Release of podocalyxin into the extracellular space. Role of metalloproteinases.

Podocalyxin (PODXL) is a type I membrane mucoprotein abundantly presented in the epithelial cells (podocytes) of kidney glomeruli where it plays an important role in maintaining the plasma filtration. PODXL is also expressed in other types of cells but its function is ignored. A recombinant soluble fragment of the PODXL ectodomain modifies the signaling of the membrane bound PODXL. Based on this antecedent, we aimed at investigating whether PODXL could be cleaved and released into the extracellular space as a soluble peptide. In this study, we used a fusion protein of human PODXL and green fluorescent protein expressed in CHO cells (CHO-PODXL-GFP) and a human tumor cell (Tera-1) inherently expressing PODXL. PODXL was detected by wide-field microscopy in the Golgi, the plasma membrane and in a vesicular form preferentially located at the leading edges of the cell and also progressing along the filopodium. We detected PODXL in the insoluble and soluble fractions of the extracellular medium of CHO-PODXL-GFP cells. Stimulation of protein kinase C (PKC) by Phorbol-12-myristate-13-acetate (PMA) enhanced the release of PODXL to the extracellular space whereas this effect was prevented either by inhibitors of PKC or specific inhibitors of matrix metalloproteinases. It is concluded that intact PODXL is released to the extracellular space as a cargo of microvesicles and also as a soluble cleaved fragment of ectodomain.

Whole-genome sequencing identifies new candidate genes for nonobstructive azoospermia.

BACKGROUND: Genetic causes that lead to spermatogenetic failure in patients with nonobstructive azoospermia (NOA) have not been yet completely established. OBJECTIVE: To identify low-frequency NOA-associated single nucleotide variants (SNVs) using whole-genome sequencing (WGS). MATERIALS AND METHODS: Men with various types of NOA (n = 39), including samples that had been previously tested with whole-exome sequencing (WES; n = 6) and did not result in diagnostic conclusions. Variants were annotated using the Ensembl Variant Effect Predictor, utilizing frequencies from GnomAD and other databases to provide clinically relevant information (ClinVar), conservation scores (phyloP), and effect predictions (i.e., MutationTaster). Structural protein modeling was also performed. RESULTS: Using WGS, we revealed potential NOA-associated SNVs, such as: TKTL1, IGSF1, ZFPM2, VCX3A (novel disease causing variants), ESX1, TEX13A, TEX14, DNAH1, FANCM, QRICH2, FSIP2, USP9Y, PMFBP1, MEI1, PIWIL1, WDR66, ZFX, KCND1, KIAA1210, DHRSX, ZMYM3, FAM47C, FANCB, FAM50B (genes previously known to be associated with infertility) and ALG13, BEND2, BRWD3, DDX53, TAF4, FAM47B, FAM9B, FAM9C, MAGEB6, MAP3K15, RBMXL3, SSX3 and FMR1NB genes, which may be involved in spermatogenesis. DISCUSSION AND CONCLUSION: In this study, we identified novel potential candidate NOA-associated genes in 29 individuals out of 39 azoospermic males. Note that in 5 out of 6 patients subjected previously to WES analysis, which did not disclose potentially causative variants, the WGS analysis was successful with NOA-associated gene findings.

Ventricular enlargement associated with the panneural ablation of the podocalyxin gene.

Podocalyxin (Podxl) is a type I membrane mucin-protein of the CD34 family abundantly expressed in kidney epithelial cells (podocytes) where it plays a crucial functional role. Podxl is also expressed in tissues other than kidney, like in brain, but its function is ignored. To investigate the functional role of podocalyxin (Podxl) in brain we produced the specific brain-ablation of the Podxl gen in mice by crossing Podxl(floxed/floxed) mice, generated in our laboratory, to mice with pan-neural expression of recombinase Cre (Cre3). Podxl(-/-) mice show no apparent behavioral phenotype but their brains showed enlargement of ventricular volumes detected in vivo by MR imaging. The pattern of brain vasculature was of normal appearance but the thickness of the main carotid artery was significantly increased. Moreover, the histological analysis showed increased number of choroidal capillaries lining the ventricular spaces. These findings are analyzed in the light of the role likely played by podocalyxin in cell migration and cell-cell recognition during brain development and also on the consistent findings of increased ventricular spaces in human pathological disorders like schizophrenia.

Experimental Strategies of Mesenchymal Stem Cell Propagation: Adverse Events and Potential Risk of Functional Changes.

