Conversion of human fibroblasts into monocyte-like progenitor cells.

Reprogramming technologies have emerged as a promising approach for future regenerative medicine. Here, we report on the establishment of a novel methodology allowing for the conversion of human fibroblasts into hematopoietic progenitor-like cells with macrophage differentiation potential. SOX2 overexpression in human fibroblasts, a gene found to be upregulated during hematopoietic reconstitution in mice, induced the rapid appearance of CD34+ cells with a concomitant upregulation of mesoderm-related markers. Profiling of cord blood hematopoietic progenitor cell populations identified miR-125b as a factor facilitating commitment of SOX2-generated CD34+ cells to immature hematopoietic-like progenitor cells with grafting potential. Further differentiation toward the monocytic lineage resulted in the appearance of CD14+ cells with functional phagocytic capacity. In vivo transplantation of SOX2/miR-125b-generated CD34+ cells facilitated the maturation of the engrafted cells toward CD45+ cells and ultimately the monocytic/macrophage lineage. Altogether, our results indicate that strategies combining lineage conversion and further lineage specification by in vivo or in vitro approaches could help to circumvent long-standing obstacles for the reprogramming of human cells into hematopoietic cells with clinical potential.

MicroRNAs contribute to induced pluripotent stem cell somatic donor memory.

Induced pluripotent stem cells (iPSCs) maintain during the first few culture passages a set of epigenetic marks and metabolites characteristic of their somatic cell of origin, a concept defined as epigenetic donor memory. These residual somatic features are lost over time after extensive culture passaging. Therefore, epigenetic donor memory may be responsible for the higher differentiation efficiency toward the tissue of origin observed in low passage iPSCs versus high passage iPSC or iPSCs derived from a different tissue source. Remarkably, there are no studies on the relevance of microRNA (miRNA) memory following reprogramming, despite the established role of these molecules in the context of pluripotency and differentiation. Using hematopoietic progenitors cells as a model, we demonstrated that miRNAs play a central role in somatic memory retention in iPSCs. Moreover, the comparison of the miRNA expression profiles among iPSCs from different sources allowed for the detection of a set of candidate miRNAs responsible for the higher differentiation efficiency rates toward blood progenitors observed in low passage iPSCs. Combining bioinformatic predictive algorithms with biological target validation, we identified miR-155 as a key player for the in vitro differentiation of iPSC toward hematopoietic progenitors. In summary, this study reveals that during the initial passages following reprogramming, iPSCs maintained the expression of a miRNA set exclusive to the original somatic population. Hence the use of these miRNAs might hold a direct application toward our understanding of the differentiation process of iPSCs toward hematopoietic progenitor cells.

Characterization of pluripotent stem cells.

Characterization of pluripotent stem cells is required for the registration of stem cell lines and allows for an impartial and objective comparison of the results obtained when generating multiple lines. It is therefore crucial to establish specific, fast and reliable protocols to detect the hallmarks of pluripotency. Such protocols should include immunocytochemistry (takes 2 d), identification of the three germ layers in in vitro-derived embryoid bodies by immunocytochemistry (immunodetection takes 3 d) and detection of differentiation markers in in vivo-generated teratomas by immunohistochemistry (differentiation marker detection takes 4 d). Standardization of the immunodetection protocols used ensures minimum variations owing to the source, the animal species, the endogenous fluorescence or the inability to collect large amounts of cells, thereby yielding results as fast as possible without loss of quality. This protocol provides a description of all the immunodetection procedures necessary to characterize mouse and human stem cell lines in different circumstances.

N-acetylcysteine protects induced pluripotent stem cells from in vitro stress: impact on differentiation outcome.

Induced pluripotent stem cells (iPSCs) have the ability to differentiate towards various cell types of the adult organism and are a potential source of transplantable material in regenerative medicine. The entire process of conversion of iPSCs into terminally differentiated cells takes place in vitro and requires long periods of time. During in vitro culture, cells are exposed to environmental factors, which are capable of decreasing cellular performance and viability. Oxidative stress is the major underlying mechanism of such negative impact of in vitro environmental factors. We aimed to study the alteration of cellular properties during in vitro hematopoietic differentiation of human iPSCs and the ability of N-acetylcysteine (NAC), a potent free radical scavenger, to prevent such alterations. IPSCs were differentiated towards hematopoietic cells in the presence of 1 mM NAC. Intracellular reactive oxygen species (ROS), nitric oxide (NO), senescence, apoptosis and mitochondrial membrane potential (MMP) were evaluated at 1 and 3 weeks of differentiation. In the course of hematopoietic differentiation of iPSCs, cells progressively accumulated intracellular ROS and NO, increased the levels of apoptosis and senescence, and showed a decrease in mitochondrial functionality. NAC supplementation reversed all these phenomena. NAC administration also improved hematopoietic differentiation of iPSCs in terms of production of CD34, CD45 and CD43 positive cells. In conclusion, when supplemented during hematopoietic differentiation of iPSCs, NAC decreased oxidative stress, rescued the decline in cellular properties induced by long-term in vitro culture and promoted hematopoietic differentiation of iPSCs.

