Addition Of Ciliary Neurotrophic Factor Research Articles

Neurotrophic factor‐secreting autologous muscle stem cell therapy for the treatment of laryngeal denervation injury

To determine if the spontaneous reinnervation that characteristically ensues after recurrent laryngeal nerve (RLN) injury could be selectively promoted and directed to certain laryngeal muscles with the use of neurotrophic factor (NF)-secreting muscle stem cell (MSC) vectors while antagonistic reinnervation is inhibited with vincristine (VNC). Basic science investigation involving primary cell cultures, gene cloning/transfer, and animal experiments. MSC survival assays were used to test multiple individual NFs in vitro. Motoneuron outgrowth assays assessed the trophic effects of identified NF on cranial nerve X (CNX)-derived motoneurons in vitro. Therapeutic NF was cloned into a lentiviral vector, and MSCs were transduced to secrete NF. Sixty rats underwent left RLN transection injury, and at 3 weeks received injections of either MSCs (n = 24), MSCs secreting NF (n = 24), or saline (n = 12) into the left thyroarytenoid muscle complex; half of the animals in the MSC groups simultaneously received left posterior cricoarytenoid injections of VNC, whereas half of the animals received saline. Ciliary neurotrophic factor (CNTF) had the greatest survival-promoting effect on MSCs in culture. The addition of CNTF (50 ng/mL) to CNX motoneuron cultures resulted in enhanced neurite outgrowth and branching. In the animal model, the injected MSCs fused with the denervated myofibers, immunohistochemistry demonstrated enhanced reinnervation based on motor endplate to nerve contact, and reverse transcriptase-polymerase chain reaction confirmed stable CNTF expression at longest follow-up (4 months) in the CNTF-secreting MSC treated groups. MSC therapy may have a future role in selectively promoting and directing laryngeal reinnervation after RLN injury.

Isolation of cortical mouse oligodendrocyte precursor cells

The reliable isolation of primary oligodendrocyte progenitors cells (OPCs) holds promise as both a research tool and putative therapy for the study and treatment of central nervous system (CNS) disease and trauma. Stringently characterized primary mouse OPCs is of additional importance due to the power of transgenics to address mechanism(s) involving single genes. In this study, we developed and characterized a reproducible method for the primary culture of OPCs from postnatal day 5–7 mouse cerebral cortex. We enriched an O4+ OPC population using Magnetic Activated Cell Sorting (MACS) technology. This technique resulted in an average yield of 3.68×105OPCs/brain. Following isolation, OPCs were glial fibrillary acidic protein− (GFAP−) and O4+. Following passage and with expansion, OPCs were O4+, A2B5+, and NG2+. Demonstrating their bi-potentiality, mouse OPCs differentiated into either more complex, highly arborized O4+ or O1+ oligodendrocytes (OLs) or GFAP+ astrocytes. This bi-potentiality is lost, however, in co-culture with rat embryonic day 15 derived dorsal root ganglia (DRG). Following 7–14 days of OPC/DRG co-culture, OPCs aligned with DRG neurites and differentiated into mature OLs as indicated by the presence of O1 and myelin basic protein (MBP) immunostaining. Addition of ciliary neurotrophic factor (CNTF) to conditioned media from OPC/DRG co-cultures improved OPC differentiation into mature O1+ and MBP+ OLs. This method allows for the study of primary mouse cortical OPC survival, maturation, and function without relying on oligosphere formation or the need for extensive passaging.

Directed differentiation of functional astroglial subtypes from human pluripotent stem cells

Regionally and functionally diverse types of astrocytes exist throughout the central nervous system and participate in nearly every aspect of normal and abnormal neural function. Therefore, human astrocyte subtypes are useful tools for understanding brain function, modulating disease processes and promoting neural regeneration. Here we describe a protocol for directed differentiation and maintenance of functional astroglia from human pluripotent stem cells in a chemically defined system. Human stem cells are first differentiated into neuroepithelial cells with or without exogenous patterning molecules (days 0-21). Regular dissociation of the neuroepithelial clusters in suspension, and in the presence of mitogens, permits generation of astroglial subtypes over a long-term expansion (days 21-90). Finally, the astroglial progenitors are either amplified for an extended time or differentiated into functional astrocytes on removal of mitogens and the addition of ciliary neurotrophic factor (days >90). This method generates robust populations of functionally diversified astrocytes with high efficiency.

