A role for polyamine regulators in ESC self-renewal

Embryonic stem cells (ESCs) depend on extensive regulatory networks to coordinate their self-renewal and differentiation. The polyamine pathway regulator AMD1 was recently implicated in ESC self-renewal and directed differentiation of ESCs to neural precursor cells (NPCs). The polyamines spermine and spermidine are essential for a wide range of biological processes, and their levels are tightly regulated. Here, we review the polyamine pathway and discuss how it can impact polyamine levels, cellular methylation and hypusinated EIF5A levels. We discuss how it could feed into regulation of ESC self-renewal and directed differentiation. We show that in addition to AMD1, a second rate-limiting enzyme in the polyamine pathway, ODC1, can also promote ESC self-renewal, and that both Amd1 and Odc1 can partially substitute for Myc during cellular reprogramming. We propose that both Amd1 and Odc1 are essential regulators of ESCs and function to ensure high polyamine levels to promote ESC self-renewal.     

Pancreatic exocrine enzyme-producing cell differentiation via embryoid bodies from human embryonic stem cells

Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all-trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.     

Contrasting and brain region-specific roles of neurogenin2 and mash1 in GABAergic neuron differentiation in vitro

We have cultivated highly uniform populations of neural precursor cells, which retain their region-specific identities, from various rat embryonic brain regions. The roles of the proneural basic-helix-loop-helix (bHLH) factors neurogenin2 (Ngn2) and Mash1 in gamma-aminobutyric acid (GABA) neuron differentiation were explored in the region-specific cultures. Consistent with previous in vivo studies, forced expression of Mash1 promoted GABA neuron formation from the precursors derived from the developing forebrains, whereas Ngn2 displayed an inhibitory role in forebrain GABA neuron differentiation. Functional analyses of mutant bHLH proteins indicated that the helix-loop-helix domains of Mash1 and Ngn2, known as the structures for protein-protein interactions, impart the distinct activities. Intriguingly, the regulatory activities of Mash1 and Ngn2 in GABA neuron differentiation from the hindbrain- and spinal cord-derived precursor cells were completely opposite of those observed in the forebrain-derived cultures: increased GABA neuron yield by Ngn2 and decreased yield by Mash1 were shown in the precursors of those posterior brain regions. No clear difference that depended on dorsal-ventral brain regions was observed in the bHLH-mediated activities. Finally, we demonstrated that Otx2, the expression of which is developmentally confined to the regions anterior to the isthmus, is a factor responsible for the anterior-posterior region-dependent opposite effects of the bHLH proteins.     

Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.     

Neurogenin 2 is required for the development of ventral midbrain dopaminergic neurons

Proneural genes are crucial regulators of neurogenesis and subtype specification in many areas of the nervous system; however, their function in dopaminergic neuron development is unknown. We report that proneural genes have an intricate pattern of expression in the ventricular zone of the ventral midbrain, where mesencephalic dopaminergic neurons are generated. Neurogenin 2 (Ngn2) and Mash1 are expressed in the ventral midline, while Ngn1, Ngn2 and Mash1 are co-localized more laterally in the ventricular zone. Ngn2 is also expressed in an intermediate zone immediately adjacent to the ventricular zone at the ventral midline. To examine the function of these genes, we analyzed mutant mice in which one or two of these genes were deleted (Ngn1, Ngn2 and Mash1) or substituted (Mash1 in the Ngn2 locus). Our results demonstrate that Ngn2 is required for the differentiation of Sox2(+) ventricular zone progenitors into Nurr1(+) postmitotic dopaminergic neuron precursors in the intermediate zone, and that it is also likely to be required for their subsequent differentiation into tyrosine hydroxylase-positive dopaminergic neurons in the marginal zone. Although Mash1 normally has no detectable function in dopaminergic neuron development, it could partially rescue the generation of dopaminergic neuron precursors in the absence of Ngn2. These results demonstrate that Ngn2 is uniquely required for the development of midbrain dopaminergic neurons.     

