Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.     

Soluble forms of NCAM and F3 neuronal cell adhesion molecules promote Schwann cell migration: identification of protein tyrosine phosphatases zeta/beta as the putative F3 receptors on Schwann cells

Neural cell adhesion molecule (NCAM) and F3 are both axonal adhesion molecules which display homophilic (NCAM) or heterophilic (NCAM, F3) binding activities and participate in bidirectional exchange of information between neurones and glial cells. Engineered Fc chimeric molecules are fusion proteins that contain the extracellular part of NCAM or F3 and the Fc region of human IgG1. Here, we investigated the effect of NCAM-Fc and F3-Fc chimeras on Schwann cell (SC) migration. Binding sites were identified at the surface of cultured SCs by chimera coated fluorospheres. The functional effect of NCAM-Fc and F3-Fc binding was studied in two different SC migration models. In the first, migration is monitored at specific time intervals inside a 1-mm gap produced in a monolayer culture of SCs. In the second, SCs from a dorsal root ganglion explant migrate on a sciatic nerve cryosection. In both systems addition of the chimeras significantly increased the extent of SC migration and this effect could be prevented by the corresponding anti-NCAM or anti-F3 blocking antibodies. Furthermore, antiproteoglycan-type protein tyrosine phosphatase zeta/beta (RPTPzeta/beta) antibodies identified the presence of RPTPzeta/beta on SCs and prevented the enhancing effect of soluble F3 on SC motility by 95%. The F3-Fc coated Sepharose beads precipitated RPTPzeta/beta from SC lysates. Altogether these data point to RPTPzeta/beta is the putative F3 receptor on SCs. These results identify F3 and NCAM receptors on SC as potential mediators of signalling occurring between axons and glial cells during peripheral nerve development and regeneration.     

An exploration of the role of Sertoli cells on fetal testis development using cell ablation strategy

Sertoli cells (SCs) are presumed to be the center of testis differentiation because they provide both structural support and biological regulation for spermatogenesis. Previous studies suggest that SCs control germ cell (GC) count and Leydig cell (LC) development in mouse testes. However, the regulatory role of SCs on peritubular myoid (PTM) cell fate in fetal testis has not been clearly reported. Here, we employed Amh‐Cre; diphtheria toxin fragment A (DTA) mouse model to selectively ablate SCs from embryonic day (E) 14.5. Results found that SC ablation in the fetal stage caused the disruption of testis cords and the massive loss of GCs. Furthermore, the number of α‐smooth muscle actin‐labeled PTM cells was gradually decreased from E14.5 and almost lost at E18.5 in SC ablation testis. Interestingly, some Ki67 and 3β‐HSD double‐positive fetal LCs could be observed in Amh‐Cre; DTA testes at E16.5 and E18.5. Consistent with this phenomenon, the messenger RNA levels of Hsd3b1, Cyp11a1, Lhr, Star and the protein levels of 3β‐HSD and P450Scc were significantly elevated by SC ablation. SC ablation appears to induce ectopic proliferation of fetal LCs although the total LC number appeared reduced. Together, these findings bring us a better understanding of SCs’ central role in fetal testis development.     

Stem-cell approaches for kidney repair: choosing the right cells

With the increasing rate of end-stage renal failure and limited alternatives for its treatment, stem cell (SC) therapy for kidney injury is urgently needed. Choosing the right SC type is the critical step in realizing the potential of this therapeutic approach. Four possible sources of SCs are envisioned for the development of this type of treatment: (i) bone-marrow-derived SCs (BMSCs), (ii) renal adult SCs, (iii) embryonic SCs and (iv) fetal renal SCs. We suggest that resident SCs recently identified in the Bowman's capsule of adult human kidneys might prospectively be the ideal cell type for treatment of both acute and chronic renal injury because they display the potential to differentiate into multiple types of renal cells. However, BMSCs also represent an attractive alternative, especially for the treatment of patients affected by acute renal failure.     

