Silica nanoparticles enhance interfacial self-adherence of a multi-layered extracellular matrix scaffold for vascular tissue regeneration.

PURPOSE: Based on the clinical need for grafts for vascular tissue regeneration, our group developed a customizable scaffold derived from the human amniotic membrane. Our approach consists of rolling the decellularized amniotic membrane around a mandrel to form a multilayered tubular scaffold with tunable diameter and wall thickness. Herein, we aimed to investigate if silica nanoparticles (SiNP) could enhance the adhesion of the amnion layers within these rolled grafts. METHODS: To test this, we assessed the structural integrity and mechanical properties of SiNP-treated scaffolds. Mechanical tests were repeated after six months to evaluate adhesion stability in aqueous environments. RESULTS: Our results showed that the rolled SiNP-treated scaffolds maintained their tubular shape upon hydration, while non-treated scaffolds collapsed. By scanning electron microscopy, SiNP-treated scaffolds presented more densely packed layers than untreated controls. Mechanical analysis showed that SiNP treatment increased the scaffold's tensile strength up to tenfold in relation to non-treated controls and changed the mechanism of failure from interfacial slipping to single-point fracture. The nanoparticles reinforced the scaffolds both at the interface between two distinct layers and within each layer of the extracellular matrix. Finally, SiNP-treated scaffolds significantly increased the suture pullout force in comparison to untreated controls. CONCLUSION: Our study demonstrated that SiNP prevents the unraveling of a multilayered extracellular matrix graft while improving the scaffolds' overall mechanical properties. In addition to the generation of a robust biomaterial for vascular tissue regeneration, this novel layering technology is a promising strategy for a number of bioengineering applications.

Silica nanoparticles enhance the cyto- and hemocompatibility of a multilayered extracellular matrix scaffold for vascular tissue regeneration.

PURPOSE: The limited availability of autologous vessels for vascular bypass surgeries is a major roadblock to treating severe cardiovascular diseases. Based on this clinical priority, our group has developed a novel engineered vascular graft by rolling human amniotic membranes into multilayered extracellular matrixes (ECM). When treated with silica nanoparticles (SiNP), these rolled scaffolds showed a significant improvement in their structural and mechanical properties, matching those from gold standard autologous grafts. However, it remained to be determined how cells respond to SiNP-treated materials. As a first step toward understanding the biocompatibility of SiNP-dosed biomaterials, we aimed to assess how endothelial cells and blood components interact with SiNP-treated ECM scaffolds. METHODS: To test this, we used established in vitro assays to study SiNP and SiNP-treated scaffolds' cyto and hemocompatibility. RESULTS: Our results showed that SiNP effects on cells were concentration-dependent with no adverse effects observed up to 10 µg/ml of SiNP, with higher concentrations inducing cytotoxic and hemolytic responses. The SiNP also enhanced the scaffold's hydrophobicity state, a feature known to inhibit platelet and immune cell adhesion. Accordingly, SiNP-treated scaffolds were also shown to support endothelial cell growth while preventing platelet and leukocyte adhesion. CONCLUSION: Our findings suggest that the addition of SiNP to human amniotic membrane extracellular matrixes improves the cyto- and hemocompatibility of rolled scaffolds and highlights this strategy as a robust mechanism to stabilize layered collagen scaffolds for vascular tissue regeneration.

RNA polymerase II pausing is essential during spermatogenesis for appropriate gene expression and completion of meiosis.

Male germ cell development requires precise regulation of gene activity in a cell-type and stage-specific manner, with perturbations in gene expression during spermatogenesis associated with infertility. Here, we use steady-state, nascent and single-cell RNA sequencing strategies to comprehensively characterize gene expression across male germ cell populations, to dissect the mechanisms of gene control and provide new insights towards therapy. We discover a requirement for pausing of RNA Polymerase II (Pol II) at the earliest stages of sperm differentiation to establish the landscape of gene activity across development. Accordingly, genetic knockout of the Pol II pause-inducing factor NELF in immature germ cells blocks differentiation to spermatids. Further, we uncover unanticipated roles for Pol II pausing in the regulation of meiosis during spermatogenesis, with the presence of paused Pol II associated with double-strand break (DSB) formation, and disruption of meiotic gene expression and DSB repair in germ cells lacking NELF.

Role for Nongenomic Estrogen Signaling in Male Fertility.

