BRD4 promotes endodermal cell fate during mammalian lung development

Reactivation of Latent HIV-1 by Inhibition of BRD4

Bromodomain-containing protein 9 (BRD9) is a specific subunit of the non-canonical SWI/SNF (ncBAF) chromatin-remodeling complex, whose function in human embryonic stem cells (hESCs) remains unclear. Here, we demonstrate that impaired BRD9 function reduces the self-renewal capacity of hESCs and alters their differentiation potential. Specifically, BRD9 depletion inhibits meso-endoderm differentiation while promoting neural ectoderm differentiation. Notably, supplementation of NODAL, TGF-β, Activin A or WNT3A rescues the differentiation defects caused by BRD9 loss. Mechanistically, BRD9 forms a complex with BRD4, SMAD2/3, β-CATENIN and P300, which regulates the expression of pluripotency genes and the activity of TGF-β/Nodal/Activin and Wnt signaling pathways. This is achieved by regulating the deposition of H3K27ac on associated genes, thus maintaining and directing hESC differentiation. BRD9-mediated regulation of the TGF-β/Activin/Nodal pathway is also demonstrated in the development of pancreatic and breast cancer cells. In summary, our study highlights the crucial role of BRD9 in the regulation of hESC self-renewal and differentiation, as well as its participation in the progression of pancreatic and breast cancers.

Embryonic stem (ES) cells require a network of transcription factors and signaling molecules to maintain their pluripotency. The transcription factor Oct4, one of pluripotency genes, is required for self-renewal and maintains the undifferentiated state of ES cells. Loss of expression of Oct4 initiates ES cell differentiation. Germ cell nuclear factor (GCNF), an orphan nuclear receptor, binds to a DR0 site within Oct4 gene promoter in human ES cells. Retinoic acid induces expression of GCNF, which binds the promoter of Oct4 gene, and accelerates the decrease of Oct4 gene expression and human ES cells differentiation. Expression of other pluripotency genes, such as Sox2, also decrease during human ES cell differentiation induced with retinoic acid. These findings increase our understanding of the mechanism of regulation of gene expression and in maintaining human ES cell pluripotence and during the differentiation process. Results 1: Tera-2 cells and human ES (H9) cells were induced to differentiate by Retinoic Acid (1μM). Expression of GCNF, Oct4, Sox2, Dax1, LRH1, SF1, Actin was determined by RT-PCR (A), and by western blot (B) in Tera-2 cells and human ES cells. GCNF expression increased by day 2 of differentiation in Tera-2 cells and human ES cells when treated with Retinoic Acid (1μM). GCNF expression is transient and decreased earlier in Tera-2 cells than in human ES cells. Expression of Oct4, Sox2 and SF-1 was gradually repressed during differentiation. Expression of the orphan receptor LRH1 increased when Tera-2 and human ES cells differentiated, and subsequently decreased. DAX1 expression was up-regulated when Tera-2 cells and human ES cells differentiate, and maintained the same level until day 6 in Tera-2 cells but gradually increased in human ES cells. Result 2: GCNF binding the DR0 site within the Oct4 promoter in Tera-2 cells and human ES cells is induced upon differentiation and is transient in nature. Detection of GCNF binding to the Oct4 promoter in vivo using Chromatin immunoprecipitation (ChIP) assays: Tera-2 cells and human ES cells were treated with Retinoic Acid (1 μM) and induced to differentiate. GCNF binding to the DR0 site of Oct4 promoter can be detected in vivo when Tera-2 cell and human ES cells differentiate, and then decreases at later stages of differentiation. Result 3: Analysis of GCNF binding to the DR0 element in the Oct4 promoter by electrophoretic mobility shift assays (EMSAs). No GCNF binding to the DR0 element was detected at day 0 in the Tera-2 cells and human ES cells. GCNF has been at the highest level of binding to the DR0 site at day 3 of RA-induced differentiation (arrow) and is shifted (arrow head) when GCNF-specific antibody is added to the EMSAs. GCNF DNA binding activity disappeared by day 5 of differentiation in Tera-2 cells. In human ES cells, GCNF displayed a higher level of binding to the DR0 site at day 3 of differentiation and decreased at day 5 of RA treatment. This result shows that GCNF directly binds to the conserved DR0 element in the Oct4 promoter in Tera-2 cells and human ES cell. (platform)

