The function of the proteasome is essential for maintenance of cellular homeostasis, and in pluripotent stem cells this has been extended to the removal of nascent proteins in a manner that restricts differentiation. In this study we show enhanced expression of genes encoding subunits of the 20s proteasome in human embryonic stem cells (hESCs) coupled to their downregulation as the cells progress into differentiation. The decrease in expression is particularly marked for the alternative catalytic subunits of the 20s proteasome variant known as the immunoproteasome indicating the possibility of a hitherto unknown function for this proteasome variant in pluripotent cells. The immunoproteasome is normally associated with antigen presenting cells where it provides peptides of an appropriate length for antibody generation: however our data suggests that it may be involved in maintaining the pluripotency in human embryonic stem cells (hESC). Selective inhibition of two immunoproteasome subunits (PSMB9 and PSMB8) results in downregulation of cell surface and transcriptional markers that characterise the pluripotent state, subtle cell accumulation in G1 at the expense of S phase and upregulation of various markers characterising the differentiated primitive and definitive lineages arising from hESC. Our data also support a different function for each of these two subunits in hESC that may be linked to their selectivity in driving proteasome mediated degradation of cell cycle regulatory components and/or differentiation inducing factors.

The therapeutic potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is vast, allowing disease modelling, drug discovery and testing and perhaps most importantly regenerative therapies. However, problems abound; techniques for cultivating self-renewing hESCs tend to give a heterogeneous population of self-renewing and partially differentiated cells and general include animal-derived products which can be cost-prohibitive for large scale production and effective lineage specific differentiation protocols also still remain relatively undefined and are inefficient in producing large amounts of cells for therapeutic use. Further, the mechanisms and signalling pathways which mediate pluripotency and differentiation are still to be fully appreciated. However, over the recent years, the development/discovery of a range of effective small molecule inhibitors/activators has had a huge impact in hESC biology. Large scale screening techniques, coupled with greater knowledge of the pathways involved, have generated pharmacological agents which can boost hESC pluripotency/self-renewal and survival and has allowed great increases in the efficiency of various differentiation protocols, while also aiding the delineation of several important signalling pathways. Within this review, we hope to describe the current uses of small molecule inhibitors/activators in hESC biology and their potential uses in the future. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Determining the molecular regulators/pathways responsible for the specification of human embryonic stem cells (hESCs) into hematopoietic precursors has far-reaching implications for potential cell therapies and disease modeling. Mouse models lacking SCL/TAL1 (stem cell leukemia/T-cell acute lymphocytic leukemia 1) do not survive beyond early embryogenesis because of complete absence of hematopoiesis, indicating that SCL is a master early hematopoietic regulator. SCL is commonly found rearranged in human leukemias. However, there is barely information on the role of SCL on human embryonic hematopoietic development. Differentiation and sorting assays show that endogenous SCL expression parallels hematopoietic specification of hESCs and that SCL is specifically expressed in hematoendothelial progenitors (CD45(-)CD31(+)CD34(+)) and, to a lesser extent, on CD45(+) hematopoietic cells. Enforced expression of SCL in hESCs accelerates the emergence of hematoendothelial progenitors and robustly promotes subsequent differentiation into primitive (CD34(+)CD45(+)) and total (CD45(+)) blood cells with higher clonogenic potential. Short-hairpin RNA-based silencing of endogenous SCL abrogates hematopoietic specification of hESCs, confirming the early hematopoiesis-promoting effect of SCL. Unfortunately, SCL expression on its own is not sufficient to confer in vivo engraftment to hESC-derived hematopoietic cells, suggesting that additional yet undefined master regulators are required to orchestrate the stepwise hematopoietic developmental process leading to the generation of definitive in vivo functional hematopoiesis from hESCs.

Chronic granulomatous disease (CGD) is an inherited disorder of phagocytes in which NADPH oxidase is defective in generating reactive oxygen species. In this study, we reprogrammed three normal unrelated patient's fibroblasts (p47(phox) and gp91(phox)) to pluripotency by lentiviral transduction with defined pluripotency factors. These induced pluripotent stem cells (iPSC) share the morphological features of human embryonic stem cells, express the key pluripotency factors, and possess high telomerase activity. Furthermore, all the iPSC lines formed embryoid bodies in vitro containing cells originating from all three germ layers and were capable of teratoma formation in vivo. They were isogenic with the original patient fibroblasts, exhibited normal karyotype, and retained the p47(phox) or gp91(pho)(x) mutations found in the patient fibroblasts. We further demonstrated that these iPSC could be differentiated into monocytes and macrophages with a similar cytokine profile to blood-derived macrophages under resting conditions. Most importantly, CGD-patient-specific iPSC-derived macrophages showed normal phagocytic properties but lacked reactive oxygen species production, which correlates with clinical diagnosis of CGD in the patients. Together these results suggest that CGD-patient-specific iPSC lines represent an important tool for modeling CGD disease phenotypes, screening candidate drugs, and the development of gene therapy.

Recent successes in the stem cell field have identified some of the key chemical and biological cues which drive photoreceptor derivation from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC); however, the efficiency of this process is variable. We have designed a three-step photoreceptor differentiation protocol combining previously published methods that direct the differentiation of hESC and hiPSC toward a retinal lineage, which we further modified with additional supplements selected on the basis of reports from the eye field and retinal development. We report that hESC and hiPSC differentiating under our regimen over a 60 day period sequentially acquire markers associated with neural, retinal field, retinal pigmented epithelium and photoreceptor cells, including mature photoreceptor markers OPN1SW and RHODOPSIN with a higher efficiency than previously reported. In addition, we report the ability of hESC and hiPSC cultures to generate neural and retinal phenotypes under minimal culture conditions, which may be linked to their ability to endogenously upregulate the expression of a range of factors important for retinal cell type specification. However, cultures that were differentiated with full supplementation under our photoreceptor-induction regimen achieve this within a significantly shorter time frame and show a substantial increase in the expression of photoreceptor-specific markers in comparison to cultures differentiated under minimal conditions. Interestingly, cultures supplemented only with B27 and/or N2 displayed comparable differentiation efficiency to those under full supplementation, indicating a key role for B27 and N2 during the differentiation process. Furthermore, our data highlight an important role for Dkk1 and Noggin in enhancing the differentiation of hESC and hiPSC toward retinal progenitor cells and photoreceptor precursors during the early stages of differentiation, while suggesting that further maturation of these cells into photoreceptors may not require additional factors and can ensue under minimal culture conditions. STEM CELLS 2012; 30:673-686.

To celebrate thirty years of peer-reviewed publication of cutting edge stem cell research in STEM CELLS, the first journal devoted to this promising field, we pause to review how far we have come in the three-decade lifetime of the Journal. To do this we will present our views of the ten most significant developments that have advanced stem cell biology where it is today. With the increasing rate of new data, it is natural that the bulk of these developments will have occurred in recent years but we must not think that stem cell biology is a young science. The idea of a stem cell has actually been around for quite a long time having appeared in the scientific literature as early as 1868 with Haeckels' concept of a stamzelle as an uncommitted or undifferentiated cell responsible for producing many types of new cells to repair the body (1), but it took many years to obtain hard evidence in support of this theory. Not until the work of James Till and Ernest McCulloch in the 1960's did we have proof of the existence of stem cells and until the derivation of embryonal carcinoma cells in the 1960's - 1970's and the first ESC in 1981, such adult or tissue specific stem cells were the only known class. The first issue of STEM CELLS was published in 1981; no small wonder that most of its papers were devoted to hematopoietic progenitors. More recently induced pluripotent stem cells (iPSC) have been developed and this is proving to be a fertile area of investigation as shown by the volume of publications appearing not only inSTEM CELLS, but in other journals over the last five years. The reader will note that many of the articles in this special issue are concerned with iPSC; however, this reflects the current surge of interest in the topic rather than any deliberate attempt to ignore other areas of stem cell investigation.

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.

Understanding the transcriptional cues that direct differentiation of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) to defined and functional cell types is essential for future clinical applications. In this study we have compared transcriptional profiles of haematopoietic progenitors derived from hESC at various developmental stages of a feeder and serum free differentiation method and show that the largest transcriptional changes occur during the first four days of differentiation. Data mining on the basis of molecular function revealed Rho-GTPase signalling as a key regulator of differentiation. Inhibition of this pathway resulted in a significant reduction in the numbers of emerging haematopoietic progenitors throughout the differentiation window, thereby uncovering a previously unappreciated role for Rho-GTPase signalling during human haematopoietic development. Our analysis indicated that SCL was the 11(th) most up-regulated transcript during the first four days of the hESC differentiation process. Overexpression of SCL in hESC promoted differentiation to meso-endodermal lineages, the emergence of haematopoietic and erythro-megakaryocytic progenitors and accelerated erythroid differentiation. Importantly, intra-splenic transplantation of SCL-overexpressing hESC derived haematopoietic cells enhanced recovery from induced acute anaemia without significant cell engraftment suggesting a paracrine mediated effect.

Human pluripotent stem cells (hPSCs) encompassing human embryonic stem cells and human induced pluripotent stem cells (hiPSCs) have a wide appeal for numerous basic biology studies and for therapeutic applications because of their potential to give rise to almost any cell type in the human body and immense ability to self-renew. Much attention in the stem cell field is focused toward the study of gene-based anomalies relating to the causative affects of human disease and their correction with the potential for patient-specific therapies using gene corrected hiPSCs. Therefore, the genetic manipulation of stem cells is clearly important for the development of future medicine. Although successful targeted genetic engineering in hPSCs has been reported, these cases are surprisingly few because of inherent technical limitations with the methods used. The development of more robust and efficient means by which to achieve specific genomic modifications in hPSCs has far reaching implications for stem cell research and its applications. Recent proof-of-principle reports have shown that genetic alterations with minimal toxicity are now possible through the use of zinc finger nucleases (ZFNs) and the inherent DNA repair mechanisms within the cell. In light of recent comprehensive reviews that highlight the applications, methodologies, and prospects of ZFNs, this article focuses on the application of ZFNs to stem cell biology, discussing the published work to date, potential problems, and future uses for this technology both experimentally and therapeutically.