Embryoid Body Differentiation Research Articles

Abstract 3334: Antiangiogenic activity of honokiol through inhibition of vascular vessel formation in the mouse embryonic stem cell-derived endothelial cells

Abstract Embryonic stem (ES) cells, which are characterized by pluripotency and self-renewal, have recently been highlighted in several aspects of drug discovery. In particular, the potential of ES cells to differentiate into specific-cell types make them an extremely useful tool in the evaluation of the biological activity of test compounds. In the present study, we employed mouse embryonic stem (mES) cell-derived embryoid bodies (EBs) to evaluate the antiangiogenic activity of natural compounds. Honokiol, a major neolignan derived from the bark of Magnolia obovata Thunberg (Magnoliaceae), was capable of inhibiting differentiation and vascular vessel formation in mES cell models. EBs were formed using hanging drop cultures. The induction of vascular formation was carried out on gelatin-coated plates in EGM-2 medium. Cell proliferation was measured by MTT assay. RT-PCR, immunocytochemistry, and Western blotting were used to measure differentiation of EBs into endothelial cells and vessel formation. The growth inhibition of honokiol in EB-derived endothelial cells was found that honokiol is more sensitive in the differentiated cells (on day 8) compared to the undifferentiated EB-derived endothelial cells (on day 1) with the IC50 values of 6.0 and 17.8 μM, respectively. Honokiol also dramatically decreased the expressions of an endothelial biomarker PECAM in the differentiated EB-derived endothelial cells (on day 11). Honokiol also suppressed the activation of p38 MAPK, Akt, ERK1/2 and SAPK/JNK in EB-derived endothelial cells. The antiangiogenic activity of honokiol is associated with the suppression of PECAM and the MAPK pathway in EB-derived endothelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3334. doi:1538-7445.AM2012-3334

The Notch Ligand Delta-Like 4 Regulates Multiple Stages of Early Hemato-Vascular Development

BackgroundIn mouse embryos, homozygous or heterozygous deletions of the gene encoding the Notch ligand Dll4 result in early embryonic death due to major defects in endothelial remodeling in the yolk sac and embryo. Considering the close developmental relationship between endothelial and hematopoietic cell lineages, which share a common mesoderm-derived precursor, the hemangioblast, and many key regulatory molecules, we investigated whether Dll4 is also involved in the regulation of early embryonic hematopoiesis.Methodology/Principal FindingsUsing Embryoid Bodies (EBs) derived from embryonic stem cells harboring hetero- or homozygous Dll4 deletions, we observed that EBs from both genotypes exhibit an abnormal endothelial remodeling in the vascular sprouts that arise late during EB differentiation, indicating that this in vitro system recapitulates the angiogenic phenotype of Dll4 mutant embryos. However, analysis of EB development at early time points revealed that the absence of Dll4 delays the emergence of mesoderm and severely reduces the number of blast-colony forming cells (BL-CFCs), the in vitro counterpart of the hemangioblast, and of endothelial cells. Analysis of colony forming units (CFU) in EBs and yolk sacs from Dll4+/− and Dll4−/− embryos, showed that primitive erythropoiesis is specifically affected by Dll4 insufficiency. In Dll4 mutant EBs, smooth muscle cells (SMCs) were seemingly unaffected and cardiomyocyte differentiation was increased, indicating that SMC specification is Dll4-independent while a normal dose of this Notch ligand is essential for the quantitative regulation of cardiomyogenesis.Conclusions/SignificanceThis study highlights a previously unnoticed role for Dll4 in the quantitative regulation of early hemato-vascular precursors, further indicating that it is also involved on the timely emergence of mesoderm in early embryogenesis.

