胚胎干细胞定向分化的研究进展

胚胎干细胞已成为组织工程、发育生物学、药物开发及基因研究的热点。本文仅就胚胎干细胞定向分化的机制、研究进展和应用前景作一综述 ,旨在进一步推动胚胎干细胞的研究工作。     

Epigenetic Control of Embryonic Stem Cell Differentiation

Pluripotent embryonic stem cells can give rise to almost all somatic cell types but this characteristic requires precise control of their gene expression patterns. The necessity of keeping the entire genome “poised” to enter into any of a number of developmental possibilities requires a unique and highly plastic chromatin organisation based around specific patterns of histone modifications although this state of affairs is normally short lived during embryonic development. By deriving embryonic stem cells from the early embryo, we can preserve the highly specialised genome organisation and this has permitted several detailed investigations into the molecular basis of pluripotency.     

The Proliferation and Differentiation of Stem Cell Journals

As scientists position themselves in translating the therapeutic potential of stem cells from laboratory to clinical applications, publishing companies have taken this rapidly evolving field as a unique opportunity to launch new journals for dissemination of stem cell research. Over the last decade, the significant increase in the number of stem cell-based journals has created a conundrum. At stake is the pressure for these new journals to build their reputation by maintaining publication standards, while at the same time attracting a cadre of stem cell researchers to consider their journals as the publication of choice. We discuss here a prophetic path of survival for these journals which likely will closely mimic the core scientific and translational value of stem cells, namely their capacity to proliferate and differentiate into something meaningful!     

Targeting Pancreatic Progenitor Cells in Human Embryonic Stem Cell Differentiation for the Identification of Novel Cell Surface Markers

New sources of beta cells are needed in order to develop cell therapies for patients with diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of stem-cell derived progenitor cells that have a natural self-expansion capacity into insulin-producing cells. In order to learn more about these progenitor cells at different stages along the differentiation process in which they become progressively more committed to the final beta cell fate, we took the approach of identifying, isolating and characterizing stage specific progenitor cells. We generated human embryonic stem cell (HESC) clones harboring BAC GFP reporter constructs of SOX17, a definitive endoderm marker, and PDX1, a pancreatic marker, and identified subpopulations of GFP expressing cells. Using this approach, we isolated a highly enriched population of pancreatic progenitor cells from hESCs and examined their gene expression with an emphasis on the expression of stage-specific cell surface markers. We were able to identify novel molecules that are involved in the pancreatic differentiation process, as well as stage-specific cell markers that may serve to define (alone or in combination with other markers) a specific pancreatic progenitor cell. These findings may help in optimizing conditions for ultimately generating and isolating beta cells for transplantation therapy.     

The Practical Consequences of a National Human Embryonic Stem Cell Registry

The executive order and issuance of federal guidelines for human embryonic stem cell research are positive developments and will produce long-term benefits by creating a new registry for hESC lines. But there may be short-term costs caused by regulatory uncertainty, procedural delay, and knock-on effects as national policies are adopted at state and local jurisdictions. Policymakers must ensure that national mechanisms of oversight for a new hESC registry are adequately funded, properly organized, transparent, and free of bureaucratic detail.     

Developments and Challenges in Human Embryonic Stem Cell Research in Spain

After years of following the trail of others, Spain is finally making a serious bid in science, specifically in regenerative medicine. In the framework of the European Union, Spain is setting up the basis for a solid collaborative network between public and private institutions, involving basic, translational, applied, technological and clinical researchers. In a society characterised by the idiom “slow but secure”, it is still too soon to see the results of the huge economic and infrastructure investment made. We present here an overview of the challenges that have been surmounted and the ones that will have to be solved in order to situate Spain as a reference country in regenerative medicine worldwide.     

Effect of a Matrigel Sandwich on Endodermal Differentiation of Human Embryonic Stem Cells

Definitive endoderm can be derived from human embryonic stem cells using low serum medium with cytokines involved in the epithelial-to-mesenchymal transition, including Activin A and Wnt3A. The purpose of this study was to develop an improved protocol that permits the induction of definitive endoderm while avoiding the high rate of cell death that often occurs with existing protocols. By including insulin and other nutrients, we demonstrate that cell viability can be preserved throughout differentiation. In addition, modifying a matrigel sandwich method previously reported to induce precardiac mesoderm allows for enhanced endodermal differentiation based on expression of endoderm-associated genes. The morphological and migratory characteristics of cells cultured by the technique, as well as gene expression patterns, indicate that the protocol can emulate key events in gastrulation towards the induction of definitive endoderm.     

Optimized Protocol for Derivation of Human Embryonic Stem Cell Lines

For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure.     

New Approaches in the Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells toward Hepatocytes

Orthotropic liver transplantation is the only established treatment for end-stage liver diseases. Utilization of hepatocyte transplantation and bio-artificial liver devices as alternative therapeutic approaches requires an unlimited source of hepatocytes. Stem cells, especially embryonic stem cells, possessing the ability to produce functional hepatocytes for clinical applications and drug development, may provide the answer to this problem. New discoveries in the mechanisms of liver development and the emergence of induced pluripotent stem cells in 2006 have provided novel insights into hepatocyte differentiation and the use of stem cells for therapeutic applications. This review is aimed towards providing scientists and physicians with the latest advancements in this rapidly progressing field.     