Production and isolation of NG2 + oligodendrocyte precursors from human embryonic stem cells in defined serum-free medium

Human embryonic stem cells (hESCs) are a promising source of oligodendrocyte precursor cells (OPCs) and oligodendrocytes. These cells can be used to repair myelin in central nervous system deficits such as multiple sclerosis or traumas such as spinal cord injury. Here, we introduce a novel differentiation method for the production of OPCs from hESCs. OPCs were differentiated as spheres in defined serum-free medium supplemented with recombinant human growth factors. A broad gene expression analysis revealed that this OPC population expressed Olig1/2, Sox10, PDGFR, Nkx2.2, Nkx6.2, oligodendrocyte-myelin glycoprotein, myelin basic protein (MBP), and proteolipid protein (PLP). According to quantitative RT-PCR analyses addition of ciliary neurotrophic factor (CNTF) upregulated the Olig2 mRNA levels in the OPC population. According to the flow cytometry analyses the OPC population was > 90% NG2-positive, > 80% PDGFR-positive, and > 60% CD44-positive, and further matured into O4- (45%) and GalC- (80%) positive oligodendrocyte populations when cultured on top of human extracellular matrix proteins, which were used instead of Matrigel. In addition, OPCs matured into myelin-forming cells when cocultured with neuronal cells. The multilayered myelin sheet formation around axons was detected with transmission electron microscopy in cocultures. Further, the OPC populations could be purified with sorting of NG2 + cells. These NG2 + cells reformed spheres that remained stable during prolonged culturing (7 weeks), and matured into GalC-positive oligodendrocytes. Importantly, these NG2 + spheres were free of pluripotent Tra1-81, Oct-4, and CD326-positive hESCs. Thus, this method is suitable for the efficient production of OPCs and in the future for therapeutic graft production.

A comparison of LEDGFp52 and CNTF on the in vitro growth of rat retinal ganglion cell neurites

Lens epithelium-derived growth factor (LEDGF) can be alternatively spliced to produce two isoforms-LEDGFp52 and LEDGFp75, however, LEDGFp52 has rarely been investigated. The LEDGFp52 protein or its monoclonal antibody was added to primary rat retinal ganglion cell cultures and their impact on neurite number and length, and the mRNA and protein expression levels of GAP-43, NF-L and MAP-2 quantified. LEDGFp52 was compared to the addition of ciliary neurotrophic factor (CNTF). LEDGFp52 protein significantly increased primary neurite growth compared to control conditions. In addition, the expression of GAP-43, NF-L and MAP2 genes and proteins were also significantly up-regulated. The positive action of the LEDGFp52 protein was similar to or more efficacious than CNTF. LEDGFp52 appears to be an important regulatory protein for the growth of cell processes.

Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells

Ciliary neurotrophic factor (CNTF) participates in retinal development by inhibiting rod differentiation and promoting bipolar and Müller cell differentiation. In order to identify genes which are regulated by CNTF in the developing retina, we carried out a subtractive hybridization study. By this approach, we identified the Pleiotrophin (Ptn) as an upregulated gene in postnatal day 0 (P0) retinal explants upon addition of CNTF. Correlation of overall expression patterns between different retinal cell markers and Ptn in situ hybridization suggest that Ptn transcripts are initially expressed in progenitor cells then in postmitotic precursors of the INL expressing the Chx10 gene, and later in some differentiated retinal Müller glial (RMG) cells and rod–bipolar cells. Overexpression of Ptn by in vitro electroporation of P0 rat retinal explants partially blocks rod differentiation and promotes bipolar cell production, similar to effects of exogenous CNTF and leukemia inhibitory factor (LIF). Furthermore, in P0 retinal explants from mice lacking Ptn, the inhibitory effect of CNTF and LIF on rod differentiation is partially reduced and the cytokine-induced bipolar cell differentiation is largely prevented. Together, these results demonstrate that influence of CNTF family of cytokines on the differentiation of late retinal progenitor cell population is partially mediated by the release of Ptn.

Long-term effects of ciliary neurotrophic factor on the survival of vasopressin magnocellular neurones in the rat supraoptic nucleus in vitro.

The use of hypothalamic organotypic cultures for the long-term study of mechanisms in magnocellular neurones (MCNs) of the hypothalamic-neurohypophysial system has been limited by the relatively poor maintenance of the vasopressin MCNs in vitro. Recent studies have shown that addition of ciliary neurotrophic factor (CNTF) to the media significantly reduced the apoptosis of both oxytocin and vasopressin MCNs. Here, we studied various temporal factors in the CNTF treatment that can influence the efficacy of MCN survival. Immunohistochemistry was used to identify and count surviving vasopressin and oxytocin MCNs in the supraoptic nucleus (SON) in hypothalamic slices cultured in the presence of CNTF (10 ng/ml media) for various time intervals, and in situ hybridization for vasopressin mRNA was used to evaluate the vasopressin mRNA gene expression in the SON under the same conditions. The presence of CNTF in the medium for 10 days produced a maximal increase in the survival of vasopressin MCNs (by 11-fold) and in the survival of oxytocin-MCNs (by approximately four-fold) over controls. These effects persisted for an additional 7-10 days even in the absence of CNTF. The ability of CNTF to increase survival of the MCNs or increase vasopressin mRNA levels in the SON required that the CNTF be present during the initial 7-10 days of culture. CNTF failed to rescue vasopressin or oxytocin MCNs when added to the media only for the last 7 days of a total of 14 days in vitro. Similar results were observed when SON vasopressin mRNA levels were measured. These results indicate that the presence of CNTF is required at the outset to rescue the vasopressin and oxytocin MCN from axotomy induced apoptosis, and that, after 10 days in CNTF, the MCNs no longer require the CNTF for survival.

