Sphere formation permits Oct4 reprogramming of ciliary body epithelial cells into induced pluripotent stem cells.
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ABSTRACT:
SUBMITTER: Ni A
PROVIDER: S-EPMC4267128 | biostudies-literature | 2014 Dec
REPOSITORIES: biostudies-literature
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Project description:Induced pluripotent stem (iPS) cells are attractive for cell replacement therapy, because they overcome ethical and immune rejection issues that are associated with embryonic stem cells. iPS cells have been derived from autonomous fibroblasts at low efficiency using multiple ectopic transcription factors. Recent evidence suggests that the epigenome of donor cell sources plays an important role in the reprogramming and differentiation characteristics of iPS cells. Thus, identification of somatic cell types that are easily accessible and are more amenable for cellular reprogramming is critical for regenerative medicine applications. Here, we identify ciliary body epithelial cells (CECs) as a new cell type for iPS cell generation that has higher reprogramming efficiency compared with fibroblasts. The ciliary body is composed of epithelial cells that are located in the anterior portion of the eye at the level of the lens and is readily surgically accessible. CECs also have a reduced reprogramming requirement, as we demonstrate that ectopic Sox2 and c-Myc are dispensable. Enhanced reprogramming efficiency may be due to increased basal levels of Sox2 in CECs. In addition, we are the first to report a cellular reprogramming haploinsufficiency observed when reprogramming with fewer factors (Oct4 and Klf4) in Sox2 hemizygous cells. Taken together, endogenous Sox2 levels are critical for the enhanced efficiency and reduced exogenous requirement that permit facile cellular reprogramming of CECs.
| S-EPMC3760062 | biostudies-literature
Project description:The activation of endogenous Oct4 transcription is a key step in the reprogramming of somatic cells into induced pluripotent stem (iPS) cells but until now it has been difficult to analyze this critical event in the reprogramming process. We have generated a transgenic mouse that expresses the tamoxifen-inducible Cre recombinase MerCreMer under the control of the endogenous Oct4 locus, enabling lineage tracing of Oct4 expression in cells in vivo or in vitro, during either reprogramming or differentiation. Using this novel resource, we have determined the timing and outcome of endogenous Oct4 induction during fibroblast reprogramming. We show that both the initiation of this key reprogramming step and the ability of cells activating endogenous Oct4 expression to complete reprogramming are not influenced by the presence of exogenous c-Myc, although the overall efficiency of the process is increased by c-Myc. Oct4 lineage tracing reveals that new reprogramming events continue to initiate over a period of 3 weeks. Furthermore, the analysis of mixed colonies, where only a subset of daughter cells induce endogenous Oct4 expression, indicates the role of unknown, stochastic events in the progression of reprogramming from the initial events to a pluripotent state. Our transgenic mouse model and cells derived from it provide powerful and precise new tools for the study of iPS cell reprogramming mechanisms and have wider implications for the investigation of the role of Oct4 during development.
| S-EPMC3626284 | biostudies-literature
Project description:A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.
| S-EPMC4489496 | biostudies-literature
Project description:The advent of induced pluripotent stem cells--generated via the ectopic overexpression of reprogramming factors such as OCT4, SOX2, KLF4, and C-MYC (OSKM) in a differentiated cell type--has enabled groundbreaking research efforts in regenerative medicine, disease modeling, and drug discovery. Although initial studies have focused on the roles of nuclear factors, increasing evidence highlights the importance of signal transduction during reprogramming. By utilizing a quantitative, medium-throughput screen to initially identify signaling pathways that could potentially replace individual transcription factors during reprogramming, we initially found that several pathways--such as Notch, Smoothened, and cyclic AMP (cAMP) signaling--were capable of generating alkaline phosphatase positive colonies in the absence of OCT4, the most stringently required Yamanaka factor. After further investigation, we discovered that cAMP signal activation could functionally replace OCT4 to induce pluripotency, and results indicate that the downstream exchange protein directly activated by cAMP (EPAC) signaling pathway rather than protein kinase A (PKA) signaling is necessary and sufficient for this function. cAMP signaling may reduce barriers to reprogramming by contributing to downstream epithelial gene expression, decreasing mesenchymal gene expression, and increasing proliferation. Ultimately, these results elucidate mechanisms that could lead to new reprogramming methodologies and advance our understanding of stem cell biology.