Estrogen actions are mediated by both nuclear (n) and membrane (m) localized estrogen receptor 1 (ESR1). Male Esr1 knockout (Esr1KO) mice lacking functional Esr1 are infertile, with reproductive tract abnormalities. Male mice expressing nESR1 but lacking mESR1 (nuclear-only estrogen receptor 1 mice) are progressively infertile due to testicular, rete testis, and efferent ductule abnormalities similar to Esr1KO males, indicating a role for mESR1 in male reproduction. The H2NES mouse expresses only mESR1 but lacks nESR1. The goal of this study was to identify the functions of mESR1 alone in mice where nESR1 was absent. Breeding trials showed that H2NES males are fertile, with decreased litter numbers but normal pup numbers/litter. In contrast to Esr1KO mice, H2NES testicular, and epididymal weights were not reduced, and seminiferous tubule abnormalities were less pronounced. However, Esr1KO and H2NES males both had decreased sperm motility and a high incidence of abnormal sperm morphology. Seminiferous tubule and rete testis dilation and decreased efferent ductule epithelial height characteristic of Esr1KO males were reduced in H2NES. Consistent with this, expression of genes involved in fluid transport and ion movement that were reduced in Esr1KO (Aqp1, Car2, Car14, Cftr) were partially or fully restored to wild-type levels in H2NES. In summary, in contrast to Esr1KO males, H2NES males are fertile and have reduced phenotypic and functional abnormalities in the testis and efferent ductules. Thus, mESR1 alone, in the absence of nESR1, can partially regulate male reproductive tract structure and function, emphasizing its importance for overall estrogen action.

Targeting estrogen signaling and biosynthesis for aged skin repair.

Non-healing skin wounds are disproportionally prevalent in older adults. Current treatments do not account for the particularities of aged skin and result in inadequate outcomes. Overall, healing chronic wounds in the elderly remains a major unmet clinical need. Estrogens play a critical role in reproduction but also have important actions in non-reproductive organs. Estrogen biosynthesis and signaling pathways are locally activated during physiological wound healing, processes that are inhibited in elderly estrogen-deprived skin. Estrogen deprivation has been shown to be a critical mediator of impaired wound healing in both postmenopausal women and aged men, and topical estrogen application reverses age-associated delayed wound healing in both elderly men and women. These data indicate that adequate estrogen biosynthesis and properly regulated estrogen signaling pathways are essential for normal wound healing and can be targeted to optimize tissue repair in the elderly. However, due to fundamental questions regarding how to safely restore estrogen signaling locally in skin wounds, there are currently no therapeutic strategies addressing estrogen deficiency in elderly chronic wounds. This review discusses established and recent literature in this area and proposes the hypothesis that estrogen plays a pleiotropic role in skin aging and that targeting estrogen signaling and biosynthesis could promote skin repair in older adults.

RNA polymerase II pausing is essential during spermatogenesis for appropriate gene expression and completion of meiosis.

Male germ cell development requires precise regulation of gene activity in a cell-type and stage-specific manner, with perturbations in gene expression during spermatogenesis associated with infertility. Here, we use steady-state, nascent and single-cell RNA sequencing strategies to comprehensively characterize gene expression across male germ cell populations, to dissect the mechanisms of gene control and provide new insights towards therapy. We discover a requirement for pausing of RNA Polymerase II (Pol II) at the earliest stages of sperm differentiation to establish the landscape of gene activity across development. Accordingly, genetic knockout of the Pol II pause-inducing factor NELF in immature germ cells blocks differentiation to mature spermatids. Further, we uncover unanticipated roles for Pol II pausing in the regulation of meiosis during spermatogenesis, with the presence of paused Pol II associated with double strand break formation by SPO11, and disruption of SPO11 expression in germ cells lacking NELF.

Subacute high-refined carbohydrate diet leads to abnormal reproductive control of the hypothalamic-pituitary axis in female rats.

We previously reported that female rats placed on a diet containing refined carbohydrates (HCD) resulted in obesity and reproductive abnormalities, such as high serum LH concentration and abnormal ovarian function. However, the impacts at the hypothalamic-pituitary (HP) function, specifically regarding pathways linked to reproductive axis modulation are unknown. In this study, we assessed whether subacute feeding with HCD results in abnormal reproductive control in the HP axis. Female rats were fed with HCD for 15 days and reproductive HP axis morphophysiology was assessed. HCD reduced hypothalamic mRNA expression (Kiss1, Lepr, and Amhr2) and increased pituitary LHß+ cells. These changes likely contribute to the increase in serum LH concentration observed in HCD. Blunted estrogen negative feedback was observed in HCD, with increased kisspeptin protein expression in the arcuate nucleus of the hypothalamus (ARH), lower LHß+ cells and LH concentration in ovariectomized (OVX)+HCD rats. Thus, these data suggest that HCD feeding led to female abnormal reproductive control of HP axis.

