Tacrolimus-Induced Neurotrophic Differentiation of Adipose-Derived Stem Cells as Novel Therapeutic Method for Peripheral Nerve Injury

Peripheral nerve injuries (PNIs) are frequent traumatic injuries across the globe. Severe PNIs result in irreversible loss of axons and myelin sheaths and disability of motor and sensory function. Schwann cells can secrete neurotrophic factors and myelinate the injured axons to repair PNIs. However, Schwann cells are hard to harvest and expand in vitro, which limit their clinical use. Adipose-derived stem cells (ADSCs) are easily accessible and have the potential to acquire neurotrophic phenotype under the induction of an established protocol. It has been noticed that Tacrolimus/FK506 promotes peripheral nerve regeneration, despite the mechanism of its pro-neurogenic capacity remains undefined. Herein, we investigated the neurotrophic capacity of ADSCs under the stimulation of tacrolimus. ADSCs were cultured in the induction medium for 18 days to differentiate along the glial lineage and were subjected to FK506 stimulation for the last 3 days. We discovered that FK506 greatly enhanced the neurotrophic phenotype of ADSCs which potentiated the nerve regeneration in a crush injury model. This work explored the novel application of FK506 synergized with ADSCs and thus shed promising light on the treatment of severe PNIs.

Microglial responses to CSF1 overexpression do not promote the expansion of other glial lineages

Background Colony-stimulating factor 1 (CSF1) expression in the central nervous system (CNS) increases in response to a variety of stimuli, and CSF1 is overexpressed in many CNS diseases. In young adult mice, we previously showed that CSF1 overexpression in the CNS caused the proliferation of IBA1+ microglia without promoting the expression of M2 polarization markers. Methods Immunohistochemical and molecular analyses were performed to further examine the impact of CSF1 overexpression on glia in both young and aged mice. Results As CSF1 overexpressing mice age, IBA1+ cell numbers are constrained by a decline in proliferation rate. Compared to controls, there were no differences in expression of the M2 markers ARG1 and MRC1 (CD206) in CSF1 overexpressing mice of any age, indicating that even prolonged exposure to increased CSF1 does not impact M2 polarization status in vivo. Moreover, RNA-sequencing confirmed the lack of increased expression of markers of M2 polarization in microglia exposed to CSF1 overexpression but did reveal changes in expression of other immune-related genes. Although treatment with inhibitors of the CSF1 receptor, CSF1R, has been shown to impact other glia, no increased expression of oligodendrocyte lineage or astrocyte markers was observed in CSF1 overexpressing mice. Conclusions Our study indicates that microglia are the primary glial lineage impacted by CSF1 overexpression in the CNS and that microglia ultimately adapt to the presence of the CSF1 mitogenic signal.

Glioblastoma Contains Topologically Distinct Proliferative and Metabolically Defined Subpopulations of Nestin- and Glut1-Expressing Cells

The difficulty in treatment of glioblastoma is a consequence of its natural infiltrative growth and the existence of a population of therapy-resistant glioma cells that contribute to growth and recurrence. To identify cells more likely to have these properties, we examined the expression in tumor specimens of several protein markers important for glioma progression including the intermediate filament protein, Nestin (NES), a glucose transporter (Glut1/SLC2A1), the glial lineage marker, glial fibrillary acidic protein, and the proliferative indicator, Ki-67. We also examined the expression of von Willebrand factor, a marker for endothelial cells as well as the macrophage/myeloid markers CD163 and CD15. Using a multicolor immunofluorescence and hematoxylin and eosin staining approach with archival formalin-fixed, paraffin embedded tissue from primary, recurrent, and autopsy IDH1 wildtype specimens combined with high-resolution tissue image analysis, we have identified highly proliferative NES(+)/Glut1(–) cells that are preferentially perivascular. In contrast, Glut1(+)/NES(–) cells are distant from blood vessels, show low proliferation, and are preferentially located at the borders of pseudopalisading necrosis. We hypothesize that Glut1(+)/NES(–) cells would be naturally resistant to conventional chemotherapy and radiation due to their low proliferative capacity and may act as a reservoir for tumor recurrence.

Wild-Type and Mutant FUS Expression Reduce Proliferation and Neuronal Differentiation Properties of Neural Stem Progenitor Cells

Nervous system development involves proliferation and cell specification of progenitor cells into neurons and glial cells. Unveiling how this complex process is orchestrated under physiological conditions and deciphering the molecular and cellular changes leading to neurological diseases is mandatory. To date, great efforts have been aimed at identifying gene mutations associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the RNA/DNA binding protein Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) have been associated with motor neuron degeneration in rodents and humans. Furthermore, increased levels of the wild-type protein can promote neuronal cell death. Despite the well-established causal link between FUS mutations and ALS, its role in neural cells remains elusive. In order to shed new light on FUS functions we studied its role in the control of neural stem progenitor cell (NSPC) properties. Here, we report that human wild-type Fused in Sarcoma (WT FUS), exogenously expressed in mouse embryonic spinal cord-derived NSPCs, was localized in the nucleus, caused cell cycle arrest in G1 phase by affecting cell cycle regulator expression, and strongly reduced neuronal differentiation. Furthermore, the expression of the human mutant form of FUS (P525L-FUS), associated with early-onset ALS, drives the cells preferentially towards a glial lineage, strongly reducing the number of developing neurons. These results provide insight into the involvement of FUS in NSPC proliferation and differentiation into neurons and glia.

