Functional high-resolution time-course expression analysis of human embryonic stem cells undergoing cardiac induction

Role of Sonic hedgehog signaling and the expression of its components in human embryonic stem cells

The goal of "Recent Developments" is to provide a concise but comprehensive overview of new advances in cardiovascular research, which we hope will keep our readers abreast of recent scientific discoveries and facilitate discussion, interpretation, and integration of the findings.This will enable readers who are not experts in a particular field to grasp the significance and effect of work performed in other fields.It is our hope and expectation that these "Recent Development" articles will help readers to gain a broader awareness and a deeper understanding of the status of research across the vast landscape of cardiovascular research-The Editors.

Stem cells of either adult or embryonic origin (embryonic stem cells, ES cells) are an unlimited source of cells that could be used for cell transplantation approaches to treat cardiac infarction and to overcome the shortage of donor organs. Whereas adult stem cell transplantation has already reached the clinical state, the research with ES cells is still in its infancy which is related to the danger of teratoma formation within the cell recipient as well as due to potential problems arising from immune responses towards the transplanted cells. Although some improvement in cardiac function has been observed so far using adult stem cell transplantation, the so far achieved benefit is not sufficient to allow routine patient treatment. Furthermore, the transdifferentiation of adult stem cells into cardiac cells that was reported in several studies could not be reproduced by other groups, and independent researchers reported that transdifferentiation was indeed a misinterpretation resulting from fusion of stem cells with adjacent cardiac cells. In contrast to adult stem cells the cardiovascular differentiation potential of ES cells is undoubted, and due to the huge proliferation capacity of ES cells this stem cell species may represent the most promising stem cell population that may be applied in future clinical approaches. However, besides problems of immune rejection which could be overcome with conventional immunosuppressants these cells bear the danger of teratoma formation which is arising from transplantation of still undifferentiated cells into the host organ. Several approaches have been elaborated during recent years to cope with teratoma generation. The most straightforward procedures to remove undifferentiated ES cells from preparations to be transplanted into organs are: antibiotic selection procedures, fluorescence‐activated cell sorting, differential centrifugation procedures, and cytotoxic antibodies directed against the undifferentiated cell population. One of the latest developments is the derivation of so called “induced ES cells”. These cells are gained by reprogramming somatic cells through introduction of stemness genes like Oct4, Sox2, Klf4, and c‐Myc. Upon transfection with these genes somatic cells achieve an ES cell phenotype and can be differentiated into cells of all three germ layers. Using induced ES cells immunological problems arising from allogeneic stem cell transplantation could be overcome since these stem cells are produced from the patients′ somatic cells thus carrying his immunological features. Besides scientific progress with the “classical” adult and ES cells, research of recent years came up with resident cardiac stem cells that reside in stem cell niches within the adult heart. It has been suggested that resident stem cells may be activated in situations of cardiac injury and may represent an intrinsic mechanism of cardiac repair, thus overcoming the previously generally accepted concept of the heart as a post‐mitotic organ. Currently researchers are trying to propagate resident cardiac stem cells in cell culture to scale up cell numbers for cell transplantation approaches. The transplantation procedures of stem cells are assisted by rapidly evolving tissue engineering procedures which utilize biodegradable scaffolds to fix transplanted cells within the transplantation site. Recent research has shown that successful engraftment of transplanted stem cell tissues into the infarcted heart can only be achieved if not only cardiomyocytes are transplanted into the area of infarction but additionally endothelial cells, smooth muscle cells and fibroblast ‐ which in concert built up the vascularized microenvironment that is necessary for cardiac cell proliferation and cell survival.