Mesenchymal stem cells (MSCs) are attractive candidates for cell-based tissue repair approaches. Hundreds of clinical trials using MSCs have been completed and many others are still being investigated. For most therapeutic applications, MSC propagation in vitro is often required. However, ex vivo culture condition is not fully physiological and may affect biological properties of MSCs including their regenerative potential. Moreover, both cell cryopreservation and labelling procedure prior to infusion may have the negative impact on their expected effect in vivo. The incidence of MSC transformation during in vitro culture should be also taken into consideration before using cells in stem cell therapy. In our review, we focused on different aspects of MSC propagation that might influence their regenerative properties of MSC. We also discussed the influence of different factors that might abolish MSC proliferation and differentiation as well as potential impact of stem cell senescence and aging. Despite of many positive therapeutic effects of MSC therapy, one has to be conscious about potential cell changes that could appear during manufacturing of MSCs.

Single-cell, high-throughput analysis of cell docking to vessel wall.

Therapeutic potential of mesenchymal stem cells (MSCs) has been reported consistently in animal models of stroke, with mechanism mainly through immunomodulation and paracrine activity. Intravenous injection has been a prevailing route for MSCs administration, but cell quantities needed when scaling-up from mouse to human are extremely high putting into question feasibility of that approach. Intra-arterial delivery directly routes the cells to the brain thus lowering the required dose. Cell engineering may additionally improve cell homing, further potentiating the value of intra-arterial route. Therefore, our goal was to create microfluidic platform for screening and fast selection of molecules that enhance the docking of stem cells to vessel wall. We hypothesized that our software will be capable of detecting distinct docking properties of naïve and ITGA4-engineered MSCs. Indeed, the cell flow tracker analysis revealed positive effect of cell engineering on docking frequency of MSCs (42% vs. 9%, engineered vs. control cells, p < 0.001). These observations were then confirmed in an animal model of focal brain injury where cell engineering resulted in improved homing to the brain. To conclude, we developed a platform to study the docking of cells to the vessel wall which is highly relevant for intraarterial cell targeting or studies on neuroinflammation.

Translation, but not transfection limits clinically relevant, exogenous mRNA based induction of alpha-4 integrin expression on human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) represent promising resource of cells for regenerative medicine in neurological disorders. However, efficient and minimally invasive methods of MSCs delivery to the brain still have to be developed. Intra-arterial route is very promising, but MSCs are missing machinery for diapedesis through blood-brain barrier. Thus, here we have tested a mRNA-based method to induce transient expression of ITGA4, an adhesion molecule actively involved in cell extravasation. We observed that transfection with an ITGA4-mRNA construct bearing a conventional cap analogue (7-methylguanosine) failed to produce ITGA4 protein, but exogenous ITGA4-mRNA was detected in transfected MSCs. This indicates that not transfection, but rather translation being the major roadblock. Stabilization of ITGA4-mRNA with SSB proteins resulted in ITGA4 protein synthesis in HEK293 cells only, whereas in MSCs, satisfactory results were obtained only after using an anti-reverse-cap-analogue (ARCA). The presence of ITGA4 protein in MSCs was transient and lasted for up to 24 h after transfection. Membranous location was confirmed by flow cytometry of viable non-permeabilized cells using anti-ITGA4 antibody. The mRNA-based expression of itga4 transgene is potentially sufficient for diapedesis after intra-arterial delivery. To conclude, mRNA-based engineering of stem cells is a rapid and integration-free method and attractive from the perspective of potential future clinical application.

Real-time MRI for precise and predictable intra-arterial stem cell delivery to the central nervous system.

Stem cell therapy for neurological disorders reached a pivotal point when the efficacy of several cell types was demonstrated in small animal models. Translation of stem cell therapy is contingent upon overcoming the challenge of effective cell delivery to the human brain, which has a volume ∼1000 times larger than that of the mouse. Intra-arterial injection can achieve a broad, global, but also on-demand spatially targeted biodistribution; however, its utility has been limited by unpredictable cell destination and homing as dictated by the vascular territory, as well as by safety concerns. We show here that high-speed MRI can be used to visualize the intravascular distribution of a superparamagnetic iron oxide contrast agent and can thus be used to accurately predict the distribution of intra-arterial administered stem cells. Moreover, high-speed MRI enables the real-time visualization of cell homing, providing the opportunity for immediate intervention in the case of undesired biodistribution.

Genetic Engineering of Mesenchymal Stem Cells to Induce Their Migration and Survival.

Mesenchymal stem cells (MSCs) are very attractive for regenerative medicine due to their relatively easy derivation and broad range of differentiation capabilities, either naturally or induced through cell engineering. However, efficient methods of delivery to diseased tissues and the long-term survival of grafted cells still need improvement. Here, we review genetic engineering approaches designed to enhance the migratory capacities of MSCs, as well as extend their survival after transplantation by the modulation of prosurvival approaches, including prevention of senescence and apoptosis. We highlight some of the latest examples that explore these pivotal points, which have great relevance in cell-based therapies.