| S-EPMC4427878 | biostudies-other
Project description:Atrionatriuretic peptide (ANP) lowers intraocular pressure in the eyes of humans and rabbits. We examined the effects of natriuretic peptides on cGMP formation and 125I-labelled-ANP binding to cultured cells derived from ciliary body epithelium, the site of aqueous humour formation in the eye. ANP, brain natriuretic peptide (BNP) and C-natriuretic peptide (CNP) at 1 microM stimulated cGMP formation 8.2(+/-1.2)-fold, 4.8(+/-0.6)-fold and 87.3(+/-12.1)-fold respectively. 125I-ANP bound to intact cells at a single site, with a dissociation constant KD=0.30+/-0.01 nM. BNP was as effective as ANP in displacing 125I-ANP, whereas CNP displaced label with a slightly higher IC50. 125I-ANP binding was displaced >95% by c-ANP, a specific ligand for natriuretic peptide C receptors (NPR-C). Cross-linking of 125I-ANP to cells labelled predominantly a protein of Mr 62000. These data suggest that 125I-ANP binding was primarily to NPR-C, whereas cGMP stimulation occurred primarily via natriuretic peptide B receptors (NPR-B). Vasopressin and histamine, both activators of the inositol phosphate/diacylglycerol phosphate pathway in non-pigmented ciliary epithelial cells, inhibited CNP stimulation of guanylate cyclase (NPR-B) and 125I-ANP binding (NPR-C) by 30-38%. Inhibition was mimicked by PMA, dioctanoylglycerol and phorbol didecanoate, whereas 4alpha phorbol didecanoate had no effect. Staurosporine and bisindolylmaleimide both blocked inhibition of 125I-ANP binding and cGMP formation by PMA. These results suggest that protein kinase C (PKC) down-regulates both NPR-B and NPR-C. PKC down-regulation of NPR-B varied inversely with CNP concentration. Inhibition by 1 microM PMA was 30.6(+/-4.0)% with 500 nM CNP, but 83.4(+/-8.8)% with 10 nM CNP, indicating that increasing CNP could partially overcome inhibition by PMA. Since extracellular CNP levels were not affected by PKC activation, the effect of PKC on NPR-B is best explained as a reduction in NPR-B affinity for CNP. NPR-C measured as 125I-ANP binding was likewise reduced 36.4(+/-5.1)% by exposure to PMA. In contrast with NPR-B inhibition, however, inhibition of NPR-C was due largely to a reduction in the number of receptor binding sites per cell rather than a reduction in receptor affinity for ligand. The data therefore suggest that both NPR-B and NPR-C are down-regulated by PKC, but that the mechanisms of down-regulation of the two receptors are different.
| S-EPMC1218400 | biostudies-other
Project description:Autologous retinal pigment epithelium (RPE) grafts derived from induced pluripotent stem cells (iPSCs) may be used to cure blinding diseases in which RPE dysfunction results in photoreceptor degeneration. Four-, two-, and one-factor-derived iPSCs (4F-, 2F-, and 1F-iPSCs, respectively) were differentiated into fully functional cuboidal pigmented cells in polarized monolayers that express RPE-specific markers. 1F-iPSCs-RPE (1F-iPS-RPE) strongly resembles primary human fetal RPE (hfRPE) based on proteomic and untargeted metabolomic analyses, and using novel in vivo imaging technology coupled with electroretinography, we demonstrated that 1F-iPS-RPE mediate anatomical and functional rescue of photoreceptors after transplantation in an animal model of RPE-mediated retinal degeneration. 1F-iPS0RPE cells were injected subretinally as a suspension and formed a monolayer dispersed between host RPE cells. Furthermore, 1F-iPS-RPE do not simply provide trophic support to rescue photoreceptors as previously speculated but actually phagocytose photoreceptor outer segments in vivo and maintain visual cycling. Thus, 1f-iPS-RPE grafts may be superior to conventional iPS-RPE for clinical use because 1F-IPS-RPE closely resemble hfRPE, mediate anatomical and functional photoreceptor rescue in vivo, and are generated using a reduced number of potentially oncogenic reprogramming factors.
