Embryonic Stem Cells In Regenerative Medicine Research Articles

Advances and complications of regenerative medicine in diabetes therapy.

The rapid development of technologies in regenerative medicine indicates clearly that their common application is not a matter of if, but of when. However, the regeneration of beta-cells for diabetes patients remains a complex challenge due to the plurality of related problems. Indeed, the generation of beta-cells masses expressing marker genes is only a first step, with maintaining permanent insulin secretion, their protection from the immune system and avoiding pathological modifications in the genome being the necessary next developments. The prospects of regenerative medicine in diabetes therapy were promoted by the emergence of promising results with embryonic stem cells (ESCs). Their pluripotency and proliferation in an undifferentiated state during culture have ensured the success of ESCs in regenerative medicine. The discovery of induced pluripotent stem cells (iPSCs) derived from the patients’ own mesenchymal cells has provided further hope for diabetes treatment. Nonetheless, the use of stem cells has significant limitations related to the pluripotent stage, such as the risk of development of teratomas. Thus, the direct conversion of mature cells into beta-cells could address this issue. Recent studies have shown the possibility of such transdifferentiation and have set trends for regeneration medicine, directed at minimizing genome modifications and invasive procedures. In this review, we will discuss the published results of beta-cell regeneration and the advantages and disadvantages illustrated by these experiments.

Embryonic Stem Cells in Development and Regenerative Medicine.

After progressive improvement in embryonic stem (ES) cell field, several studies have been conducted to explore the usage of ES cells in regenerative medicine. Unlimited self renewal and pluripoteny properties, combined with encouraging preclinical trials, remark that ES cell technology might be promising for clinical practice. ES cells, which can form three germ layers in vitro, are potential candidates to study development at the cellular and molecular level. Understanding the cell fate decision and differentiation processes during development might enable generating functional progenitor cells for tissue restoration. Progression in gene modifications and tissue engineering technology has facilitated the derivation of desired cells for therapy. Success in differentiation protocols and identification the regulatory pathways simplify the research for clinical applications. Although there are established protocols for cell differentiation in vitro and promising preclinical studies in vivo, many challenges need to be adressed before clinical translation. In this review, ES cells are discussed as a model of development in vitro and as a potential candidate for regenerative medicine. This review also dissusses current challenges for ES cell based therapy.

Pluripotent stem cells – from physiology to cell therapy, disease models and test systems

Owing to their ability to reproduce the embryonic, fetal and neonatal differentiation of all different organotypic cellular phenotypes, pluripotent stem cells represent an ideal tool to study physiological processes of embryogenesis under in vitro conditions, as well as provide the basis of cellular therapeutics to develop novel disease models and to build up test assay systems for drug discovery and toxicology. In particular, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can reproduce all organotypic electrophysiology, signalling cascades and genes involved in development (functional genomics). This spontaneously occurs within three-dimensional cell aggregates – embryoid bodies – which were developed 25 years ago. To select only one lineage (e.g. the cardiac lineage) and to allow the identification of the transplanted cells, transgenic ES and iPS cells were used. They contained a vector with two cloning sites for enhanced green fluorescent protein and a puromycin resistance gene for selection under the α -MHC (myosin heavy chain) promoter. We aimed to generate iPS cell-derived cardiomyocytes and their molecular and functional characterization in comparison with cardiomyocytes derived from established ES cells on a transcriptomic and electrophysiological level. To demonstrate the ability of ES cells for regenerative medicine and tissue repair, cardiomyocytes differentiated from ES cells were injected into the cryoinfarcted left ventricular wall of adult wild-type mice. Translation from the laboratory into the clinic is one of the remaining key issues for applied stem cell research.

