Embryoid Body Culture Research Articles

Directing Differentiation of Human Embryonic Stem Cells Toward Anterior Neural Ectoderm Using Small Molecules

Based on knowledge of early embryo development, where anterior neural ectoderm (ANE) development is regulated by native inhibitors of bone morphogenic protein (BMP) and Nodal/Activin signaling, most published protocols of human embryonic stem cell differentiation to ANE have demonstrated a crucial role for Smad signaling in neural induction. The drawbacks of such protocols include the use of an embryoid body culture step and use of polypeptide secreted factors that are both expensive and, when considering clinical applications, have significant challenges in terms of good manufacturing practices compliancy. The use of small molecules to direct differentiation of pluripotent stem cells toward a specified lineage represents a powerful approach to generate specific cell types for further understanding of biological function, for understanding disease processes, for use in drug discovery, and finally for use in regenerative medicine. We therefore aimed to find controlled and reproducible animal-component-free differentiation conditions that would use only small molecules. Here, we demonstrate that pluripotent stem cells can be reproducibly and efficiently differentiated to PAX6(+) (a marker of neuroectoderm) and OCT4(-) (a marker of pluripotent stem cells) cells with the use of potent small inhibitors of the BMP and Activin/Nodal pathways, and in animal-component-free conditions, replacing the frequently used Noggin and SB431542. We also show by transcript analysis, both at the population level and for the first time at the single-cell level, that differentiated cells express genes characteristic for the development of ANE, in particular for the development of the future forebrain.

β1-Adrenoceptor stimulation enhances the differentiation of mouse induced pluripotent stem cells into neural progenitor cells

The cyclic AMP/protein kinase A signaling pathway is thought to be involved in neural differentiation of mesenchymal stem cells. In the present study, we examined the involvement of β-adrenoceptor signaling on the differentiation of mouse induced pluripotent stem (iPS) cells into neural progenitor cells. Mouse iPS cells were cultured on ultra-low-attachment dishes to induce embryoid body (EB) formation. All-trans retinoic acid (ATRA, 1μM) and/or the β-adrenoceptor agonist l-isoproterenol (0.3 or 1μM) were added to the EB cultures for 4 days, then EBs were plated on gelatin-coated plates and cultured for 7 or 14 days. Subtype-specific antibody staining revealed that mouse iPS cells express β1-adrenoceptors predominantly. Although treatment with l-isoproterenol alone did not affect the expression of Nestin (a specific marker for neural progenitor cells), l-isoproterenol significantly enhanced ATRA-induced Nestin expression. Pretreatment of EBs with either atenolol (a selective β1-adrenoceptor antagonist) or H89 (a protein kinase A inhibitor) significantly inhibited the l-isoproterenol-enhancement of ATRA-induced Nestin expression. In addition, the l-isoproterenol treatment significantly enhanced ATRA-induced expression of NeuN (a neuron-specific nuclear protein). These findings suggest that β1-adrenoceptor stimulation enhances ATRA-induced neural differentiation of mouse iPS cells.

Cardiac Differentiation of Human Embryonic Stem Cells and their Assembly into Engineered Heart Muscle

The advent of pluripotent human embryonic stem cells has created the unique opportunity for the development of a wide variety of humanized cellular tools for basic research, as well as industrial and clinical applications. It has, however, become apparent that embryonic stem cell derivatives in classical monolayer or embryoid body culture do not resemble bona fide tissues, mainly because of their limited organotypic organization and maturation in these culture formats. This shortcoming may be addressed by tissue engineering technologies aiming at the provision of a "natural" growth environment to facilitate organotypic tissue assembly. In this unit, we provide two harmonized basic protocols for (1) cardiac differentiation of human embryonic stem cells under serum-free conditions and (2) the assembly of the stem cell-derived cardiomyocytes into engineered heart muscle. This protocol can be easily adapted to bioengineer heart muscle also from other stem cell-derived cardiomyocytes, including cardiomyocytes from human-induced pluripotent stem cells.