Human Embryonic Stem Cells Differentiated to Lung Lineage-Specific Cells Ameliorate Pulmonary Fibrosis in a Xenograft Transplant Mouse Model

BackgroundOur aim was to differentiate human (h) embryonic stem (ES) cells into lung epithelial lineage-specific cells [i.e., alveolar epithelial type I (AEI) and type II (AEII) cells and Clara cells] as the first step in the development of cell-based strategies to repair lung injury in the bleomycin mouse model of idiopathic pulmonary fibrosis (IPF). A heterogeneous population of non-ciliated lung lineage-specific cells was derived by a novel method of embryoid body (EB) differentiation. This differentiated human cell population was used to modulate the profibrotic phenotype in transplanted animals.Methodology and Principal FindingsOmission or inclusion of one or more components in the differentiation medium skewed differentiation of H7 hES cells into varying proportions of AEI, AEII, and Clara cells. ICG-001, a small molecule inhibitor of Wnt/β-catenin/Creb-binding protein (CBP) transcription, changed marker expression of the differentiated ES cells from an AEII-like phenotype to a predominantly AEI-like phenotype. The differentiated cells were used in xenograft transplantation studies in bleomycin-treated Rag2γC−/− mice. Human cells were detected in lungs of the transplanted groups receiving differentiated ES cells treated with or without ICG-001. The increased lung collagen content found in bleomycin-treated mice receiving saline was significantly reduced by transplantation with the lung-lineage specific epithelial cells differentiated from ES cells. A significant increase in progenitor number was observed in the airways of bleomycin-treated mice after transplantation of differentiated hES cells.ConclusionsThis study indicates that ES cell-based therapy may be a powerful novel approach to ameliorate lung fibrosis.

Gravity-oriented microfluidic device for uniform and massive cell spheroid formation

We propose a simple method for forming massive and uniform three-dimensional (3-D) cell spheroids in a multi-level structured microfluidic device by gravitational force. The concept of orienting the device vertically has allowed spheroid formation, long-term perfusion, and retrieval of the cultured spheroids by user-friendly standard pipetting. We have successfully formed, perfused, and retrieved uniform, size-controllable, well-conditioned spheroids of human embryonic kidney 293 cells (HEK 293) in the gravity-oriented microfluidic device. We expect the proposed method will be a useful tool to study in-vitro 3-D cell models for the proliferation, differentiation, and metabolism of embryoid bodies or tumours.

High‐Throughput Screening Assay for Embryoid Body Differentiation of Human Embryonic Stem Cells

Serum-free human pluripotent stem cell media offer the potential to develop reproducible clinically applicable differentiation strategies and protocols. The vast array of possible growth factor and cytokine combinations for media formulations makes differentiation protocol optimization both labor and cost-intensive. This unit describes a 96-well plate, 4-color flow cytometry-based screening assay to optimize pluripotent stem cell differentiation protocols. We provide conditions both to differentiate human embryonic stem cells (hESCs) into the three primary germ layers, ectoderm, endoderm, and mesoderm, and to utilize flow cytometry to distinguish between them. This assay exhibits low inter-well variability and can be utilized to efficiently screen a variety of media formulations, reducing cost, incubator space, and labor. Protocols can be adapted to a variety of differentiation stages and lineages.

Stable hydrodynamic trapping of hydrogel beads for on-chip differentiation analysis of encapsulated stem cells

This paper suggests a microbead-trapping device to distribute hydrogel beads in an array that encapsulate stem cell aggregates. Only facile pipetting of micro-bead suspension into the chip enabled trapping of hydrogel beads in microwell array within a few minutes. External hydrodynamic flow to hold beads was unnecessary because crescent weir at microwell hold the bead firmly. Tight holding of bead allowed strong washing for on-chip fluorescent staining as well as convenient culture media change. This device was applied to on-chip embryoid body (EB) formation and differentiation of mouse embryonic carcinoma (EC) cells. It is able to provide a new tool for studying the effect of chemical cues on the differentiation of stem cells encapsulated in hydrogel beads. The mouse EC cells encapsulated in the beads were cultured in the microfluidic device for 10 days to observe the effect of retinoic acid (RA) on neuronal differentiation as a model case. These experimental results verified the feasibility of long-term culture and immunostaining assay of stem cell differentiation. This device is expected to be useful tool for observing proliferation or interactions of cells in 3D microenvironment.

