The GPI-anchor biosynthesis pathway is critical for syncytiotrophoblast differentiation and placental development.

The glycosylphosphatidylinositol (GPI) biosynthetic pathway in the endoplasmic reticulum (ER) is crucial for generating GPI-anchored proteins (GPI-APs), which are translocated to the cell surface and play a vital role in cell signaling and adhesion. This study focuses on two integral components of the GPI pathway, the PIGL and PIGF proteins, and their significance in trophoblast biology. We show that GPI pathway mutations impact on placental development impairing the differentiation of the syncytiotrophoblast (SynT), and especially the SynT-II layer, which is essential for the establishment of the definitive nutrient exchange area within the placental labyrinth. CRISPR/Cas9 knockout of Pigl and Pigf in mouse trophoblast stem cells (mTSCs) confirms the role of these GPI enzymes in syncytiotrophoblast differentiation. Mechanistically, impaired GPI-AP generation induces an excessive unfolded protein response (UPR) in the ER in mTSCs growing in stem cell conditions, akin to what is observed in human preeclampsia. Upon differentiation, the impairment of the GPI pathway hinders the induction of WNT signaling for early SynT-II development. Remarkably, the transcriptomic profile of Pigl- and Pigf-deficient cells separates human patient placental samples into preeclampsia and control groups, suggesting an involvement of Pigl and Pigf in establishing a preeclamptic gene signature. Our study unveils the pivotal role of GPI biosynthesis in early placentation and uncovers a new preeclampsia gene expression profile associated with mutations in the GPI biosynthesis pathway, providing novel molecular insights into placental development with implications for enhanced patient stratification and timely interventions.

Mechanisms and function of de novo DNA methylation in placental development reveals an essential role for DNMT3B.

DNA methylation is a repressive epigenetic modification that is essential for development, exemplified by the embryonic and perinatal lethality observed in mice lacking de novo DNA methyltransferases (DNMTs). Here we characterise the role for DNMT3A, 3B and 3L in gene regulation and development of the mouse placenta. We find that each DNMT establishes unique aspects of the placental methylome through targeting to distinct chromatin features. Loss of Dnmt3b results in de-repression of germline genes in trophoblast lineages and impaired formation of the maternal-foetal interface in the placental labyrinth. Using Sox2-Cre to delete Dnmt3b in the embryo, leaving expression intact in placental cells, the placental phenotype was rescued and, consequently, the embryonic lethality, as Dnmt3b null embryos could now survive to birth. We conclude that de novo DNA methylation by DNMT3B during embryogenesis is principally required to regulate placental development and function, which in turn is critical for embryo survival.

Human adipose-derived mesenchymal stem cells accelerate decellularized neobladder regeneration.

Decellularized natural bladder matrices (neobladders) represent an exciting means to regenerate the bladder following bladder cancer-associated cystectomy. In this study, we compare the evolution of decellularized matrices with recellularized matrices by seeding it with human adipose-derived mesenchymal stem cells (ADSC) after implantation following partial cystectomy in rats. We discovered significant anatomical differences since 10 days after neobladder implantation with the ADSC-containing matrices promoting a significant recovery of mature p63- and cytokeratin 7-positive urothelium. We also discovered significantly induced expression of the vimentin mesoderm marker in the submucosal layer in ADSC-seeded matrices. Interestingly, we found a higher expression of smooth muscle actin in transversal and longitudinal smooth muscle layers with ADSC-seeded matrices. Furthermore, ADSC also showed increased vascularization and nerve innervation of the neobladder as determined by the distribution of CD31 and S100ß reactivity, respectively. We believe that ADSC and their paracrine-acting pro-regenerative secretome within decellularized matrices represent an efficient bladder substitution strategy; however, we require a fuller understanding of the mechanisms involved before clinical studies can begin.

FM19G11-Loaded Gold Nanoparticles Enhance the Proliferation and Self-Renewal of Ependymal Stem Progenitor Cells Derived from ALS Mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. In ALS mice, neurodegeneration is associated with the proliferative restorative attempts of ependymal stem progenitor cells (epSPCs) that normally lie in a quiescent in the spinal cord. Thus, modulation of the proliferation of epSPCs may represent a potential strategy to counteract neurodegeneration. Recent studies demonstrated that FM19G11, a hypoxia-inducible factor modulator, induces epSPC self-renewal and proliferation. The aim of the study was to investigate whether FM19G11-loaded gold nanoparticles (NPs) can affect self-renewal and proliferation processes in epSPCs isolated from G93A-SOD1 mice at disease onset. We discovered elevated levels of SOX2, OCT4, AKT1, and AKT3, key genes associated with pluripotency, self-renewal, and proliferation, in G93A-SOD1 epSPCs at the transcriptional and protein levels after treatment with FM19G11-loaded NPs. We also observed an increase in the levels of the mitochondrial uncoupling protein (UCP) gene in treated cells. FM19G11-loaded NPs treatment also affected the expression of the cell cycle-related microRNA (miR)-19a, along with its target gene PTEN, in G93A-SOD1 epSPCs. Overall our findings establish the significant impact of FM19G11-loaded NPs on the cellular pathways involved in self-renewal and proliferation in G93A-SOD1 epSPCs, thus providing an impetus to the design of novel tailored approaches to delay ALS disease progression.