Stem Cell and Cloning Research in Farm Animals: A Special Focus on Buffalo

Embryonic stem (ES) cell lines constitute a special class of cell lines derived from the inner cell masses of preimplantation embryos. They are special owing to their unique capability of self renewal and differentiation and propagation in culture for indefinite periods of time. Unlike cancerous cells, they main t ain stable chromosomal content and have potential to diffe r entiate into any of the 200 cell type, under appropriate culture conditions. Embryonic stem cell research has gained a tremendous momentum owing to their promises and hopes in regenerative medicine, stem cell therapy, biopharming, and usefulness as in vitro models for studying embryonic develop m ent and differen t iation. ES cell research in farm animals provides strong incentives for precise genetic mani p ulation for better health, increased disease resistance, increased milk production and desired composition, increased growth rate with improved carcass composition, enhanced reproductive perfor m ance and prolificacy. The potential benefits of using ES cells as donor cells in somatic cell nuclear transfer would boost transgenic animal production. However, with all the promises and hopes, need for more rigorous research and evidence-based therapy is the need of the time, before we could successfully embark on exploitation of ES cells in regenerative medicine. In the current review, establishment, charac t erization, applications and future challenges of farm animal ES cells are discussed in detail with a special focus on bubaline ES cell lines.

Cardiac differentiation of embryonic stem cells by substrate immobilization of insulin-like growth factor binding protein 4 with elastin-like polypeptides

The establishment of cardiomyocyte differentiation of embryonic stem cells (ESCs) is a useful strategy for cardiovascular regenerative medicine. Here, we report a strategy for cardiomyocyte differentiation of ESCs using substrate immobilization of insulin-like growth factor binding protein 4 (IGFBP4) with elastin-like polypeptides. Recently, IGFBP4 was reported to promote cardiomyocyte differentiation of ESCs through inhibition of the Wnt/β-catenin signaling. However, high amounts of IGFBP4 (approximately 1 μg/mL) were required to inhibit the Wnt/β-catenin signaling and induce differentiation to cardiomyocytes. We report herein induction of cardiomyocyte differentiation using IGFBP4-immobilized substrates. IGFBP4-immobilized substrates were created by fusion with elastin-like polypeptides. IGFBP4 was stably immobilized to polystyrene dishes through fusion of elastin-like polypeptides. Cardiomyocyte differentiation of ESCs was effectively promoted by strong and continuous inhibition of Wnt/β-catenin signaling with IGFBP4-immobilized substrates. These results demonstrated that IGFBP4 could be immobilized using fusion of elastin-like polypeptides. Our results also demonstrate that substrate immobilization of IGFBP4 is a powerful tool for differentiation of ESCs into cardiomyocytes. These findings suggest that substrate immobilization of soluble factors is a useful technique for differentiation of ESCs in regenerative medicine and tissue engineering.

Induced pluripotent stem (iPS) cells offer a powerful new tool for the life sciences

Stem cell biology started with the analysis of somatic stem cells that function to maintain the adult body. We now know that the body is maintained by regeneration of a wide range of cell types, such as skin cells, blood cells and gastrointestinal mucous cells, from somatic stem cells. This regenerative activity is essential for survival. Regenerative medicine was initiated to identify therapies that support and/or accelerate this natural regenerative ability. For example, bone marrow transplantation is a therapy for reconstituting hematopoiesis from the hematopoietic stem cells present in the donor bone marrow. The successful development of a protocol for obtaining human embryonic stem (ES) cells prompted medical scientists to utilize human ES cells for regenerative medicine. However, use of these cells raises ethical issues as they are derived from human embryos. An alternative approach using ES-like pluripotent stem cells has the considerable advantage that it does not necessitate use of human embryos. Pluripotent stem cells can be induced from terminally differentiated somatic cells by the introduction of only four defined factors. The products of this method are termed "induced pluripotent stem (iPS)" cells. iPS cells have considerable promise as a substitute for ES cells not only for regenerative medicine but also in many other fields. For example, liver and heart cells derived from iPS cells can be used in pharmaceutical research. In addition, iPS cell technology opens new avenues of disease research, for example, by construction of so-called "disease-specific iPS cells" from a patient's somatic cells.