A Disintegrin and Metalloprotease 10 (ADAM10) Is Indispensable for Maintenance of the Muscle Satellite Cell Pool

Satellite cells (SCs) are muscle-specific stem cells that are essential for the regeneration of damaged muscles. Although SCs have a robust capacity to regenerate myofibers, the number of SCs decreases with aging, leading to insufficient recovery after muscle injury. We herein show that ADAM10 (a disintegrin and metalloprotease 10), a membrane-bound proteolytic enzyme with a critical role in Notch processing (S2 cleavage), is essential for the maintenance of SC quiescence. We generated mutant mice in which ADAM10 in SCs can be conditionally abrogated by tamoxifen injection. Tamoxifen-treated mutant mice did not show any apparent defects and grew normally under unchallenged conditions. However, these mice showed a nearly complete loss of muscle regeneration after chemically induced muscle injury. In situ hybridization and flow cytometric analyses revealed that the mutant mice had significantly less SCs compared with wild type controls. Of note, we found that inactivation of ADAM10 in SCs severely compromised Notch signaling and led to dysregulated myogenic differentiation, ultimately resulting in deprivation of the SC pool in vivo. Taken together, the present findings underscore the role of ADAM10 as an indispensable component of Notch signaling in SCs and for maintaining the SC pool.     

The impact of physical,biochemical, and electrical signaling on Schwann cell plasticity

Peripheral nervous system (PNS) injuries are an ongoing health care concern. While autografts and allografts are regarded as the current clinical standard for traumatic injury, there are inherent limitations that suggest alternative remedies should be considered for therapeutic purposes. In recent years, nerve guidance conduits (NGCs) have become increasingly popular as surgical repair devices, with a multitude of various natural and synthetic biomaterials offering potential to enhance the design of conduits or supplant existing technologies entirely. From a cellular perspective, it has become increasingly evident that Schwann cells (SCs), the primary glia of the PNS, are a predominant factor mediating nerve regeneration. Thus, the development of severe nerve trauma therapies requires a deep understanding of how SCs interact with their environment, and how SC microenvironmental cues may be engineered to enhance regeneration. Here we review the most recent advancements in biomaterials development and cell stimulation strategies, with a specific focus on how the microenvironment influences the behavior of SCs and can potentially lead to functional repair. We focus on microenvironmental cues that modulate SC morphology, proliferation, migration, and differentiation to alternative phenotypes. Promotion of regenerative phenotypic responses in SCs and other non-neuronal cells that can augment the regenerative capacity of multiple biomaterials is considered along with innovations and technologies for traumatic injury.     

In Vitro and In Vivo Magnetic Resonance Tracking of Sinerem-Labeled Human Umbilical Mesenchymal Stromal Cell-Derived Schwann Cells

Tracking of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles-labeled embryonic stem cells, neural stem cells, or adult mesenchymal stem cells in vitro and in vivo by using magnetic resonance (MR) imaging have been reported. However, whether the transdifferentiated cells can be effectively labeled by USPIO has not yet been investigated. The requirement for nerve donor material evokes additional morbidity and inability to generate a sufficiently large number of cells in a short time to hamper the clinic application of Schwann cells (SCs) transplantation. These limitations may be avoided if SCs can be generated from clinically accessible sources, such as bone marrow and umbilical cord. However, a reliable means of inducing the selective differentiation of human mesenchymal stromal cells isolated from the umbilical cord (HUMSCs) into SCs in vitro has not yet been established. In this study, we induce HUMSCs into Schwann-like cells in terms of morphology, phenotype, and function by an improved protocol basing on our previous studies. Furthermore, HUMSCs-derived SCs are labeled efficiently in vitro with ultrasmall superparamagnetic iron oxide contrast agent (USPIO) Sinerem and poly-l-lysine (PLL) without affecting morphology, cell cycle, proliferation, and differentiation ability of the labeled cells between the concentration of 200 to 800 μg/ml. Importantly, when grafted into the intact cerebral cortex and striatum, the survival and migration of these Sinerem-labeled cells were observed using MRI. Our study suggest the effective concentration field for Sinerem use in tracking transdifferentiated HUMSCs, and Sinerem labeling transdifferentiated HUMSCs is feasible, efficient, and safe for MRI tracing following grafting into nervous system.     

Asymmetric ERM activation at the Schwann cell process tip is required in axon-associated motility

Axon-associated Schwann cell (SC) motility and process dynamics are crucial in the development and regeneration of the peripheral nervous system (PNS). The bipolar morphology of SCs represents an unexplored conundrum in terms of directed motility. Using fluorescence time-lapse microscopy of transfected SCs within myelinating dorsal root ganglion (DRG) explants, we demonstrate cycling of SCs between bipolar and highly motile, unipolar morphologies as a result of asymmetric process retraction and extension. Unipolar SC motility appears nucleotaxic in nature, similar to the movement of neurons on radial glia during cortical development. We also show that asymmetric process retraction is associated with the activation of ERM (ezrin/radixin/moesin) proteins and subsequent recruitment of ezrin-binding phospho-protein 50 kDa (EBP50) at the retracting process tip. This activation occurs in response to localized synthesis of phosphatidylinositol (4,5)-bisphosphate (PIP2) at this site. Finally, we demonstrate that the activation of ERM proteins at the SC process tip is essential for motility and the maintenance of SC polarity, as ERM disruption yields a dysfunctional, multi-polar cell. These results demonstrate that specializations at the tips of SC processes regulate their dynamics, which in turn is associated with directed motility in these cells.     

