Human Amniotic Fluid Stem Cells Modulate Muscle Regeneration
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ell Res C ea m rc te h S & f T Zia et al., J Stem Cell Res Ther 2016, 6:5 o h l Journal of e a DOI: 10.4172/2157-7633.1000339 r n a r p u y o J ISSN: 2157-7633 Stem Cell Research & Therapy Research Article Open Access Human Amniotic Fluid Stem Cells Modulate Muscle Regeneration After Cardiotoxin Injury in Mice Silvia Zia1, Mattia Quattrocelli2, Ester Sara Di Filippo3,4, Nikhil Sindhwani1, Francesca Bosisio5,6, Maurilio Sampaolesi2, Jan Deprest1,7* and Jaan Toelen1,7 1Cluster Organ Systems, Department of Development and Regeneration KU Leuven, Leuven, Belgium 2Translational Cardiomyology Laboratory, Stem Cell Biology and Embryology Unit, Department of Development and Regeneration, KU Leuven, Leuven, Belgium 3Department of Neuroscience Imaging and Clinical Sciences, Interuniversity Institute of Myology (IIM), University “G.d’Annunzio” Chieti-Pescara, Chieti, Italy 4Center for Excellence on Ageing (CeSI),"G.d’Annunzio" Foundation, Chieti, Italy 5Laboratory of Translational Cell and Tissue Research, KU Leuven, Leuven, Belgium 6Department of Surgical Sciences, Milano-Bicocca State University, Monza, Italy 7Division Woman and Child, University Hospitals Leuven, Leuven, Belgium Abstract Amniotic fluid stem cells (AFSc) are a very heterogeneous subtype of stem cells with a broad multi potential. They could be used to treat congenital malformations or diseases. Recently, mesoangioblasts, resident pericytes of skeletal muscles, were shown to undergo muscle differentiation in vitro and in vivo. In this study we focused on the identification of an AFS subtype with pericytic characteristics and evaluate its myogenic potential. We identified monoclonal AFSc lines expressing alkaline phosphatase activity (ALP) and the canonical pericytic markers neural-glial-2 chondroitin sulphate proteglycan (NG2), platelet derived growth factor receptor α and β (PDGFR-α, -β) and α smooth muscle actin (α-SMA). These cells were able to integrate into the newly formed myotubes when co-cultured with the C2C12 cells. To test the paracrine effects of these AFSC on muscle regeneration, we assessed their affects in a transwell assay with acutely injured myotubes. AFSc were able to modulate the expression of specific growth factors involved in muscle regeneration, such as Transforming Growth Factor β (Tgfβ), Interferon γ (Ifnγ), Hepatocyte Growth Factor (Hgf) and Matrix Metalloproteinase 2 (Mmp2). When AFSc were injected in injured muscles they ameliorated muscle repair as measured by the reduction of centronucleated fibers and fibrosis. Interestingly, the transcriptional program of growth factor response in vitro is observed in large part in the in vivo xenograft experimental model, with the extension of Myostatin and Matrix Metalloproteinase 9 (Mmp9). Our data suggest that AFSc subtype with pericytic characteristics have the ability to modulate muscle regeneration in vitro and in vivo. Keywords: Amniotic fluid; Foetal stem cells; Perycite; Muscle early stage in culture and of studying its origin. We recently described a regeneration; Stem cells monoclonal subtype of AFSc that expresses markers that are also found in the fetal urinary system [20]. These cells were able to attenuate the Introduction pathological changes of renal ischemia in a rat model. In recent years, stem cells derived from amniotic fluid have drawn The great potential of AFSc is the possibility of an autologous much attention in treatment of congenital and acquired perinatal approach for congenital malformation or predictable neonatal acquired conditions [1]. Amniotic fluid stem cells (AFSc) approximately conditions, a research topic of our group. At present, Congenital constitute 1% of the cells found in the amniotic fluid (AF). AFSc are Diaphragmatic hernia (CDH) is one of the few conditions where the a heterogeneous population of cells with multipotent characteristics, therapeutic potential of these cells has been investigated [21]. It is a retaining the ability to differentiate into different lineages such as malformation resulting in a diaphragmatic defect, with subsequent adipogenic, chondrogenic, osteogenic and neurogenic [2-4]. The origin lung hypoplasia. Both these aspects could benefit from cellular therapy of these cells remains unknown. Several methods of AFSc isolation either by paracrine effects or by construction of a bioengineered have been described and the isolation method probably influences patch for closure (necessary in around 40% of patients). Currently, the characteristics and differentiation potential of AFSs. AFSc can synthetic and biodegradable patches are used to close the defect with be cultured as a heterogeneous population [5] or isolated from early colonies consisting of two morphologically