Engineered Mesenchymal Stem Cells as an Anti-Cancer Trojan Horse.

Cell-based gene therapy holds a great promise for the treatment of human malignancy. Among different cells, mesenchymal stem cells (MSCs) are emerging as valuable anti-cancer agents that have the potential to be used to treat a number of different cancer types. They have inherent migratory properties, which allow them to serve as vehicles for delivering effective therapy to isolated tumors and metastases. MSCs have been engineered to express anti-proliferative, pro-apoptotic, and anti-angiogenic agents that specifically target different cancers. Another field of interest is to modify MSCs with the cytokines that activate pro-tumorigenic immunity or to use them as carriers for the traditional chemical compounds that possess the properties of anti-cancer drugs. Although there is still controversy about the exact function of MSCs in the tumor settings, the encouraging results from the preclinical studies of MSC-based gene therapy for a large number of tumors support the initiation of clinical trials.

Genetic Engineering of Mesenchymal Stem Cells for Regenerative Medicine.

Mesenchymal stem cells (MSCs), which can be obtained from various organs and easily propagated in vitro, are one of the most extensively used types of stem cells and have been shown to be efficacious in a broad set of diseases. The unique and highly desirable properties of MSCs include high migratory capacities toward injured areas, immunomodulatory features, and the natural ability to differentiate into connective tissue phenotypes. These phenotypes include bone and cartilage, and these properties predispose MSCs to be therapeutically useful. In addition, MSCs elicit their therapeutic effects by paracrine actions, in which the metabolism of target tissues is modulated. Genetic engineering methods can greatly amplify these properties and broaden the therapeutic capabilities of MSCs, including transdifferentiation toward diverse cell lineages. However, cell engineering can also affect safety and increase the cost of therapy based on MSCs; thus, the advantages and disadvantages of these procedures should be discussed. In this review, the latest applications of genetic engineering methods for MSCs with regenerative medicine purposes are presented.

The cerebral embolism evoked by intra-arterial delivery of allogeneic bone marrow mesenchymal stem cells in rats is related to cell dose and infusion velocity.

INTRODUCTION: Intra-arterial cell infusion is an efficient delivery route with which to target organs such as the ischemic brain. However, adverse events including microembolisms and decreased cerebral blood flow were recently reported after intra-arterial cell delivery in rodent models, raising safety concerns. We tested the hypothesis that cell dose, infusion volume, and velocity would be related to the severity of complications after intra-arterial cell delivery. METHODS: In this study, 38 rats were subjected to a sham middle cerebral artery occlusion (sham-MCAO) procedure before being infused with allogeneic bone-marrow mesenchymal stem cells at different cell doses (0 to 1.0 × 10(6)), infusion volumes (0.5 to 1.0 ml), and infusion times (3 to 6 minutes). An additional group (n = 4) was infused with 1.0 × 10(6) cells labeled with iron oxide for in vivo tracking of cells. Cells were infused through the external carotid artery under laser Doppler flowmetry monitoring 48 hours after sham-MCAO. Magnetic resonance imaging (MRI) was performed 24 hours after cell infusion to reveal cerebral embolisms or hemorrhage. Limb placing, cylinder, and open field tests were conducted to assess sensorimotor functions before the rats were perfused for histology. RESULTS: A cell dose-related reduction in cerebral blood flow was noted, as well as an increase in embolic events and concomitant lesion size, and sensorimotor impairment. In addition, a low infusion velocity (0.5 ml/6 minutes) was associated with high rate of complications. Lesions on MRI were confirmed with histology and corresponded to necrotic cell loss and blood-brain barrier leakage. CONCLUSIONS: Particularly cell dose but also infusion velocity contribute to complications encountered after intra-arterial cell transplantation. This should be considered before planning efficacy studies in rats and, potentially, in patients with stroke.

Pre- and postmortem imaging of transplanted cells.

Therapeutic interventions based on the transplantation of stem and progenitor cells have garnered increasing interest. This interest is fueled by successful preclinical studies for indications in many diseases, including the cardiovascular, central nervous, and musculoskeletal system. Further progress in this field is contingent upon access to techniques that facilitate an unambiguous identification and characterization of grafted cells. Such methods are invaluable for optimization of cell delivery, improvement of cell survival, and assessment of the functional integration of grafted cells. Following is a focused overview of the currently available cell detection and tracking methodologies that covers the entire spectrum from pre- to postmortem cell identification.

Genetic engineering of stem cells for enhanced therapy.