Human Embryonic Stem Cell-extracts Inhibit the Differentiation and Function of Monocyte-derived Dendritic Cells

Embryonic stem cells (ESC) possess inherent properties of immune privilege with the capacity to evade allogeneic immune responses. Moreover, ESCs have been shown to prevent immune activation in response to third party antigen presenting cells in vitro and have the capacity to promote allograft survival in vivo. However, clinical use of human ESCs to treat immunological disorders may risk teratoma or ectopic tissue formation. Here, we show that cellular extracts from both human and mouse ESCs retain the immune modulatory properties of intact cells. ESC-extracts that contained 12–24 μg of total protein effectively prevented T cell proliferation in allogeneic mixed lymphocyte reactions (MLR), whereas control fibroblast extracts did not affect proliferation. Cellular mechanisms underlying hESC extract-mediated immune modulation involve the maturation of monocyte derived dendritic cells (mDC). hESC extract-treated mDCs had reduced surface expression of co-stimulatory and maturation markers CD80, HLA-DR and CD83 and secreted lower levels of IL12p40. Accordingly, hESC extract-treated DCs were found to be poor stimulators of purified allogeneic T cells compared to those DCs treated with vehicle or fibroblast extracts. Our results demonstrate that ESC extracts retain the immune modulatory properties of ESCs and for the first time demonstrates that ESC derived factors can inhibit human mDC maturation and function.     

Role of BMP Signaling in Pancreatic Progenitor Differentiation from Human Embryonic Stem Cells

Transplantation of pancreatic progenitors derived from human embryonic stem cells (hESCs) is a promising way to treat diabetes. Strategies to obtain the required cell mass would rely on the up scaling of current differentiation protocols, or the proliferation of committed progenitors. We aimed at finding conditions that maintain a proliferating pancreatic progenitor pool and we assessed the role of BMP4 signaling in this process. hESCs were differentiated into PDX1 positive pancreatic progenitor stage following our established protocol with few modifications, and then the progenitor cells were passaged in a defined proliferation medium (PM). During passage, the effect of BMP4 signaling on the differentiation and proliferation of pancreatic progenitors was examined by RT-PCR and immunofluorescence analysis. We found that PDX1 positive pancreatic progenitors proliferated and gained NKX6.1 expression in the PM, whereas they failed to express NKX6.1 if BMP signaling was inhibited with Noggin. In this latter condition, part of the progenitors rather generated pro-endocrine cells denoted by NGN3 and synaptophysin expression. On the contrary, addition of BMP4 to the PM promoted the early derivation of PDX1 and NKX6.1 coexpressing pancreatic progenitors. Our findings are in line with mouse pancreas development, and indicate that BMP4 signaling is required for the derivation and maintenance of hESC-derived PDX1+NKX6.1+ pancreatic progenitors. These results are instructive for guiding the development of an efficient pancreas differentiation protocol in view of diabetes cell replacement therapy.     

Ethics and Embryonic Stem Cell Research

Ethical controversy in stem cell research arises because current methods to produce embryonic stem cell lines require the destruction of living human embryos. For this reason, there is increasing interest in developing alternative, non-embryonic sources of pluripotent stem cells. This effort is especially important in the US due to the prevailing policy against federal funding of embryo-destructive research. Altered nuclear transfer (ANT) is one of several potential methods to develop alternative sources of pluripotent stem cells. This approach employs the technique of somatic cell nuclear transfer, but the somatic cell nucleus or egg cytoplasm (or both) are first altered before the somatic cell nucleus is transferred into the oocyte. This alteration precludes the coordinated organization and developmental potential that is necessary for the resulting biological entity to be an embryo, but it still allows the entity to generate pluripotent stem cells. Proof-of-principle for one variant of ANT has been established in mice by silencing the functional expression of the gene Cdx2 in the somatic cell nucleus prior to its transfer into an enucleated egg. From the resulting non-embryonic laboratory construct, fully functional pluripotent stem cells were procured. Other more recent studies have suggested the possibility of achieving the same results by preemptively silencing maternally derived Cdx2 messenger RNA in the egg before the act of nuclear transfer. The procedure would produce the equivalent of a tissue culture of pluripotent stem cells. In contrast to the use of embryos 'left over' from clinical in vitro fertilization, ANT could produce pluripotent stem cell lines with an unlimited range of specifically selected and controlled genotypes. Such flexibility would greatly facilitate the study of disease, drug development, and toxicology testing, and may allow the production of therapeutically useful pluripotent stem cells that are immune-compatible. If developed to the point of scientific reliability, ANT would be a valuable research tool for the study of other aspects of cell development and differentiation, including gene expression patterns, imprinting, and cell-cell signaling. ANT would also help to clarify definitions and boundaries that distinguish true organisms from 'biological artifacts' and, thereby, provide moral precedent to guide future progress in developmental biology.     

Lessons from the Embryonic Neural Stem Cell Niche for Neural Lineage Differentiation of Pluripotent Stem Cells

Pluripotent stem cells offer an abundant and malleable source for the generation of differentiated cells for transplantation as well as for in vitro screens. Patterning and differentiation protocols have been developed to generate neural progeny from human embryonic or induced pluripotent stem cells. However, continued refinement is required to enhance efficiency and to prevent the generation of unwanted cell types. We summarize and interpret insights gained from studies of embryonic neuroepithelium. A multitude of factors including soluble molecules, interactions with the extracellular matrix and neighboring cells cooperate to control neural stem cell self-renewal versus differentiation. Applying these findings and concepts to human stem cell systems in vitro may yield more appropriately patterned cell types for biomedical applications.