Embryonic Stem Cells

This report demonstrates that introduction of physiologically relevant miRNAs can enhance somatic cell reprogramming. The mouse embryonic stem (ES) cell specific microRNAs (miRNA) miR-291-3p, miR-294, and miR-295 enhanced the efficiency of Klf4, Oct4 and Sox2 induced pluripotency. These miRNAs did not further enhance reprogramming in the presence of cMyc. cMyc binds the promoter of these miRNAs, suggesting that they are downstream effectors of cMyc promoted pluripotency. However, unlike exogenous cMyc, these miRNAs induced a homogeneous population of reprogrammed colonies suggesting overlapping and independent functions of cMyc and the miRNAs.

Sox2 has yet to be determined. Sox2 can act synergistically with Oct3/4 in vitro to activate Oct-Sox enhancers, which regulate the expression of pluripotent stem cellspecific genes, including Nanog, Oct3/4 and Sox2 itself. These findings suggest that Sox2 is required by ES cells for its Oct-Sox enhancer activity. Using inducible Sox2-null mouse ES cells, we show that Sox2 is dispensable for the activation of these Oct-Sox enhancers. In contrast, we demonstrate that Sox2 is necessary for regulating multiple transcription factors that affect Oct3/4 expression and that the forced expression of Oct3/4 rescues the pluripotency of Sox2-null ES cells. These results indicate that the essential function of Sox2 is to stabilize ES cells in a pluripotent state by maintaining the requisite level of Oct3/4 expression.

Neurofibromatosis type I (Nf1) is a GTPase-activating protein (GAP) that inactivates the oncoprotein Ras and plays important roles in nervous system development and learning. Alternative exon 23a falls within the Nf1 GAP domain coding sequence and is tightly regulated in favor of skipping in neurons; however, its biological function is not fully understood. Here we generated mouse embryonic stem (ES) cells with a constitutive endogenous Nf1 exon 23a inclusion, termed Nf1 23aIN/23aIN cells, by mutating the splicing signals surrounding the exon to better match consensus sequences. We also made Nf1 23aΔ/23aΔ cells lacking the exon. Active Ras levels are high in wild-type (WT) and Nf1 23aIN/23aIN ES cells, where the Nf1 exon 23a inclusion level is high, and low in Nf1 23aΔ/23aΔ cells. Upon neuronal differentiation, active Ras levels are high in Nf1 23aIN/23aIN cells, where the exon inclusion level remains high, but Ras activation is low in the other two genotypes, where the exon is skip...

Introduction 5 Cell and Gene Therapy 9 Modification of splicing: Antisense oligonucleotides and small molecules 21 Tissue Engineering 24 Small trials, difficult statistics, difficult regulation and data reproducibility. 28 Health economics of regenerative medicine 31 Regulation of stem cell therapies and regenerative medicine 41 Ethics 43 Public engagement and trust 54 Conclusions 56

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allelespecific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

Development of the nervous system begins with neural induction, which is controlled by complex signaling networks functioning in concert with one another. Fine-tuning of the bone morphogenetic protein (BMP) pathway is essential for neural induction in the developing embryo. However, the molecular mechanisms by which cells integrate the signaling pathways that contribute to neural induction have remained unclear. We find that neural induction is dependent on the Ca(2+)-activated phosphatase calcineurin (CaN). Fibroblast growth factor (FGF)-regulated Ca(2+) entry activates CaN, which directly and specifically dephosphorylates BMP-regulated Smad1/5 proteins. Genetic and biochemical analyses revealed that CaN adjusts the strength and transcriptional output of BMP signaling and that a reduction of CaN activity leads to an increase of Smad1/5-regulated transcription. As a result, FGF-activated CaN signaling opposes BMP signaling during gastrulation, thereby promoting neural induction and ...

In mammals, the X chromosome is uniquely capable of complete inactivation. Research in the past two years has validated the long-held hypothesis that the 'X-inactivation center' (Xic) controls events of X inactivation and that its resident gene Xist is not only required but is at least partially responsible for the cis-restriction of X inactivation. Progress has also been made in identifying genes within the Xic. Although Xist remains the only known required element, evidence now suggests that a separate element for X counting must exist and that the Xic may be entirely contained within a 450 kb sequence. This small region may be sufficient for both initiation and establishment of X inactivation.