Abstract 541: Nogo-B Receptor Is Essential for Embryonic Stem Cell Differentiation to Endothelial Cell Lineage and Primitive Blood Vessel Formation

Nogo isoforms-A, -B, and -C, are members of the reticulon family of proteins. Nogo-B was previously identified as a protein that is highly expressed in caveolin-1 enriched microdomains and/or lipid rafts of endothelial cells (EC) and vascular smooth muscle cells. Mice deficient in Nogo-A/B show exaggerated neointimal proliferation, abnormal remodeling and a deficit in ischemia induced arteriogenesis and angiogenesis. Nogo-B receptor (NgBR) is a type I receptor with a single transmembrane domain, and was identified as a receptor specific for Nogo-B. Our previous work has shown that Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro and intersomitic vessel formation in zebrafish via Akt pathway. Here, we further demonstrated the role of NgBR in regulating the differentiation of murine embryonic stem cells (ESC) into endothelial cell lineage and primitive blood vessel formation in 2D or 3D embryoid body culture systems. Murine NgBR gene-targeting ESC was generated by either gene-trap or homologous recombination approaches, respectively. Gene-targeting of NgBR was confirmed by Southern blot and qPCR. Homozygous knockout of NgBR in ESC results in cell apoptosis. Heterozygous knockout of NgBR does not affect ESC cell survival, but decreases the endothelial cell lineage commitment as well as reduces the formation and branching of primitive blood vessels in 2D or 3D embryoid body culture systems. However, there is no effect on the differentiation of ESC to vascular smooth muscle cell lineage. Mechanistically, NgBR has two potential regulatory roles during embryonic vasculature development. NgBR knockdown not only impairs both Nogo-B and VEGF-stimulated endothelial cell migration by abolishing Akt phosphorylation, but also decreases endothelial cell lineage commitment by delaying BMP4 production during the period of mesoderm formation. These results suggest that NgBR may be one of important genes coordinating the vasculature development.

Differentiation of Human Embryonic Stem Cells to Cardiomyocytes on Microcarrier Cultures

We have developed an improved cardiomyocyte differentiation protocol where we stabilized embryoid bodies (EB) in serum- and insulin-free medium (bSFS) supplemented with p38 MAP kinase inhibitor (SB203580) by addition of 10 µm laminin-coated positively charged (protamine sulfate derivatized TSKgel Tresyl-5PW) microcarriers. This protocol achieved a maximum 3-fold cell expansion, differentiation efficiency of 20%, and an overall cardiomyocyte yield of 3 × 10⁵ CM/ml in static conditions. In comparison, EB cultures achieved 1.5-fold cell expansion, differentiation efficiency of 15%, and an overall cardiomyocyte yield of 1.1 × 10⁵ CM/ml. The scalability of this platform was demonstrated in suspended spinner cultures, producing a maximum of 2.14 × 10⁵ CM/ml in 50-ml cultures. This yield is two-fold higher than the control static EB-based platform (1.1 × 10⁵ CM/ml), and seven-fold higher than yields reported in literature, 3.1-9 × 10⁴ CM/ml. The robustness of this protocol was tested with HES-3 and H1 cell lines.

Induction of Differentiation into Endoderm, Insulin-Secreting Cells from Mouse Embryonic Stem Cells using Activin A and Retinoic Acid

Abstract: Embryonic stem (ES) cells have been known to differentiate into various progenitor cells. In this study, we investigated the differentiation capacity of mouse ES cells into pancreatic hormone-secreting cells, and insulin-secreting cells. ES cells were cultured in Dulbecco's modified Eagle medium (DMEM) after removal of leukemia inhibitory factor (LIF) for 3 days and then transferred in DMEM supplemented with retinoic acid or activin A. When the culture of embryoid bodies (EBs) derived from ES cells in DMEM added with activin A (2 × 10-9 M or 2 × 10-10 M) for 5 days was performed, endoderm marker genes, GATA4, Sox17 and Foxa2 were expressed in the EBs. However, at 6 days of culture, expression of Sox17 was not observed. When EBs were cultured with activin A (2 × 10-9 M) for 6 days, and followed by 6 days of culture with retinoic acid (10-6 M), expression of the pancreatic cell marker genes, GATA4 and Fxa2, were continued, and pancreatic islet genes, insulins 1 and 2, glucagons and somatostatin, w...

