EGF and HB-EGF enhance the proliferation of programmable cells of monocytic origin (PCMO) through activation of MEK/ERK signaling and improve differentiation of PCMO-derived hepatocyte-like cells

Abstract Background Hepatocyte-like cells (NeoHepatocytes) generated from a peripheral blood monocyte-derived stem cell-like cell (the PCMO) are a promising alternative for primary hepatocytes in cell transplantation studies to cure liver diseases. However, to be therapeutically effective NeoHepatocytes are needed in large quantities. It was the aim of the present study to investigate i) whether the proportion of actively proliferating NeoHepatocytes can be enhanced by supplementing the PCMO differentiation medium (containing M-CSF, IL-3, and human serum) with either EGF or HB-EGF and ii) which signaling pathway underlies the promitotic effect. Results EGF and HB-EGF enhanced cell proliferation of PCMOs as demonstrated by increased expression of cycle control genes (ABL, ANAPC2, CDC2, CDK4, CDK6), phosphorylation of the retinoblastoma protein, and increased PCMO cell numbers after stimulation with EGF or HB-EGF. EGF also raised the number of monocytes expressing the proliferation marker Ki67. PCMOs expressed the EGF receptors EGFR (ERBB1) and ERBB3, and expression of both increased during PCMO generation. Phosphoimmunoblotting of PCMOs indicated that both EGF and HB-EGF activated MEK-1/2 and ERK1/2 in a concentration-dependent fashion with the effect of EGF being more prominent. EGF treatment further decreased expression of p47phox and increased that of Nanog indicating enhanced dedifferentiation and pluripotency, respectively. Treatment with both EGF and HB-EGF resulted in NeoHepatocytes with improved functional parameters. Conclusions The results suggested that the addition of EGF or HB-EGF to PCMO differentiation medium superactivates MEK/ERK signaling which then increases both PCMO proliferation, number, and functional differentiation of PCMO-derived NeoHepatocytes.</p

The mutation p. E113K in the Schiff base counterion of rhodopsin is associated with two distinct retinal phenotypes within the same family

The diagnoses of retinitis pigmentosa (RP) and stationary night blindness (CSNB) are two distinct clinical entities belonging to a group of clinically and genetically heterogeneous retinal diseases. The current study focused on the identification of causative mutations in the RP-affected index patient and in several members of the same family that reported a phenotype resembling CSNB. Ophthalmological examinations of the index patient confirmed a typical form of RP. In contrast, clinical characterizations and ERGs of another affected family member showed the Riggs-type CSNB lacking signs of RP. Applying whole exome sequencing we detected the non-synonymous substitution c.337G > A, p. E113 K in the rhodopsin (RHO) gene. The mutation co-segregated with the diseases. The identification of the pathogenic variant p. E113 K is the first description of a naturally-occurring mutation in the Schiff base counterion of RHO in human patients. The heterozygous mutation c. 337G > A in exon 1 was confirmed in the index patient as well as in five CSNB-affected relatives. This pathogenic sequence change was excluded in a healthy family member and in 199 ethnically matched controls. Our findings suggest that a mutation in the biochemically well-characterized counterion p.E113 in RHO can be associated with RP or Riggs-type CSNB, even within the same family

Cartoon to illustrate the effects of activins and TGF-βs, their receptors and target genes, and their inhibitors, on pluripotency and growth of adherently growing PCMO.

<p>Stimulatory (↓) and inhibitory (⊥) interactions are depicted by bold and dashed lines to indicate strong and weak interactions, respectively. Ab, antibody.</p

MicroRNA-24 antagonism prevents renal ischemia reperfusion injury

Ischemia-reperfusion (I/R) injury of the kidney is a major cause of AKI. MicroRNAs (miRs) are powerful regulators of various diseases. We investigated the role of apoptosis-associated miR-24 in renal I/R injury. miR-24 was upregulated in the kidney after I/R injury of mice and in patients after kidney transplantation. Cell-sorting experiments revealed a specific miR-24 enrichment in renal endothelial and tubular epithelial cells after I/R induction. In vitro, anoxia/hypoxia induced an enrichment of miR-24 in endothelial and tubular epithelial cells. Transient overexpression of miR-24 alone induced apoptosis and altered functional parameters in these cells, whereas silencing of miR-24 ameliorated apoptotic responses and rescued functional parameters in hypoxic conditions. miR-24 effects were mediated through regulation of H2A histone family, member X, and heme oxygenase 1, which were experimentally validated as direct miR-24 targets through luciferase reporter assays. In vitro, adenoviral overexpression of miR-24 targets lacking miR-24 binding sites along with miR-24 precursors rescued various functional parameters in endothelial and tubular epithelial cells. In vivo, silencing of miR-24 in mice before I/R injury resulted in a significant improvement in survival and kidney function, a reduction of apoptosis, improved histologic tubular epithelial injury, and less infiltration of inflammatory cells. miR-24 also regulated heme oxygenase 1 and H2A histone family, member X, in vivo. Overall, these results indicate miR-24 promotes renal ischemic injury by stimulating apoptosis in endothelial and tubular epithelial cell. Therefore, miR-24 inhibition may be a promising future therapeutic option in the treatment of patients with ischemic AKI

