We herein developed a protocol for the rapid procurement of adult nerve-derived Schwann cells (SCs) that was optimized to implement an immediate enzymatic dissociation of fresh nerve tissue while maintaining high cell viability, improving yields and minimizing fibroblast and myelin contamination. This protocol introduces: (1) an efficient method for enzymatic cell release immediately after removal of the epineurium and extensive teasing of the nerve fibers; (2) an adaptable drop-plating method for selective cell attachment, removal of myelin debris, and expansion of the initial SC population in chemically defined medium; (3) a magnetic-activated cell sorting purification protocol for rapid and effective fibroblast elimination; and (4) an optional step of cryopreservation for the storage of the excess of cells. Highly proliferative SC cultures devoid of myelin and fibroblast growth were obtained within three days of nerve processing. Characterization of the initial, expanded, and cryopreserved cell products confirmed maintenance of SC identity, viability and growth rates throughout the process. Most importantly, SCs retained their sensitivity to mitogens and potential for differentiation even after cryopreservation. To conclude, this easy-to-implement and clinically relevant protocol allows for the preparation of expandable homogeneous SC cultures while minimizing time, manipulation of the cells, and exposure to culture variables.
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 SCs and fibroblasts, respectively. Our analysis underscored a progenitor- or stem cell-like molecular phenotype in SCs and fibroblasts and the heterogeneity of the fibroblast populations. In addition, pathway analysis of RNA-seq data highlighted putative bidirectional networks of fibroblast-to-SC signaling that predict a complementary, yet seemingly independent contribution of SCs and fibroblasts to nerve regeneration. In sum, combining MACS with immunochemical and transcriptomics approaches provides an ideal workflow to exhaustively assess the identity, the stage of differentiation and functional features of highly purified cells from human peripheral nerve tissues.
This study was undertaken to examine the bioactivity, specificity and reversibility of lithium’s action on the growth, survival, proliferation and differentiation of cultured Schwann cells (SCs). In isolated SCs, lithium promoted a state of cell cycle arrest that featured extensive cell enlargement and c-Jun downregulation in the absence of increased expression of myelin-associated markers. In addition, lithium effectively prevented mitogen-induced S-phase entry without impairing cell viability. When lithium was administered together with differentiating concentrations of cAMP analogs, a dramatic inhibition of the expression of the master regulator of myelination Krox-20 was observed. Likewise, lithium antagonized the cAMP-dependent expression of various myelin markers such as protein zero, periaxin and galactocerebroside and allowed SCs to maintain high levels of expression of immature SC markers even in the presence of high levels of cAMP and low levels of c-Jun. Most importantly, the inhibitory action of lithium on SC proliferation and differentiation was shown to be dose dependent, specific and reversible upon removal of lithium compounds. In SC-neuron cultures, lithium suppressed myelin sheath formation while preserving axonal integrity, SC-axon contact and basal lamina formation. Lithium was unique in its ability to prevent the onset of myelination without promoting myelin degradation or SC dedifferentiation. To conclude, our results underscored an unexpected antagonistic action of lithium on SC mitogenesis and myelin gene expression. We suggest that lithium represents an attractive pharmacological agent to safely and reversibly suppress the onset of SC proliferation, differentiation and myelination while maintaining the integrity of pre-existing myelinated fibers.
Diffuse midline glioma (DMG) is a type of lethal brain tumor that develops mainly in children. The majority of DMG harbor the K27M mutation in histone H3. Oligodendrocyte progenitor cells (OPCs) in the brainstem are candidate cells-of-origin for DMG, yet there is no genetically engineered mouse model of DMG initiated in OPCs.Here, we used the RCAS/Tv-a avian retroviral system to generate DMG in
Monocytes and monocyte-derived macrophages (MDM) from blood circulation infiltrate and promote glioblastoma growth. Here we discover that glioma cells induce the expression of potent pro-inflammatory cytokine IL-1b in MDM, which engages IL-1R1 in glioma cells, activates NF-kB pathway, and subsequently leads to the induction of monocyte chemoattractant proteins (MCPs). Thus, a feedforward paracrine circuit of IL-b/IL-1R1 between the tumors and MDM creates an interdependence driving glioblastoma progression. Locally antagonizing IL-1b/IL-1R1 leads to reduced MDM infiltration, diminished tumor growth, reduced exhausted CD8+ T cells, and thereby extends the survival of tumor-bearing mice. In contrast to IL-1b, IL-1a exhibits anti-tumor effects. Genetic deletion of Il1a is associated with decreased recruitment of lymphoid cells and loss of interferon (IFN) signaling in various immune populations and subsets of malignant cells. Local antagonism of IL-1b should be considered as an effective anti-glioblastoma therapy.
Bone marrow mononuclear cells (BMMC) constitute a heterogeneous population with potential to promote tissue regeneration. For this reason, this cell fraction has recently become a therapeutic alternative to mesenchymal stem cells, as culture is not required and phenotypic transformations can be hence avoided. In this work, and in order to attain long-lasting cell labeling and study longer survival times, we used BMMC isolated from adult transgenic rats expressing GFP to reproduce our wild type model and evaluate their remyelination ability in a reversible model of Wallerian degeneration. RT-PCR and flow cytometry analysis confirmed that cells isolated from the transgenic strain exhibited similar expression levels of markers specific to multipotent progenitors (CD34, CD90 and CD105) and Schwann cells (MPZ, MBP, S100β and p75) compared to wild type BMMC. BMMC expressing GFP retained their migration capacity, arriving exclusively at the injured nerve. Most importantly, and as detected through long-lasting cell tracking, some of these BMMC settled in the demyelinated area, mingled with endogenous cells, underwent phenotypic changes and colocalized with Schwann cell markers MBP and S100β. Also worth highlighting, transgenic BMMC replicated wild type BMMC effects in terms of MBP organization and levels. On the basis of these findings, BMMC isolated from transgenic animals constitute a useful tool to evaluate their role in peripheral nervous system demyelination-remyelination and the underlying mechanisms.
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