The Müllerian ducts give rise to the female reproductive tract, including the Fallopian tubes, uterus, cervix, and anterior vagina. In male embryos, the Müllerian ducts regress, preventing the formation of female organs. We introduced the bacterial lacZ gene, encoding b-galactosidase (b-gal), into the AMHR-II locus (Amhr2) by gene targeting in mouse embryonic stem (ES) cells to mark Müllerian duct differentiation and regression. We show that Amhr2-lacZ heterozygotes express b-gal activity in an Amhr2specific pattern. In the gonads, b-gal activity was detected in Sertoli cells of the testes from 2 weeks after birth, and fetal ovaries and granulosa cells of the adult ovary. b-gal activity was first detected in the rostral mesenchyme of the Müllerian ducts at 12.5 days post coitus (dpc) in both sexes but soon thereafter expression was found along the entire length of the Müllerian ducts with higher levels initially found in males. In females, b-gal activity was restricted to one side of the ductal mesoepithelium, whereas in males b-gal expression encircled the duct. b-gal activity was also detected in the coelomic epithelium at 13.5 and 14.5 dpc. In male embryos, mesenchymal b-gal activity permitted the visualization of the temporal and spatial pattern of Müllerian duct regression. This pattern was similar to that observed using a Müllerian duct mesoepithelium lacZ reporter, indicating a coordinated loss of Müllerian duct mesoepithelium and Amhr2-expressing mesenchyme.

Her research focuses on human embryonic implantation, uterine receptivity and embryonic stem cells, leading to the publication of 26 peer-reviewed papers. She received awards from the American Fertility Society in 1997 and the Spanish Society of Obstetrics and Gynecology in 1998.

The development of a cell therapy for the neurodegenerative disorder Parkinson's disease is a realistic ambition. It is pursued by researchers and companies alike, and spans different donor tissue types of embryonic, fetal and adult origins. In this review, we briefly outline the past and current status of research and clinical trials with cell transplantation in Parkinson's disease. We discuss studies on donor tissue derived from embryonic ventral mesencephalon and assess the current research on various forms of stem cells of both embryonic and adult origins in the quest to develop a cell-based therapy for this debilitating movement disorder.

This unit describes basic techniques in human mesenchymal stem cell (hMSC) cultures. It includes protocols for the differentiation of hMSCs into osteogenic and adipogenic lineages, genetic perturbations, and phenotypic analyses. hMSCs can be differentiated with dexamethasone and β-glycerophosphate into mineralizing osteoblasts within 2 to 3 weeks, or with dexamethasone, indomethacin, and 3-isobutyl-1-methylxanthine into lipid vesicle-containing adipocytes within 1 to 2 weeks. Phenotypic changes during those highly dynamic differentiation processes can be detected by biochemical and histological assays and gene expression analyses of differentiation markers. In addition, this unit describes an electroporation method that allows the transient genetic perturbation of hMSCs. Curr. Protoc. Stem Cell Biol.

The definitive treatment for end-stage organ failure is orthotopic transplantation. However, the demand for transplantation far exceeds the number of available donor organs. A promising tissue-engineering/regenerativemedicine approach for functional organ replacement has emerged in recent years. Decellularization of donor organs such as heart, liver, and lung can provide an acellular, naturally occurring three-dimensional biologic scaffold material that can then be seeded with selected cell populations. Preliminary studies in animal models have provided encouraging results for the proof of concept. However, significant challenges for three-dimensional organ engineering approach remain. This manuscript describes the fundamental concepts of whole-organ engineering, including characterization of the extracellular matrix as a scaffold, methods for decellularization of vascular organs, potential cells to reseed such a scaffold, techniques for the recellularization process and important aspects regarding bioreactor design to support this approach. Critical challenges and future directions are also discussed.

Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by progressive and irreversible decline of memory. Neuropathological features include the progressive degeneration of cholinergic neurons in the forebrain cholinergic projection system especially nucleus basalis of Meynert (nbM). New cell therapeutic approaches for the replacement of degenerated cells are being researched. The aim of this study was to investigate the production of cholinergic neurons from mouse embryonic stem cells (ESCs) and potential for utilizing ESC-derived neuronal precursor cells (NPCs) and primed NPCs (PNPCs) for cell restorative therapy in a rodent model of AD. NPCs were produced by growth factor-mediated selection under serum-free conditions and differentiated better into cholinergic neurons when NPCs primed with Shh ($22%) in comparison with different cholinergic promoting factors. Behavioral assessment of unilateral nbM ibotenic acid-lesioned rats by Morris water maze and spatial probe test revealed a significant behavioral improvement in memory deficits following transplantation with NPCs and/or PNPCs. Immunohistochemical analysis revealed that the majority ($70%) of the NPCs and/or PNPCs retained neuronal phenotype and $40% of them had a cholinergic cell phenotype following transplantation with no tumor formation, indicating that these may be safe for transplantation. This experimental study has important implications as it suggests that the transplantation of mouse ESC-derived NPCs and/or following commitment to a cholinergic cell phenotype can promote behavioral recovery in a rodent model of AD.

