Ashraf Al Madhoun

Ascl1/Mash1 is a novel target of Gli2 during Gli2-induced neurogenesis in P19 EC cells.

The Sonic Hedgehog (Shh) signaling pathway is important for neurogenesis in vivo. Gli transcription factors, effector proteins of the Shh signaling pathway, have neurogenic properties in vivo, which are still poorly understood. To study the molecular basis of neurogenic properties of Gli2, we used a well-established embryonic stem cell model, the P19 embryonal carcinoma (EC) cell line, which can be induced to differentiate into neurons in the presence of retinoic acid (RA). We found that, in the absence of RA, overexpression of Gli2 induced P19 EC cells to differentiate into neurons, but not astrocytes during the first ten days of differentiation. To our knowledge, this is the first indication that the expression of Gli factors can convert EC cells into neurons. Furthermore, Gli2 upregulated expression of the neurogenic basic helix-loop-helix (bHLH) factors, such as NeuroD, Neurog1 and Ascl1/Mash1 in P19 EC cells. Using chromatin immunoprecipitation assays, we showed that Gli2 bound to multiple regulatory regions in the Ascl1 gene, including promoter and enhancer regions during Gli2-induced neurogenesis. In addition, Gli2 activated the Ascl1/Mash1 promoter in vitro. Using the expression of a dominant-negative form of Gli2, fused to the Engrailed repression domain, we observed a reduction in gliogenesis and a significant downregulation of the bHLH factors Ascl1/Mash1, Neurog1 and NeuroD, leading to delayed neurogenesis in P19 EC cells, further supporting the hypothesis that Ascl1/Mash1 is a direct target of Gli2. In summary, Gli2 is sufficient to induce neurogenesis in P19 stem cells at least in part by directly upregulating Ascl1/Mash1. Our results provide mechanistic insight into the neurogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo neurogenic properties.

Gli2 and MEF2C activate each other's expression and function synergistically during cardiomyogenesis in vitro

The transcription factors Gli2 (glioma-associated factor 2), which is a transactivator of Sonic Hedgehog (Shh) signalling, and myocyte enhancer factor 2C (MEF2C) play important roles in the development of embryonic heart muscle and enhance cardiomyogenesis in stem cells. Although the physiological importance of Shh signalling and MEF2 factors in heart development is well known, the mechanistic understanding of their roles is unclear. Here, we demonstrate that Gli2 and MEF2C activated each others expression while enhancing cardiomyogenesis in differentiating P19 EC cells. Furthermore, dominant-negative mutant proteins of either Gli2 or MEF2C repressed each others expression, while impairing cardiomyogenesis in P19 EC cells. In addition, chromatin immunoprecipitation (ChIP) revealed association of Gli2 to the Mef2c gene, and of MEF2C to the Gli2 gene in differentiating P19 cells. Finally, co-immunoprecipitation studies showed that Gli2 and MEF2C proteins formed a complex, capable of synergizing on cardiomyogenesis-related promoters containing both Gli- and MEF2-binding elements. We propose a model whereby Gli2 and MEF2C bind each others regulatory elements, activate each others expression and form a protein complex that synergistically activates transcription, enhancing cardiac muscle development. This model links Shh signalling to MEF2C function during cardiomyogenesis and offers mechanistic insight into their in vivo functions.

Hedgehog Signaling Regulates MyoD Expression and Activity

Background: Hedgehog (Hh) signaling regulates skeletal myogenesis; however, the molecular mechanisms involved are not fully understood. Results: Gli2, a transactivator of Hh signaling, associates with MyoD gene elements, regulating MyoD expression, and binds to MyoD protein, regulating its ability to induce myogenesis. Conclusion: Hh signaling is linked to MyoD gene expression and MyoD protein function. Significance: Novel mechanistic insight is gained into the Hh-regulated myogenesis. The inhibition of MyoD expression is important for obtaining muscle progenitors that can replenish the satellite cell niche during muscle repair. Progenitors could be derived from either embryonic stem cells or satellite cells. Hedgehog (Hh) signaling is important for MyoD expression during embryogenesis and adult muscle regeneration. To date, the mechanistic understanding of MyoD regulation by Hh signaling is unclear. Here, we demonstrate that the Hh effector, Gli2, regulates MyoD expression and associates with MyoD gene elements. Gain- and loss-of-function experiments in pluripotent P19 cells show that Gli2 activity is sufficient and required for efficient MyoD expression during skeletal myogenesis. Inhibition of Hh signaling reduces MyoD expression during satellite cell activation in vitro. In addition to regulating MyoD expression, Hh signaling regulates MyoD transcriptional activity, and MyoD activates Hh signaling in myogenic conversion assays. Finally, Gli2, MyoD, and MEF2C form a protein complex, which enhances MyoD activity on skeletal muscle-related promoters. We therefore link Hh signaling to the function and expression of MyoD protein during myogenesis in stem cells.