FRS2α-Mediated FGF Signals Suppress Premature Differentiation of Cardiac Stem Cells Through Regulating Autophagy Activity

Although the fibroblast growth factor (FGF) signaling axis plays important roles in heart development, the molecular mechanism by which the FGF regulates cardiogenesis is not fully understood. To investigate the mechanism by which FGF signaling regulates cardiac progenitor cell differentiation. Using mice with tissue-specific ablation of FGF receptors and FGF receptor substrate 2α (Frs2α) in heart progenitor cells, we demonstrate that disruption of FGF signaling leads to premature differentiation of cardiac progenitor cells in mice. Using embryoid body cultures of mouse embryonic stem cells, we reveal that FGF signaling promotes mesoderm differentiation in embryonic stem cells but inhibits cardiomyocyte differentiation of the mesoderm cells at later stages. Furthermore, we also report that inhibiting FRS2α-mediated signals increases autophagy and that activating autophagy promotes myocardial differentiation and vice versa. The results indicate that the FGF/FRS2α-mediated signals prevent premature differentiation of heart progenitor cells through suppressing autophagy. The findings provide the first evidence that autophagy plays a role in heart progenitor differentiation.

Abstract P015: Hypoxia Regulates Embryonic Cardiac Myocyte Proliferation and Differentiation

Mammalian cardiac myocytes (CM) proliferate readily during development but exit the cell cycle perinatally. The molecular mechanism for this is poorly understood could lead to new approaches for cardiac regeneration. We employed a novel bioinformatics approach to identify transcription factors that may control perinatal cell-cycle arrest and identified hypoxia inducible factor 1α (Hif1α) as a potential regulator of CM proliferation and differentiation. To test our hypothesis, e12.5d embryonic heart cells were cultured in atmospheric (17%) or hypoxic (3%) [O2] and then assessed for proliferation over 4 days. Total cell numbers were significantly increased (∼2 fold, P<0.005) in cells cultured in 3% vs. 17% [O2] after 4 days. Hypoxia increased numbers of cTn+ cells labeled with p-Histone-H3 indicating an increase in CM proliferation. Carboxyfluorescein succinimidyl ester staining indicated that the majority (70%) of cells cultured in 3% [O2] divided 2–3 times while the majority (73%) of cells in 17% [O2] divided 0–1 times after 3 days of culture. As expected, hypoxia stabilized the Hif1α protein and induced the expression of Vegf. Messenger RNA for the G1-S phase cyclin dependent kinase Ccnd2 was increased (>2 fold) in hypoxic vs. atmospheric cultured cells indicating that hypoxia my increase proliferation by regulating the expression of cell cycle machinery. Furthermore, early mesodermal transcription factors Brachyury and Mesp1 and the CM progenitor marker Nkx2.5 were induced (>5 fold) in hypoxic vs. atmospheric [O2] cultured cells suggesting that hypoxia positively regulates early mesodermal and CM progenitor cell differentiation. To validate this hypothesis, we differentiated mouse embryonic stem cells in atmospheric and hypoxic [O2] and observed similar increases in the expression of Brachyury, Mesp1 and Nkx2.5 in hypoxic differentiated cells. Transient culture (2 d) of embryoid bodies (EBs) in hypoxia increased the frequency of beating EBs and the % of cTn+ cells compared to cells differentiated in atmospheric O2. Taken together these data suggest that the hypoxic environment in which CMs develop may serve as a key signal that regulates mesodermal progenitor marker expression and CM progenitor cell differentiation and proliferation.

Abstract P023: HDAC Inhibition Promotes Cardiogenesis and the Survival of Embryonic Stem Cells Through Proteasome-Dependent Pathway