The present review addresses the issues related to innovative contributions in biotechnology and their potential role in stem cell research at present and in the future. We can expect that future developments and applications in biotechnological sciences and industry will effect the direction of emerging cellular therapies. The use of these advances may offer a unique opportunity to investigate the mechanisms related to the journey from embryonic cells or bone-marrow derived stem/progenitor cells to cardiomyocytes or endothelial cells and the molecular regulators of cell differentiation.

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by ectopic expression of four transcription factors. However, the efficiency of human iPS cell generation is extremely low and therefore elucidating the mechanisms underlying cellular reprogramming is of prime importance. We demonstrate that 8-Bromoadenosine 3′, 5′cyclic monophosphate (8-Br-cAMP) improves the reprogramming efficiency of human neonatal foreskin fibroblast (HFF1) cells transduced with the four transcription factors by 2-fold. The combination of 8-Br-cAMP and VPA synergistically increases the efficiency to 6.5-fold. The effect of 8-Br-cAMP or VPA may in part be due to the up-regulation of cytokinerelated and inflammatory pathways. Remarkably, the synergistic effect of 8-Br-cAMP and VPA on cellular reprogramming may be due to the transient decrease of p53 protein during the early stages of reprogramming. However, it could also be due to additional differentially regulated genes and pathways such as the up-regulation of cytokine-related, inflammatory pathways and self-renewal supporting gene, namely cyclinencoding CCND2, and the associated genes CCNA1 and CCNE1. Conversely, we also see the down-regulation of the p53 (CCNB2, GTSE1, SERPINE1) and cell cycle (PLK1, CCNB2) pathways. Our data demonstrates that a cyclic AMP analog, 8-Br-cAMP, enhances the efficiency of cellular reprogramming. In addition, 8-Br-cAMP and VPA have a synergistic effect on cellular reprogramming, which may be in part due to the transient down-regulation of the p53 signaling pathway during the early stages of reprogramming.

We report the characterization of a highly germline competent C57BL/6N mouse embryonic stem cell line, JM8. To simplify breeding schemes, the dominant agouti coat color gene was restored in JM8 cells by targeted repair of the C57BL/6 nonagouti mutation. These cells provide a robust foundation for large-scale mouse knockout programs that aim to provide a public resource of targeted mutations in the C57BL/6 genetic background.

Liver transplantation is still the only effective treatment for end-stage liver disease. However, because of the serious worldwide shortage of donated organs, an alternative cellular therapy would be desirable. Animal studies and preclinical trials have indicated that hepatocyte transplantation can serve as an alternative to liver transplantation. Unfortunately, however, the harvesting of hepatocytes is associated with the same problem as organ transplantation, i.e., a lack of a suitable cell source. Therefore, current stem cell technology, which is attempting to establish an unlimited supply of hepatocytes, would facilitate the clinical application of hepatocyte transplantation. This review summarizes current knowledge of embryonic and adult stem cell differentiation into hepatocytes and discusses how liver stem cells could be applied clinically in the future.

Hematopoiesis is the process in which all Blood lineages are produced from Hematopoietic Stem cells. Through series of Progenitor cells, Hematopoietic stem cells produce a large number of Adult Stem cells. Hematopoietic Stem cells have the ability of self-renewal and self-division This Divisional event results in the Differentiation of Hematopoietic Stem cells into-Lymphocytes, Granulocytes, Monocytes, etc. Progenitor cells also contain cells that act as our body's Defense mechanism. This Always active defense mechanism consists of T cells, B cells, and Natural Killer cells. These cells work together to fight against any viruses or any pathogen and destroy them or exclude them out of the body. By understanding the Hematopoietic process and Defense mechanism, development has been done in the formation of new therapies for curing specific diseases. The basic concept of Stem cell production and its classification are defined here. This paper includes the concept of the production of Progenitor cell lineages which are classified as Lymphoid Progenitor cells and Myeloid Progenitor cells. Here we discuss the Embryonic development and classification of each blood lineages.