Methylation and silencing of miRNA-124 by EVI1 and self-renewal exhaustion of hematopoietic stem cells in murine myelodysplastic syndrome.

By expressing EVI1 in murine bone marrow (BM), we previously described a myelodysplastic syndrome (MDS) model characterized by pancytopenia, dysmegakaryopoiesis, dyserythropoiesis, and BM failure. The mice invariably died 11-14 months after BM transplantation (BMT). Here, we show that a double point mutant EVI1-(1+6Mut), unable to bind Gata1, abrogates the onset of MDS in the mouse and re-establishes normal megakaryopoiesis, erythropoiesis, BM function, and peripheral blood profiles. These normal features were maintained in the reconstituted mice until the study was ended at 21 months after BMT. We also report that EVI1 deregulates several genes that control cell division and cell self-renewal. In striking contrast, these genes are normalized in the presence of the EVI1 mutant. Moreover, EVI1, but not the EVI1 mutant, seemingly deregulates these cellular processes by altering miRNA expression. In particular, the silencing of miRNA-124 by DNA methylation is associated with EVI1 expression, but not that of the EVI1 mutant, and appears to play a key role in the up-regulation of cell division in murine BM cells and in the hematopoietic cell line 32Dcl3. The results presented here demonstrate that EVI1 induces MDS in the mouse through two major pathways, both of which require the interaction of EVI1 with other factors: one, results from EVI1-Gata1 interaction, which deregulates erythropoiesis and leads to fatal anemia, whereas the other occurs by interaction of EVI1 with unidentified factors causing perturbation of the cell cycle and self-renewal, as a consequence of silencing miRNA-124 by EVI1 and, ultimately, ensues in BM failure.

Unilateral ischemic preconditioning and heterologous preconditioning in living donor liver transplantation.

Ischemic preconditioning (IP) exerts a protective effect on tissues undergoing prolonged ischemia. No studies have been performed to assess the clinical impact of IP on normal human liver used for living donor transplantation (LDLT). Heterologous preconditioning (HP) protects liver tissue as demonstrated in a rat model. Our study investigates the impact that IP and HP have on the donor and recipient liver in LDLT. Twenty candidates for living donor right hepatectomy were divided in two groups. The study group underwent 10' unilateral ischemia by clamping the right portal vein and hepatic artery at the end of the parenchymal transection. Demographics, laboratory values, biopsy studies, IL-1Ra, Ki-67, and CytoDEATH stains were compared. The results show that 10' ischemia does not exert significant clinical and laboratory changes in living donor hepatectomy candidates.

Cholera toxin inhibits IL-12 production and CD8alpha+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function.

Prior studies have demonstrated that cholera toxin (CT) and other cAMP-inducing factors inhibit interleukin (IL)-12 production from monocytes and dendritic cells (DCs). We show that CT inhibits Th1 responses in vivo in mice infected with Toxoplasma gondii. This correlated with low serum IL-12 levels and a selective reduction in the numbers of CD8alpha(+) conventional DCs (cDCs) in lymphoid organs. CT inhibited the function of interferon (IFN) regulatory factor (IRF) 8, a transcription factor known to positively regulate IL-12p35 and p40 gene expression, and the differentiation of CD8alpha(+) and plasmacytoid DCs (pDCs). Fluorescence recovery after photobleaching analysis showed that exposure to CT, forskolin, or dibutyryl (db) cAMP blocked LPS and IFN-gamma-induced IRF8 binding to chromatin. Moreover, CT and dbcAMP inhibited the binding of IRF8 to the IFN-stimulated response element (ISRE)-like element in the mouse IL-12p40 promoter, likely by blocking the formation of ISRE-binding IRF1-IRF8 heterocomplexes. Furthermore, CT inhibited the differentiation of pDCs from fms-like tyrosine kinase 3 ligand-treated bone marrow cells in vitro. Therefore, because IRF8 is essential for IL-12 production and the differentiation of CD8alpha(+) cDCs and pDCs, these data suggest that CT and other Gs-protein agonists can affect IL-12 production and DC differentiation via a common mechanism involving IRF8.

EVI1 Impairs myelopoiesis by deregulation of PU.1 function.

EVI1 is an oncogene inappropriately expressed in the bone marrow (BM) of approximately 10% of myelodysplastic syndrome (MDS) patients. This disease is characterized by severe anemia and multilineage myeloid dysplasia that are thought to be a major cause of mortality in MDS patients. We earlier reported on a mouse model that constitutive expression of EVI1 in the BM led to fatal anemia and myeloid dysplasia, as observed in MDS patients, and we subsequently showed that EVI1 interaction with GATA1 blocks proper erythropoiesis. Whereas this interaction could provide the basis for the erythroid defects in EVI1-positive MDS, it does not explain the alteration of myeloid differentiation. Here, we have examined the expression of several genes activated during terminal myelopoiesis in BM cells and identified a group of them that are altered by EVI1. A common feature of these genes is their regulation by the transcription factor PU.1. We report here that EVI1 interacts with PU.1 and represses the PU.1-dependent activation of a myeloid promoter. EVI1 does not seem to inhibit PU.1 binding to DNA, but rather to block its association with the coactivator c-Jun. After mapping the PU.1-EVI1 interaction sites, we show that an EVI1 point mutant, unable to bind PU.1, restores the activation of PU.1-regulated genes and allows a normal differentiation of BM progenitors in vitro.