Objectives: Histone deacetylase (HDAC) inhibition plays a crucial role in mediating cardiogenesis and myocardial protection, whereas HDAC degradation has recently attracted attention in mediating the biological function of HDACs. However, it remains unknown whether HDAC inhibition modulates cardiogenesis and embryonic stem cell (ESC) survival through the proteasome pathway. Methods and Results: Using the well-established mouse CGR8 mouse ESC culture, we evaluated the impact of HDAC inhibition and proteasome pathway on the cell death, viability and apoptosis in ESCs in response to oxidant stress (100μ mol/L hydrogen peroxides). We demonstrated that HDAC inhibitors, both TSA (50 nmol/L) and sodium butyrate (200 μ mol/L) that causes the pronounced reduction of HDAC4 activity, decreased cell death and increased viability of ESCs. HDAC inhibition reduced the cleaved caspase 3, 6, 9, PARP and TUNE-positive ESCs, which were abrogated with MG132 (0.5 μ mol/L), a specific proteasome inhibitor. Furthermore, we employed in vitro “hanging drop” methods to carry out two weeks of embryoid bodies (EB) culture to assess the effect of HDAC inhibition and proteasome pathway on cardiogenesis. HDAC inhibition stimulates the growth of EB, which is associated with faster spontaneous rhythmic contraction. HDAC inhibition increased the up-regulation of GATA4, MEF2, NKX2.5, cardiac actin, and α-SMA mRNA and protein levels that were abrogated by MG132. Immunostaining analysis demonstrates that trichostatin A and sodium butyrate resulted in a significant increase in cardiac lineage commitments that were blocked by the proteasome inhibition. Notably, HDAC inhibitors led to noticeable HDAC4 degradation, which was effectively prevented by MG132. Luciferase assay demonstrates an activation of MEF2 cardiac transcriptional factor by HDAC inhibition, which was repressed by MG132, revealing that the degradation of HDAC4 allows the activation of MEF2. Conclusions: Taken together, our study is the first to demonstrate that HDAC inhibition through proteasome pathway forms a novel signaling to determine the cardiac lineage commitment and elicit the survival pathway, which is dependent on specific HDAC4 degradation and subsequent MEF2 activation of ESCs.

High density cultures of embryoid bodies enhanced cardiac differentiation of murine embryonic stem cells

Murine embryonic stem cell (mESC)-derived cardiomyocytes represent a promising source of cells for use in the development of models for studying early cardiac development as well as cell-based therapies in postnatal pathologies. Here, we report a highly efficient cardiac differentiation system in which high density embryoid body (EB) cultures leads to a marked increase of cardiomyocytes production from multiple mESC lines without the addition of any cardiogenic growth factors. Our results show that high density EB cultures significantly increase the yield of functional cardiomyocytes, which express typical cardiac markers, exhibit normal rhythmic Ca 2+ transients, and respond to both β-adrenergic and electric stimulations. During the differentiation period, the inhibition of bone morphogenetic protein (BMP) signaling significantly attenuates the increase of cardiac differentiation as well as the increased expression of cardiac-specific genes, NK2 transcription factor related 5 ( Nkx2. 5) and myosin light chain 2v ( Mlc2v) by high density EB cultures. Therefore, we believe that we offer a novel and efficient means of cardiomyocyte production for practical use of mESCs in cardiac regenerative medicine.

Zfp281 functions as a transcriptional repressor for pluripotency of mouse embryonic stem cells.

Embryonic stem cells (ESCs) derived from preimplantation blastocysts have unique self-renewal and multilineage differentiation properties that are controlled by key components of a core regulatory network including Oct4, Sox2, and Nanog. Understanding molecular underpinnings of these properties requires identification and characterization of additional factors that act in conjunction with these key factors in ESCs. We have previously identified Zfp281, a Krüppel-like zinc finger transcription factor, as an interaction partner of Nanog. We now present detailed functional analyses of Zfp281 using a genetically ablated null allele in mouse ESCs. Our data show that while Zfp281 is dispensable for establishment and maintenance of ESCs, it is required for their proper differentiation in vitro. We performed microarray profiling in combination with previously published datasets of Zfp281 global target gene occupancy and found that Zfp281 mainly functions as a repressor to restrict expression of many stem cell pluripotency genes. In particular, we demonstrated that deletion of Zfp281 resulted in upregulation of Nanog at both the transcript and protein levels with concomitant compromised differentiation of ESCs during embryoid body culture. Chromatin immunoprecipitation experiments demonstrated that Zfp281 is required for Nanog binding to its own promoter, suggesting that Nanog-associated repressive complex(es) involving Zfp281 may fine-tune Nanog expression for pluripotency of ESCs.

