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Human embryonic stem cells (hESCs) differentiate into specialized cells, including midbrain dopaminergic neurons (DANs), and Non-human primates (NHPs) injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine develop some alterations observed in Parkinson's disease (PD) patients. Here, we obtained well-characterized DANs from hESCs and transplanted them into two parkinsonian monkeys to assess their behavioral and imaging changes. DANs from hESCs expressed dopaminergic markers, generated action potentials, and released dopamine (DA) in vitro. These neurons were transplanted bilaterally into the putamen of parkinsonian NHPs, and using magnetic resonance imaging techniques, we calculated the fractional anisotropy (FA) and mean diffusivity (MD), both employed for the first time for these purposes, to detect in vivo axonal and cellular density changes in the brain. Likewise, positron-emission tomography scans were performed to evaluate grafted DANs. Histological analyses identified grafted DANs, which were quantified stereologically. After grafting, animals showed signs of partially improved motor behavior in some of the HALLWAY motor tasks. Improvement in motor evaluations was inversely correlated with increases in bilateral FA. MD did not correlate with behavior but presented a negative correlation with FA. We also found higher 11C-DTBZ binding in positron-emission tomography scans associated with grafts. Higher DA levels measured by microdialysis after stimulation with a high-potassium solution or amphetamine were present in grafted animals after ten months, which has not been previously reported. Postmortem analysis of NHP brains showed that transplanted DANs survived in the putamen long-term, without developing tumors, in immunosuppressed animals. Although these results need to be confirmed with larger groups of NHPs, our molecular, behavioral, biochemical, and imaging findings support the integration and survival of human DANs in this pre-clinical PD model.

Osteosarcomas are frequently associated to a poor prognosis and a modest response to current treatments. EC-8042 is a well-tolerated mithramycin analog that has demonstrated an efficient ability to eliminate tumor cells, including cancer stem cell subpopulations (CSC), in sarcomas. In transcriptomic and protein expression analyses, we identified NOTCH1 signaling as one of the main pro-stemness pathways repressed by EC-8042 in osteosarcomas. Overexpression of NOTCH-1 resulted in a reduced anti-tumor effect of EC-8042 in CSC-enriched 3D tumorspheres cultures. On the other hand, the depletion of the NOTCH-1 downstream target HES-1 was able to enhance the action of EC-8042 on CSCs. Moreover, HES1 depleted cells failed to recover after treatment withdrawal and showed reduced tumor growth potential in vivo. In contrast, mice xenografted with NOTCH1-overexpressing cells responded worse than parental cells to EC-8042. Finally, we found that active NOTCH1 levels in sarcoma patients was associated to advanced disease and lower survival. Overall, these data highlight the relevant role that NOTCH1 signaling plays in mediating stemness in osteosarcoma. Moreover, we demonstrate that EC-8042 is powerful inhibitor of NOTCH signaling and that the anti-CSC activity of this mithramycin analog highly rely on its ability to repress this pathway., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Francisco Moris was an employee and reported ownership of stock in of EntreChem SL until April 2022. All other authors declare they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Pediatric acute myeloid leukemia (AML) is a rare and heterogeneous disease that remains the major cause of mortality in children with leukemia. To improve the outcome of pediatric AML we need to gain knowledge on the biological bases of this disease. NUP98-KDM5A (NK5A) fusion protein is present in a particular subgroup of young pediatric patients with poor outcome. We report the generation and characterization of human Embryonic Stem Cell (hESC) clonal lines with inducible expression of NK5A. Temporal control of NK5A expression during hematopoietic differentiation from hESC will be critical for elucidating its participation during the leukemogenic process., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Domingo-Reinés, Martínez-Navajas, Montes, Lamolda, Simón, Castaño, Ríos-Pelegrina, Lopez-Hidalgo, García del Moral, Marchal, Real and Ramos-Mejía.)

Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising cell therapy in regenerative medicine and for autoimmune/inflammatory diseases. However, a main hurdle for MSCs-based therapies is the loss of their proliferative potential in vitro. Here we report that glycoprotein A repetitions predominant (GARP) is required for the proliferation and survival of adipose-derived MSCs (ASCs) via its regulation of transforming growth factor-β (TGF-β) activation. Silencing of GARP in human ASCs increased their activation of TGF-β which augmented the levels of mitochondrial reactive oxygen species (mtROS), resulting in DNA damage, a block in proliferation and apoptosis. Inhibition of TGF-β signaling reduced the levels of mtROS and DNA damage and restored the ability of GARP -/low ASCs to proliferate. In contrast, overexpression of GARP in ASCs increased their proliferative capacity and rendered them more resistant to etoposide-induced DNA damage and apoptosis, in a TGF-β-dependent manner. In summary, our data show that the presence or absence of GARP on ASCs gives rise to distinct TGF-β responses with diametrically opposing effects on ASC proliferation and survival., (© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