PCMO resume proliferation during a 4-day culture period.

<p>Peripheral blood monocytes cultured in 6-well plates were cultured for up to 4 days (d) under adherent (a) or suspended (s) growth conditions and subjected on the indicated days to staining for Ki67 and immunoblotting of p21^WAF1. <i>Bar</i>, 50 μm. (A) <i>In situ</i> staining of PCMO cultures with Ki67 (red) and DAPI (blue). (B) Quantification of Ki67/DAPI-double positive cells in adhesion and suspension cultures from four different donors. Standard deviations were below 15%. *, p<0.05. (C) kinetics of p21^WAF1 expression. The bands for p21^WAF1 and α-tubulin, used as a loading control, were densitometrically analyzed. Displayed are the normalized values for p21^WAF1 relative to those on day 1 set arbitrarily at 1.0, from one representative donor out of four different donors analyzed in total.</p

Identification of the autocrine ligands that regulate pluripotency gene expression and proliferation in PCMO.

<p>Effect of SB431542 (SB), follistatin (FS), and anti-TGF-β antibody (α-TGF-β) on day 4 of culture on (A) Smad2C phosphorylation as determined by immunoblotting and ELISA (graph below blot). Co, isotype control. Numbers in brackets indicate the concentrations used (μM for SB and μg/ml for FS and α-TGF-β. (B) Oct4A and Nanog expression as determined by qPCR (graphs) and immunoblotting. Oct4 and Nanog were immunodetected in nuclear proteins (25 μg/lane) from the same day 3 (Oct4) and day 4 (Nanog) PCMO used for qPCR analysis. Successful enrichment of nuclear proteins and equal protein loading was verified with an antibody to histone H4. (C) Effect of FS, and α-TGF-β on Smad3C phosphorylation and p21^WAF1 expression in day 4 PCMO as determined by immunoblotting and ELISA (graphs below blots). (D) Ki67 expression on day 4 of culture. In (B) and (D), SB, FS, and α-TGF-β were used at concentrations of 1 μM, 0.1 μg/ml, and 5 μg/ml, respectively. ELISA data in A and C, and qPCR data in B and D are means ± SD from triplicate samples and were derived from one donor. Data are representative of four different donors. Asterisks indicate a significant difference between vehicle and SB or FS-treated cells and between Co and α-TGF-β-treated cells.</p

PCMO respond to exogenous activins and TGF-β1 with phosphorylation of Smad2.

<p>(A) PCMO were stimulated on day 4 of culture with 50 ng/ml of either activin A, activin B, or activin AB in the presence or absence of the ALK4/5/7 inhibitor SB431542 (1 μM), the p38 MAPK inhibitor SB203580 (10 μM), vehicle (dimethylsulfoxide, 0.1%), or medium alone (w/o) for 1 h followed by immunoblotting for phosphorylated Smad2C (p-Smad2), total Smad2 (Smad2), and β-actin as a loading control. (B) Responsiveness of PCMO from two different donors to TGF-β1 stimulation. PCMO were stimulated on day 4 with 5 ng/ml TGF-β1 in the presence or absence of SB431542 (1 μM), or vehicle for 1 h followed by immunoblotting for p-Smad2C and Smad2. Data in A and B are representative of four different donors.</p

Expression of cell cycle regulating genes in PCMO following neutralization of endogenous activin(s) and TGF-β(s).

<p>QPCR-based detection of the indicated genes after treatment of PCMO cultures with follistatin or anti-TGF-β antibody. Data represent the-fold stimulation relative to untreated cells (follistatin) or isotype IgG1-treated control cells (anti-TGF-β antibody) on day 4 of culture, mean ± SD. Data (means ± SD from triplicate samples) are from one donor and are representative of four different donors. ANAPC2, anaphase promoting complex subunit 2; CDC2, cell division control protein 2; CDK, cyclin-dependent kinase.</p