Nodal mutant eXtraembryonic ENdoderm (XEN) stem cells upregulate markers for the anterior visceral endoderm and impact the timing of cardiac differentiation in mouse embryoid bodies

SummaryInteractions between the endoderm and mesoderm that mediate myocardial induction are difficult to study in vivo because of the small size of mammalian embryos at relevant stages. However, we and others have demonstrated that signals from endodermal cell lines can influence myocardial differentiation from both mouse and human embryoid bodies (EBs), and because of this, assays that utilize embryonic stem (ES) cells and endodermal cell lines provide excellent in vitro models to study early cardiac differentiation. Extraembryonic endoderm (XEN) stem cells have a particular advantage over other heart-inducing cell lines in that they can easily be derived from both wild type and mutant mouse blastocysts. Here we describe the first isolation of a Nodal mutant XEN stem cell line. Nodal−/− XEN cell lines were not isolated at expected Mendelian ratios, and those that were successfully established, showed an increase in markers for the anterior visceral endoderm (AVE). Since AVE represents the heart-inducing endoderm in the mouse, cardiac differentiation was compared in EBs treated with conditioned medium (CM) collected from wild type or Nodal−/− XEN cells. EBs treated with CM from Nodal−/− cells began beating earlier and showed early activation of myocardial genes, but this early cardiac differentiation did not cause an overall increase in cardiomyocyte yield. By comparison, CM from wild type XEN cells both delayed cardiac differentiation and caused a concomitant increase in overall cardiomyocyte formation. Detailed marker analysis suggested that early activation of cardiac differentiation by Nodal−/− XEN CM caused premature differentiation and subsequent depletion of cardiac progenitors.

Small Molecules Greatly Improve Conversion of Human-Induced Pluripotent Stem Cells to the Neuronal Lineage

Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening. Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB) differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinson's disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage.

Constitutive GFP-UTF1 Expression Interferes with ES and EC Cell Differentiation

Embryonic stem cells have the ability to self-renew and can differentiate into all cell types of the three embryonic germ lineages. The undifferentiated embryonic cell Transcription Factor 1 (UTF1) gene is highly expressed in ES cells and we previously reported that UTF1 is tightly associated with chromatin and is required for differentiation of pluripotent mouseembryonic stem (ES) and embryonic carcinoma (EC) cells. In this study, we generated ES and EC cell lines constitutively expressing GFP-UTF1 to further investigate its role in differentiation. ES and EC cells constitutively expressing GFP-UTF1were suppressed in their proliferation and were still dependent on LIF for self-renewal. Embryoid body (EB) differentiation of GFP-UTF1 overexpressing ES cells showed both normal differentiation as well as a delayed or incomplete differentiation of a subset of cells. GFP-UTF1 was persistently expressed in undifferentiated cells whereas GFP-UTF1 expression was not detected in differentiated cells. Where GFP-UTF1 expressing ES cells differentiated normally in response to DMSO, EC cell differentiation was completely blocked. When ES and EC cells expressing GFP-UTF1 were treated with RA, differentiation markers were induced and endogenous UTF1 and GFP-UTF1 protein levels decreased. However, GFP-UTF1 and UTF1 (in ES cells) mRNA was still detected indicating that degradation of (GFP-)UTF1 protein preceded down regulation of (GFP-) UTF1 mRNA, suggesting that RA induced UTF1 degradation. Summarizing these data indicate that similar to UTF1 depletion, overexpression of GFP-UTF1 interfered with ES and EC cells differentiation.

