A cell cycle-dependent protein serves as a template-specific translation initiation factor

Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) and requires both eukaryotic initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). We show here that the requirements for trans-acting factors differ between related picornavirus IRESs and can account for cell type-specific differences in IRES function. The neurovirulence of Theiler's murine encephalomyelitis virus (TMEV; GDVII strain) was completely attenuated by substituting its IRES by that of foot-and-mouth disease virus (FMDV). Reconstitution of initiation using fully fractionated translation components indicated that 48S complex formation on both IRESs requires eIF2, eIF3, eIF4A, eIF4B, eIF4F, and the pyrimidine tract-binding protein (PTB) but that the FMDV IRES additionally requires ITAF(45), also known as murine proliferation-associated protein (Mpp1), a proliferation-dependent protein that is not expressed in murine brain cells. ITAF(45) did not influence assembly of 48S complexes on the TMEV IRES. Specific binding sites for ITAF(45), PTB, and a complex of the eIF4G and eIF4A subunits of eIF4F were mapped onto the FMDV IRES, and the cooperative function of PTB and ITAF(45) in promoting stable binding of eIF4G/4A to the IRES was characterized by chemical and enzymatic footprinting. Our data indicate that PTB and ITAF(45) act as RNA chaperones that control the functional state of a particular IRES and that their cell-specific distribution may constitute a basis for cell-specific translational control of certain mRNAs.     

Basic fibroblast growth factor support of human embryonic stem cell self-renewal

Human embryonic stem (ES) cells have most commonly been cultured in the presence of basic fibroblast growth factor (FGF2) either on fibroblast feeder layers or in fibroblast-conditioned medium. It has recently been reported that elevated concentrations of FGF2 permit the culture of human ES cells in the absence of fibroblasts or fibroblast-conditioned medium. Herein we compare the ability of unconditioned medium (UM) supplemented with 4, 24, 40, 80, 100, and 250 ng/ml FGF2 to sustain low-density human ES cell cultures through multiple passages. In these stringent culture conditions, 4, 24, and 40 ng/ml FGF2 failed to sustain human ES cells through three passages, but 100 ng/ml sustained human ES cells with an effectiveness comparable to conditioned medium (CM). Two human ES cell lines (H1 and H9) were maintained for up to 164 population doublings (7 and 4 months) in UM supplemented with 100 ng/ml FGF2. After prolonged culture, the cells formed teratomas when injected into severe combined immunodeficient beige mice and expressed markers characteristic of undifferentiated human ES cells. We also demonstrate that FGF2 is degraded more rapidly in UM than in CM, partly explaining the need for higher concentrations of FGF2 in UM. These results further facilitate the large-scale, routine culture of human ES cells and suggest that fibroblasts and fibro-blast-conditioned medium sustain human ES cells in part by stabilizing FGF signaling above a critical threshold.     

Hydrogels as artificial matrices for human embryonic stem cell self-renewal

Human embryonic stem cells (hESCs) have the potential to differentiate into all cell types in the body and hold great promise for regenerative medicine; however, large-scale expansion of undifferentiated hESCs remains a major challenge. Self-renewal of hESCs requires culturing these cells on either mouse or human fibroblast cells (i.e., a feeder layer of cells), or on artificial extracellular matrices (ECMs) while supplementing the media with soluble growth factors. Here we report a completely synthetic ECM system composed of a semi-interpenetrating polymer network (sIPN), a polymer hydrogel, which was designed to allow the independent manipulation of cell adhesion ligand presentation and matrix stiffness. In the short term, hESCs that were cultured on the sIPN adhered to the surface, remained viable, maintained the morphology, and expressed the markers of undifferentiated hESCs. This was the first demonstration that a completely synthetic ECM can support short-term self-renewal of hESCs.     

Ethics and embryonic stem cell research

There are important ethical issues that can be raised about embryonic stem cell research. One large ethical issue that has unfortunately arisen recently has to do with the falsification of scientific results and how, if at all, the scientific community can protect itself from such a thing. Regarding all medical research, and indeed, regarding everything having to do with health care policy, there are important ethical issues about who bears the burdens and reaps the benefits in a health care system that is fundamentally unjust, inhumane and inadequate to the needs of the growing segment of our population that is economically disadvantaged. Who is going to own this technology? Who is going to reap the economic benefits from it? How are the medical benefits going to be distributed? Yet another ethical issue is about how properly to obtain informed consent from women who volunteer to donate eggs for embryonic stem cell research, since there are some risks, but no direct benefits, to the donor.     

Human embryonic stem cell stability

Human embryonic stem cells (hESCs) are derived from human preimplantation embryos, and exhibit the defining characteristics of immortality and pluripotency. Indeed, these cell populations can be maintained for several years in continuous culture, and undergo hundreds of population doublings (see refs. 1,2). hESCs are thus likely candidates for source of cells for cell replacement therapies. Although hESC lines appear stable in their expression of cytokine markers, expression of telomerase, ability to differentiate, and maintenance of a stable karyotype, several other aspects of stability have not yet been addressed, including mitochondrial sequencing, methylation patterns, and fine resolution cytogenetic analysis. Because of the potential utility of hESCs, it will be of utmost importance to evaluate the stability of these aspects of ESC biology.     

