scRNA-Seq of Cultured Human Amniotic Fluid from Fetuses with Spina Bifida Reveals the Origin and Heterogeneity of the Cellular Content.

Amniotic fluid has been proposed as an easily available source of cells for numerous applications in regenerative medicine and tissue engineering. The use of amniotic fluid cells in biomedical applications necessitates their unequivocal characterization; however, the exact cellular composition of amniotic fluid and the precise tissue origins of these cells remain largely unclear. Using cells cultured from the human amniotic fluid of fetuses with spina bifida aperta and of a healthy fetus, we performed single-cell RNA sequencing to characterize the tissue origin and marker expression of cultured amniotic fluid cells at the single-cell level. Our analysis revealed nine different cell types of stromal, epithelial and immune cell phenotypes, and from various fetal tissue origins, demonstrating the heterogeneity of the cultured amniotic fluid cell population at a single-cell resolution. It also identified cell types of neural origin in amniotic fluid from fetuses with spina bifida aperta. Our data provide a comprehensive list of markers for the characterization of the various progenitor and terminally differentiated cell types in cultured amniotic fluid. This study highlights the relevance of single-cell analysis approaches for the characterization of amniotic fluid cells in order to harness their full potential in biomedical research and clinical applications.

The influence of CD26+ and CD26- fibroblasts on the regeneration of human dermo-epidermal skin substitutes.

CD26, also known as dipeptidyl peptidase IV (DPPIV), is a multifunctional transmembrane protein playing a significant role in the cutaneous wound healing processes in the mouse skin. However, only scarce data are available regarding the distribution and function of this protein in the human skin. Therefore, the aim of this study was to investigate the impact of CD26 deficiency in human primary fibroblasts on the regeneration of human tissue-engineered skin substitutes in vivo. Dermo-epidermal skin analogs, based on collagen type I hydrogels, were populated either with human CD26+ or CD26knockout fibroblasts and seeded with human epidermal keratinocytes. These skin substitutes were transplanted onto the back of immune-incompetent rodents. Three weeks post-transplantation, the grafts were excised and analyzed with respect to specific epidermal and dermal maturation markers. For the first time, we show here that the expression of CD26 protein in human dermis is age-dependent. Furthermore, we prove that CD26+ fibroblasts are more active in the production of extracellular matrix (ECM) both in vitro and in vivo and are necessary to achieve rapid epidermal and dermal homeostasis after transplantation.

Bio-engineering of fetal cartilage for in utero spina bifida repair.

PURPOSE: During in utero surgical spina bifida repair, a multi-layer closure is used to cover the defect. These soft tissues, however, might be not sufficient to protect the spinal cord during the future life. Our goal is to develop a more rigid protective tissue construct consisting of bioengineered cartilage and skin. METHODS: Ovine fetal chondrocytes were tested for their in vitro chondrogenic potential in three-dimensional cultures. Scaffolds based on natural biopolymers (collagen I, fibrin glue) were loaded with varying amounts of fetal chondrocytes and assessed for their ability to support cartilage formation in vitro. The bioengineered constructs were analyzed using cartilage-specific histology stainings and compared to native fetal cartilage. RESULTS: Fetal chondrocytes actively produced cartilage extracellular matrix in three-dimensional cultures, even at high passages. Among all bioengineered scaffolds, only the collagen I-based hydrogels loaded with high densities of fetal chondrocytes showed cartilage-like structure in vitro but also extensive shrinking. CONCLUSION: Fetal chondrocytes represent a good cell source for cartilage bioengineering. Collagen I scaffolds support cartilage formation in vitro, but the construct shrinking constitutes a major limitation. Future steps include the identification of suitable bioprintable materials which maintain their shape and size, as well as the analysis of the interphase between bioengineered cartilage and skin.

Advanced Drug Delivery Nanosystems for Shikonin: A Calorimetric and Electron Paramagnetic Resonance Study.

Drug delivery is considered a mature scientific and technological platform for producing innovative medicines with nanosystems composed of intelligent bio-materials that carry active pharmaceutical ingredients forming advanced drug delivery nanosystems (aDDnSs). Shikonin and its enantiomer alkannin are natural products that have been extensively studied in vitro and in vivo for, among others, their antitumor activity, and various efforts have been made to prepare shikonin-loaded drug delivery systems. This study is focused on chimeric aDDnSs and specifically on liposomal formulations combining three lipids (egg-phosphatidylcholine; dipalmitoyl phosphatidylcholine; and distearoyl phosphatidylcholine) and a hyperbranched polymer (PFH-64-OH). Furthermore, PEGylated liposomal formulations of all samples were also prepared. Calorimetric techniques and electron paramagnetic resonance were used to explore and evaluate the interactions and stability of the liposomal formulations, showing that the presence of hyperbranched polymers promote the overall stability of the chimeric aDDnSs based on the drug release profile enhancement. Furthermore, results showed that polyethylene glycol enhances drug stabilization inside the liposomes, forming a stable and promising carrier for shikonin with improved characteristics.

