Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors
ABSTRACT: Here, we report synergistic inhibition of glycogen synthase kinase 3 (GSK3), transforming growth factor β (TGF β), and Notch signaling pathways by small molecules can efficiently convert monolayer cultured hESCs into homogenous primitive neuroepithelium within one week under chemically defined condition. These primitive neuroepithelia can stably self-renew in the presence of leukemia inhibitory factor, GSK3 inhibitor (CHIR99021) and TGF β receptor inhibitor (SB431542); retain high neurogenic potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtypes; and exhibit in vivo integration. hESCs at about 20% confluence were treated with 3 μM CHIR99021, 2 μM SB431542, 0.1 μM Compound E (γ-Secretase Inhibitor XXI) in neural induction media containing Advanced DMEM/F12:Neurobasal (1:1), 1xN2, 1xB27, 1% Glutmax, 5 μg/ml BSA and 10 ng/ml hLIF, for 7 days. The culture was then split 1:3 for the next six passages using Accutase and cultured in neural induction media supplemented with 3 μM CHIR99021 and 2 μM SB431542 on X-ray inactivated MEF feeders or Matrigel-coated plates. After six passages, the cells were split 1:10 regularly. Overall design: Global gene expression analysis of primitive neural stem cells
Project description:Here, we report synergistic inhibition of glycogen synthase kinase 3 (GSK3), transforming growth factor β (TGF β), and Notch signaling pathways by small molecules can efficiently convert monolayer cultured hESCs into homogenous primitive neuroepithelium within one week under chemically defined condition. These primitive neuroepithelia can stably self-renew in the presence of leukemia inhibitory factor, GSK3 inhibitor (CHIR99021) and TGF β receptor inhibitor (SB431542); retain high neurogenic potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtypes; and exhibit in vivo integration. hESCs at about 20% confluence were treated with 3 μM CHIR99021, 2 μM SB431542, 0.1 μM Compound E (γ-Secretase Inhibitor XXI) in neural induction media containing Advanced DMEM/F12:Neurobasal (1:1), 1xN2, 1xB27, 1% Glutmax, 5 μg/ml BSA and 10 ng/ml hLIF, for 7 days. The culture was then split 1:3 for the next six passages using Accutase and cultured in neural induction media supplemented with 3 μM CHIR99021 and 2 μM SB431542 on X-ray inactivated MEF feeders or Matrigel-coated plates. After six passages, the cells were split 1:10 regularly. Global gene expression analysis of primitive neural stem cells
Project description:Throught an reiterative growth factor and chemical screening, we defined a small molecule and growth factor cocktail, including EGF, glycogen synthase kinase 3 inhibitor (CHIR99021), transforming growth factor β receptor inhibitor (e.g. E-616452), lysophosphatidic acid and sphingosine 1-phosphate, that can sustain long-term self-renewal of murine hepatoblasts under chemically defined conditions. The expandable hepatoblasts (eHBs) by this small molecule and growth factor cocktail expressed a set of genes typical of liver progenitor cells and liver development, and retained the ability to respond to liver developmental cues and produce functional hepatocytes and form bile duct-like structures. Moreover, both early- and late-passage eHBs demonstrated a similar transcriptome profile. Microarray analysis also confirmed that the gene expression of cultured cells resembled hepatoblasts. Overall design: Expandable hepatoblasts (eHBs) were routinely cultured in basal medium (DMEM/F12 medium supplemented with insulin-transferrin-sodium selenite, 5 mM nicotinamide, 30 μg/ml 2-phospho-L-ascorbic acid and 50 μg/ml bovine serum albumin human recombinant albumin and 1% penicillin/streptomycin including 10 ng/ml EGF, 3 μM CHIR99021, 2 μM E-616452, 5 μM LPA and 0.5 μM S1P, on Matrigel or laminin-coated surface. The culture was split 1:6 using Accutase. Total RNA was prepared from passage 5 and passage 20 of eHBs (passage 5 and passage 20 of eHBs were derived from two independent experiments) using the RNeasy Plus Mini Kit.
Project description:Analysis of genes induced by CHIR99021 CHIR99021 is a inhibitor of glycogen synthase kinase 3 (GSK3) and can promote B6 mESC sef-renewal when combined with LIF in serum condition. Total RNA obtained from B6 mESCs treated with LIF or LIF/CHIR99021 for 12 hours.
Project description:In this study we showed that rat XEN cells grown in the presence of a GSK3 inhibitor exhibited enhanced formation of cell contacts and decreased motility. In contrast, treatment with forskolin induced the PE formation and epithelial-mesenchymal transition (EMT) in rat XEN cells. Using microarray and real-time PCR assays, we found that VE versus PE formation of rat XEN cells was correlated with change in expression levels of VE or PE marker genes. Similar to forskolin, EMT was prompted upon treatment of rat XEN cells with recombinant parathyroid hormone related peptide (PTHRP), an activator of the cAMP pathway in vivo. Taken together, our data suggest that rat XEN cells are PrE-like cells. The activation of Wnt pathway in rat XEN cells leads to the acquisition of VE characteristics, whereas the activation of the PTHRP/cAMP pathway leads to EMT and the formation of PE. Rat XEN cells were cultured in four different conditions with 3 parallels for each condition: (1-3) without treatments control, (4-6) 2 days treated with CHIR alone, (7-9) 1 day treated with CHIR and 1 day with both CHIR and forskolin, and (10-12) 1 day treated with forskolin alone. In all samples on DAY1 cells were plated, at DAY2 cells were cultured in usual conditions (DMEM F12 medium and 10% Fetal Bovine Serum, both from Sigma) and inhibitor of GSK3 kinase 3 μM CHIR99021 (Axon Medchem) was added in samples 4-9. On DAY3 all cells were cultured in medium with 0.1% serum to exclude the influence of serum and in CONTROL (1-3) cells were cultured without experimental treatments, in CHIR (4-6) cells were further cultured with 3 μM CHIR99021, in CHIR plus FORSKOLIN cells were cultured with 3 μM CHIR99021 and 10μM Forskolin (Sigma), in FORSKOLIN cells were cultured for 1 day with 10 μM Forskolin. On DAY4 RNA was isolated.
