Mitochondrial Routing of Glucose and Sucrose Polymers after Pinocytotic Uptake: Avenues for Drug Delivery

Mitochondria are key organelles organizing cellular metabolic flux. Therefore, a targeted drug delivery to mitochondria promises the advancement of medicine in fields that are associated with mitochondrial dysfunction. However, successful mitochondrial drug delivery is limited by complex transport steps across organelle membranes and fast drug efflux in cases of multidrug resistance. Strategies to deliver small-molecular-weight drugs to mitochondria are very limited, while the use of complex polymeric carriers is limited by a lack of clinical feasibility. We show here that clinically established macromolecules such as a sucrose copolymer (Ficoll 70/400 kDa) and polyglucose (dextran 70/500 kDa) are micropinocytosed swiftly by mesenchymal stem cells and subsequently routed to mitochondria. The intracellular level of Ficoll appears to decrease over time, suggesting that it does not persist within cells. After coupling to polysucrose, the low-molecular-weight photodynamic drug Rose Bengal reached mitochondria and thus exhibited an increased destructive potential after laser excitation. These findings support new opportunities to deliver already clinically approved drugs to mitochondria

Three-Dimensional Environment Sustains Hematopoietic Stem Cell Differentiation into Platelet-Producing Megakaryocytes

<div><p>Hematopoietic stem cells (HSC) differentiate into megakaryocytes (MK), whose function is to release platelets. Attempts to improve <i>in vitro</i> platelet production have been hampered by the low amplification of MK. Providing HSC with an optimal three-dimensional (3D) architecture may favor MK differentiation by mimicking some crucial functions of the bone marrow structure. To this aim, porous hydrogel scaffolds were used to study MK differentiation from HSC as well as platelet production. Flow cytometry, qPCR and perfusion studies showed that 3D was suitable for longer kinetics of CD34⁺ cell proliferation and for delayed megakaryocytic differentiation far beyond the limited shelf-life observed in liquid culture but also increased production of functional platelets. We provide evidence that these 3D effects were related to 1) persistence of MK progenitors and precursors and 2) prolongation of expression of EKLF and c-myb transcription factors involved in early MK differentiation. In addition, presence of abundant mature MK with increased ploidy and impressive cytoskeleton elongations was in line with expression of NF-E2 transcription factor involved in late MK differentiation. Platelets produced in flow conditions were functional as shown by integrin αIIbβ3 activation following addition of exogenous agonists. This study demonstrates that spatial organization and biological cues synergize to improve MK differentiation and platelet production. Thus, 3D environment constitutes a powerful tool for unraveling the physiological mechanisms of megakaryopoiesis and thrombopoiesis in the bone marrow environment, potentially leading to an improved amplification of MK and platelet production.</p></div

In situ characterization of differentiation markers and ploidy of mature MK.

<p><b>(A)</b> Immunofluorescence staining of CD41 (green) and CD42b (red) cells growing inside pores of 3D (I) and in liquid culture (II). Nuclear staining of cell grown in 3D (III) and liquid culture (IV) with YOYO-1 marker (white). All images were acquired 12 days after seeding using the Leica 510 confocal microscope with 40X Plan-NeoFluar objective lens. Scale bar = 10 μm. <b>(B)</b> Representative flow cytometry ploidy analysis of CD41⁺/CD42b⁺ UCB cells from 3D and liquid culture, 11 days after seeding. <b>(C)</b> Ploidy analysis of CD41⁺/CD42b⁺ UCB cells in 3D (black bars) compared to liquid culture (white bars), 11 days after seeding. Data are means ± SEM of 3 independent experiments. *p<0.05. Abbreviation: UCB, umbilical cord blood.</p

Cell morphology in 3D and in liquid culture.

<p><b>(A)</b> Cell proliferation 12 days after cell seeding, relative to the initial cell number/scaffold. <b>(B)</b> Cell proliferation and differentiation in 3D between day 2 and day 36. <b>(C)</b> Cell proliferation and differentiation in liquid culture on days 2, 12 and 16. All images were acquired using the Axiovert 135 transmission optical microscope with 20X Plasdic magnification. Scale bar = 20 μm.</p

Characterization of MK differentiation as a function of time.

