Inhibition of miRNA-212/132 improves the reprogramming of fibroblasts into induced pluripotent stem cells by de-repressing important epigenetic remodelling factors.

MicroRNAs (miRNAs) repeatedly have been demonstrated to play important roles in the generation of induced pluripotent stem cells (iPSCs). To further elucidate the molecular mechanisms underlying transcription factor-mediated reprogramming we have established a model, which allows for the efficient screening of whole libraries of miRNAs modulating the generation of iPSCs from murine embryonic fibroblasts. Applying this model, we identified 14 miRNAs effectively inhibiting iPSC generation, including miR-132 and miR-212. Intriguingly, repression of these miRNAs during iPSC generation also resulted in significantly increased reprogramming efficacy. MiRNA target evaluation by qRT-PCR, Western blot, and luciferase assays revealed two crucial epigenetic regulators, the histone acetyl transferase p300 as well as the H3K4 demethylase Jarid1a (KDM5a) to be directly targeted by both miRNAs. Moreover, we demonstrated that siRNA-mediated knockdown of either p300 or Jarid1a recapitulated the miRNA effects and led to a significant decrease in reprogramming efficiency. Thus, conducting a full library miRNA screen we here describe a miRNA family, which markedly reduces generation of iPSC and upon inhibition in turn enhances reprogramming. These miRNAs, at least in part, exert their functions through repression of the epigenetic modulators p300 and Jarid1a, highlighting these two molecules as an endogenous epigenetic roadblock during iPSC generation.

Lost in translation: pluripotent stem cell-derived hematopoiesis.

Pluripotent stem cells (PSCs) such as embryonic stem cells or induced pluripotent stem cells represent a promising cell type to gain novel insights into human biology. Understanding the differentiation process of PSCs in vitro may allow for the identification of cell extrinsic/intrinsic factors, driving the specification process toward all cell types of the three germ layers, which may be similar to the human in vivo scenario. This would not only lay the ground for an improved understanding of human embryonic development but would also contribute toward the generation of novel cell types used in cell replacement therapies. In this line, especially the developmental process of mesodermal cells toward the hematopoietic lineage is of great interest. Therefore, this review highlights recent progress in the field of hematopoietic specification of pluripotent stem cell sources. In addition, we would like to shed light on emerging factors controlling primitive and definitive hematopoietic development and to highlight recent approaches to improve the differentiation potential of PSC sources toward hematopoietic stem/progenitor cells. While the generation of fully defined hematopoietic stem cells from PSCs remains challenging in vitro, we here underline the instructive role of cell extrinsic factors such as cytokines for the generation of PSC-derived mature hematopoietic cells. Thus, we have comprehensively examined the role of cytokines for the derivation of mature hematopoietic cell types such as macrophages, granulocytes, megakaryocytes, erythrocytes, dendritic cells, and cells of the B- and T-cell lineage.

Large-scale hematopoietic differentiation of human induced pluripotent stem cells provides granulocytes or macrophages for cell replacement therapies.

Interleukin-3 (IL-3) is capable of supporting the proliferation of a broad range of hematopoietic cell types, whereas granulocyte colony-stimulating factor (G-CSF) and macrophage CSF (M-CSF) represent critical cytokines in myeloid differentiation. When this was investigated in a pluripotent-stem-cell-based hematopoietic differentiation model, IL-3/G-CSF or IL-3/M-CSF exposure resulted in the continuous generation of myeloid cells from an intermediate myeloid-cell-forming complex containing CD34(+) clonogenic progenitor cells for more than 2 months. Whereas IL-3/G-CSF directed differentiation toward CD45(+)CD11b(+)CD15(+)CD16(+)CD66b(+) granulocytic cells of various differentiation stages up to a segmented morphology displaying the capacity of cytokine-directed migration, respiratory burst response, and neutrophil-extracellular-trap formation, exposure to IL-3/M-CSF resulted in CD45(+)CD11b(+)CD14(+)CD163(+)CD68(+) monocyte/macrophage-type cells capable of phagocytosis and cytokine secretion. Hence, we show here that myeloid specification of human pluripotent stem cells by IL-3/G-CSF or IL-3/M-CSF allows for prolonged and large-scale production of myeloid cells, and thus is suited for cell-fate and disease-modeling studies as well as gene- and cell-therapy applications.

Effects of genistein and estrogen receptor subtype-specific agonists in ArKO mice following different administration routes.

We have scrutinized the effects of the phytoestrogen genistein and three synthetic estrogen receptor agonists, 17 alpha-ethynylestradiol (EE), propylpyrazole-triol (PPT) and diarylpropionitrile (DPN) in the completely estrogen-free background of aromatase knockout (ArKO) mice by means of two routes of substance administration: oral via diet (per os; po) or subcutaneous injection (sc) with the intention to evaluate the ArKO mice as sensitive model organism for uterotrophic assays. Additionally, we were aiming to qualitatively analyze effects resulting from oral administration path, in particular for PPT and DPN. Therefore, we analyzed the resulting uterine wet weights (UWW) and epithelial heights as physiological endpoints of function as well as the gonadotropin levels. Moreover, the gene expression profiles of estrogen receptors as well as important uterine and ovarian estrogen-response genes were investigated by real-time PCR. The uterus of ArKO mice responded very sensitive upon the substitution with EE (sc 5 microg/kg BW; po 50 microg/kg BW) in a proliferative manner. This was evaluated inter alia by increased UWW and by up-regulation of the expression of proliferation-associated and estrogen-response genes. It is important to note, that ER alpha and ER beta-agonist, PPT and DPN respectively (po 5mg/kg BW and sc 0.5mg/kg BW), have only been used for sc applications so far. Here, effects resulting from oral application were qualitatively described and evaluated for their applicability. The UWW and expression of proliferation-associated genes were increased following both po and sc treatment with PPT. In contrast, DPN did not exert an increase of the UWW, but a significant decrease of proliferation-associated gene and protein expression. Additionally, a substantial hypoplasia was detectable in the uterine cross-sections of DPN-treated mice. On the other hand, the phytoestrogen genistein (sc 10mg/kg BW; po 70 mg/kg BW) did not cause detectable uterotrophic responses or large changes of uterine and ovarian gene expression profiles under the applied experimental conditions, but significantly reduced the elevated gonadotropin levels of ArKO mice. In summary, we showed the utility of ArKO mice to detect ER-specific effects, in particular those of PPT and DPN also when applied orally.