Sertoli cells require TDP-43 to support spermatogenesis†.

TAR DNA binding protein of 43 kD (TDP-43) is an evolutionarily conserved, ubiquitously expressed transcription factor and RNA-binding protein with major human health relevance. TDP-43 is present in Sertoli and germ cells of the testis and is aberrantly expressed in the sperm of infertile men. Sertoli cells play a key role in spermatogenesis by offering physical and nutritional support to male germ cells. The current study investigated the requirement of TDP-43 in Sertoli cells. Conditional knockout (cKO) of TDP-43 in mouse Sertoli cells caused failure of spermatogenesis and male subfertility. The cKO mice showed decreased testis weight, and low sperm count. Testis showed loss of germ cell layers, presence of vacuoles, and sloughing of round spermatids, suggesting loss of contact with Sertoli cells. Using a biotin tracer, we found that the blood-testis barrier (BTB) was disrupted as early as postnatal day 24 and worsened in adult cKO mice. We noted aberrant expression of the junction proteins connexin-43 (gap junction) and N-cadherin (ectoplasmic specialization). Oil Red O staining showed a decrease in lipid droplets (phagocytic function) in tubule cross-sections, Sertoli cells cytoplasm, and in the lumen of seminiferous tubules of cKO mice. Finally, qRT-PCR showed upregulation of genes involved in the formation and/or maintenance of Sertoli cell junctions as well as in the phagocytic pathway. Sertoli cells require TDP-43 for germ cell attachment, formation and maintenance of BTB, and phagocytic function, thus indicating an essential role for TDP-43 in the maintenance of spermatogenesis.

Loss of TDP-43 in male germ cells causes meiotic failure and impairs fertility in mice.

Meiotic arrest is a common cause of human male infertility, but the causes of this arrest are poorly understood. Transactive response DNA-binding protein of 43 kDa (TDP-43) is highly expressed in spermatocytes in the preleptotene and pachytene stages of meiosis. TDP-43 is linked to several human neurodegenerative disorders wherein its nuclear clearance accompanied by cytoplasmic aggregates underlies neurodegeneration. Exploring the functional requirement for TDP-43 for spermatogenesis for the first time, we show here that conditional KO (cKO) of the Tardbp gene (encoding TDP-43) in male germ cells of mice leads to reduced testis size, depletion of germ cells, vacuole formation within the seminiferous epithelium, and reduced sperm production. Fertility trials also indicated severe subfertility. Spermatocytes of cKO mice showed failure to complete prophase I of meiosis with arrest at the midpachytene stage. Staining of synaptonemal complex protein 3 and γH2AX, markers of the meiotic synaptonemal complex and DNA damage, respectively, and super illumination microscopy revealed nonhomologous pairing and synapsis defects. Quantitative RT-PCR showed reduction in the expression of genes critical for prophase I of meiosis, including Spo11 (initiator of meiotic double-stranded breaks), Rec8 (meiotic recombination protein), and Rad21L (RAD21-like, cohesin complex component), as well as those involved in the retinoic acid pathway critical for entry into meiosis. RNA-Seq showed 1036 upregulated and 1638 downregulated genes (false discovery rate <0.05) in the Tardbp cKO testis, impacting meiosis pathways. Our work reveals a crucial role for TDP-43 in male meiosis and suggests that some forms of meiotic arrest seen in infertile men may result from the loss of function of TDP-43.

Characterization of rodent Sertoli cell primary cultures.

Sertoli cells play a vital role in spermatogenesis by offering physical and nutritional support to the differentiating male germ cells. They form the blood-testis barrier and secrete growth factors essential for germ cell differentiation. Sertoli cell primary cultures are critical for understanding the regulation of spermatogenesis; however, obtaining pure cultures has been a challenge. Rodent Sertoli cell isolation protocols do not rule out contamination by the interstitial or connective tissue cells. Sertoli cell-specific markers could be helpful, but there is no consensus. Vimentin, the most commonly used marker, is not specific for Sertoli cells since its expression has been reported in peritubular myoid cells, mesenchymal stem cells, fibroblasts, macrophages, and endothelial cells, which contaminate Sertoli cell preparations. Markers based on transcription and growth factors also have limitations. Thus, the impediment to obtaining pure Sertoli cell cultures pertains to both the method of isolation and marker usage. The aim of this review is to discuss improvements to current methods of rodent Sertoli cell primary cultures, assess the properties of prepubertal versus mature Sertoli cell cultures, and propose steps to improve cellular characterization. Potential benefits of using contemporary approaches, including lineage tracing, specific cell ablation, and RNA-seq for obtaining Sertoli-specific transcript markers are discussed. Evaluating the specificity and applicability of these markers at the protein level to characterize Sertoli cells in culture would be critical. This review is expected to positively impact future work using primary cultures of rodent Sertoli cells.