A Novel Signaling by Vitamin A/Retinol Promotes Self Renewal of Mouse Embryonic Stem Cells by Activating PI3K/Akt Signaling Pathway via Insulin-Like Growth Factor-1 Receptor

Pluripotent embryonic stem (ES) cells are a potential source of all types of cells for regenerative medicine. ES cells maintain pluripotency through a complex interplay of different signaling pathways and transcription factors, including leukemia inhibitory factor (LIF), Nanog, Sox2, and Oct3/4. Nanog, however, plays a key role in maintaining the pluripotency of mouse and human ES cells. Phosphoinositde 3-kinase (PI3K) signaling pathway which is activated in response to growth factors and cytokines also plays a critical role in promoting the survival and proliferation of ES cells. Our earlier studies revealed that retinol, the alcohol form of vitamin A, enhances the expression of Nanog and prevents differentiation of ES cells in long-term cultures. Normally vitamin A/retinol is associated with cell differentiation via its potent metabolite, retinoic acid. Thus far, no direct function has been ascribed to retinol itself. In this study, we demonstrate for the first time that retinol directly activates phosphoinositide three (PI3) kinase signaling pathway through IGF-1 receptor/insulin receptor substrate one (IRS-1) by engaging Akt/PKB-mTORC1 mammalian target of rapamycin-2 (mammalian target of rapamycin complex 2), indicating a growth factor-like function of vitamin A. Furthermore, ES cells do not express enzymes to metabolize retinol into retinoic acid and lack receptors for retinol transport into the cytoplasm, indicating that retinol signaling is independent of retinoic acid. This study presents a novel system to investigate how extracellular signals control the self renewal of ES cells which will be important for high-quality ES cells for regenerative medicine.

New method for forming large embryoid bodies using the wall of the culture dish along with an analysis of their structural characteristics.

Early embryonic stem (EES) cells, which were established from 2 cell stage embryos obtained from ddY mice, had similar characteristics as embryonic stem (ES) cells. These cells were maintained in an undifferentiated stage in growth media supplemented with leukemia inhibitory factor (LIF) and were capable of differentiating into triploblastic tissues under various growth factors. It has been known that normal sized embryoid bodies (EBs) are formed by removing LIF. In this study, large EBs gradually formed along the side wall of a culture dish, particularly at the boundary between the air and the growth medium when cells were cultured for a considerable period of time and without subculturing. We call this method the "wall adhesion culture" procedure. The method itself is simple and do not need any instruments except plastic dishes because only the side walls of the dishes were utilized. The mean thickness of the large EBs was about 1.5 mm 3 months after establishing the static culture. Their surface was covered with a monolayer of cells and they contained an eosinophilic cell matrix. By electron microscopy, some characteristic structures was observed, such as intracisternal A particles which were present inside the swelling of the rough endoplasmic reticulum. Since many tissues derived from ES cells are obtained through EBs, it is expected that efficient acquisition of sufficient quantities of these structures using the wall adhesion culture procedure will be a shortcut for using ES cells in regenerative medicine.

Engineered embryonic stem cells for regnerative nedicine

Due to their ability to reproduce the embryological differentiation of nearly all different cellular phenotypes, embryonic stem (ES) cells represent an ideal tool to study processes of embryogenesis under in vitro conditions, in particular the signalling cascades and genes involved in the functional development (functional genomics), as well as to provide a new source for cellular replacement therapy. We cultivated ES cells in three dimensional cell aggregates (embryoid bodies), where they differentiate into derivatives of all three germ layers. To select only one lineage, e.g. the cardiac lineage, and to allow the identification of the transplanted cells, transgenic ES cells were used. They contained a bicistronic vector with two cloning sites for EGFP and an antibiotics resistance for selective selection both under the α-MHC promoter. To demonstrate the ability of ES cells for regenerative medicine and tissue repair, cardiomyocytes differentiated from ES cells were injected into the cryoinfarcted left ventricular wall of adult wild type mice. Immunological cross reactions were avoided by using the same inbred mouse strain. EGFP positive transplanted cardiomyocytes could easily be detected in the native heart at different intervals after operation. The cells were found to engraft and to differentiate into adult-like cardiomyocytes as confirmed by cross striation after immunostaining with a-actinin. These data were corroborated by patch clamp experiments on isolated EGFP positive cardiomyocytes at different time points after operation. The transplanted cells displayed ventricular action potentials and β-adrenergicas well as muscarinic regulation. When survival was investigated in a large colony of mice, the control group had an almost double mortality rate as compared to the transplanted mice. Our data show engraftment and differentiation of embryonic cardiomyocytes after transplantation into cryoinfarcted areas of the heart. Before a clinical use of human ES cells for therapeutic trials in humans two major prerequisites must be fulfilled: (i) they must be safe, i.e. the development of tumours because of the high proliferative potential must be omitted and (ii) the rejection of the transplanted cells must be prevented. For the criterion (i) the technology of “lineage selection” appears optimal, for the criterion (ii) reprogramming techniques are now tested. Although autologous bone marrow cells appear ideal to circumvent rejection, we were not able to demonstrate a transdifferentiate into cardiomyocytes. Hence emphasis is made on reprogrammed human ES cells. The complexity of questions to be answered makes it necessary to establish closer collaborations between various disciplines including cell-, molecular-, developmentalbiology, molecular genetics, biochemistry, immunology, as well as clinical disciplines with medical basic sciences, experimental surgery and bioengineering together with cell culture techniques and material sciences. Cell Research (2008) 18:s111. doi: 10.1038/cr.2008.201; published online 4 August 2008