Translating Human Embryonic Stem Cells from 2-Dimensional to 3-Dimensional Cultures in a Defined Medium on Laminin- and Vitronectin-Coated Surfaces

While defining the environment for human embryonic stem cell (hESC) culture on 2-dimensional (2D) surfaces has made rapid progress, the industrial-scale implementation of this technology will benefit from translating this knowledge into a 3-dimensional (3D) system, thus enabling better control, automation, and volumetric scale-up in bioreactors. The current study describes a system with defined conditions that are capable of supporting the long-term 2D culture of hESCs and the transposing of these conditions to 3D microcarrier (MC) cultures. Vitronectin (VN) and laminin (LN) were chosen as matrices for the long-term propagation of hESCs in a defined culture medium (STEMPRO(®)) for conventional 2D culture. Adsorption of these proteins onto 2D tissue culture polystyrene (TCPS) indicated that surface density saturation of 510 and 850 ng/cm(2) for VN and LN, respectively, was attained above 20 μg/mL deposition solution concentration. Adsorption of these proteins onto spherical (97±10 μm), polystyrene MC followed a similar trend and coating surface densities of 450 and 650 ng/cm(2) for VN and LN, respectively, were used to support hESC propagation. The long-term expansion of hESCs was equally successful on TCPS and MC, with consistently high expression (>90%) of pluripotent markers (OCT-4, MAB-84, and TRA-1-60) over 20 passages and maintenance of karyotypic normality. The average fold increase in cell numbers on VN-coated MC per serial passage was 8.5±1.0, which was similar to LN-coated MC (8.5±0.9). Embryoid body differentiation assays and teratoma formation confirmed that hESCs retained the ability to differentiate into lineages of all 3 germ layers, thus demonstrating the first translation to a fully defined MC-based environment for the expansion of hESCs.

211 REPROGRAMMING OF PORCINE NEURAL PROGENITOR CELLS TO INDUCED PLURIPOTENT STEM CELL-LIKE CELLS

Human-induced pluripotent stem cells (iPSC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC would be highly useful for pre-clinical safety testing by autologous transplantation in a porcine biomedical model. However, a major impediment is that currently, continuous expression of reprogramming factors is required to maintain the pluripotent state of porcine iPSC. In the mouse, neural progenitor cells (NPC) have proved to be highly amenable to reprogramming due to their partly stem-like epigenetic state and expression of pluripotency-related genes such as SOX2. The objective of this study was to establish iPSC from porcine epiblast-derived NPC by use of a tetracycline-inducible Tet-ON approach. A total of 1.5 × 105 porcine NPC at passage 6 (Rasmussen et al. 2011) were transduced O/N with 0.5 mL of active virus containing the following porcine pluripotency genes: pOCT4 (pO); pOCT4 and pKLF4 (pOK); pOCT4 and pC-MYC (pOM); pOCT4, pC-MYC and pKLF4 (pOMK) or polycistronic pOCT4, pSOX2, pC-MYC and pKLF4 (pOSMK); all including 0.25 mL of transactivator (rtTA). After 3 days, the cells were trypsinized and passaged to MEF feeder cells and cultured in iPSC medium containing DMEM/F12, 20% KSR, 1% NEAA, 10 μM β-Me, 20 ng mL–1 human bFGF and 2 μg mL–1 doxycycline. On Day 8, tightly packed colonies of cells presenting an embryonic stem cell-like morphology were visible in the pOM, pOMK and pOSMK combinations. In contrast, colonies were not observed with the pO and pOK combination. On Day 14, several iPSC-like colonies were manually picked and subcultured on MEF feeder cells in iPSC medium. Two lines from the pOSMK combination were capable of prolonged clonal propagation while maintaining an ESC-like morphology. However, when doxycycline was removed from the culture medium, growth arrest and spontaneous differentiation occurred. The iPSC-like lines expressed OCT4, SOX2, C-MYC and KLF4, as evaluated by immunocytochemistry and expression of NANOG, SSEA-1 and SSEA-4 was also confirmed, demonstrating activation of endogenous pluripotency genes. The iPSC-like lines were capable of forming embryoid bodies (EB) without addition of doxycycline and in vitro differentiation of EB in medium containing DMEM and 15% FCS confirmed the presence of meso- (SMA) and endodermal (AFP) derivatives by immunocytochemistry. Furthermore, co-culture experiments with MS5 stromal cells in medium containing DMEM, 15% KSR and 150 ng mL–1 human Noggin resulted in differentiation into neuroectoderm (NESTIN and SOX2), as well as more mature neurons (TUJI and GFAP). The porcine iPSC-like lines could serve as an excellent platform for optimizing culture conditions, which may sustain the pluripotency network in the pig and could be applied for autologous stem cell transplantation in a porcine model for evaluation of safety and efficacy. The Danish Agency for Science, Technology and Innovation, the Danish National Advanced Technology Foundation as well as the EU projects, EU FP7 (PartnErS, PIAP-GA-2008-218205; PluriSys, HEALTH-2007-B-223485).