Human and mouse embryonic stem (ES) cells have the capacity to differentiate into derivatives of all three germ layers, suggesting novel therapies for degenerative, metabolic, and traumatic disorders. ES-based regenerative medicine will be further advanced by the development of reliable methods for transgene introduction and expression. Here, we show infection of human and mouse embryonic stem (ES) cells with two of the most popular vectors in gene transfer, adenovirus type 5 (Ad5) and adeno-associated virus (AAV; serotypes 2, 4, and 5). All vectors express the nuclear-localized marker gene beta-galactosidase expressed from the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Both Ad5 and AAV2 infected human and mouse ES cells and gave rise to beta-galactosidase expression. AAV4 and 5 did not yield detectable levels of beta-galactosidase expression. Quantitative PCR analysis of virally infected human and mouse ES cells revealed that only Ad5 and AAV2 are capable of transducing both cell-types. No viral DNA was detected in cells infected with either AAV4 or AAV5. Infection and subsequent differentiation of mouse and human ES cells with Ad5 showed that beta-galactosidase-expressing cells were restricted to cells in the interior of the embryoid body mass. No beta-galactosidase expression was observed in AAV-infected cells following differentiation. There was no difference in morphology or differentiation patterns between infected and noninfected differentiating mouse and human ES cells. Differentiation of hES cells prior to infection led to transduction of neuronally differentiated cells with good efficiency using all vectors. These data show that Ad5- and AAV2-based vectors are capable of infecting both human and mouse ES cells, in both their undifferentiated and differentiated states, whereas AAV4 and AAV5 can infect human and mouse ES cells only following differentiation.

Retinal pigment epithelium cells derived from human embryonic stem cells (ESCs) could be useful for restoring retinal function in age-related macular degeneration. However the use of non-human feeder cells to support the growth of ESCs for clinical applications raises the concern of possible contamination because of direct contact between animal and human cells. In this study, we produced human ESCs using human fibroblast feeder layers isolated from foreskin and abdominal tissues. Using this system, human ESCs differentiated into retinal pigment epithelium cells in differentiation medium. Seven human ESC lines were established from 18 blastocysts. These human ESCs showed normal morphology, expressed all expected cell surface markers, had the ability to form embryoid bodies upon culture in vitro and teratomas after injection into SCID mice, and differentiated further into derivatives of all three germ layers. Under conditions of committed differentiation, these human ESCs could differentiate into retinal pigment epithelium cells after 2 months in culture. The results of this study demonstrated that human foreskin/abdominal fibroblasts have the potential to support the derivation and long-term culture of human ESCs, which can then be used to generate retinal pigment epithelium cells with characteristic morphology and molecular markers. This technique avoids the concerns of contamination from animal feeder layers during human ESC derivation, culture and differentiation, and will thus facilitate the development of retinal pigment epithelium cell transplantation therapy.

SummaryThe gap in knowledge of the molecular mechanisms underlying differentiation of human pluripotent stem cells (hPSCs) into the mesenchymal cell lineages hinders the application of hPSCs for cell-based therapy. In this study, we identified a critical role of muscle segment homeobox 2 (MSX2) in initiating and accelerating the molecular program that leads to mesenchymal stem/stromal cell (MSC) differentiation from hPSCs. Genetic deletion of MSX2 impairs hPSC differentiation into MSCs. When aided with a cocktail of soluble molecules, MSX2 ectopic expression induces hPSCs to form nearly homogeneous and fully functional MSCs. Mechanistically, MSX2 induces hPSCs to form neural crest cells, an intermediate cell stage preceding MSCs, and further differentiation by regulating TWIST1 and PRAME. Furthermore, we found that MSX2 is also required for hPSC differentiation into MSCs through mesendoderm and trophoblast. Our findings provide novel mechanistic insights into lineage specification of hPSCs to MSCs and effective strategies for applications of stem cells for regenerative medicine.