Rapid and Efficient Generation of Functional Motor Neurons From Human Pluripotent Stem Cells Using Gene Delivered Transcription Factor Codes

Stem cell-derived motor neurons (MNs) are increasingly utilized for modeling disease in vitro and for developing cellular replacement strategies for spinal cord injury and diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Human embryonic stem cell (hESC) differentiation into MNs, which involves retinoic acid (RA) and activation of the sonic hedgehog (SHH) pathway is inefficient and requires up to 60 days to develop MNs with electrophysiological properties. This prolonged differentiation process has hampered the use of hESCs, in particular for high-throughput screening. We evaluated the MN gene expression profile of RA/SHH-differentiated hESCs to identify rate-limiting factors involved in MN development. Based on this analysis, we developed an adenoviral gene delivery system encoding for MN inducing transcription factors: neurogenin 2 (Ngn2), islet-1 (Isl-1), and LIM/homeobox protein 3 (Lhx3). Strikingly, delivery of these factors induced functional MNs with mature electrophysiological properties, 11-days after gene delivery, with >60-70% efficiency from hESCs and human induced pluripotent stem cells (hiPSCs). This directed programming approach significantly reduces the time required to generate electrophysiologically-active MNs by approximately 30 days in comparison to conventional differentiation techniques. Our results further exemplify the potential to use transcriptional coding for rapid and efficient production of defined cell types from hESCs and hiPSCs.

Smad1 signaling restricts hematopoietic potential after promoting hemangioblast commitment

AbstractBone morphogenetic protein (BMP) signaling regulates embryonic hematopoiesis via receptor-mediated activation of downstream SMAD proteins, including SMAD1. In previous work, we showed that Smad1 expression is sufficient to enhance commitment of mesoderm to hemangioblast fate. We also found indirect evidence to support a subsequent repressive function for Smad1 in hematopoiesis. To test this hypothesis directly, we developed a novel system allowing temporal control of Smad1 levels by conditional knockdown in embryonic stem cell derivatives. Depletion of Smad1 in embryoid body cultures before hemangioblast commitment limits hematopoietic potential because of a block in mesoderm development. Conversely, when Smad1 is depleted in FlK1+ mesoderm, at a stage after hemangioblast commitment, the pool of hematopoietic progenitors is expanded. This involves enhanced expression levels for genes specific to hematopoiesis, including Gata1, Runx1 and Eklf, rather than factors required for earlier specification of the hemangioblast. The phenotype correlates with increased nuclear SMAD2 activity, indicating molecular cross-regulation between the BMP and TGF-β signaling pathways. Consistent with this mechanism, hematopoiesis was enhanced when Smad2 was directly expressed during this same developmental window. Therefore, this study reveals a temporally defined function for Smad1 in restricting the expansion of early hematopoietic progenitors.

Sustained embryoid body formation and culture in a non-laborious three dimensional culture system for human embryonic stem cells

Pluripotent human embryonic stem cell (hESC) lines are a promising model system in developmental and tissue regeneration research. Differentiation of hESCs towards the three germ layers and finally tissue specific cell types is often performed through the formation of embryoid bodies (EBs) in suspension or hanging droplet culture systems. However, these systems are inefficient regarding embryoid body (EB) formation, structural support to the EB and long term differentiation capacity. The present study investigates if agarose, as a semi solid matrix, can facilitate EB formation and support differentiation of hESC lines. The results showed that agarose culture is able to enhance EB formation efficiency with 10% and increase EB growth by 300%. The agarose culture system was able to maintain expression of the three germ layers over 8weeks of culture. All of the four hESC lines tested developed EBs in the agarose system although with a histological heterogeneity between cell lines as well as within cell lines. In conclusion, a 3-D agarose culture of spherical hESC colonies improves EB formation and growth in a cost effective, stable and non-laborious technique.