Longitudinal tracking of human fetal cells labeled with super paramagnetic iron oxide nanoparticles in the brain of mice with motor neuron disease

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival.     

成年猴雪旺细胞的在体增殖和体外迁移的研究

为了探讨成年猴雪旺氏细胞的在体增殖和体外迁移的能力,我们对用神经结扎术结扎的A组6只3-13岁雄性恒河猴的腓肠神经进行植块培养,部分细胞培养在聚酯纤维上,2-4周后作抗S-100抗体免疫组化染色和电镜观察;B组2只未做结扎的新生猴腓肠神经培养作为对照.结果显示A组雪旺氏细胞平均在培养的第5天从神经段中迁出,年幼者早于成年猴;细胞在纤维上以螺旋状向前迁移;雪旺氏细胞抗S-100蛋白抗体染色阳性;电镜显示,雪旺氏细胞包卷纤维,但是,未见髓鞘形成.B组神经段培养2周仍无雪旺氏细胞迁出.研究表明,结扎神经使其发生瓦勒氏变性,经植块培养、纯化,能够获得可用于移植的成年猴的雪旺氏细胞.     

The Role of Symmetric Stem Cell Divisions in Tissue Homeostasis

Successful maintenance of cellular lineages critically depends on the fate decision dynamics of stem cells (SCs) upon division. There are three possible strategies with respect to SC fate decision symmetry: (a) asymmetric mode, when each and every SC division produces one SC and one non-SC progeny; (b) symmetric mode, when 50% of all divisions produce two SCs and another 50%—two non-SC progeny; (c) mixed mode, when both the asymmetric and two types of symmetric SC divisions co-exist and are partitioned so that long-term net balance of the lineage output stays constant. Theoretically, either of these strategies can achieve lineage homeostasis. However, it remains unclear which strategy(s) are more advantageous and under what specific circumstances, and what minimal control mechanisms are required to operate them. Here we used stochastic modeling to analyze and quantify the ability of different types of divisions to maintain long-term lineage homeostasis, in the context of different control networks. Using the example of a two-component lineage, consisting of SCs and one type of non-SC progeny, we show that its tight homeostatic control is not necessarily associated with purely asymmetric divisions. Through stochastic analysis and simulations we show that asymmetric divisions can either stabilize or destabilize the lineage system, depending on the underlying control network. We further apply our computational model to biological observations in the context of a two-component lineage of mouse epidermis, where autonomous lineage control has been proposed and notable regional differences, in terms of symmetric division ratio, have been noted—higher in thickened epidermis of the paw skin as compared to ear and tail skin. By using our model we propose a possible explanation for the regional differences in epidermal lineage control strategies. We demonstrate how symmetric divisions can work to stabilize paw epidermis lineage, which experiences high level of micro-injuries and a lack of hair follicles as a back-up source of SCs.     

In-Vivo Imaging of Cell Migration Using Contrast Enhanced MRI and SVM Based Post-Processing

The migration of cells within a living organism can be observed with magnetic resonance imaging (MRI) in combination with iron oxide nanoparticles as an intracellular contrast agent. This method, however, suffers from low sensitivity and specificty. Here, we developed a quantitative non-invasive in-vivo cell localization method using contrast enhanced multiparametric MRI and support vector machines (SVM) based post-processing. Imaging phantoms consisting of agarose with compartments containing different concentrations of cancer cells labeled with iron oxide nanoparticles were used to train and evaluate the SVM for cell localization. From the magnitude and phase data acquired with a series of T2*-weighted gradient-echo scans at different echo-times, we extracted features that are characteristic for the presence of superparamagnetic nanoparticles, in particular hyper- and hypointensities, relaxation rates, short-range phase perturbations, and perturbation dynamics. High detection quality was achieved by SVM analysis of the multiparametric feature-space. The in-vivo applicability was validated in animal studies. The SVM detected the presence of iron oxide nanoparticles in the imaging phantoms with high specificity and sensitivity with a detection limit of 30 labeled cells per mm³, corresponding to 19 μM of iron oxide. As proof-of-concept, we applied the method to follow the migration of labeled cancer cells injected in rats. The combination of iron oxide labeled cells, multiparametric MRI and a SVM based post processing provides high spatial resolution, specificity, and sensitivity, and is therefore suitable for non-invasive in-vivo cell detection and cell migration studies over prolonged time periods.     