Tissue engineering scaffolds are designed to influence the physical, chemical and biological environment surrounding a cell population. In this review we focus on our own work and introduce a range of strategies and materials used for tissue engineering, including the sources of cells suitable for tissue engineering: embryonic stem cells, bone marrow-derived mesenchymal stem cells and cord-derived mesenchymal stem cells. Furthermore, we emphasize the developments in custom scaffold design and manufacture, highlighting laser sintering, supercritical carbon dioxide processing, growth factor incorporation and zoning, plasma modification of scaffold surfaces, and novel multi-use temperature-sensitive injectable materials.

Osterix (Osx) is a transcription factor required for the differentiation of preosteoblasts into fully functioning osteoblasts. However, the pattern of Osx activation during preosteoblast differentiation and maturation has not been clearly defined. Our aim was to study Osx activation during these processes in osteoblasts differentiating from murine and human embryonic stem cells (ESC). To do this, we constructed an Osx-GFP fusion protein reporter system to track Osx translocation within the cells. The distribution of Osx-GFP at representative stages of differentiation was also investigated by screening primary osteoblasts, mesenchymal stem cells, synoviocytes, and pre-adipocytes. Our experiments revealed that Osx-GFP protein was detectable in the cytoplasm of cultured, differentiated ESC 4 days after plating of enzymatically dispersed embryoid bodies. Osterix-GFP protein became translocated into the nucleus on day 7 following transfer of differentiated ESC to osteogenic medium. After 14 days of differentiation, cells showing nuclear translocation of Osx-GFP formed rudimentary bone nodules that continued to increase in number over the following weeks (through day 21). We also found that Osx translocated into the nuclei of mesenchymal stem cells (C3H10T1/2) and pre-osteoblasts (MC3T3-E1) and showed partial activation in pre-adipocytes (MC3T3-L1). These data suggest that Osx activation occurs at a very early point in the differentiation of the mesenchymal-osteoblastic lineage.

The heart is the first visible organ during vertebrate embryogenesis. It originates from the most anterior part of lateral plate mesoderm. Cardiac progenitor cells (CPCs) are divided into two major populations, occupying the first and second heart fields during development. First heart field CPCs are labelled with Nkx2-5 and/or Tbx5 and are mainly destined to from the left ventricle and atria of the heart, while the second heart field CPCs, labelled with Isl1, will give rise to the right ventricle and the outflow tract. To date, it has been shown that Nkx2-5+ CPCs  are able to give rise to smooth muscle cells and cardiomyocytes, while Isl1+ CPCs are multipotent able to differentiate to cardiomyocytes, smooth muscle and endothelial cells. There has been no report examining the potential of Tbx5+ cells as CPCs, to date.
To better understand CPCs onset, we have performed single cell cDNA analysis on cells from the early allantoic bud (EB) stage to early headfold (EHF) stage on embryonic day (E) 7.5. This study showed that Tbx5+, Nkx2.5+ and Tbx5+/Nkx2.5+ cells are the three major CPC subpopulations. As heart development proceeds, the Tbx5+ cell population begins to diminish, with the Tbx5+/Nkx2.5+ to steadily increase while approaching the late allantoic bud (LB) stage. By the early somite stage, no Tbx5+ cells are present. To test the hypothesis of whether Tbx5+/Nkx2.5+ CPCs descend from Tbx5+ CPCs, we generated a tamoxifen-inducible BAC Tbx5(CreERT2/+);Rosa26RYFP/- transgenic mouse. With this system, we are able to trace Tbx5+ CPCs in vivo as well as deriving ES cell lines from these transgenic mice Tbx5+ CPCs for tracing analysis in vitro. We will present our findings with this newly established experimental system.

The last decade has witnessed the development of cell-based therapy as a major biomedical research area, including the treatment of musculoskeletal diseases. Both differentiated and undifferentiated stem cells have been used as starting cell sources. In particular, the use of multipotent adult mesenchymal stem cells holds great promise for future therapeutic strategies. In addition to the cell type used, the cell delivery system is also of critical importance in cell-based therapy. Cell delivery may be achieved by direct cell injection or by grafting engineered constructs derived by cell seeding into natural or synthetic biomaterial scaffolds. While direct injection is the most direct and convenient means of cell delivery, the latter approach is capable of producing three-dimensional engineered tissues with mechanical properties compatible with those of various musculoskeletal tissues. This review will focus on the functional approach of using biomaterial scaffold materials as cell carriers for musculoskeletal applications, as well as the use of cell-based gene therapy for tissue engineering and regeneration.