Pulmonary Hypoplasia in Mice Lacking Tumor Necrosis Factor-α Converting Enzyme Indicates an Indispensable Role for Cell Surface Protein Shedding during Embryonic Lung Branching Morphogenesis

Human Embryonic Stem Cells . Edited by J. Odorico, S. Zhang and R. Pedersen . (Pp. xxii + 391 , illustrated , ISBN 18599 62785 , £80 hardback.) Abingdon, UK : BIOS Scientific/Garland Science . It is now nearly seven years since the first description of human embryonic stem (ES) cell lines was published by Jamie Thomson. That event, closely apposed to the reports of the cloning of sheep (‘Dolly’), and then mice, by somatic nuclear transfer, catalysed substantial interest in the idea of regenerative medicine and the possibility that stem cells of all sorts might be harnessed for the replacement of tissues lost to accident or disease. At about the same time, a number of papers appeared, reporting that various somatic stem cells – which were previously thought to be committed to lineages from within their tissue of origin – might indeed exhibit substantial plasticity and be able to generate terminally differentiated cells corresponding to a much wider range of cell types. Thus, the current era of stem cell biology was born, though the concept of stem cells and their application in medicine is much older. One consequence of this resurgent interest in stem cell biology has been the publishing of several multi-author volumes devoted to stem cells and their potential. The volume Human Embryonic Stem Cells, edited by J. Odorico, S. Zhang and R. Pedersen, is the latest in a series produced by different publishing houses over the past few years. Paradoxically, however, as Jamie Thomson points out in his foreword to the present volume, progress in the field has been slow over this time. Prospects for adult stem cells have been dimmed by controversy over the phenomenon of ‘plasticity’, with the suggestion that at least some of the reported cases might be artefactual, depending upon cell fusion or rare transdifferentiation events. At the same time, progress with human ES cells was initially hindered by the difficulty of access to established lines, and the complexity of maintaining and expanding the cultures when they could be obtained. Although mouse ES cells have been available for over 20 years, they have mostly been used as tools to produce transgenic mice. With notable exceptions, few have investigated their cell biology for its own sake, and so there has been relatively little experience from studies of mouse ES cells to guide the study of their human counterparts. The development of ES cell lines was a systematic progression from the study of teratocarcinomas in the 1970s, by those who thought that these tumours might provide key insights into the mechanisms of embryonic development. It is then perhaps ironic that clues about how to control the behaviour of human ES cells in culture are now being provided by our detailed knowledge of developmental genetics, especially of the mouse. However, the human ES cell field is changing. Many laboratories in many countries have now derived human ES cell lines. In the current International Stem Cell Initiative, a collaborative venture to compare the properties of human ES cell lines derived worldwide, 75 independent lines derived in 17 laboratories and ten countries have been enrolled in the study. Meanwhile, reports are now beginning to appear in prominent journals, addressing the molecular mechanisms of human ES cell proliferation and differentiation – not merely describing their characteristics and potential for diverse differentiation. The present volume, unlike many of its competitors, focuses explicitly on human ES cells, and may be thought to mark the end of the first phase of human ES cell research. It brings together a series of authors who have contributed significantly to the field and it balances chapters discussing the basic biology of ES cells with others discussing their differentiation along specific lineages and eventual application. Despite the focus on ES cells, two early chapters provide useful comparative reviews of recent studies of adult stem cell plasticity and of mesenchymal stem cells. Further chapters address potential issues that will need to be addressed as derivatives of human ES cells are developed for eventual clinical application. Included here are two interesting and useful reviews of the ethical, legal and intellectual property aspects of human ES cell research and application. Undoubtedly, research in the new field of human ES cell biology is beginning to advance rapidly, so that it might be considered that the present volume will become rapidly obsolete. However, its well-organized and well-written chapters provide a valuable reference to the current state of the field for newcomers and established human ES cell researchers alike. Although our understanding of molecular mechanisms and the means of manipulating human ES cells is likely to change significantly in the future, much of what is contained in this volume will remain fundamental, and so of value to those working in this exciting new area of research for quite a few years to come.