CNTF inhibits high voltage activated Ca2+ currents in fetal mouse cortical neurones.

Neurotrophic factors yield neuroprotection by mechanisms that may be related to their effects as inhibitors of apoptosis as well as their effects on ion channels. The effect of ciliary neurotrophic factor (CNTF) on high-threshold voltage-activated Ca channels in cultured fetal mouse brain cortical neurones was investigated. Addition of CNTF into serum-free growth medium resulted in delayed reduction of the Ca2+ currents. The currents decreased to 50% after 4 h and stabilized at this level during incubation with CNTF for 48 h. Following removal of CNTF the inhibition was completely reversed after 18 h. CNTF reduced the current of all pharmacological subtypes of Ca channels as shown by use of selective blockers of L, N, and P/Q type Ca channels (nifedipine, omega-conotoxin MVIIA, omega-agatoxin IVA). The Ca channel depression was mediated via the CNTF receptor, because enzymatic cleavage of the alpha-subunit glycerophosphatidylinositol anchor of the receptor eliminated the response. The CNTF effect was not elicited through pertussis toxin-sensitive G proteins. Other neurotrophic factors like neurotrophin-3 and insulin-like growth factor-I had no effect on the Ca2+ currents. These results may have important implications for the possible functions of CNTF in the nervous system, such as altered synaptic activity, neuronal excitability and susceptibility to brain ischaemia.

Serum and CNTF Stimulate Oligodendroglia and Reduce Fiber Outgrowth from Striatal Cultures

Organotypic slice cultures of newborn rat striatal tissue displayed an exceptionally dense and fasciculated outgrowth of GABAergic fibers when grown in a chemically defined medium, compared to serum-containing medium. The enhanced fiber growth was not the result of an increased density of GABAergic neurons in the cultures, but coincided with a marked reduction of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase)-immunoreactive cells within and around the cultures. An inverse, causal relationship between the number of CNPase-positive cells, presumably of oligodendroglial lineage, and GABAergic fiber outgrowth was further evidenced by the observation that addition of ciliary neurotrophic factor (CNTF) to the chemically defined medium resulted in both an increase in CNPase-positive cells and a decrease in GABAergic fiber outgrowth. The observations suggest that CNTF and serum indirectly inhibit axonal growth by stimulating oligodendroglial cells.

Ciliary neurotrophic factor supports p75NGFR-immunoreactive non-cholinergic, but not cholinergic, developing septal neurons in vitro.

Ciliary neurotrophic factor is known to exert both survival and differentiative actions on a number of neuronal populations of the peripheral and central nervous systems. In this study we have compared the trophic effects of ciliary neurotrophic factor and nerve growth factor on developing septal neurons of the rat in vitro. Fetal septal neurons were grown in vitro under glass coverslips in sandwich culture. Septal cultures grown for 14 days in the continual presence of nerve growth factor contain a population of cholinergic neurons that stain intensely for the low-affinity nerve growth factor receptor (p75NGFR), choline acetyltransferase and acetylcholinesterase. Without added nerve growth factor, few neurons stain for these markers. Ciliary neurotrophic factor addition for 14 days from plating in the absence of exogenous nerve growth factor results in the appearance of a population of neurons that stains for p75NGFR. This population is similar in number to that seen in nerve growth factor-treated cultures but is not immunoreactive for choline acetyltransferase and is significantly smaller in mean cross-sectional area. Delayed addition of nerve growth factor to ciliary neurotrophic factor-supported cultures at 14 days for a further seven days fails to induce choline acetyltransferase immunoreactivity in these p75NGFR-positive septal neurons. In cultures grown in the continual presence of nerve growth factor from plating, removal of nerve growth factor and addition of nerve growth factor antibodies at 14 days results in the death of over 80% of the cholinergic neurons after a further four days. Addition of ciliary neurotrophic factor during the period of nerve growth factor withdrawal appears to preserve a p75NGFR-positive, choline acetyltransferase-negative neuronal population. However, seven day re-addition of nerve growth factor to ciliary neurotrophic factor-treated, nerve growth factor-withdrawn cultures fails to induce choline acetyltransferase immunoreactivity in the ciliary neurotrophic factor-supported p75NGFR-positive septal neurons. Simultaneous treatment of cultures with both ciliary neurotrophic factor and nerve growth factor for 14 days from plating approximately doubles the number of p75NGFR-positive neurons relative to cultures treated with either ciliary neurotrophic factor or nerve growth factor alone, but the number of choline acetyltransferase-positive neurons in these cultures is not significantly greater than that found in cultures treated solely with nerve growth factor. These results suggest that ciliary neurotrophic factor does not support the survival and differentiation of developing septal cholinergic neurons in vitro, but can support the development of a p75NGFR-immunoreactive population of non-cholinergic septal neurons.