Recent advances in human embryonic stem cell (hESC) biology now offer an alternative cell source for tissue engineers, as these cells are capable of proliferating indefinitely and differentiating to many clinically relevant cell types. Novel culture methods capable of exerting spatial and temporal control over the stem cell microenvironment allow for more efficient expansion of hESCs, and significant advances have been made toward improving our understanding of the biophysical and biochemical cues that direct stem cell fate choices. Effective production of lineage specific progenitors or terminally differentiated cells enables researchers to incorporate hESC derivatives into engineered tissue constructs. Here, we describe current efforts using hESCs as a cell source for tissue engineering applications, highlighting potential advantages of hESCs over current practices as well as challenges which must be overcome.

Estradiol protects dopamine neurons of the substantia nigra from toxic insults. Such neurons succumb in Parkinson's disease; one strategy for restoring dopamine deficiency is cell therapy with neurons differentiated from embryonic stem cells. We investigated the effects of 17b-estradiol on dopaminergic induction of embryonic stem cells using the 5-stage protocol. Cells were incubated with different steroid concentrations during the proliferation (stage 4) or differentiation (stage 5) phases. Estradiol added at nM concentrations only during stage 4 increases the proliferation of dopaminergic precursors expressing Lmx1a, inducing a higher proportion of dopamine neurons at stage 5. These actions were mediated by activation of estrogen receptors, because co-incubation of cells with estradiol and ICI 182,780 completely abolished the positive effect on both proliferation of committed precursors, and subsequent differentiation to dopaminergic neurons. Our results suggest that estradiol should be useful in producing higher proportions of dopamine neurons from embryonic stem cells aimed for treating Parkinson's disease.

The focus of regenerative medicine is rebuilding damaged tissues by cell transplantation or implantation of bioartificial tissues. In either case, therapies focus on adult stem cells (ASCs) and embryonic stem cells (ESCs) as cell sources. Here we review four topics based on these two cell sources. The first compares the current performance of ASCs and ESCs as cell transplant therapies and the drawbacks of each. The second explores somatic cell nuclear transfer (SCNT) as a method to derive ESCs that will not be immunorejected.

Stem cell research is very promising. The use of human embryos has been confronted with objections based on ethical and religious positions. The recent production of reprogrammed adult (induced pluripotent) cells does not – in the opinion of scientists – reduce the need to continue human embryonic stem cell research. So the debate continues.Islam always encouraged scientific research, particularly research directed toward finding cures for human disease. Based on the expectation of potential benefits, Islamic teachings permit and support human embryonic stem cell research. The majority of Muslim scholars also support therapeutic cloning. This permissibility is conditional on the use of supernumerary early pre-embryos which are obtained during infertility treatment in vitro fertilization (IVF) clinics. The early pre-embryos are considered in Islamic jurisprudence as worthy of respect but do not have the full sanctity offered to the embryo after implantation in the uterus and especially after ensoulment.In this paper the Islamic positions regarding human embryonic stem cell research and therapeutic cloning are reviewed in some detail, whereas positions in other religious traditions are mentioned only briefly.The status of human embryonic stem cell research and therapeutic cloning in different countries, including the USA and especially in Muslim countries, is discussed.

Type 1 diabetes is characterized by near total absence of pancreatic b cells. Current treatments consisting of insulin injections and islet transplantation are clinically unsatisfactory. In order to develop a cure for type 1 diabetes, we must find a way to reverse autoimmunity, which underlies b cell destruction, as well as an effective strategy to generate new b cells. This article reviews the different approaches that are being taken to produce new b cells. Much emphasis has been placed on selecting the right non-b cell population, either in vivo or in vitro, as the starting material. Different cell types, including adult stem cells, other types of progenitor cells in situ, and even differentiated cell populations, as well as embryonic stem cells and induced pluripotent stem cells, will require different methods for islet and b cell induction. We discussed the pros and cons of the different strategies that are being used to re-invent the pancreatic b cell. (Translational Research 2010;156:169e179) Abbreviations: DDC ¼ 3,5-diethoxycarbonyl-1,4-dihydrocollidine; ES ¼ embryonic stem; ESC ¼ embryonic stem cells; FBS ¼ fetal bovine serum; FGAd ¼ first generation adenovirus; FGF10 ¼ fibroblast growth factor 10; GCNF ¼ germ cell nuclear factor; GSIS ¼ glycemia stimulated insulin secretion; HDAd ¼ helper-dependent adenovirus; hESC ¼ human embryonic stem cells; iPSC ¼ induced-pluripotent stem cells; Klf4 ¼ Kruppel-like factor 4; MSC ¼ mesenchymal stem cell; MEF ¼ mouse embryonic fibroblasts; Ngn3 ¼ neurogenin 3; Oct4 ¼ Octamer-4; Pdx1 ¼ pancreatic and duodenal homeobox 1; RA ¼ retinoic acid; SCNT ¼ somatic cell nuclear transfer; Shh ¼ sonic hedgehog; STZ ¼ streptozotocin; TGFb ¼ transforming growth factor-b