Skin and corneal cellular therapy using embryonic stem cells: how far are we?

Adult stem cell therapy is efficiently applied to severe skin burn patients but with some important limitations. A major challenge remains with regards to the maintenance of the stem cell’s self-renewal and pluripotency ex vivo. Human embryonic stem (ES) cells that are derived from the inner cell mass of the developing blastocyst were successfully isolated for the first time in 1998. These cells can be passaged in culture as undifferentiated pluripotent colonies and may also enter lineage-specific differentiation under the appropriate signals. Thus, human ES cells are considered to be a promising source for future regenerative medicine. Herein, we review the most recent advances and achievements as well as important challenges and obstacles that must be resolved before using ES cells for regenerative medicine of cutaneous and corneal epithelium. Recently, ES cells have been successfully differentiated into pure progenitor cell populations of epidermal lineage but future attempts are still required for man...

Generation of Multipotential Mesendodermal Progenitors from Mouse Embryonic Stem Cells via Sustained Wnt Pathway Activation

Pluripotent embryonic stem cells (ESCs) are capable of differentiating into cell types belonging to all three germ layers within the body, which makes them an interesting and intense field of research. Inefficient specific differentiation and contamination with unwanted cell types are the major issues in the use of ESCs in regenerative medicine. Lineage-specific progenitors generated from ESCs could be utilized to circumvent the issue. We demonstrate here that sustained activation of the Wnt pathway (using Wnt3A or an inhibitor of glycogen synthase kinase 3beta) in multiple mouse and human ESCs results in meso/endoderm-specific differentiation. Using monolayer culture conditions, we have generated multipotential "mesendodermal progenitor clones" (MPC) from mouse ESCs by sustained Wnt pathway activation. MPCs express increased levels of meso/endodermal and mesendodermal markers and exhibit a stable phenotype in culture over a year. The MPCs have enhanced potential to differentiate along endothelial, cardiac, vascular smooth muscle, and skeletal lineages than undifferentiated ESCs. In conclusion, we demonstrate that the Wnt pathway activation can be utilized to generate lineage-specific progenitors from ESCs, which can be further differentiated into desired organ-specific cells.

Potential of embryonic stem cells

Embryonic stem (ES) cells are pluripotent cell lines established from undifferentiated embryonic cells characterized by nearly unlimited self-renewal and differentiation capacity. During differentiation in vitro, ES cells were found to be able to develop into specialized somatic cells types and to recapitulate processes of early embryonic development. These properties allow to use ES cells as model system for studying early embryonic development by gain- or loss-of-function approaches, or to investigate the effects of drugs and environmental factors on differentiation and cell function in embryotoxicity and pharmacology. Now, ES cells derived of human blastocysts may be used for the generation of somatic precursor or differentiated cells in cell and tissue therapy. The review presents data of mouse ES cell differentiation and gives an outlook on future perspectives and problems of using human ES cells in regenerative medicine.