Neural differentiation of human embryonic stem cells as an in vitro tool for the study of the expression patterns of the neuronal cytoskeleton during neurogenesis

The neural differentiation of human embryonic stem cells (ESCs) is a potential tool for elucidating the key mechanisms involved in human neurogenesis. Nestin and β-III-tubulin, which are cytoskeleton proteins, are marker proteins of neural stem cells (NSCs) and neurons, respectively. However, the expression patterns of nestin and β-III-tubulin in neural derivatives from human ESCs remain unclear. In this study, we found that neural progenitor cells (NPCs) derived from H9 cells express high levels of nestin and musashi-1. In contrast, β-III-tubulin was weakly expressed in a few NPCs. Moreover, in these cells, nestin formed filament networks, whereas β-III-tubulin was distributed randomly as small particles. As the differentiation proceeded, the nestin filament networks and the β-III-tubulin particles were found in both the cell soma and the cellular processes. Moreover, the colocalization of nestin and β-III-tubulin was found mainly in the cell processes and neurite-like structures and not in the cell soma. These results may aid our understanding of the expression patterns of nestin and β-III-tubulin during the neural differentiation of H9 cells.     

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.     

Activation of GSK3β by Sirt2 Is Required for Early Lineage Commitment of Mouse Embryonic Stem Cell

Sirt2, a member of the NAD⁺-dependent protein deacetylase family, is increasingly recognized as a critical regulator of the cell cycle, cellular necrosis and cytoskeleton organization. However, its role in embryonic stem cells (ESCs) remains unclear. Here we demonstrate that Sirt2 is up-regulated during RA (retinoic acid)-induced and embryoid body (EB) differentiation of mouse ESCs. Using lentivirus-mediated shRNA methods, we found that knockdown of Sirt2 compromises the differentiation of mouse ESCs into ectoderm while promoting mesoderm and endoderm differentiation. Knockdown of Sirt2 expression also leads to the activation of GSK3β through decreased phosphorylation of the serine at position 9 (Ser9) but not tyrosine at position 216 (Tyr216). Moreover, the constitutive activation of GSK3β during EB differentiation mimics the effect of Sirt2 knockdown, while down-regulation of GSK3β rescues the effect of Sirt2 knockdown on differentiation. In contrast to the effect on lineage differentiation, Sirt2 knockdown and GSK3β up-regulation do not change the self-renewal state of mouse ESCs. Overall, our report reveals a new function for Sirt2 in regulating the proper lineage commitment of mouse ESCs.     

DEPTOR Is a Stemness Factor That Regulates Pluripotency of Embryonic Stem Cells

The mammalian target of rapamycin (mTOR) pathway regulates stem cell regeneration and differentiation in response to growth factors, nutrients, cellular energetics, and various extrinsic stressors. Inhibition of mTOR activity has been shown to enhance the regenerative potential of pluripotent stem cells. DEPTOR is the only known endogenous inhibitor of all known cellular mTOR functions. We show that DEPTOR plays a key role in maintaining stem cell pluripotency by limiting mTOR activity in undifferentiated embryonic stem cells (ESCs). DEPTOR levels dramatically decrease with differentiation of mouse ESCs, and knockdown of DEPTOR is sufficient to promote ESC differentiation. A strong decrease in DEPTOR expression is also observed during human ESCs differentiation. Furthermore, reduction in DEPTOR level during differentiation is accompanied by a corresponding increase in mTOR complex 1 activity in mouse ESCs. Our data provide evidence that DEPTOR is a novel stemness factor that promotes pluripotency and self-renewal in ESCs by inhibiting mTOR signaling.     

Neurogenin 2 has an essential role in development of the dentate gyrus

The dentate gyrus (DG) of the hippocampus has a central role in learning and memory in adult rodents. The DG is generated soon after birth, although new neurons continue to be generated in the DG throughout life. The proneural factors Mash1 (Ascl1) and neurogenin 2 (Ngn2) are expressed during formation of the DG but their role in the development of this structure has not yet been addressed. Here, we show that Ngn2 is essential for the development of the DG. Ngn2 mutant mice have fewer DG progenitors and these cells present defects in neuronal differentiation. By contrast, the DG is normal in Mash1 mutant mice at birth, and loss of both Mash1 and Ngn2 does not aggravate the defect observed in Ngn2 single mutants. These data establish a unique role of Ngn2 in DG neurogenesis during development and raise the possibility that Ngn2 has a similar function in adult neurogenesis.     

An Engineered N-Cadherin Substrate for Differentiation,Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors

For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and β-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell—material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.     