Scientists at Duke University Medical Center have demonstrated they can grow human stem cells in the laboratory by blocking an enzyme that naturally triggers stem cells to mature and differentiate into specialized cells. The discovery may enable scientists to rapidly grow stem cells and transplant them into patients with blood disorders, immune defects and select genetic diseases, said the Duke researchers.Stem cells are the most flexible cells in the body, continually dividing into new stem cells or into specialized cells that carry out specific roles in the body. But little is known about how stem cells choose their fate. The Duke team focused on "hematopoietic" or blood stem cells.In their study, the investigators discovered that an enzyme, aldehyde dehydrogenase (ALDH), stimulates hematopoietic stem cells to mature and transform into blood or immune cells, a process called differentiation. They inhibited this enzyme in stem cell cultures and successfully increased the number of stem cells by 3.4 fold. Moreover, they demonstrated the new stem cells were capable of fully rebuilding the blood-forming and immune systems of immune-deficient mice.Results of the study are published on line and will be published in the August 1, 2006, issue of the Proceedings of the National Academy of Sciences."Our ability to treat human diseases is limited by our knowledge of how human stem cells determine their fate -- that is, whether they maintain their ability to self-renew or whether they go on to become specialized cells," said John Chute, M.D., associate professor of medicine in the Duke Adult Bone Marrow and Stem Cell Transplant Program. "Unraveling the pathways that regulate self-renewal or differentiation in human stem cells can facilitate our ability to expand the growth of human stem cells for therapeutic uses."Currently, patients who require stem cell transplants are given either bone marrow from adult donors, umbilical cord blood derived from newborn babies, or stem cells from blood. But stem cells are scarce, representing less than 0.01 percent of the bone marrow cell population. Likewise, cord blood units frequently lack sufficient numbers of stem cells to rebuild a patient's decimated immune system.Efforts to grow human hematopoietic stem cells in the laboratory have proven extraordinarily difficult, Chute said, because growth factors in culture make stem cells rapidly differentiate. The scientists searched for ways to block a stem cell's natural propensity to differentiate without promoting uncontrolled growth. The researchers focused on the ALDH enzyme because it is a telltale "marker" that distinguishes stem cells from other blood and immune cells. Moreover, it is known to play an essential role in the body's production of retinoic acids, which regulate cell differentiation in a variety of tissues. Yet how ALDH functions in stem cells remained unknown, Chute said.The scientists began by analyzing how stem cells behave under normal circumstances when grown in culture. They mixed together purified human stem cells with growth factors that induce stem cells to mature and differentiate. As expected, the stem cells showed a marked decline in number as they differentiated into other types of specialized cells. By day seven, all stem cells had disappeared from culture.The scientists then added an inhibitor of ALDH to the stem cell cultures, and they found that half of the stem cells maintained their immature and undifferentiated status. Moreover, adding the inhibitor caused a 3.4-fold increase in stem cell numbers within seven days.Next, the scientists transplanted the cultured stem cells into immune-deficient mice to determine how the stem cells would behave. The new population of stem cells migrated to the bone marrow as expected and successfully "engrafted," or took hold in the bone marrow, where they began to produce new blood and immune cells. "ALDH appears to play a fundamental role in the differentiation program of human hematopoietic stem cells," Chute said. "Inhibition of this enzyme facilitates the expansion of human hematopoietic stem cells in culture." Chute said their results reveal a unique role for both ALDH and the process of retinoic acid signaling in human stem cells. Chute and colleague DonaldMcDonnell, Ph.D., professor of pharmacology and cancer biology, are currently testing whether they can directly block the retinoic acid receptors in stem cells and produce a comparable expansion of human stem cells.The investigators plan to develop a clinical trial to test their approach to expand human stem cells for therapeutic purposes.For more information contact: Becky Levine; Tel.: 919.660.1308 or 919.684.4148; Email: levin005@mc.duke.edu

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

### H11 Kinase Promotes or Inhibits Cardiac Cell Growth and Survival, Depending on its Subcellular Distribution Christophe Depre, Anna Zajac, Nadia Hedhli, Chull Hong, Jing Liu, Guiping Yang, Li Wang, Huacheng Dai, Univ. Medicine New Jersey, Newark, NJ; Thomas Wagner, Greenville Hosp System, Greenville, SC; Stephen F Vatner; Univ. Medicine New Jersey, Newark, NJ H11 kinase is a protein of unknown function mainly expressed in heart and skeletal muscle. Its expression is markedly increased in the ischemic heart together with an array of cytoprotective and anti-apoptotic genes, which supposes that H11 kinase promotes cell survival. Paradoxically, overexpression of H11 kinase in other cell types induces apoptosis. Therefore, the goal of the present study was to determine how H11 kinase affects cardiac cell survival and death, using both a cardiac-specific transgenic (TG) mouse model and cardiac cell culture. After 45 min coronary occlusion in vivo, infarct size in hearts from TG mice (7-fold overexpression) was reduced by 80% compared to wild type (P<0.01). This protection was related to a significant (P<0.05) increase in the expression of proteins promoting cell survival, including heat-shock proteins (HSP70, mmDJA4), phospho-Akt, the mTOR targets p70S6K and 4EBP-1, and the glucose transporter GLUT1. Surprisingly, although overexpression of H11 kinase is protective in the TG animal, ischemia reduced H11 kinase expression by 60% in wild type mice (P<0.01 versus sham). To reconcile this paradox, we performed a subcellular fractionation, which revealed that H11 kinase is expressed in both the cytosol and the nucleus. In wild type mice, the cytosolic expression of H11 kinase during ischemia was reduced by more than 80%, whereas the nuclear expression was unaffected. In the TG mouse, overexpression of H11 kinase accumulated as a nuclear protein, which therefore mediates the survival effect. To understand the role of the cytosolic form of the protein, we transfected isolated cardiac myocytes with increasing doses of adenovirus (3 MOI to 100 MOI) harboring the …