Mouse Sertoli cells isolation by lineage tracing and sorting.

Sertoli cells play a key role in spermatogenesis by supporting the germ cells throughout differentiation. The isolation of Sertoli cells is essential to study their functions. However, the close contact of Sertoli cells with other testicular cell types and the high proliferation of contaminating cells are obstacles to obtain pure primary cultures. Current rodent Sertoli cell isolation protocols result in enriched, rather than pure Sertoli cells. Therefore, novel approaches are necessary to improve the purity of Sertoli cell primary cultures. The goal of this study is to obtain pure mouse Sertoli cells using lineage tracing and fluorescence-activated cell sorting (FACS). We bred the Amh-Cre mouse line with tdTomato line to generate mice constitutively expressing red fluorescence specifically in Sertoli cells. Primary cultures of Sertoli cells isolated from prepubertal mice showed that 79% of cells expressed tdTomato, as evaluated by fluorescence microscopy and flow cytometry; however, nearly all adherent cells were positive for vimentin. Most of the tomato-negative cells expressed α-smooth muscle actin (α-SMA), a peritubular myoid cell marker, but double-negative populations were also present. These findings suggest that vimentin lacks Sertoli cell-specificity and that α-SMA is not adequate to identify all of the contaminating cells. Upon FACS sorting; however, virtually 100% of the cells were tdTomato positive, expressed vimentin, but not α-SMA. Prepubertal mice yielded a higher number of Sertoli cells compared to adults, but both could be adequately sorted. In conclusion, our study shows that lineage tracing and sorting is an efficient strategy for acquiring pure populations of murine Sertoli cells.

Skin wound healing in humans and mice: Challenges in translational research.

Despite the great progress in translational research concerning skin wound healing in the last few decades, no animal model fully predicts all clinical outcomes. The mouse is the most commonly used model, as it is easy to maintain and standardize, and is economically accessible. However, differences between murine and human skin repair, such as the contraction promoted by panniculus carnosus and the role of specific niches of skin stem cells, make it difficult to bridge the gap between preclinical and clinical studies. Therefore, this review highlights the particularities of each species concerning skin morphophysiology, immunology, and genetics, which is essential to properly interpret findings and translate them to medicine.

Mesenchymal and induced pluripotent stem cells: general insights and clinical perspectives.

Mesenchymal stem cells have awakened a great deal of interest in regenerative medicine due to their plasticity, and immunomodulatory and anti-inflammatory properties. They are high-yield and can be acquired through noninvasive methods from adult tissues. Moreover, they are nontumorigenic and are the most widely studied. On the other hand, induced pluripotent stem (iPS) cells can be derived directly from adult cells through gene reprogramming. The new iPS technology avoids the embryo destruction or manipulation to generate pluripotent cells, therefore, are exempt from ethical implication surrounding embryonic stem cell use. The pre-differentiation of iPS cells ensures the safety of future approaches. Both mesenchymal stem cells and iPS cells can be used for autologous cell transplantations without the risk of immune rejection and represent a great opportunity for future alternative therapies. In this review we discussed the therapeutic perspectives using mesenchymal and iPS cells.

Reproductive stem cell differentiation: extracellular matrix, tissue microenvironment, and growth factors direct the mesenchymal stem cell lineage commitment.

The mesenchymal stem cells (MSCs) have awakened interest in regenerative medicine due to its high capability to proliferate and differentiate in multiple specialized lineages under defined conditions. The reproductive system is considered a valuable source of MSCs, which needs further investigations. Many factors have been reported as critical for these cell lineage specification and determination. In this review, we discuss the main effects of extracellular matrix or tissue environment and growth factors in the cell lineage commitment, including the reproductive stem cells. The MSCs responses to culture medium stimuli or to soluble factors probably occur through several intracellular activation pathways. However, the molecular mechanisms in which the cells respond to these mechanical or chemical perturbations remain elusive. Recent findings suggest a synergic effect of microenvironment and soluble cell culture factors affecting cell differentiation. For future applications in cell therapy, protocols of reproductive MSCs differentiation must be established.

Morphological characterization of the progenitor blood cells in canine and feline umbilical cord.

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells.