<p>Cells were left untreated (control) or stimulated with CPT-cAMP, EPAC-cAMP or PKA-cAMP a... more <p>Cells were left untreated (control) or stimulated with CPT-cAMP, EPAC-cAMP or PKA-cAMP at a concentration of 200 µM each, unless otherwise indicated. Cells were analyzed for the incorporation of [<sup>3</sup>H]-thymidine (A) and the expression of myelin associated markers (C-D) 3 days after cAMP administration. The activity of PKA was determined by <i>in </i><i>vitro</i> phosphorylation assays (B, upper panel) and the immunodetection of phosphorylated PKA-specific substrates (C, bottom panels) in SCs stimulated for 30 min with the indicated analogs of cAMP. The activity of EPAC (Rap1 assays) was determined under identical experimental conditions (B, lower panels). As shown in C and D, EPAC-cAMP and PKA-cAMP failed to mimic the action of CPT-cAMP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082354#pone-0082354-g005" target="_blank">Figure 5C</a>) or db-cAMP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082354#pone-0082354-g002" target="_blank">Figure 2</a>) either if provided alone or in combination (not shown). Curiously, PKA-cAMP modestly increased P<sub>0</sub> and reduced c-Jun expression without increasing Krox-20 or reducing GFAP expression (D). </p
<p>SC-DRG neuron cultures were left untreated (control) or stimulated with CPT-cAMP, EPAC-c... more <p>SC-DRG neuron cultures were left untreated (control) or stimulated with CPT-cAMP, EPAC-cAMP or PKA-cAMP (200 µM each, unless otherwise indicated) for 3 days (A) or 12 days (B) prior to analysis by [<sup>3</sup>H]-thymidine incorporation (A) and immunofluorescence microscopy (B), respectively. Experimental conditions and analysis were identical to those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082354#pone-0082354-g004" target="_blank">Figure 4</a>. In B, images of myelinating co-cultures are shown at low (top panels) and high (bottom panels) magnifications, respectively. EPAC-cAMP significantly reduced proliferation of axon-contacting SCs (A). However, it did not mimic the effect of CPT-cAMP and db-cAMP (not shown) on inducing the formation of myelin sheaths (B). The strong pro-differentiating effect of CPT-cAMP is reflected by the occurrence of O1 positive, MBP positive cells throughout the co-culture system (B).</p
<p>The figure depicts the temporal progression of phenotypic changes during cAMP-induced di... more <p>The figure depicts the temporal progression of phenotypic changes during cAMP-induced differentiation in isolated nerve-derived SC cultures. SCs were subjected to mitogen and serum starvation (Methods) prior to treatment with CPT-cAMP (250 μM) for 3 days unless otherwise indicated. The control condition in this and subsequent figures refers to cells that were incubated in the absence of cAMP-stimulating agents for the whole time course of the experiment. In panels B-C, the control (C) condition refers to cells that were fixed or lysed for analysis at the time of stimulation (day/hour zero). Cells were analyzed for the expression of c-Jun, Krox-20, O1 and MBP by immunofluorescence microscopy and western blot, as marked in the figure. In A, SCs were co-stained with the SC-specific marker S-100 to label all cells and reveal the changes in cell morphology that occur in response to prolonged CPT-cAMP stimulation. The schematic diagram (D) depicts the temporal course of changes during cAMP-induced differentiation as revealed by this and our previous time course studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116948#pone.0116948.ref008" target="_blank">8</a>]. Arrowheads in A-B point out to representative Krox-20 positive cells (nuclear localization) that fail to express O1. Cells that labeled positive for O1 and negative for Krox-20 were not found. Of note, staining with MBP antibodies did not detect a specific signal in either control or cAMP-treated cells (B, quantification shown on the right). All antibodies used are indicated in the figure; nuclei were labeled with DAPI (blue) in this and all subsequent figures.</p
<p>Forskolin was provided to SC-neuron cultures together with ascorbate essentially as desc... more <p>Forskolin was provided to SC-neuron cultures together with ascorbate essentially as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116948#pone.0116948.g008" target="_blank">Fig. 8</a>. Low magnification composites of representative cultures (A) are shown along with selected high magnification areas (B) to reveal the effect of the indicated treatments on O1 and MBP expression. The quantitative analysis provided in panel C confirmed that similar to CPT-cAMP, forskolin effectively enhanced O1 and MBP expression without concurrently increasing the MBP/O1 ratio. The DAPI channel in A (bottom panel) is shown to serve as an indication of unchanged cell density in forskolin-treated cultures. The arrowheads and arrows (B, left panels) point out to representative O1 positive cells exhibiting thin and thick myelin, respectively, as denoted by the intensity of MBP positive myelin segments. This heterogeneity of MBP staining in individual cells was not evident in forskokin-treated cultures (B, right panels), possibly due to increased synchronization in the myelination process.</p
<p>Cells that were subjected to the same experimental treatments as in <a href="htt... more <p>Cells that were subjected to the same experimental treatments as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116948#pone.0116948.g002" target="_blank">Fig. 2</a> were fixed and analyzed by TEM (Methods). Higher magnification images of selected areas (boxes) are shown to better resolve the interaction between SC processes (SC) and axons (ax). As opposed to ascorbate, CPT-cAMP treatment did not support the ensheathment of axons, the formation of a basal lamina, the deposition of extracellular collagen fibers (arrowheads) or the production of myelin membranes (bracket). Similar to the control condition (left panels), CPT-cAMP-treated SCs (cAMP) associated with multiple low diameter axons leaving many neurites deprived of direct contact with SC processes. The asynchronous responses in myelination induced by ascorbate are illustrated by the multiple ensheathment profiles shown in the upper right panel, as follows: (1) – No ensheathment; (2) Partial ensheathment; (3) Complete ensheathment, no myelin; (4) Complete ensheathment, myelin.</p
The isolation of well formed crystals of the biomineral whewellite (monohydrated calcium oxalate)... more The isolation of well formed crystals of the biomineral whewellite (monohydrated calcium oxalate) from Opuntia microdasys, a cactaceae species found in central Mexico, is described. The morphology of the crystals was investigated by means of electron microscopy. Infrared spectroscopic measurements allow an unambiguous characterization of the nature of the crystals. This is the first report of the presence of a biomineral in this species.
<p>Experimental conditions were identical to those of <a href="http://www.plosone.o... more <p>Experimental conditions were identical to those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116948#pone.0116948.g005" target="_blank">Fig. 5</a>. In A, low magnification composites of representative cultures stained with O1 (green), neurofilament (red) and DAPI (blue) are shown to reveal the effect of the indicated treatments on O1 expression with respect to the location of the neuronal bodies (white ovals), the extension of the neurite substrate (neurofilament, NF) and the distribution of the SCs (DAPI). A quantitative analysis of O1 and MBP expression is provided in B (Methods). This analysis confirmed that cAMP enhanced the total number of O1 and MBP positive cells without concomitantly increasing the MBP/O1 ratio. Selected areas within the center (a, b) and periphery (c) of the axonal outgrowth are shown at higher magnification in C and D, respectively, to reveal details of the morphology of the cells, the relationship to axons and the co-localization of O1 (green) and MBP (red). Whereas O1 positive cells could be found throughout the culture system, MBP positive cells were found exclusively within the axonal network (C, D). Images taken at the frontier of the axonal outgrowth (D) revealed that MBP rather than O1 expression was restricted to those SCs that established a direct contact with the axons, as revealed by neurofilament staining (dotted lines). The boxed area in panel D was enlarged to better resolve the distribution of the MBP staining with respect to the position of the axons (NF, arrows), the plasma membrane (O1) and the nuclei of the cells (DAPI). In these and all subsequent graphs, bar heights are means of triplicate determinations; error bars represent S.D, and * represents statistical significance for p < 0.05.</p
The benefits of transplanting cultured Schwann cells (SCs) for the treatment of spinal cord injur... more The benefits of transplanting cultured Schwann cells (SCs) for the treatment of spinal cord injury (SCI) have been systematically investigated in experimental animals since the early 1990s. Importantly, human SC (hSC) transplantation for SCI has advanced to clinical testing and safety has been established via clinical trials conducted in the USA and abroad. However, multiple barriers must be overcome to enable accessible and effective treatments for SCI patients. This review presents available information on hSC transplantation for SCI with the intention to uncover gaps in our knowledge and discuss areas for future development. To this end, we introduce the historical progression of the work that supports existing and prospective clinical initiatives and explain the reasons for the choice of hSCs while also addressing their limitations as cell therapy products. A search of the relevant literature revealed that rat SCs have served as a preclinical model of reference since the onset o...