REPROGRAMMING OF PORCINE EPIBLAST-DERIVED NEURAL PROGENITOR CELLS TO PLURIPOTENCY

Human induced pluripotent stem cells (iPSC) and neural progenitor cells (NPC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC and NPC would be highly useful for pre-clinical safety testing by autologous transplantation in a porcine biomedical model. The objective of this study was to establish iPSC from porcine epiblast-derived NPC by use of a tetracycline-inducible Tet-ON approach. A total of 1.5 × 105 porcine NPC at passage 6 (Rasmussen et al. 2011) were transduced O/N with 0.5 ml active virus containing the following porcine pluripotency genes: pOCT4 (pO); pOCT4 and pKLF4 (pOK); pOCT4 and pC-MYC (pOM); pOCT4, pC-MYC, and pKLF4 (pOMK) or polycistronic pOCT4, pSOX2, pC-MYC, and pKLF4 (pOSMK); all including 0.25 ml transactivator (rtTA). After 3 days, the cells were trypsinized and passaged to MEF feeder cells and cultured in iPSC medium containing DMEM/F12, 20% KSR, 1% NEAA, 10 μM β-Me, 20 ng mL–1 human bFGF and 2 μg mL–1 doxycycline. On Day 8, tightly packed colonies of cells presenting an embryonic stem cell-like morphology were visible in the pOM, pOMK, and pOSMK combinations. In contrast, colonies were not observed with the pO and pOK combination. On Day 14, several iPSC-like colonies were manually picked and sub-cultured on MEF feeder cells in iPSC medium. Two lines from the pOSMK combination were capable of prolonged clonal propagation while maintaining an ESC-like morphology. However, when doxycycline was removed from the culture medium, growth arrest and spontaneous differentiation occurred. The iPSC-like lines expressed OCT4, SOX2, C-MYC, and KLF4, as evaluated by immunocytochemistry, and expression of NANOG, SSEA-1, and SSEA-4 was also confirmed, demonstrating activation of endogenous pluripotency genes. The iPSC-like lines were capable of forming embryoid bodies (EB) without addition of doxycycline and in vitro differentiation of EB in medium containing DMEM and 15% FCS confirmed the presence of meso- (SMA) and endodermal (AFP) derivatives by immunocytochemistry. Furthermore, co-culture experiments with MS5 stromal cells in medium containing DMEM, 15% KSR, and 150 ng mL–1 human Noggin resulted in differentiation into neuroectoderm (NESTIN and SOX2), as well as more mature neurons (TUJI and GFAP). The latter resulted in establishment of new NPC lines. The system can be used to study mechanisms involved in the early transition from pluripotency to multipotency in the pig and the reversal of the process caused by reprogramming. The Danish Agency for Science, Technology and Innovation, the Danish National Advanced Technology Foundation as well as the EU projects, EU FP7 Stem Cell Project “PartnErS” (218205; 204, 523) and EU FP7 Stem Cell Project “PluriSys” (223485).

INS GFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells

Aims/hypothesisWe aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS+) cells derived in vitro.MethodsHomologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS+ cells.ResultsDifferentiation of INS GFP/w hESCs using published protocols demonstrated that all GFP+ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP+ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS GFP/w hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP+ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP+ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS+ cells.Conclusions/interpretation INS GFP/w hESCs are a valuable tool for investigating the nature of early INS+ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS+ cells from differentiating hESCs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-011-2379-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Discrimination of primitive endoderm in embryoid bodies by Raman microspectroscopy

Embryoid bodies (EBs), derived from aggregated embryonic stem (ES) cells, are capable of differentiating into all three germ layers, including the endoderm, mesoderm, and ectoderm. The initial stage of EB differentiation is the formation of a primitive endoderm (PE) layer located at the periphery of the aggregate. Raman microspectroscopy was employed to segregate PE cells from undifferentiated ES cells. The Raman spectra of the PE cells of the periphery of EBs, formed upon the withdrawal of leukemia inhibitory factor (LIF), were compared with those of the undifferentiated ES cells of the core of cell aggregates, formed in the presence of LIF. It was noticed that the PE cells have high contents of proteins and low contents of nucleic acids, lipids, and carbohydrates compared with ES cells. Also, we established the presence of another population of PE cells located in the core of the EBs. In addition, we identified some specific Raman markers to distinguish PE cells from ES cells (e.g., I(1003)/I(937)). This is the first study to investigate the PE cells of live EBs and define some Raman markers to distinguish them from undifferentiated ES cells.

JAK/STAT Signal Transduction Pathway Activation During Hematopoietic Differentiation From Human Embryonic Stem Cells (hESC)

Abstract 2346The ability to produce hematopoietic cells from human embryonic stem cells (hESC) has been demonstrated, using different multistage culture systems with multiple growth factor combinations. However, very little is understood about the molecular mechanisms that regulate the differentiation from hESC to hematopoietic stem and progenitor cells and further to specific lineages of differentiated hematopoietic cells. Among many signaling pathways involved in stem and progenitor cell differentiation, the JAK/STAT pathways are known to play critical roles in hematopoietic stem cell maintenance and hematopoietic differentiation. STAT3 activation is known to be essential for maintenance of murine ESC, but not human ESC, but it appears not to play a major role in myeloid cell differentiation. Although different levels of JAK2 and STAT5 signaling are important for erythroid and megakaryocytic differentiation, JAK/STAT signaling is not thought to play a role in hESC maintenance or differentiation and is not known to be essential for early stages of differentiation to hematopoietic stem and progenitor cells (HSC/HPC). We have established a serum-free, feeder cell-free system for maintaining hESC (H1 and H9 cells) and for differentiating the hESC to embryoid bodies (EB), from which end-stage hematopoietic cells, notably megakaryocytes and platelets, are produced. We used a multi-stage culture system to produce megakaryocytes and platelets from EBs, including 2 days with vascular endothelial growth factor (VEGF) and bone morphogenic protein (BMP4), 2 more days with VEGF, BMP4, stem cell factor (SCF), Flt3 ligand (FL), and thrombopoietin (TPO), 10 days with VEGF, BMP4, TPO, SCF, FL, IL3, IL6, and IL11, and 2–6 weeks with TPO, SCF, FL, IL3, IL6, and IL11. We used serial western blots, immunofluorescence with confocal microscopy and systematically observed changes of JAK/STAT signal transduction molecule activation. We found a consistent, dynamic change of STAT5 protein phosphorylation during the hematopoietic differentiation process. Interestingly, although JAK2, STAT3 and STAT5 protein were present, and JAK2 and STAT3 were phosphorylated in hESC, there was no evidence of STAT5 phosphorylation/activation in the undifferentiated hESC. During the early phases of differentiation of hESC-derived EBs toward hematopoietic progenitors in the presence of hematopoiesis-related cytokines, STAT5 was phosphorylated and activated in CD34+ HSCs and in CD61+/CD235a (glycophorin A)+ or CD41+/CD235a+ early megakaryocytic/erythroid progenitor cells (MEP). Although there was no detectable change in total STAT5 protein expression levels through hematopoietic differentiation, there was a slowly progressive decrease in phosphorylated/activated STAT5 with further maturation to megakaryocytes that express CD42b+, platelet factor 4, and von Willebrand factor and form proplatelets and platelets. Thus, in spite of hESC containing abundant phosphorylated JAK2, which is a known activator of STAT5, there was no phosphorylation/activation of STAT5 in undifferentiated hESC or early EBs. However, STAT5 became phosphorylated/activated early in hematopoiesis and declined over the course of progressive differentiation along the megakaryocytic lineage. These findings imply that activated JAK2 does not drive the activation of STAT5 that is an early event in differentiation from EBs and mesoderm to HSC and HPC in vitro. Disclosures:No relevant conflicts of interest to declare.