Development is a highly ordered process that institutes considerable changes in cell cycle structure of the different cells composing the embryo. Cell fate is decided at early stages of embryogenesis in embryonic stem cells (ESCs). These cells are derived from the inner cell mass of the primordial embryo and can give rise to all cell lineages of the 3 primary germ layers. The cell cycle of ESCs is uncommonly rapid compared with a wide range of somatic cells due to shorter gap phases, especially G1, resulting in the characteristic high proportion of cells in S phase. Previous observations have highlighted the importance of a rapid and contracted cell cycle for ESCs maintenance. A high proliferation rate was shown to be required for maintenance of ESC identity,1 and increased susceptibility to differentiate occurs during the contracted G1 phase.2 Very recently, the molecular mechanism underlying cell fate decisions of self-renewing ESCs was analyzed in more detail and shown to originate at specific points within this critical retracted G1 phase,3 establishing a connection between the mechanisms that control the cell cycle, cell fate decisions, and loss of pluripotency. ESCs differentiation induces massive changes in the nuclear protein landscape, surprisingly reported to be largely regulated at the post-transcriptional level rather than by direct regulation of gene expression levels by transcription factors,4 as previously postulated by various studies in diverse model organisms. One prominent protein post-translational modification is ubiquitylation, which regulates abundance and/or localization of target proteins altering their function, inasmuch regulating a multitude of cellular processes operating in most biological systems. Ubiquitylation is performed in 3 main consecutive steps catalyzed by E1, E2, and E3 enzymes, and in many cases the specificity of the reaction is provided by the E3 ligase complex, which conjugates activated ubiquitin to target substrates. The reverse reaction consists of removing ubiquitin from target proteins and is catalyzed by deubiquitylating enzymes (Dubs). Interestingly, converging evidences indicate that in ESCs, pluripotency is under tight control of the ubiquitin-dependent degradation pathways and proteasomal activity.5,6 In these studies, substrates and activity of the proteasome were shown to be different in ESCs compared with differentiated cells, reflecting differentiation-induced adaptations. For example, Nanog and Oct4, 2 key transcription factors involved in maintenance of pluripotency, were shown to have short half-lives in ESCs. Given that the activity of degradation pathways are commonly coupled to cell cycle stage and progression, it is not unlikely that both Nanog and Oct4 may be degraded in a cell cycle-dependent manner. The ubiquitin proteasome degradation pathway governs major cell cycle events including G1 to S transition and anaphase. It is interesting to note that both in human and mouse ESCs, substrates of the anaphase-promoting complex/cyclosome (APC/C) have recently been identified as critical players in pluripotency maintenance.3,6 APC/C activity is elevated in late anaphase and persists through progression of the G1 phase. In human ESCs, Cyclin D levels are maximal at the G1/S transition and inhibit endoderm differentiation by inducing CDK4/6-dependent phosphorylation of Smad2 and Smad3 transcription factors, clearing them from chromatin and blocking expression of endoderm genes. In mouse ESCs, we have reported that post-translational control of Cdc25A, a cell cycle master regulator, by the Dub3 deubiquitylase, is essential for pluripotency maintenance by control of Cdc25A protein abundance. Indeed, the balance of ubiquitylating and deubiquitylating activities on Cdc25A is tipped toward deubiquitylation due to high levels of Dub3. In addition failure to reduce Dub3 levels during differentiation does not affect the onset of differentiation; however, it results in cell lethality at a time of cell cycle remodelling. This provides one molecular mechanism of how the cell cycle impacts cell fate decisions through contraction of the G1 phase and relaxation of the G1/S checkpoint.6 In addition, the activity of SCF (Skip1/Cul1/F-box protein)-mediated degradation pathway, which directly controls abundance of 2 major cell cycle regulators, the p21 and p27 proteins, is itself under tight control of APC/CCdh1. Altogether, these observations emphasize the importance of the ubiquitin signaling pathways and associated Dub activities during development that remove ubiquitin from substrates and regulate proteasomal function. These new observations tentatively provide insight into the regulation of midblastula transition onset in Drosophila, in which, similarly to mESCs, post-translational modifications of Cdc25A and not changes of its mRNA expression have also appeared as main determinants of cell cycle changes and fate decisions.7 Altogether, these data converge to the concept that developmentally regulated, post-translational control of key cell cycle substrates and proteasomal activity are essential for regulating cell fate decisions of pluripotent stem cells and during development (Fig. 1). Given that the degradation pathway targeting substrates for degradation and associated proteasomal activity itself are cell cycle-dependent, it seems relevant to dissect the regulatory mechanisms that govern and institute these proteasome-dependent degradation pathways in an unperturbed pluripotent cell cycle. Figure 1. Schematic representation of developmental transitions mediated by protein post-translational modifications (PTMs). In Drosophila, PTMS are implicated in pre- to post-midblastula transition (MBT). The enzyme(s) responsible for Drosophila ...

