression patterns. The pluripotency genes OCT4 and NANOG were downregulated with cardiac differentiation, but the downregulation was blunted in the iPS cell lines because of residual transgene expression. Proliferation of iPS and ES cell-derived cardiomyocytes based on 5-bromodeoxyuridine labeling was similar, and immunocytochemistry of isolated cardiomyocytes revealed indistinguishable sarcomeric organizations. Electrophysiology studies indicated that iPS cells have a capacity like ES cells for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics. Both iPS and ES cell-derived cardiomyocytes exhibited responsiveness to beta-adrenergic stimulation manifest by an increase in spontaneous rate and a decrease in action potential duration. We conclude that human iPS cells can differentiate into functional cardiomyocytes, and thus iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.

Blimp1 regulates development of the posterior forelimb, caudal pharyngeal arches, heart and sensory vibrissae in mice.

The zinc-finger transcriptional repressor Blimp1 (Prdm1) controls gene expression patterns during differentiation of B lymphocytes and regulates epigenetic changes required for specification of primordial germ cells. Blimp1 is dynamically expressed at diverse tissue sites in the developing mouse embryo, but its functional role remains unknown because Blimp1 mutant embryos arrest at E10.5 due to placental insufficiency. To explore Blimp1 activities at later stages in the embryo proper, here we used a conditional inactivation strategy. A Blimp1-Cre transgenic strain was also exploited to generate a fate map of Blimp1-expressing cells. Blimp1 plays essential roles in multipotent progenitor cell populations in the posterior forelimb, caudal pharyngeal arches, secondary heart field and sensory vibrissae and maintains key signalling centres at these diverse tissues sites. Interestingly, embryos carrying a hypomorphic Blimp1gfp reporter allele survive to late gestation and exhibit similar, but less severe developmental abnormalities, whereas transheterozygous Blimp1(gfp/-) embryos with further reduced expression levels, display exacerbated defects. Collectively, the present experiments demonstrate that Blimp1 requirements in diverse cell types are exquisitely dose dependent.

Loss of invariant chain protects nonobese diabetic mice against type 1 diabetes.

The invariant (Ii) chain acts as an essential chaperone to promote MHC class II surface expression, Ag presentation, and selection of CD4(+) T cells. We have examined its role in the development of type 1 diabetes in NOD mice and show that Ii chain-deficient NOD mice fail to develop type 1 diabetes. Surprisingly, Ii chain functional loss fails to disrupt in vitro presentation of islet Ags, in the context of NOD I-A(g7) molecules. Moreover, pathogenic effector cells could be shown to be present in Ii chain-deficient NOD mice because they were able to transfer diabetes to NOD.scid recipients. The ability of these cells to transfer diabetes was markedly enhanced by depletion of CD25 cells coupled with in vivo anti-CD25 treatment of recipient mice. The numbers of CD4(+)CD25(+)Foxp3(+) T cells in thymus and periphery of Ii chain-deficient NOD mice were similar to those found in normal NOD mice, in contrast to conventional CD4(+) T cells whose numbers were reduced. This suggests that regulatory T cells are unaffected in their selection and survival by the absence of Ii chain and that an alteration in the balance of effector to regulatory T cells contributes to diabetes prevention.

BMP4 substitutes for loss of BMP7 during kidney development.

Functional inactivation of divergent bone morphogenetic proteins (BMPs) causes discrete disturbances during mouse development. BMP4-deficient embryos display mesodermal patterning defects at early post-implantation stages, whereas loss of BMP7 selectively disrupts kidney and eye morphogenesis. Whether these distinct phenotypes simply reflect differences in expression domains, or alternatively intrinsic differences in the signaling properties of these ligands remains unknown. To address this issue, we created embryos exclusively expressing BMP4 under control of the BMP7 locus. Surprisingly, this novel knock-in allele efficiently rescues kidney development. These results demonstrate unequivocally that these structurally divergent BMP family members, sharing only minimal sequence similarity can function interchangeably to activate all the essential signaling pathways for growth and morphogenesis of the kidney. Thus, we conclude that partially overlapping expression patterns of BMPs serve to modulate strength of BMP signaling rather than create discrete fields of ligands with intrinsically different signaling properties.