Improving murine embryonic stem cell differentiation into cardiomyocytes with neuregulin-1: differential expression of microRNA

Identification of factors that direct embryonic stem (ES) cell (ESC) differentiation into functional cardiomyocytes is essential for successful use of ESC-based therapy for cardiac repair. Neuregulin-1 (NRG1) and microRNA play important roles in the cardiac differentiation of ESCs. Understanding how NRG1 regulates microRNA will provide new mechanistic insights into the role of NRG1 on ESCs. It may also lead to the discovery of novel microRNAs that are important for ESC cardiac differentiation. The objective of this study was to assess the microRNA expression profile during NRG1-induced ESC cardiac differentiation. Murine ESCs were incubated with a recombinant NRG1β or an inhibitor of ErbB2 or ErbB4 during hanging drop-induced cardiac differentiation. The expression of cardiac-specific markers and microRNAs was analyzed by RT-PCR and microRNA array, respectively. We found that the expression of NRG1 and the ErbB receptors was increased during hanging drop-induced cardiac differentiation of ESCs. NRG1 stimulation during a specific developmental window enhanced, while inhibition of the ErbB2 or ErbB4 receptor inhibited, cardiac differentiation of ESCs. NRG1 increased the expression of mmu-miR-296-3p and mmu-miR-200c*, and decreased mmu-miR-465b-5p. Inhibition of mmu-miR-296-3p or mmu-miR-200c* decreased, while inhibition of mmu-miR-465-5p increased, the differentiation of ESCs into the cardiac lineage. This is the first report demonstrating that microRNAs are differentially regulated by NRG1-ErbB signaling during cardiac differentiation of ESCs. This study has also identified new microRNAs that are important for ESC cardiac differentiation.     

The Plasminogen Activation System Modulates Differently Adipogenesis and Myogenesis of Embryonic Stem Cells

Regulation of the extracellular matrix (ECM) plays an important functional role either in physiological or pathological conditions. The plasminogen activation (PA) system, comprising the uPA and tPA proteases and their inhibitor PAI-1, is one of the main suppliers of extracellular proteolytic activity contributing to tissue remodeling. Although its function in development is well documented, its precise role in mouse embryonic stem cell (ESC) differentiation in vitro is unknown. We found that the PA system components are expressed at very low levels in undifferentiated ESCs and that upon differentiation uPA activity is detected mainly transiently, whereas tPA activity and PAI-1 protein are maximum in well differentiated cells. Adipocyte formation by ESCs is inhibited by amiloride treatment, a specific uPA inhibitor. Likewise, ESCs expressing ectopic PAI-1 under the control of an inducible expression system display reduced adipogenic capacities after induction of the gene. Furthermore, the adipogenic differentiation capacities of PAI-1^−/− induced pluripotent stem cells (iPSCs) are augmented as compared to wt iPSCs. Our results demonstrate that the control of ESC adipogenesis by the PA system correspond to different successive steps from undifferentiated to well differentiated ESCs. Similarly, skeletal myogenesis is decreased by uPA inhibition or PAI-1 overexpression during the terminal step of differentiation. However, interfering with uPA during days 0 to 3 of the differentiation process augments ESC myotube formation. Neither neurogenesis, cardiomyogenesis, endothelial cell nor smooth muscle formation are affected by amiloride or PAI-1 induction. Our results show that the PA system is capable to specifically modulate adipogenesis and skeletal myogenesis of ESCs by successive different molecular mechanisms.     

Regulatory role of Sirt1 on the gene expression of fatty acid‐binding protein 3 in cultured porcine adipocytes

To investigate whether Sirt1 could modulate fatty acid‐binding protein 3 (FABP3), we treated porcine adipocytes either with the Sirt1 inhibitor nicotinamide (NAM), with the Sirt1 activator resveratrol (RES), or by knockdown of Sirt1 by Sirt1‐siRNA. NAM or knockdown with Sirt1‐siRNA significantly inhibited Sirt1 mRNA expression, while increasing FABP3 mRNA levels. RES or RES + Sirt1‐siRNA treatments further proved that Sirt1 negatively regulated FABP3 gene expression in adipocytes. We also found a similar Sirt1 regulation pattern for PPARγ to that of FABP3 in adipocytes. Furthermore, NAM/RES + PPARγ‐siRNA treatments showed that Sirt1 may regulate the FABP3 gene expression partly through the PPARγ‐mediated signals. In summary, Sirt1 regulates the expression of FABP3 gene in adipocytes, and PPARγ apparently plays an important role in this process. J. Cell. Biochem. 107: 984–991, 2009. © 2009 Wiley‐Liss, Inc.