Step-wise divergence of primitive and definitive haematopoietic and endothelial cell lineages during embryonic stem cell differentiation

BACKGROUND: The developmental processes leading from the mesoderm to primitive and definitive haematopoietic and endothelial lineages, although of great importance, are still poorly defined. Recent studies have suggested a model in which common precursors give rise to endothelial progenitors and haematopoietic progenitors, the latter subsequently generating both primitive and definitive haematopoietic lineages. However, this model is contradicted by findings that suggest the emergence of haematopoietic cells from the endothelial lineage. RESULTS: We found sequential steps in the differentiation of FLK1+ mesoderm into haematopoietic and endothelial lineages in an in vitro differentiation system of embryonic stem (ES) cells: (i) the GATA-1+ subset of FLK1+ mesodermal cells loses the capacity to give rise to endothelial cells and is restricted to primitive erythroid, macrophage and definitive erythroid progenitors; (ii) the remaining GATA-1- cells give rise to VE-cadherin+ endothelial cells; and subsequently (iii) multiple definitive haematopoietic progenitors and endothelial cells branch off from a subset of VE-cadherin+ cells. CONCLUSIONS: These observations strongly suggest that the divergence of primitive and multilineage definitive haematopoietic/endothelial lineages occurs first, and then multilineage definitive haematopoietic progenitors arise from VE-cadherin+ endothelial cells in the development of haematopoietic and endothelial cells.     

Identification and characterization of hemoangiogenic progenitors during cynomolgus monkey embryonic stem cell differentiation

We identified intermediate-stage progenitor cells that have the potential to differentiate into hematopoietic and endothelial lineages from nonhuman primate embryonic stem (ES) cells. Sequential fluorescence-activated cell sorting and immunostaining analyses showed that when ES cells were cultured in an OP9 coculture system, both lineages developed after the emergence of two hemoangiogenic progenitor-bearing cell fractions, namely, vascular endothelial growth factor receptor (VEGFR)-2(high) CD34(-) and VEGFR-2(high) CD34(+) cells. Exogenous vascular endothelial growth factor increased the proportion of VEGFR-2(high) cells, particularly that of VEGFR-2(high) CD34(+) cells, in a dose-dependent manner. Although either population of VEGFR-2(high) cells could differentiate into primitive and definitive hematopoietic cells (HCs), as well as endothelial cells (ECs), the VEGFR-2(high) CD34(+) cells had greater hemoangiogenic potential. Both lineages developed from VEGFR-2(high) CD34(-)or VEGFR-2(high) CD34(+) precursor at the single-cell level, which strongly supports the existence of hemangioblasts in these cell fractions. Thus, this culture system allows differentiation into the HC and EC lineages to be defined by surface markers. These observations should facilitate further studies both on early developmental processes and on regeneration therapies in human.     

胚胎干细胞、成体干细胞的基础研究及应用

对干细胞的研究现状及进展进行了综述。讨论了胚胎干细胞和成体干细胞的定义及判别标准,干细胞的分离及生物学特性,干细胞生长分化及定向诱导分化的条件,成体干细胞可塑性定义、机制及判定标准,干细胞的应用及存在的问题。     

Derivation of a xeno-free human embryonic stem cell line

Elimination of all animal material during both the derivation and long-term culture of human embryonic stem cells (hESCs) is necessary prior to future application of hESCs in clinical cell therapy. The potential consequences of transplanting xeno-contaminated hESCs into patients, such as an increased risk of graft rejection [Stem Cells 2006; 24:221-229] and the potential transfer of nonhuman pathogens, make existing hESC lines unsuitable for clinical applications. To avoid xeno-contamination during derivation and culture of hESCs, we first developed a xeno-free medium supplemented with human serum, which supports long-term (>50 passages) culture of hESCs in an undifferentiated state. To enable derivation of new xeno-free hESCs, we also established xeno-free human foreskin fibroblast feeders and replaced immunosurgery, which involves the use of guinea pig complement, with a modified animal-product-free derivation procedure. Here, we report the establishment and characterization (>20 passages) of a xeno-free pluripotent diploid normal hESC line, SA611.     

Development of a European human embryonic stem cell registry

The number of human embryonic stem cell (hESC) lines that are available and that are subsequently being used in numerous research projects is increasing steadily. However, there is little coordination of hESC line derivation, and comparative information on the characteristics and quality of these cells is sparse. Obtaining consistent information on hESCs is hampered further by legislative fragmentation, particularly in Europe. Recognizing these obstacles, the European Commission has set up a Human Embryonic Stem Cell Registry (hESCreg) to make hESCs and their characterizing information accessible and to ensure that the results of research become more quickly available to the public. The primary objectives of hESCreg are to provide freely accessible information on existing hESC lines, their derivation, molecular characteristics, use and quality. Successful research with listed hESC lines will be used to evaluate clinical potential and thus directly influence policy decisions. The developing integration with other initiatives, such as characterization projects, registries and cell banks, is expected to lead to a common and internationally accepted central reference. The hESCreg provides a first step in this direction and might grow into an internationally funded and administered project.