ADNP syndrome is an intellectual disability associated with Autism spectrum disorder caused by mutations in ADNP. We generated an iPSC line from an ADNP syndrome pediatric patient harboring the mutation p.Trp719* (GENYOi004-A). Peripheral blood mononuclear cells were reprogrammed using a non-transmissible form of Sendai viruses expressing the four Yamanaka factors (Oct3/4, SOX2, KLF4 and c-MYC). Characterization of GENYOi004-A included mutation analysis of ADNP by allele-specific PCR, genetic identity by Short Tandem Repeats polymorphism profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and pluripotency studies in vivo. GENYOi004-A will be useful to evaluate ADNP syndrome alterations at early developmental stages., (Copyright © 2019. Published by Elsevier B.V.)

The Notch ligand DLL4 has key roles during embryonic development of different tissues, but most of the data comes from animal models. Here we describe the generation and characterization of 2 human Pluripotent Stem Cell (hPSC) lines that overexpress DLL4, as well as the two corresponding control hPSC lines. DLL4 expression can be detected at the mRNA and protein level, and does not affect the pluripotency of the cells. These hPSC lines can be used to study the role of DLL4 during human embryonic development., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Bernard Soulier Syndrome (BSS) is a rare autosomal platelet disorder characterized by mutations in the von Willebrand factor platelet receptor complex GPIb-V-IX. In this work we have generated an induced pluripotent stem cell (BSS3-PBMC-iPS4F8) from peripheral blood mononuclear cells of a BSS patient with a p.Phe55Ser mutation in the GPIX gene. Characterization of BSS3-PBMC-iPS4F8 showed that these cells maintained the original mutation present in the BSS patient, expressed pluripotent stem cell markers and were able to differentiate into the three germline layers. This new iPSC line will contribute to better understand the biology of BSS disease., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Pediatric Acute Megakaryoblastic Leukemia not associated to Down Syndrome (non-DS AMKL) is a rare disease with a dismal prognosis. Around 15% of patients carry the chromosomal translocation t(1;22) that originates the fusion oncogene RBM15-MKL1, which is linked to an earlier disease onset (median of 6months of age) and arises in utero. Here we report the generation of two hPSC cell lines constitutively expressing the oncogene RBM15-MKL1, resulting in an increased expression of known RBM15-MKL1 gene targets. These cell lines represent new disease models of pediatric AMKL to study the impact of the RBM15-MKL1 oncogene on human embryonic hematopoietic development., (Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.)

Bernard Soulier Syndrome (BSS) is an inherited rare platelet disorder characterized by mutations in the platelet glycoprotein complex GPIb-IX-V. We generated an induced pluripotent stem cell (iPSC) line from a BSS patient with a mutation p.Asn45Ser in the GPIX locus (BSS2-PBMC-iPS4F24). Peripheral blood mononuclear cells were reprogrammed using non-integrative viral transduction. Characterization of BSS2-PBMC-iPS4F24 included mutational analysis of GPIX locus, analysis of conventional pluripotency-associated factors at mRNA and protein level and in vitro and in vivo differentiation studies. This iPSC line will provide a powerful tool to study the biology of BSS disease., (Copyright © 2016 Michael Boutros, German Cancer Research Center, Heidelberg, Germany. Published by Elsevier B.V. All rights reserved.)

Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming "boosters" also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell therapy in experimental models of Parkinson's disease replaces the lost dopamine neurons (DAN), but we still need improved methods to guide dopaminergic axons (DAx) of grafted neurons to make proper connections. The protein Semaphorin 3C (Sema3C) attracts DAN axons and enhances their growth. In this work, we show that the hydrogel PuraMatrix, a self-assembling peptide-based matrix, incorporates Sema3C and releases it steadily during 4 weeks. We also tested if hydrogel-delivered Sema3C attracts DAx using a system of rat midbrain explants embedded in collagen gels. We show that Sema3C released by this hydrogel attracts DAx, in a similar way to pretectum, which is known to attract growing DAN axons. We assessed the effect of Sema3C on the growth of DAx using microfluidic devices. DAN from rat midbrain or those differentiated from human embryonic stem cells showed enhanced axonal extension when exposed to hydrogel-released Sema3C, similar to soluble Sema3C. Notably, DAN of human origin express the cognate Sema3C receptors, Neuropilin1 and Neuropilin2. These results show that PuraMatrix is able to incorporate and release Sema3C, and such delivery guides and promotes the axonal growth of DAN. This biocompatible hydrogel might be useful as a Sema3C carrier for in vivo studies in parkinsonian animal models.