Targeting Nanocarriers with Anisamide: Fact or Artifact?

Encapsulating chemotherapeutics in nanoparticles can reduce the side effects of intravenous administration and improve their antitumor efficacy. Additionally, surface decoration of the nanocarriers with tumor-targeting ligands may enhance their specificity for cancer cells overexpressing the corresponding ligand-binding counterpart. The focus here is on anisamide, a low-molecular-weight benzamide derivative used as a tumor-directing moiety in functionalized nanosystems, based on its alleged interaction with Sigma receptors. The scintigraphic agents that initially inspired the use of anisamide for tumor targeting are described, and the published anisamide-tethered nanocarrier formulations are reviewed, together with a critical overview of the ligand's tumor-targeting properties. Moreover, anisamide's putative but dubious cellular target, the Sigma-1 receptor, is discussed with regard to its subcellular localization and implications in cancer. Data from in vivo studies reveal that the effect of anisamide on the antitumor efficacy of the decorated nanosystems varies considerably among the published reports. Together with the evidence questioning the interaction of anisamide with the Sigma receptors, the variability of anisamide's effect on the tumor deposition and the antitumor efficacy of the decorated drug carriers calls into question the extent of the ligand's tumor-targeting effect. Further research is necessary to elucidate the ligand's utility in tumor targeting.

Regulation of lymphangiogenesis in the diaphragm by macrophages and VEGFR-3 signaling.

Lymphatic vessels play important roles in fluid drainage and in immune responses, as well as in pathological processes including cancer progression and inflammation. While the molecular regulation of the earliest lymphatic vessel differentiation and development has been investigated in much detail, less is known about the control and timing of lymphatic vessel maturation in different organs, which often occurs postnatally. We investigated the time course of lymphatic vessel development on the pleural side of the diaphragmatic muscle in mice, the so-called submesothelial initial diaphragmatic lymphatic plexus. We found that this lymphatic network develops largely after birth and that it can serve as a reliable and easily quantifiable model to study physiological lymphangiogenesis in vivo. Lymphangiogenic growth in this tissue was highly dependent on vascular endothelial growth factor receptor (VEGFR)-3 signaling, whereas VEGFR-1 and -2 signaling was dispensable. During diaphragm development, macrophages appeared first in a linearly arranged pattern, followed by ingrowth of lymphatic vessels along these patterned lines. Surprisingly, ablation of macrophages in colony-stimulating factor-1 receptor (Csf1r)-deficient mice and by treatment with a CSF-1R-blocking antibody did not inhibit the general lymphatic vessel development in the diaphragm but specifically promoted branch formation of lymphatic sprouts. In agreement with these findings, incubation of cultured lymphatic endothelial cells with conditioned medium from P7 diaphragmatic macrophages significantly reduced LEC sprouting. These results indicate that the postnatal diaphragm provides a suitable model for studies of physiological lymphangiogenic growth and maturation, and for the identification of modulators of lymphatic vessel growth.

Findings questioning the involvement of Sigma-1 receptor in the uptake of anisamide-decorated particles.

Anisamide is a small benzamide previously suggested as a tumor-targeting ligand for nanocarriers and it has been shown to enhance tumor uptake in vitro as well as in vivo when grafted on the nanoparticle surface. Anisamide has been hypothesized to interact with the Sigma-1 receptor, based on the binding of larger benzamides, which contain anisamide in their structure, to this receptor. However, the interaction between anisamide and Sigma-1 receptor has never been thoroughly studied. We developed fluorescent PEGylated particles decorated with anisamide, which were preferentially taken up in vitro by melanoma cells compared to macrophages. The anisamide-decorated particles were used to study their interaction with the Sigma-1 receptor. The absence of competition of Sigma-1 receptor ligands for the particle uptake was a first indication that the receptor might not be involved in the uptake process. In addition, the extent of particle uptake did not correlate with the levels of cellular expression of Sigma-1 receptor in the cell models tested. Immunostaining of the receptor on melanoma cells revealed intracellular localization, indirectly excluding the possibility of anisamide binding to the receptor when grafted on the particles. All these data question the previously suggested Sigma-1 receptor-mediated uptake of the anisamide-decorated particles, a finding which may have an impact on the use of anisamide as a targeting ligand.