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Alternative Routes to Induced Pluripotent Stem Cells Revealed by Reprogramming of the Neural Lineage.

ABSTRACT: During the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells, the activation of pluripotency genes such as NANOG occurs after the mesenchymal to epithelial transition. Here we report that both adult stem cells (neural stem cells) and differentiated cells (astrocytes) of the neural lineage can activate NANOG in the absence of cadherin expression during reprogramming. Gene expression analysis revealed that only the NANOG+E-cadherin+ populations expressed stabilization markers, had upregulated several cell cycle genes; and were transgene independent. Inhibition of DOT1L activity enhanced both the numbers of NANOG+ and NANOG+E-cadherin+ colonies in neural stem cells. Expressing SOX2 in MEFs prior to reprogramming did not alter the ratio of NANOG colonies that express E-cadherin. Taken together these results provide a unique pathway for reprogramming taken by cells of the neural lineage.

SUBMITTER: Jackson SA

PROVIDER: S-EPMC4788781 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Alternative Routes to Induced Pluripotent Stem Cells Revealed by Reprogramming of the Neural Lineage.

Jackson Steven A SA Olufs Zachariah P G ZP Tran Khoa A KA Zaidan Nur Zafirah NZ Sridharan Rupa R

Stem cell reports 20160218 3

During the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells, the activation of pluripotency genes such as NANOG occurs after the mesenchymal to epithelial transition. Here we report that both adult stem cells (neural stem cells) and differentiated cells (astrocytes) of the neural lineage can activate NANOG in the absence of cadherin expression during reprogramming. Gene expression analysis revealed that only the NANOG+E-cadherin+ populations expressed stabili ...[more]

PMID: 26905202

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Alternative routes to induced pluripotent stem cells revealed by reprogramming of neural lineage

Project description:During the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells, the activation of pluripotency genes such as Nanog occurs after the mesenchymal to epithelial transition (MET). Here we report that both adult stem cells (neural stem cells- NSCs) and differentiated cells (astrocytes) of the neural lineage can activate Nanog in the absence of cadherin expression during reprogramming. Gene expression analysis revealed that only the Nanog+Ecadherin+ populations expressed stabilization markers and had upregulated several cell cycle genes; and were transgene independent. Inhibition of Dot1L activity, which has previously been shown to increase MET, enhanced both the numbers of Nanog+ and Nanog+E-cadherin+ colonies, suggesting a MET independent pathway for activation of Nanog in NSCs. Expressing Sox2 in MEFs prior to reprogramming does not alter the ratio of Nanog colonies that express E-cadherin obtained. Taken together these results provide a novel pathway for reprogramming taken by cells of the neural lineage. Neural Stem Cells (NSCs) were induced to reprogram by the induction of Oct4, Sox2, c-Myc and Klf4. Reprogramming colonies that expressed either Nanog alone (N+E-) or Nanog and E-cadherin (N+E+) were sorted by flow cytometry and used as input for RNA-sequencing. There are 3 replicates each for the N+E- and N+E+ samples.

2016-02-18 | E-GEOD-76130 | biostudies-arrayexpress

Alternative routes to induced pluripotent stem cells revealed by reprogramming of neural lineage

2016-02-18 | GSE76130 | GEO

Lineage-specific purification of neural stem/progenitor cells from differentiated mouse induced pluripotent stem cells.

Project description:Since induced pluripotent stem (iPS) cells have differentiation potential into all three germ layer-derived tissues, efficient purification of target cells is required in many fields of iPS research. One useful strategy is isolation of desired cells from differentiated iPS cells by lineage-specific expression of a drug-resistance gene, followed by drug selection. With this strategy, we purified neural stem/progenitor cells (NSCs), a good candidate source for regenerative therapy, from differentiated mouse iPS cells. We constructed a bicistronic expression vector simultaneously expressing blasticidin S resistance gene and DsRed under the control of tandem enhancer of a 257-base pair region of nestin second intron, an NSC-specific enhancer. This construct was efficiently inserted into the iPS genome by piggyBac transposon-mediated gene transfer, and the established subclone was differentiated into NSCs in the presence or absence of blasticidin S. Consequently, incubation with blasticidin S led to purification of NSCs from differentiated iPS cells. Our results suggest that a lineage-specific drug selection strategy is useful for purification of NSCs from differentiated iPS cells and that this strategy can be applied for the purification of other cell types.

| S-EPMC3673754 | biostudies-literature

Switching cell fate: the remarkable rise of induced pluripotent stem cells and lineage reprogramming technologies.