Mice exclusively expressing the short isoform of Smad2 develop normally and are viable and fertile.

Smad2 and Smad3 are closely related effectors of TGFbeta/Nodal/Activin-related signaling. Smad3 mutant mice develop normally, whereas Smad2 plays an essential role in patterning the embryonic axis and specification of definitive endoderm. Alternative splicing of Smad2 exon 3 gives rise to two distinct protein isoforms. The short Smad2(Deltaexon3) isoform, unlike full-length Smad2, Smad2(FL), retains DNA-binding activity. Here, we show that Smad2(FL) and Smad2(Deltaexon3) are coexpressed throughout mouse development. Directed expression of either Smad2(Deltaexon3) or Smad3, but not Smad2(FL), restores the ability of Smad2-deficient embryonic stem (ES) cells to contribute descendants to the definitive endoderm in wild-type host embryos. Mice engineered to exclusively express Smad2(Deltaexon3) correctly specify the anterior-posterior axis and definitive endoderm, and are viable and fertile. Moreover, introducing a human Smad3 cDNA into the mouse Smad2 locus similarly rescues anterior-posterior patterning and definitive endoderm formation and results in adult viability. Collectively, these results demonstrate that the short Smad2(Deltaexon3) isoform or Smad3, but not full-length Smad2, activates all essential target genes downstream of TGFbeta-related ligands, including those regulated by Nodal.

Dissecting MHC class II export, B cell maturation, and DM stability defects in invariant chain mutant mice.

Invariant (Ii) chain loss causes defective class II export, B cell maturation, and reduced DM stability. In this study, we compare Ii chain and class II mutant mouse phenotypes to dissect these disturbances. The present results demonstrate that ER retention of alphabeta complexes, and not beta-chain aggregates, disrupts B cell development. In contrast, we fail to detect class II aggregates in Ii chain mutant thymi. Ii chain loss in NOD mice leads to defective class II export and formation of alphabeta aggregates, but in this background, downstream signals are misregulated and mature B cells develop normally. Finally, Ii chain mutant strains all display reduced levels of DM, but mice expressing either p31 or p41 alone, and class II single chain mutants, are indistinguishable from wild type. We conclude that Ii chain contributions as a DM chaperone are independent of its role during class II export. This Ii chain/DM partnership favors class II peptide loading via conventional pathway(s).

DM loss in k haplotype mice reveals isotype-specific chaperone requirements.

DM actions as a class II chaperone promote capture of diverse peptides inside the endocytic compartment(s). DM mutant cells studied to date express class II bound by class II-associated invariant chain-derived peptide (CLIP), a short proteolytic fragment of the invariant chain, and exhibit defective peptide-loading abilities. To evaluate DM functional contributions in k haplotype mice, we engineered a novel mutation at the DMa locus via embryonic stem cell technology. The present experiments demonstrate short-lived A(k)/CLIP complexes, decreased A(k) surface expression, and enhanced A(k) peptide binding activities. Thus, we conclude that DM loss in k haplotype mice creates a substantial pool of empty or loosely occupied A(k) conformers. On the other hand, the mutation hardly affects E(k) activities. The appearance of mature compact E(k) dimers, near normal surface expression, and efficient Ag presentation capabilities strengthen the evidence for isotype-specific DM requirements. In contrast to DM mutants described previously, partial occupancy by wild-type ligands is sufficient to eliminate antiself reactivity. Mass spectrometry profiles reveal A(k)/CLIP and a heterogeneous collection of relatively short peptides bound to E(k) molecules. These experiments demonstrate that DM has distinct roles depending on its specific class II partners.