Mesenchymal differentiation propensity of a human embryonic stem cell line

To characterize basal differentiation tendencies of a human embryonic stem (hES) cell line, KCL-002. In vitro specification and differentiation of hES cells were carried out using embryoid body (EB) cultures and tests of pluripotency and in vivo differentiation were performed by teratoma assays in SCID mice. Real-time PCR, immunohistochemistry, flow cytometry and histological analyses were used to identify expression of genes and proteins associated with the ectodermal, endodermal and mesodermal germ layers. Undifferentiated KCL-002 cells expressed characteristic markers of pluripotent stem cells such as Nanog, Sox-2, Oct-4 and TRA 1-60. When differentiated in vitro as EB cultures, expression of pluripotency, endodermal and ectodermal markers decreased rapidly. In contrast, mesodermal and mesenchymal markers such as VEGFR-2, α-actin and vimentin increased during EB differentiation as shown by qPCR, immunostaining and flow cytometric analyses. Teratoma formation in SCID mice demonstrated the potential to form all germ layers in vivo with a greater proportion of the tumours containing mesenchymal derivatives. The data presented suggest that the KCL-002 hES cell line is pluripotent and harbours a bias in basal differentiation tendencies towards mesodermal and mesenchymal lineage cells. Characterizing innate differentiation propensities of hES cell lines is important for understanding heterogeneity between different cell lines and for further studies aimed at deriving specific lineages from hES cells.

In vitro enhanced differentiation of neural networks in ES gut-like organ from mouse ES cells by a 5-HT 4-receptor activation

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster, a “gut”, from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited various types of spontaneous movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (ICC) (c-kit, a transmembrane receptor that has tyrosine kinase activity, positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified, but enteric neural networks were not identified. In the present study, we succeeded in forming dense enteric neural networks by a 5-HT 4-receptor (SR4) agonist, mosapride citrate (1–10 μM) added only during EB formation. Addition of an SR4-antagonist, GR113808 (10 μM) abolished the SR4-agonist-induced formation of enteric neural networks. The SR4-agonist (1 μM) up-regulated the expression of mRNA of SR4 and the SR4-antagonist abolished this upregulation. 5-HT per se exerted similar effects to those of SR4-agonist, though less potent. These results suggest SR4-agonist differentiated enteric neural networks, mediated via activation of SR4 in the ES gut.

Influencing Hematopoietic Differentiation of Mouse Embryonic Stem Cells using Soluble Heparin and Heparan Sulfate Saccharides

Heparan sulfate proteoglycans (HSPG) encompass some of the most abundant macromolecules on the surface of almost every cell type. Heparan sulfate (HS) chains provide a key interaction surface for the binding of numerous proteins such as growth factors and morphogens, helping to define the ability of a cell to respond selectively to environmental cues. The specificity of HS-protein interactions are governed predominantly by the order and positioning of sulfate groups, with distinct cell types expressing unique sets of HS epitopes. Embryos deficient in HS-synthesis (Ext1(-/-)) exhibit pre-gastrulation lethality and lack recognizable organized mesoderm and extraembryonic tissues. Here we demonstrate that embryonic stem cells (ESCs) derived from Ext1(-/-) embryos are unable to differentiate into hematopoietic lineages, instead retaining ESC marker expression throughout embryoid body (EB) culture. However hematopoietic differentiation can be restored by the addition of soluble heparin. Consistent with specific size and composition requirements for HS:growth factor signaling, chains measuring at least 12 saccharides were required for partial rescue of hematopoiesis with longer chains (18 saccharides or more) required for complete rescue. Critically N- and 6-O-sulfate groups were essential for rescue. Heparin addition restored the activity of multiple signaling pathways including bone morphogenic protein (BMP) with activation of phospho-SMADs re-established by the addition of heparin. Heparin addition to wild-type cultures also altered the outcome of differentiation, promoting hematopoiesis at low concentrations, yet inhibiting blood formation at high concentrations. Thus altering the levels of HS and HS sulfation within differentiating ESC cultures provides an attractive and accessible mechanism for influencing cell fate.

Fetal Stromal Niches Enhance Human Embryonic Stem Cell–Derived Hematopoietic Differentiation and Globin Switch