The Identities of Membrane Steroid Receptors, 2003
1α,25-dihydroxy-vitamin D3 (lα,25(OH)2D3; calcitriol) and estrogen (17β-estradiol) act, as other ... more 1α,25-dihydroxy-vitamin D3 (lα,25(OH)2D3; calcitriol) and estrogen (17β-estradiol) act, as other steroid hormones, through two different mechanisms. In addition to regulating expression of target genes via their specific nuclear receptors (VDR and ER, respectively), both hormones induce fast, non transcriptional responses involving stimulation of transmembrane signal transduction pathways. The rapid nature and specificity by which 1α,25(OH)2D3 and 17β-estradiol trigger the activation of second messengers has led to the concept that interaction with a plasma membrane receptor is responsible for the initiation of their effects. However, there is controversy over its molecular characteristics. Among several models for non-genomic steroid receptor identity, has been proposed the existence of membrane-associated forms of either the classical receptors or alternatively of novel 1α,25(OH)2D3 and 17β- estradiol binding proteins. In this chapter we report the presence of the nuclear VDR and ER in the plasma membrane of avian muscle and mammalian breast cells and furnish data suggesting that they may be involved in non-genomic signalling by their cognate ligands.
N-cadherin and beta-catenin are involved in cell adhesion and cell cycle in tumor cells and neura... more N-cadherin and beta-catenin are involved in cell adhesion and cell cycle in tumor cells and neural crest. Both are expressed at key stages of Schwann cell (SC) development, but little is known about their function in the SC lineage. We studied the role of these molecules in adult rat derived SC-embryonic dorsal root ganglion cocultures by using low-Ca(2+) conditions and specific blocking antibodies to interfere with N-cadherin function and by using small interfering RNA (siRNA) to decrease beta-catenin expression in both SC-neuron cocultures and adult rat-derived SC monocultures. N-cadherin blocking conditions decreased SC-axon association and reduced axon-induced SC proliferation. In SC monocultures, beta-catenin reduction diminished the proliferative response of SCs to the mitogen beta1-heregulin, and, in SC-DRG cocultures, beta-catenin reduction inhibited axon-contact-dependent SC proliferation. Stimulation of SC cultures with beta1-heregulin increased total beta-catenin protein amount, phosphorylation of GSK-3beta and beta-catenin presence in nuclear extracts. In conclusion, our findings suggest a previously unrecognized contribution of beta-catenin and N-cadherin to axon-induced SC proliferation.
The isolation of well formed crystals of the biomineral weddellite (calcium oxalate dihydrate) fr... more The isolation of well formed crystals of the biomineral weddellite (calcium oxalate dihydrate) from Chamaecereus silvestrii, a Cactaceae species found in the northern part of Argentina, is described. Infrared spectroscopic measurements allow an unambiguous characterization of the nature of the crystals. This is the first report of the presence of a biomineral in this plant species.
Background Heregulin is a ligand for the protooncogene product ErbB/HER that acts as a key mitog... more Background Heregulin is a ligand for the protooncogene product ErbB/HER that acts as a key mitogenic factor for human Schwann cells (hSCs). Heregulin is required for sustained hSC growth in vitro but must be thoroughly removed before cell collection for transplantation due to potential safety concerns. The goal of this study was to develop simple cell-based assays to assess the effectiveness of heregulin addition to and removal from aliquots of hSC culture medium. These bioassays were based on the capacity of a β1-heregulin peptide to elicit ErbB/HER receptor signaling in adherent ErbB2+/ErbB3+ cells. Results Western blotting was used to measure the activity of three different β1-heregulin/ErbB-activated kinases (ErbB3/HER3, ERK/MAPK and Akt/PKB) using phospho-specific antibodies against key activating residues. The duration, dose-dependency and specificity of β1-heregulin-initiated kinase phosphorylation were investigated, and controls were implemented for assay optimization and r...