Hypoxia induces early neurogenesis in human fetal neural stem cells by activating the WNT pathway.

Fetal neural stem cells (FNSCs) are present in the brain of human fetuses that differentiate into cells of neuronal and glial lineages. Difference in oxygen concentration between maternal and fetal circulation indicates that the developing fetus may be exposed to lower oxygen concentrations compared to adults. This physiological hypoxia may influence the growth and differentiation of the FNSCs. This study aimed to evaluate the effect of hypoxia on the differentiation potential of human FNSCs isolated from the sub-ventricular zone of aborted fetal brains (n=5). FNSCs were isolated, expanded, and characterized by Nestin and Sox2 expression, using immunocytochemistry and flowcytometry respectively. These FNSCs were exposed to 20% oxygen (normoxia) and 0.2% oxygen (hypoxia) concentrations for 48 hours, and hypoxia exposure (n=5) was evaluated by a panel of markers (CA9, PGK1 and VEGF). Whole human genome transcriptomic analyses (Genespring GX13) of FNSCs exposed to hypoxia (Agilent 4x44K human array slides), highlighted that genes associated with neurogenesis were getting enriched. The pathway analysis of these enriched genes (using Metacore) showed that WNT signaling played a role in determining the cell fate of FNSCs exposed to hypoxic environment. Microarray analyses was validated using neuronal and glial lineage commitment markers such as Ngn1, Ngn2, ASCL1, DCX, GFAP, Olig2 and Nkx2.2 using qPCR (n=9). This demonstrated upregulation of the neuronal commitment markers on hypoxia exposure, while no change was observed in astrocytic and oligodendrocyte lineage commitment markers. Increased expression of downstream targets of the WNT signaling pathway, TCF4 and ID2, by qPCR (n=9), indicated its involvement in mediating neuronal differentiation on exposure to hypoxia.

C21orf91 Regulates Oligodendroglial Precursor Cell Fate—A Switch in the Glial Lineage?

Neuropathological diseases of the central nervous system (CNS) are frequently associated with impaired differentiation of the oligodendroglial cell lineage and subsequent alterations in white matter structure and dynamics. Down syndrome (DS), or trisomy 21, is the most common genetic cause for cognitive impairments and intellectual disability (ID) and is associated with a reduction in the number of neurons and oligodendrocytes, as well as with hypomyelination and astrogliosis. Recent studies mainly focused on neuronal development in DS and underestimated the role of glial cells as pathogenic players. This also relates to C21ORF91, a protein considered a key modulator of aberrant CNS development in DS. We investigated the role of C21orf91 ortholog in terms of oligodendrogenesis and myelination using database information as well as through cultured primary oligodendroglial precursor cells (OPCs). Upon modulation of C21orf91 gene expression, we found this factor to be important for accurate oligodendroglial differentiation, influencing their capacity to mature and to myelinate axons. Interestingly, C21orf91 overexpression initiates a cell population coexpressing astroglial- and oligodendroglial markers indicating that elevated C21orf91 expression levels induce a gliogenic shift towards the astrocytic lineage reflecting non-equilibrated glial cell populations in DS brains.

Lin28 Inhibits the Differentiation from Mouse Embryonic Stem Cells to Glial Lineage Cells through Upregulation of Yap1

The RNA-binding protein Lin28 regulates neurogliogenesis in mammals, independently of the let-7 microRNA. However, the detailed regulatory mechanism remains obscured. Here, we established Lin28a or Lin28b overexpression mouse embryonic stem cells (ESCs) and found that these cells expressed similar levels of the core pluripotent factors, such as Oct4 and Sox2, and increased Yap1 but decreased lineage-specific markers compared to the control ESCs. Further differentiation of these ESCs to neuronal and glial lineage cells revealed that Lin28a/b overexpression did not affect the expression of neuronal marker βIII-tubulin, but dramatically inhibited the glial lineage markers, such as Gfap and Mbp. Interestingly, overexpression of Yap1 in mouse ESCs phenocopied Lin28a/b overexpression ESCs by showing defect in glial cell differentiation. Inhibition of Yap1/Tead-mediated transcription with verteporfin partially rescued the differentiation defect of Lin28a/b overexpression ESCs. Mechanistically, we demonstrated that Lin28 can directly bind to Yap1 mRNA, and the induction of Yap1 by Lin28a in mESCs is independent of Let7. Taken together, our results unravel a novel Lin28-Yap1 regulatory axis during mESC to glial lineage cell differentiation, which may shed light on glial cell generation in vitro.