Stem cell therapy is a promising strategy for overcoming the limitations of current treatment methods. The modification of stem cell properties may be necessary to fully exploit their potential. Genetic engineering, with an abundance of methodology to induce gene expression in a precise and well-controllable manner, is particularly attractive for this purpose. There are virus-based and non-viral methods of genetic manipulation. Genome-integrating viral vectors are usually characterized by highly efficient and long-term transgene expression, at a cost of safety. Non-integrating viruses are also highly efficient in transduction, and, while safer, offer only a limited duration of transgene expression. There is a great diversity of transfectable forms of nucleic acids; however, for efficient shuttling across cell membranes, additional manipulation is required. Both physical and chemical methods have been employed for this purpose. Stem cell engineering for clinical applications is still in its infancy and requires further research. There are two main strategies for inducing transgene expression in therapeutic cells: transient and permanent expression. In many cases, including stem cell trafficking and using cell therapy for the treatment of rapid-onset disease with a short healing process, transient transgene expression may be a sufficient and optimal approach. For that purpose, mRNA-based methods seem ideally suited, as they are characterized by a rapid, highly efficient transfection, with outstanding safety. Permanent transgene expression is primarily based on the application of viral vectors, and, due to safety concerns, these methods are more challenging. There is active, ongoing research toward the development of non-viral methods that would induce permanent expression, such as transposons and mammalian artificial chromosomes.

Megakaryocyte gene targeting mediated by restricted expression of recombinase Cre.

The availability of mice with tissue-specific expression of recombinase Cre is the limiting step for a successful gene targeting by the Cre-LoxP methodology. This work aimed at generating transgenic mice with restricted expression of recombinase Cre in megakaryocytes and platelets, driven by the promoter of the αIIb gene (mαIIb-cre). Mice oocytes were microinjected with a 4.1 Kb construct comprising a 2.7 Kb promoter fragment of the glycoprotein αIIb gene, linked to the Cre-cDNA and followed by the polyA tail of the SV40. We found four mice with positive DNA genotype and three probable sites of genomic integration of the transgene. Only two of the founders showed presence of Cre-mRNA and production of Cre protein, restricted to megakaryocytes. The activity of Cre in mediating gene targeting was assessed by crossing mαIIb-cre mice to Cre-reporter mice (ROSA26-lacZ). The activity of ß-galactosidase, detected only in megakaryocytes, was sufficient to generate intense staining of X-Gal in hepatic haematopoietic islands of 14.5 dpc fetuses, in bone marrow megakaryocytes and platelets from adult mice as well as in vitro cultured megakaryocytes differentiated from bone marrow hematopoietic stem cells. Moreover, the recombinase activity was sufficient to produce the specific gene targeting of a floxed CD40L allele in megakaryocytes. The mαIIb-cre transgenic mice with restricted production of Cre in megakaryocytes, offers a selective, alternative, new tool for the genetic analysis of platelet pathophysiology.

Overexpression of podocalyxin in megakaryocytes and platelets decreases the bleeding time and enhances the agonist-induced aggregation of platelets.

Podocalyxin (PODXL) is a 145KDa sialoprotein abundantly expressed in the glycocalix of the intraglomerular kidney epithelial cells, essential in maintaining a normal renal function. PODXL is also found in vascular endothelial cells, megakaryocytes and platelets. The function of PODXL in platelets is ignored; however, its surface exposure upon platelet activation suggests its participation in controlling the hemostasis. We have generated mice (pralphaIIb-PODXL) overexpressing PODXL specifically in megakaryocytes , either alone or as a fusion protein with green fluorescent protein. The transgenic mice showed a phenotype characterized by decreased bleeding time, mild rebleeding and enhanced platelets aggregation upon agonist stimulation. The cytohematological exams as well as the prothrombin time (PT) and (APTT) tests did not differ from the control group. The biochemical analysis showed only a discrete hyperlipemia and a rise in plasma uric acid levels in the transgenic mice. The present data seem to indicate that PODXL may act as a costimulator of agonists in the activation of platelets and formation of a stable thrombus.

Screening of the myelin protein zero gene in patients with Charcot-Marie-Tooth disease.

The myelin protein zero gene (MPZ) coding for the most abundant protein of the peripheral myelin was shown to be mutated in Charcot-Marie-Tooth type 1B disease (CMT1B). Later on MPZ mutations have been shown in axonal type of CMT (CMT2). Recently three novel MPZ gene mutations were reported in congenital hypomyelinating neuropathy (CHN). In contrast to the previously reported studies, focused on CMT1B disease, we aimed to analyze the coding and promoter sequences of the MPZ gene in a group of patients with three CMT phenotypes i.e.: CMT1, CMT2 and CHN. Over 500 PCR products were screened by single strand conformation polymorphism analysis (SSCP) and heteroduplex analysis (HA). In one CMT2 family we founded the E56K mutation in the MPZ gene and in one CHN patient the T124K substitution was detected. In agreement with previously reported studies we conclude that MPZ gene screening should be performed for wide phenotype spectrum of CMT.