Adult Schwann cell (SC) cultures are usually derived from nerves subjected to a lengthy step of p... more Adult Schwann cell (SC) cultures are usually derived from nerves subjected to a lengthy step of pre-degeneration to facilitate enzymatic digestion and recovery of viable cells. To overcome the need for pre-degeneration, we developed a method that allows the isolation of adult rat sciatic nerve SCs immediately after tissue harvesting. This method combines the advantages of implementing a rapid enzymatic dissociation of the nerve fibers and a straightforward separation of cells versus myelin that improves both cell yield and viability. Essentially, the method consists of (1) acute dissociation with collagenase and dispase immediately after removal of the epineurium layer and extensive teasing of the nerve fibers, (2) removal of myelin debris by selective attachment of the cells to a highly adhesive poly-L-lysine/laminin substrate, (3) expansion of the initial SC population in medium containing chemical mitogens, and (4) preparation of cryogenic stocks for transfer or delayed experimentation. This protocol allows for the procurement of homogeneous SC cultures deprived of myelin and fibroblast growth as soon as 3-4 days after nerve tissue dissection. SC cultures can be used as such for experimentation or subjected to consecutive rounds of expansion prior to use, purification, or cryopreservation.
To date, magnetic-activated cell sorting (MACS) remains a powerful method to isolate distinct cel... more To date, magnetic-activated cell sorting (MACS) remains a powerful method to isolate distinct cell populations based on differential cell surface labeling. Optimized direct and indirect MACS protocols for cell immunolabeling are presented here as methods to divest Schwann cell (SC) cultures of contaminating cells (specifically, fibroblast cells) and isolate SC populations at different stages of differentiation. This chapter describes (1) the preparation of single-cell suspensions from established human and rat SC cultures, (2) the design and application of cell selection strategies using SC-specific (p75NGFR, O4, and O1) and fibroblast-specific (Thy-1) markers, and (3) the characterization of both the pre- and post-sorting cell populations. A simple protocol for the growth of hybridoma cell cultures as a source of monoclonal antibodies for cell surface immunolabeling of SCs and fibroblasts is provided as a cost-effective alternative for commercially available products. These steps allow for the timely and efficient recovery of purified SC populations without compromising the viability and biological activity of the cells.
Nerve-derived human Schwann cell (SC) cultures are irreplaceable models for basic and translation... more Nerve-derived human Schwann cell (SC) cultures are irreplaceable models for basic and translational research but their use can be limited due to the risk of fibroblast overgrowth. Fibroblasts are an ill-defined population consisting of highly proliferative cells that, contrary to human SCs, do not undergo senescence in culture. We initiated this study by performing an exhaustive immunological and functional characterization of adult nerve-derived human SCs and fibroblasts to reveal their properties and optimize a protocol of magnetic-activated cell sorting (MACS) to separate them effectively both as viable and biologically competent cells. We next used immunofluorescence microscopy imaging, flow cytometry analysis and next generation RNA sequencing (RNA-seq) to unambiguously characterize the post-MACS cell products. High resolution transcriptome profiling revealed the identity of key lineage-specific transcripts and the clearly distinct neural crest and mesenchymal origin of human S...
Schwann cell (SC) cultures from experimental animals and human donors can be prepared using nearl... more Schwann cell (SC) cultures from experimental animals and human donors can be prepared using nearly any type of nerve at any stage of maturation to render stage- and patient-specific populations. Methods to isolate, purify, expand in number, and differentiate SCs from adult, postnatal and embryonic sources are efficient and reproducible as these have resulted from accumulated refinements introduced over many decades of work. Albeit some exceptions, SCs can be passaged extensively while maintaining their normal proliferation and differentiation controls. Due to their lineage commitment and strong resistance to tumorigenic transformation, SCs are safe for use in therapeutic approaches in the peripheral and central nervous systems. This review summarizes the evolution of work that led to the robust technologies used today in SC culturing along with the main features of the primary and expanded SCs that make them irreplaceable models to understand SC biology in health and disease. Tradit...
<p>Cells were left untreated (control) or stimulated with CPT-cAMP, EPAC-cAMP or PKA-cAMP a... more <p>Cells were left untreated (control) or stimulated with CPT-cAMP, EPAC-cAMP or PKA-cAMP at a concentration of 200 µM each, unless otherwise indicated. Cells were analyzed for the incorporation of [<sup>3</sup>H]-thymidine (A) and the expression of myelin associated markers (C-D) 3 days after cAMP administration. The activity of PKA was determined by <i>in </i><i>vitro</i> phosphorylation assays (B, upper panel) and the immunodetection of phosphorylated PKA-specific substrates (C, bottom panels) in SCs stimulated for 30 min with the indicated analogs of cAMP. The activity of EPAC (Rap1 assays) was determined under identical experimental conditions (B, lower panels). As shown in C and D, EPAC-cAMP and PKA-cAMP failed to mimic the action of CPT-cAMP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082354#pone-0082354-g005" target="_blank">Figure 5C</a>) or db-cAMP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082354#pone-0082354-g002" target="_blank">Figure 2</a>) either if provided alone or in combination (not shown). Curiously, PKA-cAMP modestly increased P<sub>0</sub> and reduced c-Jun expression without increasing Krox-20 or reducing GFAP expression (D). </p
<p>SC-DRG neuron cultures were left untreated (control) or stimulated with CPT-cAMP, EPAC-c... more <p>SC-DRG neuron cultures were left untreated (control) or stimulated with CPT-cAMP, EPAC-cAMP or PKA-cAMP (200 µM each, unless otherwise indicated) for 3 days (A) or 12 days (B) prior to analysis by [<sup>3</sup>H]-thymidine incorporation (A) and immunofluorescence microscopy (B), respectively. Experimental conditions and analysis were identical to those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082354#pone-0082354-g004" target="_blank">Figure 4</a>. In B, images of myelinating co-cultures are shown at low (top panels) and high (bottom panels) magnifications, respectively. EPAC-cAMP significantly reduced proliferation of axon-contacting SCs (A). However, it did not mimic the effect of CPT-cAMP and db-cAMP (not shown) on inducing the formation of myelin sheaths (B). The strong pro-differentiating effect of CPT-cAMP is reflected by the occurrence of O1 positive, MBP positive cells throughout the co-culture system (B).</p
<p>The figure depicts the temporal progression of phenotypic changes during cAMP-induced di... more <p>The figure depicts the temporal progression of phenotypic changes during cAMP-induced differentiation in isolated nerve-derived SC cultures. SCs were subjected to mitogen and serum starvation (Methods) prior to treatment with CPT-cAMP (250 μM) for 3 days unless otherwise indicated. The control condition in this and subsequent figures refers to cells that were incubated in the absence of cAMP-stimulating agents for the whole time course of the experiment. In panels B-C, the control (C) condition refers to cells that were fixed or lysed for analysis at the time of stimulation (day/hour zero). Cells were analyzed for the expression of c-Jun, Krox-20, O1 and MBP by immunofluorescence microscopy and western blot, as marked in the figure. In A, SCs were co-stained with the SC-specific marker S-100 to label all cells and reveal the changes in cell morphology that occur in response to prolonged CPT-cAMP stimulation. The schematic diagram (D) depicts the temporal course of changes during cAMP-induced differentiation as revealed by this and our previous time course studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116948#pone.0116948.ref008" target="_blank">8</a>]. Arrowheads in A-B point out to representative Krox-20 positive cells (nuclear localization) that fail to express O1. Cells that labeled positive for O1 and negative for Krox-20 were not found. Of note, staining with MBP antibodies did not detect a specific signal in either control or cAMP-treated cells (B, quantification shown on the right). All antibodies used are indicated in the figure; nuclei were labeled with DAPI (blue) in this and all subsequent figures.</p
<p>Forskolin was provided to SC-neuron cultures together with ascorbate essentially as desc... more <p>Forskolin was provided to SC-neuron cultures together with ascorbate essentially as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116948#pone.0116948.g008" target="_blank">Fig. 8</a>. Low magnification composites of representative cultures (A) are shown along with selected high magnification areas (B) to reveal the effect of the indicated treatments on O1 and MBP expression. The quantitative analysis provided in panel C confirmed that similar to CPT-cAMP, forskolin effectively enhanced O1 and MBP expression without concurrently increasing the MBP/O1 ratio. The DAPI channel in A (bottom panel) is shown to serve as an indication of unchanged cell density in forskolin-treated cultures. The arrowheads and arrows (B, left panels) point out to representative O1 positive cells exhibiting thin and thick myelin, respectively, as denoted by the intensity of MBP positive myelin segments. This heterogeneity of MBP staining in individual cells was not evident in forskokin-treated cultures (B, right panels), possibly due to increased synchronization in the myelination process.</p
<p>Cells that were subjected to the same experimental treatments as in <a href="htt... more <p>Cells that were subjected to the same experimental treatments as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116948#pone.0116948.g002" target="_blank">Fig. 2</a> were fixed and analyzed by TEM (Methods). Higher magnification images of selected areas (boxes) are shown to better resolve the interaction between SC processes (SC) and axons (ax). As opposed to ascorbate, CPT-cAMP treatment did not support the ensheathment of axons, the formation of a basal lamina, the deposition of extracellular collagen fibers (arrowheads) or the production of myelin membranes (bracket). Similar to the control condition (left panels), CPT-cAMP-treated SCs (cAMP) associated with multiple low diameter axons leaving many neurites deprived of direct contact with SC processes. The asynchronous responses in myelination induced by ascorbate are illustrated by the multiple ensheathment profiles shown in the upper right panel, as follows: (1) – No ensheathment; (2) Partial ensheathment; (3) Complete ensheathment, no myelin; (4) Complete ensheathment, myelin.</p
The isolation of well formed crystals of the biomineral whewellite (monohydrated calcium oxalate)... more The isolation of well formed crystals of the biomineral whewellite (monohydrated calcium oxalate) from Opuntia microdasys, a cactaceae species found in central Mexico, is described. The morphology of the crystals was investigated by means of electron microscopy. Infrared spectroscopic measurements allow an unambiguous characterization of the nature of the crystals. This is the first report of the presence of a biomineral in this species.
<p>Experimental conditions were identical to those of <a href="http://www.plosone.o... more <p>Experimental conditions were identical to those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116948#pone.0116948.g005" target="_blank">Fig. 5</a>. In A, low magnification composites of representative cultures stained with O1 (green), neurofilament (red) and DAPI (blue) are shown to reveal the effect of the indicated treatments on O1 expression with respect to the location of the neuronal bodies (white ovals), the extension of the neurite substrate (neurofilament, NF) and the distribution of the SCs (DAPI). A quantitative analysis of O1 and MBP expression is provided in B (Methods). This analysis confirmed that cAMP enhanced the total number of O1 and MBP positive cells without concomitantly increasing the MBP/O1 ratio. Selected areas within the center (a, b) and periphery (c) of the axonal outgrowth are shown at higher magnification in C and D, respectively, to reveal details of the morphology of the cells, the relationship to axons and the co-localization of O1 (green) and MBP (red). Whereas O1 positive cells could be found throughout the culture system, MBP positive cells were found exclusively within the axonal network (C, D). Images taken at the frontier of the axonal outgrowth (D) revealed that MBP rather than O1 expression was restricted to those SCs that established a direct contact with the axons, as revealed by neurofilament staining (dotted lines). The boxed area in panel D was enlarged to better resolve the distribution of the MBP staining with respect to the position of the axons (NF, arrows), the plasma membrane (O1) and the nuclei of the cells (DAPI). In these and all subsequent graphs, bar heights are means of triplicate determinations; error bars represent S.D, and * represents statistical significance for p < 0.05.</p
The benefits of transplanting cultured Schwann cells (SCs) for the treatment of spinal cord injur... more The benefits of transplanting cultured Schwann cells (SCs) for the treatment of spinal cord injury (SCI) have been systematically investigated in experimental animals since the early 1990s. Importantly, human SC (hSC) transplantation for SCI has advanced to clinical testing and safety has been established via clinical trials conducted in the USA and abroad. However, multiple barriers must be overcome to enable accessible and effective treatments for SCI patients. This review presents available information on hSC transplantation for SCI with the intention to uncover gaps in our knowledge and discuss areas for future development. To this end, we introduce the historical progression of the work that supports existing and prospective clinical initiatives and explain the reasons for the choice of hSCs while also addressing their limitations as cell therapy products. A search of the relevant literature revealed that rat SCs have served as a preclinical model of reference since the onset o...
The Identities of Membrane Steroid Receptors, 2003
1α,25-dihydroxy-vitamin D3 (lα,25(OH)2D3; calcitriol) and estrogen (17β-estradiol) act, as other ... more 1α,25-dihydroxy-vitamin D3 (lα,25(OH)2D3; calcitriol) and estrogen (17β-estradiol) act, as other steroid hormones, through two different mechanisms. In addition to regulating expression of target genes via their specific nuclear receptors (VDR and ER, respectively), both hormones induce fast, non transcriptional responses involving stimulation of transmembrane signal transduction pathways. The rapid nature and specificity by which 1α,25(OH)2D3 and 17β-estradiol trigger the activation of second messengers has led to the concept that interaction with a plasma membrane receptor is responsible for the initiation of their effects. However, there is controversy over its molecular characteristics. Among several models for non-genomic steroid receptor identity, has been proposed the existence of membrane-associated forms of either the classical receptors or alternatively of novel 1α,25(OH)2D3 and 17β- estradiol binding proteins. In this chapter we report the presence of the nuclear VDR and ER in the plasma membrane of avian muscle and mammalian breast cells and furnish data suggesting that they may be involved in non-genomic signalling by their cognate ligands.
N-cadherin and beta-catenin are involved in cell adhesion and cell cycle in tumor cells and neura... more N-cadherin and beta-catenin are involved in cell adhesion and cell cycle in tumor cells and neural crest. Both are expressed at key stages of Schwann cell (SC) development, but little is known about their function in the SC lineage. We studied the role of these molecules in adult rat derived SC-embryonic dorsal root ganglion cocultures by using low-Ca(2+) conditions and specific blocking antibodies to interfere with N-cadherin function and by using small interfering RNA (siRNA) to decrease beta-catenin expression in both SC-neuron cocultures and adult rat-derived SC monocultures. N-cadherin blocking conditions decreased SC-axon association and reduced axon-induced SC proliferation. In SC monocultures, beta-catenin reduction diminished the proliferative response of SCs to the mitogen beta1-heregulin, and, in SC-DRG cocultures, beta-catenin reduction inhibited axon-contact-dependent SC proliferation. Stimulation of SC cultures with beta1-heregulin increased total beta-catenin protein amount, phosphorylation of GSK-3beta and beta-catenin presence in nuclear extracts. In conclusion, our findings suggest a previously unrecognized contribution of beta-catenin and N-cadherin to axon-induced SC proliferation.
The isolation of well formed crystals of the biomineral weddellite (calcium oxalate dihydrate) fr... more The isolation of well formed crystals of the biomineral weddellite (calcium oxalate dihydrate) from Chamaecereus silvestrii, a Cactaceae species found in the northern part of Argentina, is described. Infrared spectroscopic measurements allow an unambiguous characterization of the nature of the crystals. This is the first report of the presence of a biomineral in this plant species.
Background Heregulin is a ligand for the protooncogene product ErbB/HER that acts as a key mitog... more Background Heregulin is a ligand for the protooncogene product ErbB/HER that acts as a key mitogenic factor for human Schwann cells (hSCs). Heregulin is required for sustained hSC growth in vitro but must be thoroughly removed before cell collection for transplantation due to potential safety concerns. The goal of this study was to develop simple cell-based assays to assess the effectiveness of heregulin addition to and removal from aliquots of hSC culture medium. These bioassays were based on the capacity of a β1-heregulin peptide to elicit ErbB/HER receptor signaling in adherent ErbB2+/ErbB3+ cells. Results Western blotting was used to measure the activity of three different β1-heregulin/ErbB-activated kinases (ErbB3/HER3, ERK/MAPK and Akt/PKB) using phospho-specific antibodies against key activating residues. The duration, dose-dependency and specificity of β1-heregulin-initiated kinase phosphorylation were investigated, and controls were implemented for assay optimization and r...
Adult Schwann cell (SC) cultures are usually derived from nerves subjected to a lengthy step of p... more Adult Schwann cell (SC) cultures are usually derived from nerves subjected to a lengthy step of pre-degeneration to facilitate enzymatic digestion and recovery of viable cells. To overcome the need for pre-degeneration, we developed a method that allows the isolation of adult rat sciatic nerve SCs immediately after tissue harvesting. This method combines the advantages of implementing a rapid enzymatic dissociation of the nerve fibers and a straightforward separation of cells versus myelin that improves both cell yield and viability. Essentially, the method consists of (1) acute dissociation with collagenase and dispase immediately after removal of the epineurium layer and extensive teasing of the nerve fibers, (2) removal of myelin debris by selective attachment of the cells to a highly adhesive poly-L-lysine/laminin substrate, (3) expansion of the initial SC population in medium containing chemical mitogens, and (4) preparation of cryogenic stocks for transfer or delayed experimentation. This protocol allows for the procurement of homogeneous SC cultures deprived of myelin and fibroblast growth as soon as 3-4 days after nerve tissue dissection. SC cultures can be used as such for experimentation or subjected to consecutive rounds of expansion prior to use, purification, or cryopreservation.
To date, magnetic-activated cell sorting (MACS) remains a powerful method to isolate distinct cel... more To date, magnetic-activated cell sorting (MACS) remains a powerful method to isolate distinct cell populations based on differential cell surface labeling. Optimized direct and indirect MACS protocols for cell immunolabeling are presented here as methods to divest Schwann cell (SC) cultures of contaminating cells (specifically, fibroblast cells) and isolate SC populations at different stages of differentiation. This chapter describes (1) the preparation of single-cell suspensions from established human and rat SC cultures, (2) the design and application of cell selection strategies using SC-specific (p75NGFR, O4, and O1) and fibroblast-specific (Thy-1) markers, and (3) the characterization of both the pre- and post-sorting cell populations. A simple protocol for the growth of hybridoma cell cultures as a source of monoclonal antibodies for cell surface immunolabeling of SCs and fibroblasts is provided as a cost-effective alternative for commercially available products. These steps allow for the timely and efficient recovery of purified SC populations without compromising the viability and biological activity of the cells.
Nerve-derived human Schwann cell (SC) cultures are irreplaceable models for basic and translation... more Nerve-derived human Schwann cell (SC) cultures are irreplaceable models for basic and translational research but their use can be limited due to the risk of fibroblast overgrowth. Fibroblasts are an ill-defined population consisting of highly proliferative cells that, contrary to human SCs, do not undergo senescence in culture. We initiated this study by performing an exhaustive immunological and functional characterization of adult nerve-derived human SCs and fibroblasts to reveal their properties and optimize a protocol of magnetic-activated cell sorting (MACS) to separate them effectively both as viable and biologically competent cells. We next used immunofluorescence microscopy imaging, flow cytometry analysis and next generation RNA sequencing (RNA-seq) to unambiguously characterize the post-MACS cell products. High resolution transcriptome profiling revealed the identity of key lineage-specific transcripts and the clearly distinct neural crest and mesenchymal origin of human S...
Schwann cell (SC) cultures from experimental animals and human donors can be prepared using nearl... more Schwann cell (SC) cultures from experimental animals and human donors can be prepared using nearly any type of nerve at any stage of maturation to render stage- and patient-specific populations. Methods to isolate, purify, expand in number, and differentiate SCs from adult, postnatal and embryonic sources are efficient and reproducible as these have resulted from accumulated refinements introduced over many decades of work. Albeit some exceptions, SCs can be passaged extensively while maintaining their normal proliferation and differentiation controls. Due to their lineage commitment and strong resistance to tumorigenic transformation, SCs are safe for use in therapeutic approaches in the peripheral and central nervous systems. This review summarizes the evolution of work that led to the robust technologies used today in SC culturing along with the main features of the primary and expanded SCs that make them irreplaceable models to understand SC biology in health and disease. Tradit...
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Papers by Paula Monje