Engineering Artificial Signaling Centers to Polarize Embryoid Body Differentiation

Embryonic stem (ES) cells differentiating as aggregates self-organize dependent on Wnt signaling that is initially localized to discrete sites in the aggregate. As differentiation proceeds, Wnt signaling expands to most of the aggregates, thus resulting in widespread differentiation of mesendodermal progenitors. This process resembles primitive streak formation, but the lack of organized positional information makes the differentiating aggregates develop in a disorganized fashion. Here, we report that exogenous, cellular signaling sources can control the site where differentiation initiates in ES cell aggregates. Fibroblasts engineered to express cadherins are assembled with ES cells to form composite aggregates where the fibroblasts are positioned as a discrete pole. When engineered to express secreted Wnt agonists or antagonists, this pole functions to localize signaling in a way that polarizes the differentiating aggregates. The use of cell adhesion molecules to control morphology of developing stem cell aggregates should be widely applicable in tissue engineering.

Directed Differentiation of Human Embryonic Stem Cells to Interrogate the Cardiac Gene Regulatory Network

The limited ability of the heart to regenerate has prompted development of new systems to produce cardiomyocytes for therapeutics. While differentiation of human embryonic stem cells (hESCs) into cardiomyocytes has been well documented, the process remains inefficient and/or expensive, and progress would be facilitated by better understanding the early genetic events that cause cardiac specification. By maintaining a transgenic cardiac-specific MYH6-monomeric red fluorescent protein (mRFP) reporter hESC line in conditions that promote pluripotency, we tested the ability of combinations of 15 genes to induce cardiac specification. Screening identified GATA4 plus TBX5 as the minimum requirement to activate the cardiac gene regulatory network and produce mRFP(+) cells, while a combination of GATA4, TBX5, NKX2.5, and BAF60c (GTNB) was necessary to generate beating cardiomyocytes positive for cTnI and α-actinin. Including the chemotherapeutic agent, Ara-C, from day 10 of induced differentiation enriched for cTnI/α-actinin double positive cells to 45%. Transient expression of GTNB for 5-7 days was necessary to activate the cardiogenesis through progenitor intermediates in a manner consistent with normal heart development. This system provides a route to test the effect of different factors on human cardiac differentiation and will be useful in understanding the network failures that underlie disease phenotypes.