Project description:Cell reprogramming, in which a differentiated cell is made to switch its fate, is an emerging field with revolutionary prospects in biotechnology and medicine. The recent discovery of induced pluripotency by means of in vitro reprogramming has made way for unprecedented approaches for regenerative medicine, understanding human disease and drug discovery. Moreover, recent studies on regeneration and repair by direct lineage reprogramming in vivo offer an attractive novel alternative to cell therapy. Although we continue to push the limits of current knowledge in the field of cell reprogramming, the mechanistic elements that underlie these processes remain largely elusive. This article reviews landmark developments in cell reprogramming, current knowledge, and technological developments now on the horizon with significant promise for biomedical applications.

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Notch promotes neural lineage entry by pluripotent embryonic stem cells.

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Reprogramming of pancreatic beta cells into induced pluripotent stem cells.

Project description:Induced pluripotent stem (iPS) cells have been derived from fibroblast, stomach, and liver cultures at extremely low frequencies by ectopic expression of the transcription factors Oct4, Sox2, c-myc, and Klf4, a process coined direct or in vitro reprogramming [1-8]. iPS cells are molecularly and functionally highly similar to embryonic stem cells (ESCs), including their ability to contribute to all tissues as well as the germline in mice. The heterogeneity of the starting cell populations and the low efficiency of reprogramming suggested that a rare cell type, such as an adult stem cell, might be the cell of origin for iPS cells and that differentiated cells are refractory to reprogramming. Here, we used inducible lentiviruses [9] to express Oct4, Sox2, c-myc, and Klf4 in pancreatic beta cells to assess whether a defined terminally differentiated cell type remains amenable to reprogramming. Genetically marked beta cells gave rise to iPS cells that expressed pluripotency markers, formed teratomas, and contributed to cell types of all germ layers in chimeric animals. Our results provide genetic proof that terminally differentiated cells can be reprogrammed into pluripotent cells, suggesting that in vitro reprogramming is not restricted to certain cell types or differentiation stages.

| S-EPMC2819222 | biostudies-literature

Reprogramming of Cancer Cells into Induced Pluripotent Stem Cells Questioned.

Project description:Background and objectivesSeveral recent studies have claimed that cancer cells can be reprogrammed into induced pluripotent stem cells (iPSCs). However, in most cases, cancer cells seem to be resistant to cellular reprogramming. Furthermore, the underlying mechanisms of limited reprogramming in cancer cells are largely unknown. Here, we identified the candidate barrier genes and their target genes at the early stage of reprogramming for investigating cancer reprogramming.MethodsWe tried induction of pluripotency in normal human fibroblasts (BJ) and both human benign (MCF10A) and malignant (MCF7) breast cancer cell lines using a classical retroviral reprogramming method. We conducted RNA-sequencing analysis to compare the transcriptome of the three cell lines at early stage of reprogramming.ResultsWe could generate iPSCs from BJ, whereas we were unable to obtain iPSCs from cancer cell lines. To address the underlying mechanism of limited reprogramming in cancer cells, we identified 29 the candidate barrier genes based on RNA-sequencing data. In addition, we found 40 their target genes using Cytoscape software.ConclusionsOur data suggest that these genes might one of the roadblock for cancer cell reprogramming. Furthermore, we provide new insights into application of iPSCs technology in cancer cell field for therapeutic purposes.

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Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1.

Project description:Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4.

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Probabilistic Modeling of Reprogramming to Induced Pluripotent Stem Cells.

Project description:Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is typically an inefficient and asynchronous process. A variety of technological efforts have been made to accelerate and/or synchronize this process. To define a unified framework to study and compare the dynamics of reprogramming under different conditions, we developed an in silico analysis platform based on mathematical modeling. Our approach takes into account the variability in experimental results stemming from probabilistic growth and death of cells and potentially heterogeneous reprogramming rates. We suggest that reprogramming driven by the Yamanaka factors alone is a more heterogeneous process, possibly due to cell-specific reprogramming rates, which could be homogenized by the addition of additional factors. We validated our approach using publicly available reprogramming datasets, including data on early reprogramming dynamics as well as cell count data, and thus we demonstrated the general utility and predictive power of our methodology for investigating reprogramming and other cell fate change systems.

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