TNF-alpha converting enzyme (TACE)-mediated cell surface protein ectodomain cleavage constitutes an important cellular regulatory mechanism during mammalian lung development. Herein, we have found that TAPI, a synthetic inhibitor of TACE, inhibits embryonic mouse lung branching morphogenesis in culture. To further investigate the biological significance of TACE as a shedding enzyme during early lung organogenesis, we have devised an antisense oligonucleotide to specifically block endogenous TACE gene expression at both transcriptional and translational levels in embryonic mouse lung explant culture. Addition of TACE antisense oligonucleotide resulted in a concentration-dependent reduction in lung branching morphogenesis in culture, whereas both scrambled and sense control oligonucleotides showed no adverse effects on lung growth. Furthermore, both aquaporin-5 (Aqp5) and surfactant protein-C (SP-C) mRNA expression and protein immunoreactivity were significantly inhibited in cultured mouse lungs treated with TACE antisense oligonucleotide, indicating defective epithelial cell differentiation in embryonic lungs with decreased TACE expression. TACE is known to be involved in the proteolytic release of TGF-alpha, an EGF family stimuli critical for lung growth and maturation. We therefore tested the possibility that a lack of diffusable TGF-alpha, due to TACE deficiency, contributes to the inhibitory lung morphogenesis in the presence of TACE antisense oligonucleotide in lung culture. Soluble TGF-alpha, when included in the lung culture, rescued the TACE antisense oligonucleotide-treated lungs from inhibition of both lung branching morphogenesis and lung epithelial cell differentiation, suggesting an impaired release of circulating regulators necessary for lung development in the absence of TACE gene expression. Our findings provide evidence that TACE-mediated membrane protein shedding is indispensable for normal lung branching morphogenesis and cytodifferentiation, probably through regulating the availability of positive cytokines/growth factors essential for lung organogenesis such as TGF-alpha.

Carbon nanomaterials have been introduced as a scaffold for various biological applications due to their unique physical and electrical properties. Here we studied carbon nanotubes (CNTs) and carbon nanofibers (CNFs) as scaffold materials for the differentiation of human embryonic stem cells (hESCs) towards photoreceptor precursor cells (PRPs). We report on their cytoxicity, their effect on cell morphology, cell-surface interface and the differentiation process. To this end, hESCs were differentiated into PRPs on carbon nanofibers (CNFs), long horizontal CNTs (LHCNTs), vertically aligned CNTs (VACNTs) or glass (control) surfaces. The differentiated cells were investigated by immunohistochemistry, fluorescence imaging and electron microscopy. Our results revealed that the investigated nanomaterials were not cytotoxic to the cells during the differentiation process. The surface interface effect on the cells was apparent, affecting cell directionality, migration and morphology. Interestingly, cell fate was not dependent on the substrate type, as inferred from the similar dynamics of the loss of pluripotency and the comparable expression levels of the photoreceptor marker Crx for all investigated substrates. These results are important for better understanding the effect of nanomaterial surface interaction with differentiating neural cells in general, and for future use of these materials as scaffolds for differentiating photoreceptors for vision restoration in particular.

Mammalian lung development starts from a specific cluster of endodermal cells situated within the ventral foregut region. With the orchestrating of delicate choreography of transcription factors, signaling pathways, and cell-cell communications, the endodermal diverticulum extends into the surrounding mesenchyme, and builds the cellular and structural basis of the complex respiratory system. This review provides a comprehensive overview of the current molecular insights of mammalian lung development, with a particular focus on the early stage of lung cell fate differentiation and spatial patterning. Furthermore, we explore the implications of several congenital respiratory diseases and the relevance to early organogenesis. Finally, we summarize the unprecedented knowledge concerning lung cell compositions, regulatory networks as well as the promising prospect for gaining an unbiased understanding of lung development and lung malformations through state-of-the-art single-cell omics.

Transcription elongation has been recognized as a rate-limiting step for the expression of signal-inducible genes. Through recruitment of positive transcription elongation factor P-TEFb, the bromodomain-containing protein BRD4 plays critical roles in regulating the transcription elongation of a vast array of inducible genes that are important for multiple cellular processes. The diverse biological roles of BRD4 have been proposed to rely on its functional transition between chromatin targeting and transcription regulation. The signaling pathways and the molecular mechanism for regulating this transition process, however, are largely unknown. Here, we report a novel role of phosphorylated Ser(10) of histone H3 (H3S10ph) in governing the functional transition of BRD4. We identified that the acetylated lysines 5 and 8 of nucleosomal histone H4 (H4K5ac/K8ac) is the BRD4 binding site, and the protein phosphatase PP1α and class I histone deacetylase (HDAC1/2/3) signaling pathways are essential for the stress-induced BRD4 release from chromatin. In the unstressed state, phosphorylated H3S10 prevents the deacetylation of nucleosomal H4K5ac/K8ac by HDAC1/2/3, thereby locking up the majority of BRD4 onto chromatin. Upon stress, PP1α-mediated dephosphorylation of H3S10ph allows the deacetylation of nucleosomal H4K5ac/K8ac by HDAC1/2/3, thereby leading to the release of chromatin-bound BRD4 for subsequent recruitment of P-TEFb to enhance the expression of inducible genes. Therefore, our study revealed a novel mechanism that the histone cross-talk between H3S10ph and H4K5ac/K8ac connects PP1α and HDACs to govern the functional transition of BRD4. Combined with previous studies on the regulation of P-TEFb activation, the intricate signaling network for the tight control of transcription elongation is established.