<p><b>(A, C, E)</b> Frequency of non-megakaryocytic cells (CD41^-/CD42b^-), MK precursors (CD41⁺/CD42b^-) and mature MK (CD41⁺/CD42b⁺) in 3D (closed circles, dotted lines) and liquid culture (open squares, full lines) between day 6 and day 36. Data are means ± SEM of 3 independent experiments.*p<0.05. In 3D, late time points (D23 and D36) were compared to day 6. <b>(B)</b> Histogram representation of total CD41^-/CD42b^- cell number in 3D (black bars) and liquid culture (white bars) at different days of culture. <b>(D)</b> Histogram representation of total CD41⁺/CD42b^- cell number in 3D (black bars) and liquid culture (white bars) at different days of culture. <b>(F)</b> Histogram representation of total CD41⁺/CD42b⁺ cell number in 3D (black bars) and liquid culture (white bars) at different days of culture. Total cell number of each MK population was determined by multiplying the total cell number by the frequency of each MK population. Data are means ± SEM of 3 independent experiments. In 3D, late time points (D23 and D36) were compared to day 7. *p<0.05. <b>(G)</b> CD41/CD34 dot plots of one representative experiment of 3 independent experiments in 3D and liquid culture on days 9, 16 and 23. Abbreviation: TCN, total cell number.</p

MK deformation and platelet production in flow conditions.

<p><b>(A)</b> Stages of MK deformations and reorganization into proplatelets and platelets at 20 minutes of perfusion in 3D (left panel) and in liquid culture (right panel) using Bioflux microfluidic platform to increase platelet formation from mature MK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136652#pone.0136652.ref010" target="_blank">10</a>]. Images were acquired using the Axiovert 135 transmission optical microscope with 20X Plasdic magnification. Bar = 20 μm. <b>(B)</b> Histogram representation of proplatelet/platelet numbers as a percentage of total adherent cells per field recovered from mature MK obtained in 3D (black bars) and liquid culture (white bars). Data are means ± SEM of 3 independent experiments.*p<0.05.</p

Representative images of immune-staining for cells in contact with the biomaterial using primary antibodies for von Willebrand Factor (A–C), Bone Morphogenetic Protein 2 (D–F) and CD 68 (G–I).

<p>Positive cells appear in brown and nuclei in dark blue.</p

Representative images of histological staining of defects that were left empty (A–C, G–I) and for defects that received beads as implants (D–F, J–O).

<p>Calcified sections were stained with Von Kossa (A–F), while decalcified sections were stained with Masson’s Trichrome (G–O). Increase of mineralization is visible over time when comparing sections obtained at day 15 (A, D, G, J) with sections of day 30 (B, E, H, K) or those of day 70 (C, F, I, L). Areas of defects are marked with the dotted white line (A–L) and higher magnifications of the orange squares in J–L are shown in M–O.</p

High-Resolution Cellular MRI: Gadolinium and Iron Oxide Nanoparticles for in-Depth Dual-Cell Imaging of Engineered Tissue Constructs

Recent advances in cell therapy and tissue engineering opened new windows for regenerative medicine, but still necessitate innovative noninvasive imaging technologies. We demonstrate that high-resolution magnetic resonance imaging (MRI) allows combining cellular-scale resolution with the ability to detect two cell types simultaneously at any tissue depth. Two contrast agents, based on iron oxide and gadolinium oxide rigid nanoplatforms, were used to “tattoo” endothelial cells and stem cells, respectively, with no impact on cell functions, including their capacity for differentiation. The labeled cells’ contrast properties were optimized for simultaneous MRI detection: endothelial cells and stem cells seeded together in a polysaccharide-based scaffold material for tissue engineering appeared respectively in black and white and could be tracked, at the cellular level, both <i>in vitro</i> and <i>in vivo</i>. In addition, endothelial cells labeled with iron oxide nanoparticles could be remotely manipulated by applying a magnetic field, allowing the creation of vessel substitutes with in-depth detection of individual cellular components

The morphology of microspheres was analyzed by scanning electron microscopy (A).

<p>After rehydration in PBS, porosity of hydrated scaffolds was observed with an Environmental Scanning Electron Microscopy (B). Back-scattered Electron Microscopy images of dry beads show pores and overall surface properties (C) and the distribution of nHA aggregates within the structure (D).</p