Transcription profiling by array of human cells of neuroectodermal fate induced from embryonic stem cells by small molecules.
ABSTRACT: Here we demonstrate that hES cells can be reproducibly and efficiently induced to PAX6+ and OCT4- cells with the use of small molecules in animal-component free condition. We are also showing by whole genome microarray and single cell QPCR that induced cells express genes characteristic for the development of anterior neuroectoderm, in particular development of future forebrain. Note: The samples included in this experiment were processed in 3 separate batches and we performed a batch correction step in the analysis as indicated in the transformation protocol.
Project description:Retinal pigment epithelium (RPE) cell integrity is critical to the maintenance of retinal function. Many retinopathies such as age-related macular degeneration (AMD) are caused by the degeneration or malfunction of the RPE cell layer. Replacement of diseased RPE with healthy, stem cell derived RPE is a potential therapeutic strategy for treating AMD. Human embryonic stem cells (hESC) differentiated into RPE progeny have potential to provide an unlimited supply of cells for transplantation but challenges around scalability and efficiency of the differentiation process still remain. Using hESC-derived RPE as a cellular model, we sought to understand mechanisms that could be modulated to increase RPE yield following differentiation. Our data show that activation of the cAMP pathway increases proliferation of dissociated RPE in culture, in part through inhibition of TGFβ signalling. This in turn results in enhanced uptake of epithelial identity. In line with these findings, targeted manipulation of the TGFβ pathway with small molecules produces an increase in efficiency of RPE re-epithelialization. Taken together, these data highlight mechanisms that promote epithelial fate acquisition in stem cell derived RPE. Modulation of these pathways has potential to favorably impact upon scalability and clinical translation of hESC-derived RPE as a cell therapy. A sample of Gene Pool™ cDNA, from human fetal normal brain tissue (Invitrogen D8830-01) is included for reference.
Project description:Development of efficient and reproducible conditions for directed differentiation of pluripotent stem cells into specific cell types is important not only to understand early human development but also to enable more practical applications, such as in vitro models of disease, drug discovery, and cell therapies. The differentiation of stem cells to retinal pigment epithelium (RPE) in particular holds promise as a source of cells for therapeutic replacement in age-related macular degeneration. Here we show development of a robust and efficient method to derive RPE with high reproducibility in an adherent, monolayer system using sequential inhibition and activation of the Activin and BMP signalling pathways. We use whole genome transcript analysis to characterize cells at different stages of differentiation to gain further understanding of the developmental dynamics and fate specification of RPE.
Project description:Safety is the principle consideration with any clinical program, for which hESC and their derived products hold specific challenges. Differentiated cell products derived from hESC must be free from pluripotent cells as these could potentially form teratomas. One relevant clinical program is transplantation of retinal pigment epithelial cells (RPE) derived from hESC. This has potential for halting visual decline in conditions where the RPE layer is damaged such as age-related macular degeneration (AMD). In this study we show that whole genome gene expression analysis of SHEF1.3 starting material and the P0 pigmented RPE foci shows that the two cell types are distinct.
Project description:Retinal Pigment Epithelial (RPE) cells are located behind the retina and are critical for photoreceptor survival. Loss of RPE is associated with several pathogenic conditions such as Age Related Macular Degeneration and Retinitis Pigmentosa. RPE derived from human embryonic stem cells (hESC) offer a potential source for producing these cells for therapy. Here we report the molecular and cellular characterization of RPE differentiated from hESC. hESC derived RPE are capable of proliferation and lose their epithelial characteristics before becoming confluent and re-differentiating back into their typical pigmented, cobblestoned appearance. During the proliferative phase, they adopt a mesenchymal morphology and express mesenchymal markers. Our results demonstrate that this apparent Epithelial-Mesenchymal Transition is not regulated by the classical EMT transcription factors SNAIL and SLUG. Furthermore, it is possible to regulate RPE de-differentiation and re-differentiation by modulating the Wnt and BMP pathway respectively. These findings further our understanding of the genesis and expansion of RPE which is essential for their therapeutic use.
