15 research outputs found
Correction: Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential
[This corrects the article DOI: 10.1371/journal.pone.0137999.]
Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential
A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM). We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs) onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-to-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol
Monoclonal Antibody-Targeted Fluorescein-5-isothiocyanate-Labeled Biomimetic Nanoapatites: A Promising Fluorescent Probe for Imaging Applications
Multifunctional biomimetic nanoparticles (NPs) are acquiring increasing interest as carriers in medicine and basic research since they can efficiently combine labels for subsequent tracking, moieties for specific cell targeting, and bioactive molecules, e.g., drugs. In particular, because of their easy synthesis, low cost, good biocompatibility, high resorbability, easy surface functionalization, and pH-dependent solubility, nanocrystalline apatites are promising candidates as nanocarriers. This work describes the synthesis and characterization of bioinspired apatite nanoparticles to be used as fluorescent nanocarriers targeted against the Met/hepatocyte growth factor receptor, which is considered a tumor associated cell surface marker of many cancers. To this aim the nanoparticles have been labeled with Fluorescein-5-isothiocyanate (FITC) by simple isothermal adsorption, in the absence of organic, possibly toxic, molecules, and then functionalized with a monoclonal antibody (mAb) directed against such a receptor. Direct labeling of the nanoparticles allowed tracking the moieties with spatiotemporal resolution and thus following their interaction with cells, expressing or not the targeted receptor, as well as their fate in vitro. Cytofluorometry and confocal microscopy experiments showed that the functionalized nanocarriers, which emitted a strong fluorescent signal, were rapidly and specifically internalized in cells expressing the receptor. Indeed, we found that, once inside the cells expressing the receptor, mAb-functionalized FITC nanoparticles partially dissociated in their two components, with some mAbs being recycled to the cell surface and the FITC-labeled nanoparticles remaining in the cytosol. This work thus shows that FITC-labeled nanoapatites are very promising probes for targeted cell imaging applications
Telomere length (A) and telomerase expression (B).
<p>Variation in spheroids and reseeded spheroids vs monolayers. Data expressed as mean ± SD. (<b>C</b>) Cell spreading from spheroids after reseeding on culture 2D plates. Scale bar 120 μm.</p
Migratory activity of hCPCs kept as monolayers or of cells migrated from seeded spheroids (wound healing assay).
<p>(<b>A</b>) Quiescent cell monolayers were “wounded” with a pipette tip and incubated for 24 hours in the absence (C-) or presence of 20% FBS (C+) or of different concentrations of the HGF receptor agonist mAb DO-24 (5, 20, 80 nM respectively). (<b>B</b>) Quantitative analysis calculated as % variation of cell numbers in the rectangles depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137999#pone.0137999.g005" target="_blank">Fig 5A</a> relative to untreated samples. The assay was repeated three times with triplicates and one representative assay is reported. Images were taken at 5x magnification. After 24 hours healing was significantly induced by 20% FBS and mAb DO-24 treatments. Cells derived from spheroids (dark grey bars) were more efficient than cells from 2D cultures at any condition (light grey bars).</p
Primary antibodies used for immunofluorescence.
<p>Primary antibodies used for immunofluorescence.</p
Gene expression of different growth factors, their receptors, ECM molecules, adhesion/invasiveness components and cardiac markers in spheroids and monolayer cultures.
<p>Data from qPCR are expressed as fold variation of gene expression in spheroids normalized <i>vs</i> monolayers (line). Data are expressed as mean ± SD of at least three experiments. Significant difference of p≤0.05 is indicated as * and p≤0.001 is indicated as **.</p
<i>In vivo</i> transplantation of spheroids in myocardium-injured mice.
<p>(<b>A</b>) Evaluation of cardiotoxin-induced injury in the myocardium wall at two magnifications (right) compared to healthy myocardium (left) in Hematoxylin-Eosin staining. (<b>B</b>) Myocardium sections from myocardium injured mice transplanted with spheroids were stained with TO-PRO3 (blue) to show all nuclei, anti-human nuclei antibody (green), and connexin-43 or phalloidin (red) for actin and analysed by confocal microscopy. Representative experiments out of three performed are shown. The circle and arrows show the engrafted spheroid 1 day after the injection and the dispersed hCPCs 7 days after the injection, respectively.</p