Notch1 signaling regulates chondrogenic lineage determination through Sox9 activation

Notch signaling is involved in several cell lineage determination processes during embryonic development. Recently, we have shown that Sox9 is most likely a primary target gene of Notch1 signaling in embryonic stem cells (ESCs). By using our in vitro differentiation protocol for chondrogenesis from ESCs through embryoid bodies (EBs) together with our tamoxifen-inducible system to activate Notch1, we analyzed the function of Notch signaling and its induction of Sox9 during EB differentiation towards the chondrogenic lineage. Temporary activation of Notch1 during early stages of EB, when lineage determination occurs, was accompanied by rapid and transient Sox9 upregulation and resulted in induction of chondrogenic differentiation during later stages of EB cultivation. Using siRNA targeting Sox9, we knocked down and adjusted this early Notch1-induced Sox9 expression peak to non-induced levels, which led to reversion of Notch1-induced chondrogenic differentiation. In contrast, continuous Notch1 activation during EB cultivation resulted in complete inhibition of chondrogenic differentiation. Furthermore, a reduction and delay of cardiac differentiation observed in EBs after early Notch1 activation was not reversed by siRNA-mediated Sox9 knockdown. Our data indicate that Notch1 signaling has an important role during early stages of chondrogenic lineage determination by regulation of Sox9 expression.

Induction of LYVE-1/stabilin-2-positive liver sinusoidal endothelial-like cells from embryoid bodies by modulation of adrenomedullin-RAMP2 signaling

Embryonic stem cells (ESCs) are a useful source for various cell lineages. So far, however, progress toward reconstitution of mature liver morphology and function has been limited. We have shown that knockout mice deficient in adrenomedullin (AM), a multifunctional endogenous peptide, or its receptor-activity modifying protein (RAMP2) die in utero due to poor vascular development and hemorrhage within the liver. In this study, using embryoid bodies (EBs)-culture system, we successfully induced liver sinusoidal endothelial-like cells by modulation of AM–RAMP2. In an EB differentiation system, we found that co-administration of AM and SB431542, an inhibitor of transforming growth factor β (TGFβ) receptor type 1, markedly enhanced differentiation of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)/stabilin-2-positive endothelial cells. These cells showed robust endocytosis of acetylated low-density lipoprotein (Ac-LDL) and upregulated expression of liver sinusoidal endothelial cells (LSECs)-specific markers, including factor 8 (F8), Fc-γ receptor 2b (Fcgr2b), and mannose receptor C type 1 (Mrc1), and also possessed fenestrae-like structure, a key morphological feature of LSECs. In RAMP2-null liver, by contrast, LYVE-1 was downregulated in LSECs, and the sinusoidal structure was disrupted. Our findings highlight the importance of AM–RAMP2 signaling for development of LSECs.

Dynamic Behavior and Spontaneous Differentiation of Mouse Embryoid Bodies on Hydrogel Substrates of Different Surface Charge and Chemical Structures

Differentiation of embryoid bodies (EBs) into particular cell lineages has been extensively studied. There is an increasing interest in the effect of soft hydrogel scaffolds on the behavior of EBs, such as the initial adhesion, dynamic morphology change, and differentiation. In this study, without adding any other bioactive factors in the serum-containing medium, dynamic behaviors of mouse EBs loaded on the surface of hydrogels with different surface charge and chemical structures are investigated. EBs adhered quickly to negatively charged poly(sodium p-styrene sulfonate) (PNaSS) hydrogels, which facilitates EBs spreading, migration, and differentiation into three germ layers with high efficiency of cardiomyocytes differentiation, similar to that on gelatin coated polystyrene (PS) culture plate. While on neutral poly(acrylamide) (PAAm) hydrogels, EBs maintained the initial spherical morphology with high expression of pluripotency-related markers in the short culture periods, and then showed the significantly greater levels of selected endoderm markers after long-time culture. EBs cultured on negatively charged poly(2-acrylamido-2-methyl-propane sulfonic acid sodium salt) (PNaAMPS) gels demonstrated the analogous behaviors with that of neutral PAAm gels at early differentiation phase (day 4+1). Then, their adhesion, spreading and differentiation were quite similar to that on negatively charged PNaSS gels. The correlation between surface properties of hydrogels and EBs differentiation was discussed.