We generated an induced pluripotent stem cell (iPSC) line from a Bernard-Soulier Syndrome (BSS) patient carrying the mutation p.Trp71Arg in the GPIX locus (BSS1-PBMC-iPS4F4). Peripheral blood mononuclear cells (PBMCs) were reprogrammed using heat sensitive non-integrative Sendai viruses containing the reprogramming factors Oct3/4, SOX2, KLF4 and c-MYC. Successful silencing of the exogenous reprogramming factors was checked by RT-PCR. Characterization of BSS1-PBMC-iPS4F4 included mutation analysis of GPIX locus, Short Tandem Repeats (STR) profiling, alkaline phosphatase enzymatic activity, analysis of conventional pluripotency-associated factors at mRNA and protein level and in vivo differentiation studies. BSS1-PBMC-iPS4F4 will provide a powerful tool to study BSS., (Copyright © 2016 Roslin Cells Ltd. Published by Elsevier B.V. All rights reserved.)

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.

The molecular determinants regulating the specification of human embryonic stem cells (hESCs) into hematopoietic cells remain elusive. HOXA9 plays a relevant role in leukemogenesis and hematopoiesis. It is highly expressed in hematopoietic stem and progenitor cells (HSPCs) and is downregulated upon differentiation. Hoxa9-deficient mice display impaired hematopoietic development, and deregulation of HOXA9 expression is frequently associated with acute leukemia. Analysis of the genes differentially expressed in cord blood HSPCs vs hESC-derived HSPCs identified HOXA9 as the most downregulated gene in hESC-derived HSPCs, suggesting that expression levels of HOXA9 may be crucial for hematopoietic differentiation of hESC. Here we show that during hematopoietic differentiation of hESCs, HOXA9 expression parallels hematopoietic development, but is restricted to the hemogenic precursors (HEP) (CD31(+)CD34(+)CD45(-)), and diminishes as HEPs differentiate into blood cells (CD45(+)). Different gain-of-function and loss-of-function studies reveal that HOXA9 enhances hematopoietic differentiation of hESCs by specifically promoting the commitment of HEPs into primitive and total CD45(+) blood cells. Gene expression analysis suggests that nuclear factor-κB signaling could be collaborating with HOXA9 to increase hematopoietic commitment. However, HOXA9 on its own is not sufficient to confer in vivo long-term engraftment potential to hESC-hematopoietic derivatives, reinforcing the idea that additional molecular regulators are needed for the generation of definitive in vivo functional HSPCs from hESC., (© 2014 by The American Society of Hematology.)

Access to healthy or diseased human neural tissue is a daunting task and represents a barrier for advancing our understanding about the cellular, genetic, and molecular mechanisms underlying neurogenesis and neurodegeneration. Reprogramming of somatic cells to pluripotency by transient expression of transcription factors was achieved a few years ago. Induced pluripotent stem cells (iPSC) from both healthy individuals and patients suffering from debilitating, life-threatening neurological diseases have been differentiated into several specific neuronal subtypes. An alternative emerging approach is the direct conversion of somatic cells (i.e., fibroblasts, blood cells, or glial cells) into neuron-like cells. However, to what extent neuronal direct conversion of diseased somatic cells can be achieved remains an open question. Optimization of current expansion and differentiation approaches is highly demanded to increase the differentiation efficiency of specific phenotypes of functional neurons from iPSCs or through somatic cell direct conversion. The realization of the full potential of iPSCs relies on the ability to precisely modify specific genome sequences. Genome editing technologies including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat/CAS9 RNA-guided nucleases have progressed very fast over the last years. The combination of genome-editing strategies and patient-specific iPSC biology will offer a unique platform for in vitro generation of diseased and corrected neural derivatives for personalized therapies, disease modeling and drug screening., (© 2014 The Authors. Stem Cells Published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

The realization of human embryonic stem cells (hESC) as a model for human developmental hematopoiesis and in potential cell replacement strategies relies on an improved understanding of the extrinsic and intrinsic factors regulating hematopoietic-specific hESC differentiation. Human mesenchymal stem cells (hMSCs) are multipotent cells of mesodermal origin that form a part of hematopoietic stem cell niches and have an important role in the regulation of hematopoiesis through production of secreted factors and/or cell-to-cell interactions. We have previously shown that hESCs may be successfully maintained feeder free using hMSC-conditioned media (MSC-CM). Here, we hypothesized that hESCs maintained in MSC-CM may be more prone to differentiation toward hematopoietic lineage than hESCs grown in standard human foreskin fibroblast-conditioned media. We report that specification into hemogenic progenitors and subsequent hematopoietic differentiation and clonogenic progenitor capacity is robustly enhanced in hESC lines maintained in MSC-CM. Interestingly, co-culture of hESCs on hMSCs fully abrogates hematopoietic specification of hESCs, thus suggesting that the improved hematopoietic differentiation is mediated by MSC-secreted factors rather than by MSC-hESC physical interactions. To investigate the molecular mechanism involved in this process, we analyzed global (LINE-1) methylation and genome-wide promoter DNA methylation. hESCs grown in MSC-CM showed a decrease of 17% in global DNA methylation and a promoter DNA methylation signature consisting of 45 genes commonly hypomethylated and 102 genes frequently hypermethylated. Our data indicate that maintenance of hESCs in MSC-CM robustly augments hematopoietic specification and that the process seems mediated by MSC-secreted factors conferring a DNA methylation signature to undifferentiated hESCs which may influence further predisposition toward hematopoietic specification.