Project description:Serum-starved, quieced HSVSMC (human saphenous vein smooth muscle cells, isolated from the great saphenous vein of coronary artery bypass graft patients) were treated with recombinant human TGFbeta-1 (10ng/mL) in the presence or absence of ALK5 kinase inhibitor SB-525334 (10uM) or ALK1/2 kinase inhibitor KO2288, https://doi.org/10.1371/journal.pone.0062721, (10uM). Gene expression analysis was performed on RNA extracted from cells at the 24h timepoint. RNA samples from three independent experiments (performed on cells from three different patients) were analysed. Quiesced, unstimulated cells were used as a negative control.
Project description:Human trisomies can alter cellular phenotypes and produce congenital abnormalities such as Down Syndrome (DS). Here we have generated induced pluripotent stem cells (iPSCs) from DS fibroblasts, and introduced a TKNEO transgene into one copy of chromosome 21 by gene targeting. When selecting against TKNEO, spontaneous chromosome loss was the most common cause for survival, with a frequency of ~10-4, while point mutations, epigenetic silencing, and TKNEO deletions occurred at lower frequencies in this unbiased comparison of inactivating mutations. Mitotic recombination events resulting in extended loss of heterozygosity were not observed in DS iPSCs. The disomic cells that we derived proliferated faster and produced more endothelia in vivo than their otherwise isogenic trisomic counterparts, but hematopoietic differentiation, pluripotency and survival were statistically unchanged. Our study describes the first targeted removal of a human trisomy, which could prove useful in both clinical and research applications. RNA samples were from two trisomic iPSC clone (C2-4, C3-5) and four derived disomic subclones (C2-4-3, C2-4-4 and C3-5-11, C3-5-13). Duplicate RNA samples from human embyonic stem cells (H1) were included as control.
Project description:New vaccine design approaches would be greatly facilitated by a better understanding of the early systemic changes, and those that occur at the site of injection, responsible for the installation of a durable and oriented protective response. We performed a detailed characterization of very early infection and host response events following the intradermal administration of the modified vaccinia virus Ankara attenuated vaccine in non-human primates. Integrated analysis of the data obtained from in vivo imaging, histology, flow cytometry, multiplex cytokine, and transcriptomic analysis using tools derived from systems biology, such as correlation networks, showed a strong early local and systemic inflammatory response that peaked at 24 h, which was then progressively replaced by an adaptive response during the installation of the host response to the vaccine. Such comprehensive approaches should improve our understanding of how to effectively orientate the immune response, and could contribute to rational vaccine development.
Project description:Characterization of the gene expression changes accompanying the differentiation of hPSC-sensory from embryonic stem cells through to neuronal precursor cells. We also compare the time course gene expression profile to that of the relevant primary human tissue, human dorsal root ganglia (hDRG). A reference brain sample is also included.
Project description:The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their HLA genes. This has led to the proposed use of histocompatible, patient-specific stem cells, however the preparation of many different stem cell lines for clinical use is a daunting task. Here we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I-negative cells by targeted disruption of both alleles of the B2M gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8+ T cell responses were reduced in class I-negative cells that had undergone differentiation in embryoid bodies. These B2M-/- ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines. Total RNA, 4 samples were analyzed as follows: 2 replicates for the hESC H1 wt ( rep1 and rep2) and 2 independent clones of hESC B2M knock-out
Project description:To gain insights into which pathways might be dysregulated in JMML, we compared the transcriptome profiles among hiPSC and sorted CD33+ myeloid cells from control and patient samples. NS/JMML-derived CD33+ myeloid cells showed dysregulation in major biological processes. Moreover, a consistent expression pattern among sets of genes related to pluripotency and myeloid regulation was observed in the control, NS and NS/JMML CD33+ myeloid cells further supporting generally successful myelopoiesis. Total RNA obtained from hiPSC and sorted CD33+ myeloid cells (at day 14 of differentiation) from control and patient samples