distinct adherent cell types, high recurrence rates [22]. However, a tissue-engineered patch with termed as spindle-shaped (SS) and round-shaped (RS) [6]. A small fraction of cells have been isolated for the expression of the marker c-Kit (stem cell factor receptor) [2] and shown to be positive for *Corresponding author: Jan Deprest, Division Woman and Child, University Hospitals pluripotency markers [7-11]. AFSc integrated and participated in the Leuven, Leuven, Belgium, Tel: 0496 239 992; E-mail: [email protected] regeneration of damaged organs, such as lung [12], kidney [13], heart Received April 27, 2016; Accepted May 17, 2016; Published May 25, 2016 [14,15] and liver [16,17]. However, aforementioned methodologies Citation: Zia S, Quattrocelli M, Filippo ESD, Sindhwani N, Bosisio F, et al. (2016) do not distinguish among AFSc origins. A potential answer to this Human Amniotic Fluid Stem Cells Modulate Muscle Regeneration After Cardiotoxin unsolved question is to derive a monoclonal population of stem cells Injury in Mice. J Stem Cell Res Ther 6: 339. doi:10.4172/2157-7633.1000339 by means of a single “starter cell” method. AFSc colonies derived from Copyright: © 2016 Zia S, et al. This is an open-access article distributed under one single cell in primary culture were mechanically selected to derive the terms of the Creative Commons Attribution License, which permits unrestricted a monoclonal population [18,19]. The advantage of this methodology use, distribution, and reproduction in any medium, provided the original author and is the possibility of obtaining a monoclonal population of cells at a very source are credited. J Stem Cell Res Ther ISSN: 2157-7633 JSCRT, an open access journal Volume 6 • Issue 5 • 1000339 Citation: Zia S, Quattrocelli M, Filippo ESD, Sindhwani N, Bosisio F, et al. (2016) Human Amniotic Fluid Stem Cells Modulate Muscle Regeneration After Cardiotoxin Injury in Mice. J Stem Cell Res Ther 6: 339. doi:10.4172/2157-7633.1000339 Page 2 of 9 addition of stem cells could provide a more effective and long-lasting 24 h later, the medium was replaced with chondrogenic differentiation solution [23,24]. medium (Invitrogen). Chondrogenic differentiation was determined by Alcian Blue staining (Sigma-Aldrich). In this study, we focused on the identification of a AFSc pool suitable for muscle repair. Recently mesangioblast, a pericytic sub-population Cell lines derived from different gestational weeks were tested of mesenchymal-like cells that associated with the microvasculature for Alkaline Phosphatase activity by enzymatic colorimetric assay wall, have been shown to be able to undergo the skeletal muscle lineage (SIGMAFAST BCIP/NBT tablet, Sigma Aldrich, Diegem, Belgium). and restore muscle functionality [25,26]. Herein we demonstrate that Cells were grown at 100% of confluence and then fixed with 4% of some AFS pools show pericytic characteristics hence have the ability PFA for 10 minutes. The staining was then performed following to integrate in myotubes in vitro and to improve muscle regeneration manufacturer’s protocol. Signal was quantified using ImageJ software process in a mouse model of acute muscle damage in vivo. (Colour deconvolution plugin) in order to measure the percentage of the area stained. Materials and Methods To test whether AFSc share similar characteristics with pericytes, AFSc culture and characterization platelet derived growth factor receptor β (PDGFR-β) (clone 958, Santa Cruz Biotechnology, Heidelberg, Germany) and α (Millipore, Human AFSc were derived and characterized following previously Overijse, Belgium), α-smooth muscle actin (αSMA) (Dako, Heverlee, reported conditions [18]. Briefly, amniotic fluid (AF) was received Belgium) and neural-glial-2 chondroitin sulphate proteglycan (NG2) from consenting women between 15-22 weeks of pregnancy (research (Millipore, Overijse, Belgium) were detected by immunofluorescence. program approved by the Ethics Committee of the UZ Leuven). AF was For the immunofluorescence staining, cells were rinsed twice with cold filtered with a 40 µm strainer (BD bioscience, Erembodegem, Belgium) PBS, and then subjected to fixation in 1% PFA in PBS for 10 min at and centrifuged. The cell pellet was re-suspended in expansion room temperature (RT). Cells were then permeabilized with a solution medium consisting of α-MEM (Invitrogen, Ghent, Belgium), 15% containing 0.2% triton and 2% BSA in PBS for 10 min. Unspecific foetal bovine serum (Invitrogen, Ghent, Belgium), 1% L-glutamine reactions were blocked with 10% donkey serum (Sigma Aldrich) in (Invitrogen, Ghent, Belgium), 1% penicillin/streptomycin (Invitrogen, PBS for 20 min. Cells were then incubated with primary antibody Ghent, Belgium) and 18% Chang B and 2% Chang C (Irvine Scientific, over night at 4°C. After 3 washes 1% BSA in PBS, Alexa Fluor