Glycoengineering Human Neural and Adipose Stem Cells with Novel Thiol-Modified N-Acetylmannosamine (ManNAc) Analogs

This report describes novel thiol-modified N-acetylmannosamine (ManNAc) analogs that extend metabolic glycoengineering (MGE) applications of Ac5ManNTGc, a non-natural monosaccharide that metabolically installs the thio-glycolyl of sialic acid into human glycoconjugates. We previously found that Ac5ManNTGc elicited non-canonical activation of Wnt signaling in human embryoid body derived (hEBD) cells but only in the presence of a high affinity, chemically compatible scaffold. Our new analogs Ac5ManNTProp and Ac5ManNTBut overcome the requirement for a complementary scaffold by displaying thiol groups on longer, N-acyl linker arms, thereby presumably increasing their ability to interact and crosslink with surrounding thiols. These new analogs showed increased potency in human neural stem cells (hNSCs) and human adipose stem cells (hASCs). In the hNSCs, Ac5ManNTProp upregulated biochemical endpoints consistent with Wnt signaling in the absence of a thiol-reactive scaffold. In the hASCs, both Ac5ManNTProp and Ac5ManNTBut suppressed adipogenic differentiation, with Ac5ManNTBut providing a more potent response, and they did not interfere with differentiation to a glial lineage (Schwann cells). These results expand the horizon for using MGE in regenerative medicine by providing new tools (Ac5ManNTProp and Ac5ManNTBut) for manipulating human stem cells.

MODL-16. ABEMACICLIB, A SELECTIVE CDK4/6 INHIBITOR, RESTRICTS GROWTH OF PEDIATRIC GLIAL-LINEAGE TUMORS IN VITRO AND IN VIVO

BACKGROUND Glial-lineage tumors constitute a heterogeneous group of neoplasms, comprising gliomas, oligodendrogliomas, and ependymomas, which account for 40%–50% of all pediatric central nervous system tumors. Advances in modern neuro-oncological therapeutics are aimed at improving neoadjuvant chemotherapy and deferring radiotherapy because radiation exposure may cause long-term side effects on the developing brain in young children. Despite aggressive treatment, more than half the high-grade gliomas (pHGGs) and one-third of ependymomas exhibit recurrence within 2 years of initial treatment. METHODS By using integrated bioinformatics and through experimental validation, we found that at least one gene among CCND1, CDK4, and CDK6 was overexpressed in pHGGs and ependymomas. RESULTS The use of abemaciclib, a highly selective CDK4/6 inhibitor, effectively inhibited cell proliferation and reduced the expression of cell cycle–related and DNA repair–related gene expression, which was determined through RNA-seq analysis. The efficiency of abemaciclib was validated in vitro in pHGGs and ependymoma cells and in vivo by using subcutaneously implanted ependymoma cells from patient-derived xenograft (PDX) in mouse models. Abemaciclib demonstrated the suppression of RB phosphorylation, downstream target genes of E2F, G2M checkpoint, and DNA repair, resulting in tumor suppression. CONCLUSION Abemaciclib showed encouraging results in preclinical pediatric glial-lineage tumors models and represented a potential therapeutic strategy for treating challenging tumors in children.

Retinal Ependymoma: A Rare Extra-Axial Presentation

Introduction/Objective Ependymomas are well-demarcated and slow-growing neuroepithelial neoplasms that comprise 3–9% of primary CNS tumors. The vast majority of ependymomas arise either intracranially, mostly in children, or in the spinal cord and are associated with ependymal lining. Histologic hallmarks are perivascular pseudorosettes, ependymal rosettes and alternating zones of nuclear crowding and nuclear free zones composed of coarse cell processes. In high grade ependymomas increased mitoses, necrosis and nuclear pleomorphism may be seen. Methods We present the case of a 63-year-old woman in with a past medical history of retinopathy of prematurity, glaucoma and right eye enucleation. She presented with a painful blind left eye refractory to medical treatment and subsequently underwent left eye enucleation. Results On histologic examination, an incidental retinal ependymoma was identified. The neoplastic cells were fusiform and had long coarsely fibrillar cell processes. Characteristic periodicity of nuclear crowding and scarcity was observed. In places, neoplastic cell processes extended radially to delicate and sometimes hyalinized blood vessels, forming so-called perivascular pseudorosettes. Definite ependymal rosettes were not recognized in the examined sections. Stigmata of chronicity were found, such as ischemic type necrosis, blood vessels with dystrophic calcifications and foci of ossification replete with fibroadipose tissue in the marrow spaces. The neoplastic cells labeled with antibodies against glial fibrillary acidic protein (GFAP), thus confirming glial lineage in the neoplastic cell. Additionally, there was scattered intracytoplasmic expression of epithelial membrane antigen (EMA) in a dot-like or circular pattern. The latter is well described in ependymomas and often used to support the diagnosis. Conclusion Ependymomas rarely occur at extracranial sites, such as the chest, abdomen and pelvis. We are presenting the fourth case of retinal ependymoma reported in literature. The tumor had classical immunomorphologic findings.