Plasma Rich in Growth Factors Induces Cell Proliferation, Migration, Differentiation, and Cell Survival of Adipose-Derived Stem Cells.

Adipose-derived stem cells (ASCs) are a promising therapeutic alternative for tissue repair in various clinical applications. However, restrictive cell survival, differential tissue integration, and undirected cell differentiation after transplantation in a hostile microenvironment are complications that require refinement. Plasma rich in growth factors (PRGF) from platelet-rich plasma favors human and canine ASC survival, proliferation, and delaying human ASC senescence and autophagocytosis in comparison with serum-containing cultures. In addition, canine and human-derived ASCs efficiently differentiate into osteocytes, adipocytes, or chondrocytes in the presence of PRGF. PRGF treatment induces phosphorylation of AKT preventing ASC death induced by lethal concentrations of hydrogen peroxide. Indeed, AKT inhibition abolished the PRGF apoptosis prevention in ASC exposed to 100 µM of hydrogen peroxide. Here, we show that canine ASCs respond to PRGF stimulus similarly to the human cells regarding cell survival and differentiation postulating the use of dogs as a suitable translational model. Overall, PRGF would be employed as a serum substitute for mesenchymal stem cell amplification to improve cell differentiation and as a preconditioning agent to prevent oxidative cell death.

Human Suprapatellar Fat Pad-Derived Mesenchymal Stem Cells Induce Chondrogenesis and Cartilage Repair in a Model of Severe Osteoarthritis.

Cartilage degeneration is associated with degenerative bone and joint processes in severe osteoarthritis (OA). Spontaneous cartilage regeneration is extremely limited. Often the treatment consists of a partial or complete joint implant. Adipose-derived stem cell (ASC) transplantation has been shown to restore degenerated cartilage; however, regenerative differences of ASC would depend on the source of adipose tissue. The infra- and suprapatellar fat pads surrounding the knee offer a potential autologous source of ASC for patients after complete joint substitution. When infrapatellar- and suprapatellar-derived stromal vascular fractions (SVF) were compared, a significantly higher CD105 (+) population was found in the suprapatellar fat. In addition, the suprapatellar SVF exhibited increased numbers of colony formation units and a higher population doubling in culture compared to the infrapatellar fraction. Both the suprapatellar- and infrapatellar-derived ASC were differentiated in vitro into mature adipocytes, osteocytes, and chondrocytes. However, the suprapatellar-derived ASC showed higher osteogenic and chondrogenic efficiency. Suprapatellar-derived ASC transplantation in a severe OA mouse model significantly diminished the OA-associated knee inflammation and cartilage degenerative grade, significantly increasing the production of glycosaminoglycan and inducing endogenous chondrogenesis in comparison with the control group. Overall, suprapatellar-derived ASC offer a potential autologous regenerative treatment for patients with multiple degenerative OA.

Platelet-rich plasma and adipose-derived mesenchymal stem cells for regenerative medicine-associated treatments in bottlenose dolphins (Tursiops truncatus).

Dolphins exhibit an extraordinary capacity to heal deep soft tissue injuries. Nevertheless, accelerated wound healing in wild or captive dolphins would minimize infection and other side effects associated with open wounds in marine animals. Here, we propose the use of a biological-based therapy for wound healing in dolphins by the application of platelet-rich plasma (PRP). Blood samples were collected from 9 different dolphins and a specific and simple protocol which concentrates platelets greater than two times that of whole blood was developed. As opposed to a commonly employed human protocol for PRP preparation, a single centrifugation for 3 minutes at 900 rpm resulted in the best condition for the concentration of dolphin platelets. By FACS analysis, dolphin platelets showed reactivity to platelet cell-surface marker CD41. Analysis by electron microscopy revealed that dolphin platelets were larger in size than human platelets. These findings may explain the need to reduce the duration and speed of centrifugation of whole blood from dolphins to obtain a 2-fold increase and maintain proper morphology of the platelets. For the first time, levels of several growth factors from activated dolphin platelets were quantified. Compared to humans, concentrations of PDGF-BB were not different, while TGFß and VEGF-A were significantly lower in dolphins. Additionally, adipose tissue was obtained from cadaveric dolphins found along the Spanish Mediterranean coast, and adipose-derived mesenchymal stem cells (ASCs) were successfully isolated, amplified, and characterized. When dolphin ASCs were treated with 2.5 or 5% dolphin PRP they exhibited significant increased proliferation and improved phagocytotic activity, indicating that in culture, PRP may improve the regenerative capacity of ASCs. Taken together, we show an effective and well-defined protocol for efficient PRP isolation. This protocol alone or in combination with ASCs, may constitute the basis of a biological treatment for wound-healing and tissue regeneration in dolphins.