Hematopoiesis during mammalian embryonic development has been perceived as a migratory phenomenon, from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), and subsequently, the fetal bone marrow. In this study, we investigated the effects of primary stromal cells from fetal hematopoietic niches and their conditioned media (CM), applied singly or in sequential orders, on induction of human embryonic stem cells, H1, H9, and H14 lines, to hematopoietic cells. Our results demonstrated that stromal support of FL, AGM + FL, and AGM + FL + fetal bone marrow significantly increased the proliferation of embryoid bodies (EB) at day 18 of hematopoietic induction in the presence of thrombopoietin, stem cell factor, and Flt-3 ligand. AGM + FL also increased hematopoietic colony-forming unit (CFU) formation. CM did not enhance EB proliferation but CM of FL and AGM + FL significantly increased the density of total CFU and early erythroid (burst-forming unit) progenitors. Increased commitment to the hematopoietic lineage was demonstrated by enhanced expressions of CD45, alpha-, beta-, and gamma-globins in CFU at day 32, compared with EB at day 18. CM of FL significantly increased these globin expressions, indicating enhanced switches from embryonic to fetal and adult erythropoiesis. Over 50% and 10% of cells derived from CFU expressed CD45 and beta-globin proteins, respectively. Expressions of hematopoietic regulatory genes (Bmi-1, β-Catenin, Hox B4, GATA-1) were increased in EB or CFU cultures supported by FL or sequential CM. Our study has provided a strategy for derivation of hematopoietic cells from embryonic stem cells under the influence of primary hematopoietic niches and CM, particularly the FL.

P38 Mitogen-Activated Protein Kinase Controls a Switch Between Cardiomyocyte and Neuronal Commitment of Murine Embryonic Stem Cells by Activating Myocyte Enhancer Factor 2C-Dependent Bone Morphogenetic Protein 2 Transcription

Many studies have shown that it is possible to use culture conditions to direct the differentiation of murine embryonic stem (ES) cells into a variety of cell types, including cardiomyocytes and neurons. However, the molecular mechanisms that control lineage commitment decisions by ES cells remain poorly understood. In this study, we investigated the role of the 3 major mitogen-activated protein kinases (MAPKs: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) in ES cell lineage commitment and showed that the p38 MAPK-specific inhibitor SB203580 blocks the spontaneous differentiation of ES cells into cardiomyocytes and instead induces the differentiation of these ES cells into neurons. Robust p38 MAPK activity between embryoid body culture days 3 and 4 is crucial for cardiomyogenesis of ES cells, and specific inhibition of p38 MAPK activity at this time results in ES cell differentiation into neurons rather than cardiomyocytes. At the molecular level, inhibition of p38 MAPK activity suppresses the expression of bmp-2 mRNA, whereas treatment of ES cells with bone morphogenetic protein 2 (BMP-2) inhibits the neurogenesis induced by SB203580. Further, luciferase reporter assays and chromatin immunoprecipitation experiments showed that BMP-2 expression in ES cells is regulated directly by the transcription factor myocyte enhancer factor 2C, a well-known substrate of p38 MAPK. Our findings reveal the molecular mechanism by which p38 MAPK activity in ES cells drives their commitment to differentiate preferentially into cardiomyocytes, and the conditions under which these same cells might develop into neurons.

Embryoid body culture of mouse embryonic stem cells using microwell and micropatterned chips

The proliferation and differentiation properties of embryoid bodies (EB) from mouse embryonic stem (ES) cells were compared under two microchip conditions: microwell chip and micropatterned chip. The microwell chip contained 270 microwells (diameter, 600 μm; depth, 600 μm) on a polymethylmethacrylate plate and was surface-modified with polyethylene glycol (PEG) to render it non-adhesive. The micropatterned chip contained 270 gelatin spots (diameter, 200 μm) as the cell adhesion area on a glass plate; the region lacking these spots was PEG-modified to render it non-adhesive. The ES cells spontaneously formed the EBs from cell aggregates in each microwell in the chip. In contrast, cells inoculated onto the patterned chip formed a monolayer on the gelatin spots and gradually proliferated to form EBs. The EBs in the patterned chip maintained the high cell growth rate and the expression of endoderm (TTR and AFP) and mesoderm (Nkx2.5, αMHC, Flk1, and PDGFRβ) markers was increased, and these cell properties were similar to the previous methods (hanging drop and round-bottomed 96-well plate cultures). In contrast, the proliferation of ES cells in the microwell chip was lower than in the patterned chip and previous methods, and the EB differentiation proceeded slowly and only formed a small amount of endoderm. These results indicate that the difference of EB generating process in the microchip cultures may affect to the proliferation and differentiation of ES cells, and the existence of microwell structure in the microchip downregulates the cell proliferation and the differentiated progress of ES cells.