BackgroundPluripotent embryonic stem (ES) cells, which have the capacity to give rise to all tissue types in the body, show great promise as a versatile source of cells for regenerative therapy. However, the basic mechanisms of lineage specification of pluripotent stem cells are largely unknown, and generating sufficient quantities of desired cell types remains a formidable challenge. Small molecules, particularly those that modulate key developmental pathways like the bone morphogenetic protein (BMP) signaling cascade, hold promise as tools to study in vitro lineage specification and to direct differentiation of stem cells toward particular cell types.Methodology/ Principal FindingsWe describe the use of dorsomorphin, a selective small molecule inhibitor of BMP signaling, to induce myocardial differentiation in mouse ES cells. Cardiac induction is very robust, increasing the yield of spontaneously beating cardiomyocytes by at least 20 fold. Dorsomorphin, unlike the endogenous BMP antagonist Noggin, robustly induces cardiomyogenesis when treatment is limited to the initial 24-hours of ES cell differentiation. Quantitative-PCR analyses of differentiating ES cells indicate that pharmacological inhibition of BMP signaling during the early critical stage promotes the development of the cardiomyocyte lineage, but reduces the differentiation of endothelial, smooth muscle, and hematopoietic cells.Conclusions/ SignificanceAdministration of a selective small molecule BMP inhibitor during the initial stages of ES cell differentiation substantially promotes the differentiation of primitive pluripotent cells toward the cardiomyocytic lineage, apparently at the expense of other mesodermal lineages. Small molecule modulators of developmental pathways like dorsomorphin could become versatile pharmacological tools for stem cell research and regenerative medicine.

The successful culture and characterization of human embryonic stem cells (hESCs) in 1998 [1] made unlimited human cell manufacture a plausible goal, ushering in a new era of science and technology, and promising to change the face of medicine. More than a decade of research later, significant advancements have been made in our understanding of the factors that govern hESC pluripotency and lineage-specific differentiation. Additional investigations into the safety and efficacy of transplanted hESC progeny in animal models of neural injury and retinal degenerative disease have brought the field to the brink of clinical trials. However, no sooner did the scientific, medical and lay communities become familiar with hESCs than a new source of pluripotent stem cells (PSCs) appeared on the scene. In 2007, the Yamanaka and Thomson laboratories independently published methods whereby human somatic cells could be reprogrammed to a pluripotent state by misexpressing a surprisingly small number of genes [2,3]. The resulting human induced PSCs (hiPSCs) appeared to have all the desirable attributes of hESCs without the ethical issues surrounding the use of human embryos. Furthermore, hiPSCs could be derived from individual patients, making it possible to develop customized stem cell therapies and generate disease-specific stem cell lines. Now that a number of laboratories have had the opportunity to evaluate both sources of PSCs, it is reasonable to examine the early findings, and ask how hiPSCs and hESCs have compared with regard to their capacity for directed cell differentiation. While several cell types could be used as readouts for such a comparison, the retinal lineage possesses a number of features that qualifies it as a robust and reliable barometer to assess the efficiency of targeted hESC and hiPSC differentiation. The retina has long served as a model of neural development owing to its accessibility, relatively simple architecture and conserved developmental program, as well as the availability of specific markers for some of its cellular constituents, such as photoreceptors and retinal pigment epithelium (RPE). Owing in large part to these advantages, a tremendous amount of information is available regarding the mechanisms, sequence of events and time course of vertebrate retinogenesis. Such knowledge is useful when evaluating the ability of PSC lines to differentiate along the retinal lineage, and, more importantly, for intervening when the process veers from the desired course. Numerous groups, including our own, have demonstrated that differentiating hESCs mimic the stepwise development of retinal cells in vivo [4–6]. Furthermore, hESCs appear to respond to secreted morphogens in a manner predicted by studies of vertebrate neural induction and retinogenesis. In particular, blockade of bone morphogenetic protein and canonical Wnt signaling is known to be important for neural and retinal patterning, and many retinal differentiation protocols call for antagonists of one or both of these pathways to be included in the culture medium [4,6]. Differentiation protocols that do not add bone morphogenetic protein or Wnt inhibitors likely rely upon their endogenous expression and secretion in culture, which could, at least partially, account for the observed ‘default’ production of early anterior neural and retinal cells [5]. In fact, numerous hESC lines, including the widely used H1 (WA01) and H9 (WA09) WiCell lines, generate anterior neuroectodermal derivatives with little, if any, manipulation of culture conditions. For example, overgrown hESC cultures will undergo spontaneous differentiation and generate patches of pigmented RPE, often in large abundance [7].