Significant increase of self-renewal in hematopoietic cells after forced expression of EVI1.

EVI1 was first identified as a preferential integration site of ecotropic retroviruses in the MDS1/EVI1 genomic locus leading to myeloid tumors in susceptible mice. Later studies showed that retroviral integration in the MDS1/EVI1 locus results in the emergence of a non-malignant dominant hematopoietic stem cell clone in non-susceptible mice strains, in non-human primates, and in patients, suggesting that a gene encoded by the locus could affect the self-renewal potential of a cell. The locus encodes two genes. One of them, EVI1, has long been associated with myeloid leukemia. To understand whether EVI1 has a role in self-renewal control, we forcibly expressed EVI1 in the bone marrow progenitors of two mice strains that differ in their proliferation and self-renewal potential. By comparing the response of the hematopoietic cells to EVI1, we conclude that EVI1 has a role in prolonging the self-renewal potential of the cells and that this ability of EVI1 is limited and modulated by inherent characteristics of the cells.

Point mutations in two EVI1 Zn fingers abolish EVI1-GATA1 interaction and allow erythroid differentiation of murine bone marrow cells.

EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.

EVI1 and hematopoietic disorders: history and perspectives.

The ecotropic viral integration site 1 (EVI1) gene was identified almost 20 years ago as the integration site of an ecotropic retrovirus leading to murine myeloid leukemia. Since its identification, EVI1 has slowly been recognized as one of the most aggressive oncogenes associated with human leukemia. Despite the effort of many investigators, still very little is known about this gene. The mechanism by which EVI1 operates in the transformation of hematopoietic cells is not known, but it is clear that EVI1 upregulates cell proliferation, impairs cell differentiation, and induces cell transformation. In this review, we summarize the biochemical properties of EVI1 and the effects of EVI1 in biological models.

Partner-regulated interaction of IFN regulatory factor 8 with chromatin visualized in live macrophages.

IFN regulatory factor (IRF) 8 is a transcription factor that directs macrophage differentiation. By fluorescence recovery after photobleaching, we visualized the movement of IRF8-GFP in differentiating macrophages. Recovery data fitted to mathematical models revealed two binding states for IRF8. The majority of IRF8 was highly mobile and transiently interacted with chromatin, whereas a small fraction of IRF8 bound to chromatin more stably. IRF8 mutants that did not stimulate macrophage differentiation showed a faster recovery, revealing little interaction with chromatin. A macrophage activation signal by IFN-gamma/LPS led to a global slowdown of IRF8 movement, leading to increased chromatin binding. In fibroblasts where IRF8 has no known function, WT IRF8 moved as fast as the mutants, indicating that IRF8 does not interact with chromatin in these cells. However, upon introduction of IRF8 binding partners, PU.1 and/or IRF1, the mobility of IRF8 was markedly reduced, producing a more stably bound component. Together, IRF8-chromatin interaction is dynamic in live macrophages and influenced by partner proteins and immunological stimuli.

Comparison between the surface plasmon resonance (SPR) and the quartz crystal microbalance (QCM) method in a structural analysis of human endothelin-1.

In this study, an automated surface plasmon resonance (SPR)-based biosensor was compared with a quartz crystal microbalance (QCM) biosensor. The two biosensor systems were used for characterizing a site-directed monoclonal antibody (mAb), raised against the C-terminal heptapeptide ET-1(15-21) of the human endothelin (ET-1). The mAb was characterized by its capacity for binding to ET-1, ET-3, Big.ET-1(22-38), the C-terminal (ET-1(15-21), ET-1(16-21), ET-1(17-21)), and six derivates of ET-1(16-21), each containing a substitution with alanine (Ala) of a single aminoacid from position 16-21, respectively. The mAb reacted well with ET-1 and its fragments ET-1(15-21), ET-1(16-21), ET-1(17-21), but showed only a partial cross-reaction with ET-3, and did not bind human Big.ET-1(22-38). The Ala substitution on position 16,17, or 19 of ET-1(16-21) did not affect the antibody binding capacity of the hexapaptide ET-1(16-21). On the contrary, Ala substitution or Asp(18), Ile(20) and particularly Trp(21), inhibited its immunoreactivity. Thus the C-terminal represents an immunodominant epitope in ET-1 and is important for antibody binding. The SPR and QCM response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. With regard to the fundamental problem of comparing different measurement principles, we found a good correlation between results obtained using the BIA technology and the QCM.