Skeletal myosin light chain kinase regulates skeletal myogenesis by phosphorylation of MEF2C

The MEF2 factors regulate transcription during cardiac and skeletal myogenesis. MEF2 factors establish skeletal muscle commitment by amplifying and synergizing with MyoD. While phosphorylation is known to regulate MEF2 function, lineage‐specific regulation is unknown. Here, we show that phosphorylation of MEF2C on T80 by skeletal myosin light chain kinase (skMLCK) enhances skeletal and not cardiac myogenesis. A phosphorylation‐deficient MEF2C mutant (MEFT80A) enhanced cardiac, but not skeletal myogenesis in P19 stem cells. Further, MEFT80A was deficient in recruitment of p300 to skeletal but not cardiac muscle promoters. In gain‐of‐function studies, skMLCK upregulated myogenic regulatory factor (MRF) expression, leading to enhanced skeletal myogenesis in P19 cells and more efficient myogenic conversion. In loss‐of‐function studies, MLCK was essential for efficient MRF expression and subsequent myogenesis in embryonic stem (ES) and P19 cells as well as for proper activation of quiescent satellite cells. Thus, skMLCK regulates MRF expression by controlling the MEF2C‐dependent recruitment of histone acetyltransferases to skeletal muscle promoters. This work identifies the first kinase that regulates MyoD and Myf5 expression in ES or satellite cells.

Multi-Lineage Differentiation of Human Umbilical Cord Wharton’s Jelly Mesenchymal Stromal Cells Mediates Changes in the Expression Profile of Stemness Markers

Wharton’s Jelly- derived Mesenchymal stem cells (WJ-MSCs) have gained interest as an alternative source of stem cells for regenerative medicine because of their potential for self-renewal, differentiation and unique immunomodulatory properties. Although many studies have characterized various WJ-MSCs biologically, the expression profiles of the commonly used stemness markers have not yet been addressed. In this study, WJ-MSCs were isolated and characterized for stemness and surface markers expression. Flow cytometry, immunofluorescence and qRT-PCR analysis revealed predominant expression of CD29, CD44, CD73, CD90, CD105 and CD166 in WJ-MSCs, while the hematopoietic and endothelial markers were absent. Differential expression of CD 29, CD90, CD105 and CD166 following adipogenic, osteogenic and chondrogenic induction was observed. Furthermore, our results demonstrated a reduction in CD44 and CD73 expressions in response to the tri-lineage differentiation induction, suggesting that they can be used as reliable stemness markers, since their expression was associated with undifferentiated WJ-MSCs only.

Testosterone enhances cardiomyogenesis in stem cells and recruits the androgen receptor to the MEF2C and HCN4 genes

Since a previous study (Goldman-Johnson et al., 2008 [4]) has shown that androgens can stimulate increased differentiation of mouse embryonic stem (mES) cells into cardiomyocytes using a genomic pathway, the aim of our study is to elucidate the molecular mechanisms regulating testosterone-enhanced cardiomyogenesis. Testosterone upregulated cardiomyogenic transcription factors, including GATA4, MEF2C, and Nkx2.5, muscle structural proteins, and the pacemaker ion channel HCN4 in a dose-dependent manner, in mES cells and P19 embryonal carcinoma cells. Knock-down of the androgen receptor (AR) or treatment with anti-androgenic compounds inhibited cardiomyogenesis, supporting the requirement of the genomic pathway. Chromatin immunoprecipitation (ChIP) studies showed that testosterone enhanced recruitment of AR to the regulatory regions of MEF2C and HCN4 genes, which was associated with increased histone acetylation. In summary, testosterone upregulated cardiomyogenic transcription factor and HCN4 expression in stem cells. Further, testosterone induced cardiomyogenesis, at least in part, by recruiting the AR receptor to the regulatory regions of the MEF2C and HCN4 genes. These results provide a detailed molecular analysis of the function of testosterone in stem cells and may offer molecular insight into the role of steroids in the heart.

Novel G6B gene variant causes familial autosomal recessive thrombocytopenia and anemia.