a front page story in The New York Times ([22][1]) at the end of March 2001 reported that “the basis of a possible revolution in treating heart attack patients has been laid by three reports of using stem cells from bone marrow to repair heart tissue in animals.” Those three reports ([11][2], [

The Promise and Perils of Stem Cell Therapeutics

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kitpos) cells. The adult heart indeed contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kitpos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kitpos sorting. The blood/endothelial lineage-committed (Lineagepos) CD45posc-kitpos cardiac cells were compared to CD45neg(Lineageneg/Linneg) c-kitpos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kitpos cardiac cells are blood/endothelial lineage-committed CD45posCD31posc-kitpos cells. In contrast, the LinnegCD45negc-kitpos cardiac cell cohort, which represents ⩽10% of the total c-kitpos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kitneg and the blood/endothelial lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived clones, which represent only 1–2% of total c-kitpos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linnegc-kitpos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC’s myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kitpos cardiac cells were injected. Thus, among the cardiac c-kitpos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

Comparison of the differentiation potential of neural crest derived progenitor cells from apical papilla (dNC-PCs) and stem cells from exfoliated deciduous teeth (SHED) into mineralising cells

In recent years stem and progenitor cells have been identified in the heart, changing our understanding and therapeutic approach of heart disease. These cells can proliferate and differentiate in vitro into beating cardiomyocytes, endothelial and vascular smooth muscle cells, providing the potential for tissue regeneration and repair. microRNAs (miRs) have been identified as master switches controlling proliferation and differentiation, and in particular as key regulators of stem cells and cardiac development and function. Modulation of miR-regulated gene expression networks holds the potential to control cell fate and proliferation, with predictable biotechnological and therapeutic applications. We have characterized the expression profile of a subset of 100 miRs with reported functions in stem cell and tissue differentiation in mouse Sca1+ cardiac progenitor cells (CPCs). CPC miR expression profiles were compared with bone marrow mesenchymal stem cells (MSCs) and mouse embryonic heart (E=9) to obtain insights into the origins and function of this rare cell population. Within the miR panel that was studied, a subset with relative high expression levels in CPCs was identified, which may act as negative regulators of differentiation and proliferation. Although the overall expression profile of Sca1+ CPCs is closer to BM-MSCs, the most highly expressed miRs in CPCs are distinctive and predicted to target key genes involved in the control of cell proliferation and adhesion, vascular function and cardiomyocyte differentiation. Our results provide novel insights into the gene expression networks active in CPCs, which will now be explored in functional studies to identify key regulators of proliferation and differentiation.

Recently, various kinds of tissue specific stem cells, such as hematopoietic stem cells or neural stem cells, have been identified. It has been well known the embryoid body formed by aggregated embryonic stem (ES) cells differentiate into cardiac muscle cells, but cardiac progenitor/stem cells, which are supposed to exist during cardiac differentiation, have not been identified. Here, we intended to characterize cardiac progenitor/stem cells from ES cells using FACS sorting system. We considered that flk 1 might be a good marker for isolating cardiac progenitor/stem cells. Because flk 1 is one of the early markers of the anterior lateral mesoderm where vasculogenesis and also cardiomyogenesis originate from paired regions of the cardiac crescent. Differentiation of ES cells was induced in suspension culture in medium without leukemia inhibitory factor (LIF) . At 3.75 day from induction, the cells were stained with anti-flk 1 antibody, and cells with flk 1+ or fikl- were sorted respectively. Each subpopulation was co-cultured on OP 9 stromal cell. The differentiation of cardiac muscle cells was examined at day 10. Simultaneously, the gene expression analysis for Nkx-2.5 was carried out using RT-PCR.No colonies of contracting cardiac muscle cells were identified on tissue culture plate or type 4-collagen corted plates. Contracting muscle cells were identified only after plating on OP 9 cells, although no colonies on NIH 3 T 3 cells. These results suggest that OP 9 cells supply a signal indispensable for cardiac cell differentiation. However, there were no significant difference in numbers of contracting colonies and expression of Nkx 2.5 between flk 1 + and flk 1- cells. In this sense, the significance of flk 1 for cardiac cell differentiation still requires further discussion. Further examination remains to be done about signaling molecules for cardiac muscle cell differentiation.