| S-EPMC3328503 | biostudies-literature
Project description:Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) eliminates many epigenetic modifications that characterize differentiated cells. In this study, we tested whether functional differences between chronic obstructive pulmonary disease (COPD) and non-COPD fibroblasts could be reduced utilizing this approach. Primary fibroblasts from non-COPD and COPD patients were reprogrammed to iPSCs. Reprogrammed iPSCs were positive for oct3/4, nanog, and sox2, formed embryoid bodies in vitro, and induced teratomas in nonobese diabetic/severe combined immunodeficient mice. Reprogrammed iPSCs were then differentiated into fibroblasts (non-COPD-i and COPD-i) and were assessed either functionally by chemotaxis and gel contraction or for gene expression by microarrays and compared with their corresponding primary fibroblasts. Primary COPD fibroblasts contracted three-dimensional collagen gels and migrated toward fibronectin less robustly than non-COPD fibroblasts. In contrast, redifferentiated fibroblasts from iPSCs derived from the non-COPD and COPD fibroblasts were similar in response in both functional assays. Microarray analysis identified 1,881 genes that were differentially expressed between primary COPD and non-COPD fibroblasts, with 605 genes differing by more than twofold. After redifferentiation, 112 genes were differentially expressed between COPD-i and non-COPD-i with only three genes by more than twofold. Similar findings were observed with microRNA (miRNA) expression: 56 miRNAs were differentially expressed between non-COPD and COPD primary cells; after redifferentiation, only 3 miRNAs were differentially expressed between non-COPD-i and COPD-i fibroblasts. Interestingly, of the 605 genes that were differentially expressed between COPD and non-COPD fibroblasts, 293 genes were changed toward control after redifferentiation. In conclusion, functional and epigenetic alterations of COPD fibroblasts can be reprogrammed through formation of iPSCs.
| S-EPMC3949084 | biostudies-literature
Project description:During embryonic development, epithelial cell blocks called somites are periodically formed according to the segmentation clock, becoming the foundation for the segmental pattern of the vertebral column. The process of somitogenesis has recently been recapitulated with murine and human pluripotent stem cells. However, an in vitro model for human somitogenesis coupled with the segmentation clock and epithelialization is still missing. Here, we report the generation of human somitoids, organoids that periodically form pairs of epithelial somite-like structures. Somitoids display clear oscillations of the segmentation clock that coincide with the segmentation of the presomitic mesoderm. The resulting somites show anterior-posterior and apical-basal polarities. Matrigel is essential for epithelialization but dispensable for the differentiation into somite cells. The size of somites is rather constant, irrespective of the initial cell number. The amount of WNT signaling instructs the proportion of mesodermal lineages in somitoids. Somitoids provide a novel platform to study human somitogenesis.
| S-EPMC9050736 | biostudies-literature
Project description:BackgroundInduced pluripotent stem cells (iPSCs) hold tremendous potential, both as a biological tool to uncover the pathophysiology of disease by creating relevant human cell models and as a source of cells for cell-based therapeutic applications. Studying the reprogramming process will also provide significant insight into tissue development.ObjectiveWe sought to characterize the derivation of iPSC lines from nasal epithelial cells (NECs) isolated from nasal mucosa samples of children, a highly relevant and easily accessible tissue for pediatric populations.MethodsWe performed detailed comparative analysis on the transcriptomes and methylomes of NECs, iPSCs derived from NECs (NEC-iPSCs), and embryonic stem cells (ESCs).ResultsNEC-iPSCs express pluripotent cell markers, can differentiate into all 3 germ layers in vivo and in vitro, and have a transcriptome and methylome remarkably similar to those of ESCs. However, residual DNA methylation marks exist, which are differentially methylated between NEC-iPSCs and ESCs. A subset of these methylation markers related to epithelium development and asthma and specific to NEC-iPSCs persisted after several passages in vitro, suggesting the retention of an epigenetic memory of their tissue of origin. Our analysis also identified novel candidate genes with dynamic gene expression and DNA methylation changes during reprogramming, which are indicative of possible roles in airway epithelium development.ConclusionNECs are an excellent tissue source to generate iPSCs in pediatric asthmatic patients, and detailed characterization of the resulting iPSC lines would help us better understand the reprogramming process and retention of epigenetic memory.
| S-EPMC4289122 | biostudies-literature
Project description:The primary cilium is a critical sensory organelle that is built of axonemal microtubules ensheathed by a ciliary membrane. In polarized epithelial cells, primary cilia reside on the apical surface and must extend these microtubules directly into the extracellular space and remain a stable structure. However, the factors regulating cross-talk between ciliation and cell polarization, as well as axonemal microtubule growth and stabilization in polarized epithelia are not fully understood. In this study, we find TTLL12, a previously uncharacterized member of the Tubulin Tyrosine Ligase-Like (TTLL) family, localizes to the base of primary cilia and is required for cilia formation in polarized renal epithelial cells. We also show that TTLL12 directly binds to the /-tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications (PTMs). While all other TTLLs catalyze the addition of glutamate or glycine to microtubule C-terminal tails, TTLL12 uniquely affects tubulin PTMs by promoting both microtubule lysine acetylation and arginine methylation. Together, this work identifies a novel microtubule regulator and provides insight into the requirements for apical extracellular axoneme formation.
| S-SCDT-10_1038-S44319-023-00005-5 | biostudies-other