To characterize the underlying genetic and molecular defects in a consanguineous family with lifelong blood disorder manifested with thrombocytopenia (low platelets count) and anemia.

The Rise and the Fall of Betatrophin/ANGPTL8 as an Inducer of β-Cell Proliferation

Diabetes is a global health problem that is caused by impaired insulin production from pancreatic β-cells. Efforts to regenerate β-cells have been advancing rapidly in the past two decades with progress made towards identifying new agents that induce β-cells regeneration. ANGPTL8, also named betatrophin, has been recently identified as a hormone capable of inducing β-cells proliferation and increasing β-cells mass in rodents. Its discovery has been cherished as a breakthrough and a game changer in the field of β-cells regeneration. Initially, ANGPTL8 has been identified as atypical member of the angiopoietin-like protein family as a regulator of triglyceride in plasma through its interaction with ANGPTL3 and its regulation of lipoprotein lipase activity. In this review, we will review literature on the proposed role of ANGPTL8 in β-cells proliferation, the controversy regarding this role, and the emerging data questioning its involvement in β-cells proliferation. Additionally we will discuss new clinical data that describes its role in diabetes and the putative therapeutic targeting of this protein.

Dental pulp pluripotent-like stem cells (DPPSC), a new stem cell population with chromosomal stability and osteogenic capacity for biomaterials evaluation

BackgroundBiomaterials are widely used to regenerate or substitute bone tissue. In order to evaluate their potential use for clinical applications, these need to be tested and evaluated in vitro with cell culture models. Frequently, immortalized osteoblastic cell lines are used in these studies. However, their uncontrolled proliferation rate, phenotypic changes or aberrations in mitotic processes limits their use in long-term investigations. Recently, we described a new pluripotent-like subpopulation of dental pulp stem cells derived from the third molars (DPPSC) that shows genetic stability and shares some pluripotent characteristics with embryonic stem cells. In this study we aim to describe the use of DPPSC to test biomaterials, since we believe that the biomaterial cues will be more critical in order to enhance the differentiation of pluripotent stem cells.MethodsThe capacity of DPPSC to differentiate into osteogenic lineage was compared with human sarcoma osteogenic cell line (SAOS-2). Collagen and titanium were used to assess the cell behavior in commonly used biomaterials. The analyses were performed by flow cytometry, alkaline phosphatase and mineralization stains, RT-PCR, immunohistochemistry, scanning electron microscopy, Western blot and enzymatic activity. Moreover, the genetic stability was evaluated and compared before and after differentiation by short-comparative genomic hybridization (sCGH).ResultsDPPSC showed excellent differentiation into osteogenic lineages expressing bone-related markers similar to SAOS-2. When cells were cultured on biomaterials, DPPSC showed higher initial adhesion levels. Nevertheless, their osteogenic differentiation showed similar trend among both cell types. Interestingly, only DPPSC maintained a normal chromosomal dosage before and after differentiation on 2D monolayer and on biomaterials.ConclusionsTaken together, these results promote the use of DPPSC as a new pluripotent-like cell model to evaluate the biocompatibility and the differentiation capacity of biomaterials used in bone regeneration.

The Effect of Commercially Available Endodontic Cements and Biomaterials on Osteogenic Differentiation of Dental Pulp Pluripotent-Like Stem Cells

The aim of this study is to compare the osteogenic differentiation capacity of the dental pulp pluripotent-like stem cells (DPPSCs) using conditional media pretreated with ProRoot-MTA, Biodentine (BD) or the newly manufactured pure Portland cement Med-PZ (MZ). DPPSCs, isolated from human third molars, are the most relevant cell model to draw conclusions about the role of biomaterials on dental tissue regeneration. Cytotoxicity, alkaline phosphatase (ALP) activity, and calcium deposition analysis were evaluated at different differentiation time points. Gene expression of key osteogenic markers (RUNX2, Collagen I and Osteocalcin) was determined by qRT-PCR analysis. The osteogenic capacity of cells cultured in conditioned media prepared from MZ or MTA cements was comparable. BD conditioned media supported cell proliferation but failed to induce osteogenesis. Relative to controls and other cements, high osteogenic gene expression was observed in cultures pre-treated with the novel endodontic cement MZ. In conclusion, the in vitro behavior of a MZ- endodontic cement was evaluated, showing similar enhanced cell proliferation compared to other commercially available cements but with an enhanced osteogenic capacity with prospective potential as a novel cement for endodontic treatments.