Project description:The objective of this study was to investigate the roles of GSK3 inhibitor CHIR99021 and MEK inhibitor PD0325901 on 2i-adapted mouse embryonic stem cells (ESCs) in serum-free conditions.Canonical Wnt signaling supports the pluripotency of mouse ESCs but also promotes differentiation of early mammalian cell lineages. To explain these paradoxical observations, we explored the gene regulatory networks at play. Canonical Wnt signaling is intertwined with the pluripotency network comprising Nanog, Oct4, and Sox2 in mouse ESCs. In defined media supporting the derivation and propagation of mouse ESCs, Tcf3 and β-catenin interact with Oct4; Tcf3 binds to Sox motif within Oct-Sox composite motifs that are also bound by Oct4-Sox2 complexes. Further, canonical Wnt signaling up-regulates the activity of the Pou5f1 distal enhancer via the Sox motif in mouse ESCs. When viewed in the context of published studies on Tcf3 and β-catenin mutants, our findings suggest that Tcf3 counters pluripotency by competition with Sox2 at these sites, and Tcf3 inhibition is blocked by β-catenin entry into this complex. Wnt pathway stimulation also triggers β-catenin association at regulatory elements with classic Lef/Tcf motifs associated with differentiation programs. The failure to activate these targets in the presence of a MEK/ERK inhibitor essential for mouse ESC culture suggests that MEK/ERK signaling and canonical Wnt signaling combine to mouse promote ESC differentiation. Triplicates of mouse embryonic stem cells cultured under the following conditions: 1) CHIR99021+PD0325901+LIF; 2) CHIR99021+PD0325901; 3) CHIR99021; 4) PD0325901; 5) DMSO
Project description:1 million human pluripotent cells cultured as suspension aggregates (hES3-NKX2.5 cell line) were treated with 0, 7.5 or 15µM CHIR99021 in 1 or 3 ml RPMI/B27 (w/o insulin) for 24h to induce the differentiation of human pluripotent stem cells (hPSCs). CHIR99021 is a potent GSK3 inhibitor that results in prominent WNT pathway activation and thereby induces mesendodermal differentiation.
Project description:Human ES (H9) cells were directed towards a neuromesodermal progenitor-like cell state and these cells were then subsequently differentiated towards a neural cell fate. Human ES cells (H9) were differentiated into neuromesodermal progenitor-like cells by culturing in Neurobasal/1x N2/1x B27 medium (N2/B27) supplemented with 20 ng/ml bFgf and 3 μM CHIR99021 for 3 days and exposure to dual SMAD inhibition (dSMADi) (Noggin 50 ng/ml and the TGFb receptor type 1 inhibitor SB431542 10 μM) during day 3 (D3). Transcriptome analysis was then carried out following a selection procedure to enrich for NMP-like cells (sD3/NMP-like). This involved use of a hES (H9) cell line engineered with CRISPR-Cas9 to express GFP under the control of the endogenous Nkx1.2 promoter. At the end of day 3 cells were selected for high GFP expression, as high Nkx1.2 transcription is characteristic of NMP cell populations in mouse and chick embryos. These cells were then lysed and RNA extracted for RNASeq. Humans ES cells (H9) differentiated into NMP-like cells as above (without selection) were also allowed to develop further on day 4 (in the presence of dSMADi and Retinoic acid (RA) 100nM, in N2/B27) and then in RA alone until end of day 8 (D8). These cells were then lysed and RNA extracted for RNASeq.
Project description:Bone morphogenetic protein (BMP) signaling is known to support differentiation of human embryonic stem cells (hESCs) into mesoderm and extraembryonic lineages, whereas other signaling pathways can largely influence this lineage specification. Here, we set out to reinvestigate the influence of ACTIVIN/NODAL and fibroblast growth factor (FGF) pathways on the lineage choices made by hESCs during BMP4-driven differentiation. We show that BMP activation, coupled with inhibition of both ACTIVIN/NODAL and FGF signaling, induces differentiation of hESCs, specifically to βhCG hormone-secreting multinucleated syncytiotrophoblast and does not support induction of embryonic and extraembryonic lineages, extravillous trophoblast, and primitive endoderm. It has been previously reported that FGF2 can switch BMP4-induced hESC differentiation outcome to mesendoderm. Here, we show that FGF inhibition alone, or in combination with either ACTIVIN/NODAL inhibition or BMP activation, supports hESC differentiation to hCG-secreting syncytiotrophoblast. We show that the inhibition of the FGF pathway acts as a key in directing BMP4-mediated hESC differentiation to syncytiotrophoblast. Human embryonic Stem Cells (hESCs) were treated under defined conditions (N2B27) with BMP4 (B), SB431542 (SB) (ACTIVIN/NODAL inhibitor), SU5402 (SU) (FGFR1 inhibitor), FGF2 (F) either alone or in various combinations as mentioned, followed by isolation of total RNA.
Project description:Human pluripotent cells were reset to ground state pluripotency by transient overexpression of NANOG and KLF2 and subsequent inhibition of ERK and protein kinase C. Transcriptional profiling of reset cells and conventional pluripotent stem cell cultures was carried out by RNA-seq, in tandem with mouse embryonic stem cells propagated under similar conditions to assess the combinatorial effects of MEK inhibitor PD0325901, GSK3 inhibitor CHIR99021 and PKC inhibitor Go6983.