雪旺氏细胞与同种异体骨支架的体外共培养研究

目的:探讨雪旺细胞(Schwann’s cells,SCs)在同种异体骨支架上的生物相容性,体外构建组织工程骨神经化模型。方法:利用新鲜人体骨骼制备同种异体骨支架材料,检测其物理性能;采用优化方法提取新生SD大鼠坐骨、臂丛神经培养SCs,实验分为三维培养实验组(SCs+同种异体骨)、二维培养对照组(SCs+胶原玻片),S-100抗体免疫荧光染色鉴定SCs纯度;细胞计数法检测两组细胞增殖特点;细胞接种后第3、7天取样,扫描电镜观察。结果:同种异体骨支架具有良好的三维孔隙结构,适宜细胞贴附生长;S-100免疫荧光染色证实SCs纯度95%;扫描电镜检测显示两组SCs均可正常粘附增殖,细胞间排布规律相似,培养早期实验组SCs胞体更加细长,伪足更加明显,随着培养时间的延长表现出较强的迁移能力;细胞增殖检测:两组SCs生长曲线特征基本一致,支架材料对SCs无毒性作用。结论:同种异体骨支架SCs具有良好的生物相容性,其三维立体多孔结构有利于SCs的粘附与迁移,初步构建了体外组织工程骨神经化模型。     

From Cell Differentiation to Cell Collectives: Bacillus subtilis Uses Division of Labor to Migrate

The organization of cells, emerging from cell–cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called “van Gogh bundles”) of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.     

Up-Regulation of Cdc37 Contributes to Schwann Cell Proliferation and Migration After Sciatic Nerve Crush

Cell division cycle protein 37 (Cdc37), a molecular chaperone takes part in a series of cellular processes including cell signal transduction, cell cycle progression, cell proliferation, cell motility, oncogenesis and malignant progression. It can not only recruit immature protein kinases to HSP90 but also work alone. Cdc37 was reported to be associated with neurogenesis, neurite outgrowth, axon guidance and myelination. However, the roles of Cdc37 on Schwann cells (SC) after peripheral nerve injury (PNI) remain unknown. In this study, we found that the expression of Cdc37 increased and reached the peak at 1 week after sciatic nerve crush (SNC), which was consistent with that of proliferation cell nuclear antigen. Immunofluorescence verified that Cdc37 co-localized with SC in vivo and in vitro. Intriguingly, Cdc37 protein level was potentiated in the model of TNF-α-induced SC proliferation. Moreover, we found that Cdc37 silencing impaired proliferation of SC in vitro. Moreover, Cdc37 suppression attenuated kinase signaling pathways of Raf–ERK and PI3K/AKT which are crucial cell signaling for SC proliferation. Finally, we found that Cdc37 silencing inhibited SC migration in vitro. In conclusion, we demonstrated that the way Cdc37 contributed to SC proliferation is likely via activating kinase signaling pathways of Raf–ERK and PI3K/AKT, and CDC37 was also involved in SC migration after SNC.     

A method for obtaining Schwann cell cultures from adult rabbit nerve based on "in vitro" pre-degeneration and neuregulin treatment

Schwann cells (SCs) are basic elements for cell therapy and tissue engineering in the central and peripheral nervous system. Therefore, the development of a reliable method to obtain SC cultures is required. For possible therapeutic applications the cultures need to produce a sufficiently large number of SCs with a high level of purity in a relatively short period of time. To increase SC yield and purity we pre-degenerated pieces of 1-2 mm of adult rabbit sciatic nerves by incubating them for seven days in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, penicillin/streptomycin and NRG1-β1. Following pre-degeneration the nerve pieces were dissociated and then cultured for 6 or 15 days in the same culture medium. After 6 days of culture we obtained around 9.5x103 cells/mg with approximately 94% SCs (S-100 positive) purity. After 15 days of culture the yield was about 80x103 cells/mg and the purity was approximately 75%. Pre-degeneration and subsequent culture of small pieces of adult nerve with NRG1-β1 supplemented medium increased the number of SCs and restricted the overgrowth of fibroblast-like cells.