Objectives: Signaling by fibroblast growth factor (FGF) receptor (FGFR) 2IIIb regulates branching morphogenesis in the mammalian lung. FGFR2IIIb is primarily expressed in epithelial cells, whereas its ligands, FGF-10 and keratinocyte growth factor (KGF; FGF-7), are expressed in mesenchymal cells. FGF-10 null mice lack lungs, whereas KGF null animals have normal lung development, indicating that FGF-10 regulates lung branching morphogenesis. In this study, we determined the effects of FGF-10 on lung branching morphogenesis and accompanying gene expression in cultures of embryonic rat lungs. Methods: Embryonic day 14 rat lungs were cultured with FGF-10 (0–250 ng/ml) in the absence or presence of heparin (30 ng/ml) for 4 days. Gene expression profiles were analyzed by Affymetrix microchip array including pathway analysis. Some of these genes, functionally important in FGF-10 signaling, were further analyzed by Northern blot, real-time PCR, in situ hybridization and immunohistochemistry. Results: Exogenous FGF-10 inhibited branching and induced cystic lung growth only in cultures containing heparin. In total, 252 upregulated genes and 164 downregulated genes were identified, and these included Spry1 (Sprouty-1), Spry2 (Sprouty-2), Spred-1, Bmp4 (bone morphogenetic protein-4, BMP-4), Shh (sonic hedgehog, SHH), Pthlh (parathyroid hormone-related protein, PTHrP), Dusp6 (MAP kinase phosphatase-3, MKP-3) and Clic4 (chloride intracellular channel-4, CLIC-4) among the upregulated genes and Igf1 (insulin-like growth factor-1, IGF-1), Tcf21 (POD), Gyg1 (glycogenin 1), Sparc (secreted protein acidic and rich in cysteine, SPARC), Pcolce (procollagen C-endopeptidase enhancer protein, Pro CEP) and Lox (lysyl oxidase) among the downregulated genes. Gsk3β and Wnt2, which are involved in canonical Wnt signaling, were up- and downregulated, respectively. Conclusions: Unlike FGF-7, FGF-10 effects on lung branching morphogenesis are heparin-dependent. Sprouty-2, BMP-4, SHH, IGF-1, SPARC and POD are known to regulate branching morphogenesis; however, potential roles of CLIC-4 and MKP-3 in lung branching morphogenesis remain to be investigated. FGF-10 may also function in regulating branching morphogenesis or inducing cystic lung growth by inhibiting Wnt2/β-catenin signaling.

Cell transplantation is being extensively explored as a means of treating many human degenerative diseases. The leukodystrophies are examples of neurological disorders where new therapeutic strategies, either cellular or molecular, will be required to repair the central nervous system (CNS) of affected patients. Much hope is being pinned on the use of human embryonic stem (ES) cells as the exogenous source of neurons and glia to replace dysfunctional or dying cells in the CNS. In the case of leukoencephalopathies, the goal is to generate oligodendrocytes or other myelinating cells such as Schwann cells from ES cells, to myelinate or remyelinate CNS axons on transplantation. Experimental data suggests that mouse ES cells have this capacity, but at present differentiation of oligodendrocytes in sufficient numbers from human ES cells is not possible. It may in fact be more feasible to isolate oligodendrocytes from human neural stem cells derived from the fetal brain, but the source of these is in short supply and, like that of ES cells, is ethically controversial. None the less, it appears certain that either of these two sources will eventually give rise to sufficient numbers of neural stem cells or oligodendrocyte progenitors that have greater capacity for repair than such cells derived from the adult brain. Once the primary technical issues concerning human ES cell differentiation have been overcome, the most likely first clinical target will be Pelizaeus-Merzbacher disease. However, widespread dissemination of cells throughout the CNS may be required for functional improvement; hence diseases such as adrenoleukodystrophy may also be considered as therapeutic targets.