Despite the improvements in the human embryonic stem cell (hESC) culture systems, very similar conditions to those used to maintain hESCs on mouse feeders are broadly applied to culture methods based on human feeders. Indeed, basic fibroblast growth factor (bFGF), a master hESC-sustaining factor, is still added in nearly all medium formulations for hESC propagation. Human foreskin fibroblasts (HFFs) and mesenchymal stem cells (MSCs) used as feeders have recently been reported to support hESC growth without exogenous bFGF. However, whether hESCs may be maintained undifferentiated without exogenous bFGF using media conditioned (CM) by human feeders remains elusive. We hypothesize that HFFs and hMSCs are likely to be functionally different and therefore the mechanisms by which HFF-CM and MSC-CM support undifferentiated growth of hESCs may differ. We have thus determined whether HFF-CM and/or MSC-CM sustain feeder-free undifferentiated growth of hESC without exogenous supplementation of bFGF. We report that hMSCs synthesize higher levels of endogenous bFGF than HFFs. Accordingly and in contrast to HFF-CM, MSC-CM produced without the addition of exogenous bFGF supports hESC pluripotency and culture homeostasis beyond 20 passages without the need of bFGF supplementation. hESCs maintained without exogenous bFGF in MSC-CM retained hESC morphology and expression of pluripotency surface markers and transcription factors, formed teratomas, and showed spontaneous and lineage-directed in vitro differentiation capacity. Our data indicate that MSC-CM, but not HFF-CM, provides microenvironment cues supporting feeder-free long-term maintenance of pluripotent hESCs and obviates the requirement of exogenous bFGF at any time.

Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation.

Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.

Global mechanisms defining the gene expression programs specific for hematopoiesis are still not fully understood. Here, we show that promoter DNA demethylation is associated with the activation of hematopoietic-specific genes. Using genome-wide promoter methylation arrays, we identified 694 hematopoietic-specific genes repressed by promoter DNA methylation in human embryonic stem cells and whose loss of methylation in hematopoietic can be associated with gene expression. The association between promoter methylation and gene expression was studied for many hematopoietic-specific genes including CD45, CD34, CD28, CD19, the T cell receptor (TCR), the MHC class II gene HLA-DR, perforin 1 and the phosphoinositide 3-kinase (PI3K) and results indicated that DNA demethylation was not always sufficient for gene activation. Promoter demethylation occurred either early during embryonic development or later on during hematopoietic differentiation. Analysis of the genome-wide promoter methylation status of induced pluripotent stem cells (iPSCs) generated from somatic CD34(+) HSPCs and differentiated derivatives from CD34(+) HSPCs confirmed the role of DNA methylation in regulating the expression of genes of the hemato-immune system, and indicated that promoter methylation of these genes may be associated to stemness. Together, these data suggest that promoter DNA demethylation might play a role in the tissue/cell-specific genome-wide gene regulation within the hematopoietic compartment.

Oct4 is a transcription factor that has been associated with pluripotency and fate determination in the initial cell lineages of mammals. On the other hand, Pou2, the ortholog of Oct4 in zebrafish, serves additional later functions during brain development acting as a differentiation switch. In mice, Oct4 is expressed throughout the neural plate of embryos until embryonic day (E) 8.0. In this study, we produced transgenic mouse embryos that ubiquitously express Oct4 and analyzed the consequences during development. We show that, at E8.0, a higher dosage of Oct4 in the neuroectoderm is sufficient to transiently alter mid-hindbrain patterning and produced a strong up-regulation of Pax2, indicating that Oct4 can regulate this gene in vivo. After E9.5, ectopic Oct4 in this region produced cell death and affected the development of the forebrain, suggesting that, at these later stages, Oct4 down-regulation is necessary for normal development to proceed. The phenotype of the transgenic embryos was also accompanied with an increase of Fgf8 expression in several of its endogenous domains, suggesting the possibility that Oct4 can participate in the regulation of expression of this ligand. Our observations support the hypothesis that Oct4, like zebrafish Pou2, has a conserved function during early brain patterning in mouse.