Epigenetics refers to mitotically and/or meiotically heritable variations in gene expression that are not caused by changes in DNA sequence. Epigenetic mechanisms regulate all biological processes from conception to death, including genome reprogramming during early embryogenesis and gametogenesis, cell differentiation and maintenance of a committed lineage. Key epigenetic players are DNA methylation and histone post-translational modifications, which interplay with each other, with regulatory proteins and with non-coding RNAs, to remodel chromatin into domains such as euchromatin, constitutive or facultative heterochromatin and to achieve nuclear compartmentalization. Besides epigenetic mechanisms such as imprinting, chromosome X inactivation or mitotic bookmarking which establish heritable states, other rapid and transient mechanisms, such as histone H3 phosphorylation, allow cells to respond and adapt to environmental stimuli. However, these epigenetic marks can also have long-term effects, for example in learning and memory formation or in cancer. Erroneous epigenetic marks are responsible for a whole gamut of diseases including diseases evident at birth or infancy or diseases becoming symptomatic later in life. Moreover, although epigenetic marks are deposited early in development, adaptations occurring through life can lead to diseases and cancer. With epigenetic marks being reversible, research has started to focus on epigenetic therapy which has had encouraging success. As we witness an explosion of knowledge in the field of epigenetics, we are forced to revisit our dogma. For example, recent studies challenge the idea that DNA methylation is irreversible. Further, research on Rett syndrome has revealed an unforeseen role for methyl-CpG-binding protein 2 (MeCP2) in neurons.

Since the first cell therapeutic study to repair articular cartilage defects in the knee in 1994, several clinical studies have been reported. An overview of the results of clinical studies did not conclusively show improvement over conventional methods, mainly because few studies reach level I of evidence for effects on middle or long term. However, these explorative trials have provided valuable information about study design, mechanisms of repair and clinical outcome and have revealed that much is still unknown and further improvements are required. Furthermore, cellular and molecular studies using new technologies such as cell tracking, gene arrays and proteomics have provided more insight in the cell biology and mechanisms of joint surface regeneration. Besides articular cartilage, cartilage of other anatomical locations as well as progenitor cells are now considered as alternative cell sources. Growth Factor research has revealed some information on optimal conditions to support cartilage repair. Thus, there is hope for improvement. In order to obtain more robust and reproducible results, more detailed information is needed on many aspects including the fate of the cells, choice of cell type and culture parameters. As for the clinical aspects, it becomes clear that careful selection of patient groups is an important input parameter that should be optimized for each application. In addition, the study outcome parameters should be improved. Although reduced pain and improved function are, from the patient's perspective, the most important outcomes, there is a need for more structure/tissuerelated outcome measures. Ideally, criteria and/or markers to identify patients at risk and responders to treatment are the ultimate goal for these more sophisticated regenerative approaches in joint surface repair in particular, and regenerative medicine in general.

Embryonic stem cells (ESCs) offer a powerful in vitro model to study mechanisms implicated in cell fate decision. Developmental pathways by which pluripotent ESCs become committed to specific lineages are reflected in dynamic changes of signaling and transcriptional programs. However, the mechanisms that govern the regulatory intracellular networks underlying lineage fate decisions and differentiation programs remain poorly understood and differ significantly in different species. In this review we analyze the current understanding of the signaling mechanisms and transcriptional regulation of differentiation of murine and human ESCs into the mesoderm.

Since the initial discoveries of human embryonic and induced pluripotent stem cells, many strategies have been developed to utilize the potential of these cells for translational research and disease modeling. The success of these aims and the development of future applications in this area will depend on the ability to generate high-quality and large numbers of differentiated cell types that genetically, epigenetically, and functionally mimic the cells found in the body. In this review, we highlight the current strategies used to maintain stem cell pluripotency (a measure of stem cell quality), as well as provide an overview of the various differentiation strategies being used to generate cells from all three germ lineages. We also discuss the particular considerations that must be addressed when utilizing these cells for translational therapy, and provide an example of a cell type currently used in clinical trials. The major challenge in regenerative medicine and disease modeling ...