Johana Tello Velasquez

Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles

We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures.

Low-Dose Curcumin Stimulates Proliferation, Migration and Phagocytic Activity of Olfactory Ensheathing Cells

One of the promising strategies for neural repair therapies is the transplantation of olfactory ensheathing cells (OECs) which are the glial cells of the olfactory system. We evaluated the effects of curcumin on the behaviour of mouse OECs to determine if it could be of use to further enhance the therapeutic potential of OECs. Curcumin, a natural polyphenol compound found in the spice turmeric, is known for its anti-cancer properties at doses over 10 µM, and often at 50 µM, and it exerts its effects on cancer cells in part by activation of MAP kinases. In contrast, we found that low-dose curcumin (0.5 µM) applied to OECs strikingly modulated the dynamic morphology, increased the rate of migration by up to 4-fold, and promoted significant proliferation of the OECs. Most dramatically, low-dose curcumin stimulated a 10-fold increase in the phagocytic activity of OECs. All of these potently stimulated behavioural characteristics of OECs are favourable for neural repair therapies. Importantly, low-dose curcumin gave a transient activation of p38 kinases, which is in contrast to the high dose curcumin effects on cancer cells in which these MAP kinases tend to undergo prolonged activation. Low-dose curcumin mediated effects on OECs demonstrate cell-type specific stimulation of p38 and ERK kinases. These results constitute the first evidence that low-dose curcumin can modulate the behaviour of olfactory glia into a phenotype potentially more favourable for neural repair and thereby improve the therapeutic use of OECs for neural repair therapies.

Olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system.

During development of the primary olfactory system, axon targeting is inaccurate and axons inappropriately project within the target layer or overproject into the deeper layers of the olfactory bulb. As a consequence there is considerable apoptosis of primary olfactory neurons during embryonic and postnatal development and axons of the degraded neurons need to be removed. Olfactory ensheathing cells (OECs) are the glia of the primary olfactory nerve and are known to phagocytose axon debris in the adult and postnatal animal. However, it is unclear when phagocytosis by OECs first commences. We investigated the onset of phagocytosis by OECs in the developing mouse olfactory system by utilizing two transgenic reporter lines: OMP‐ZsGreen mice which express bright green fluorescent protein in primary olfactory neurons, and S100β‐DsRed mice which express red fluorescent protein in OECs. In crosses of these mice, the fate of the degraded axon debris is easily visualized. We found evidence of axon degradation at embryonic day (E)13.5. Phagocytosis of the primary olfactory axon debris by OECs was first detected at E14.5. Phagocytosis of axon debris continued into the postnatal animal during the period when there was extensive mistargeting of olfactory axons. Macrophages were often present in close proximity to OECs but they contributed only a minor role to clearing the axon debris, even after widespread degeneration of olfactory neurons by unilateral bulbectomy and methimazole treatment. These results demonstrate that from early in embryonic development OECs are the primary phagocytic cells of the primary olfactory nerve. J. Comp. Neurol. 523:479–494, 2015.

Mechanism of impaired microtubule-dependent peroxisome trafficking and oxidative stress in SPAST-mutated cells from patients with Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is an inherited neurological condition that leads to progressive spasticity and gait abnormalities. Adult-onset HSP is most commonly caused by mutations in SPAST, which encodes spastin a microtubule severing protein. In olfactory stem cell lines derived from patients carrying different SPAST mutations, we investigated microtubule-dependent peroxisome movement with time-lapse imaging and automated image analysis. The average speed of peroxisomes in patient-cells was slower, with fewer fast moving peroxisomes than in cells from healthy controls. This was not because of impairment of peroxisome-microtubule interactions because the time-dependent saltatory dynamics of movement of individual peroxisomes was unaffected in patient-cells. Our observations indicate that average peroxisome speeds are less in patient-cells because of the lower probability of individual peroxisome interactions with the reduced numbers of stable microtubules: peroxisome speeds in patient cells are restored by epothilone D, a tubulin-binding drug that increases the number of stable microtubules to control levels. Patient-cells were under increased oxidative stress and were more sensitive than control-cells to hydrogen peroxide, which is primarily metabolised by peroxisomal catalase. Epothilone D also ameliorated patient-cell sensitivity to hydrogen-peroxide. Our findings suggest a mechanism for neurodegeneration whereby SPAST mutations indirectly lead to impaired peroxisome transport and oxidative stress.

Differing phagocytic capacities of accessory and main olfactory ensheathing cells and the implication for olfactory glia transplantation therapies.

The rodent olfactory systems comprise the main olfactory system for the detection of odours and the accessory olfactory system which detects pheromones. In both systems, olfactory axon fascicles are ensheathed by olfactory glia, termed olfactory ensheathing cells (OECs), which are crucial for the growth and maintenance of the olfactory nerve. The growth-promoting and phagocytic characteristics of OECs make them potential candidates for neural repair therapies such as transplantation to repair the injured spinal cord. However, transplanting mixed populations of glia with unknown properties may lead to variations in outcomes for neural repair. As the phagocytic capacity of the accessory OECs has not yet been determined, we compared the phagocytic capacity of accessory and main OECs in vivo and in vitro. In normal healthy animals, the accessory OECs accumulated considerably less axon debris than main OECs in vivo. Analysis of freshly dissected OECs showed that accessory OECs contained 20% less fluorescent axon debris than main OECs. However, when assayed in vitro with exogenous axon debris added to the culture, the accessory OECs phagocytosed almost 20% more debris than main OECs. After surgical removal of one olfactory bulb which induced the degradation of main and accessory olfactory sensory axons, the accessory OECs responded by phagocytosing the axon debris. We conclude that while accessory OECs have the capacity to phagocytose axon debris, there are distinct differences in their phagocytic capacity compared to main OECs. These distinct differences may be of importance when preparing OECs for neural transplant repair therapies.

The serrulatane diterpenoid natural products RAD288 and RAD289 stimulate properties of olfactory ensheathing cells useful for neural repair therapies

Olfactory ensheathing cells (OECs) are being trialled for cell transplantation therapies for neural repair as they have unique properties which can enhance neuron regeneration. However, improvements in cell viability, proliferation and migration are needed to enhance therapeutic outcomes. Growth factors can enhance cell activity, but they can also induce side effects as they can act on numerous cell types. An alternative approach is to identify natural products (NPs) that more selectively activate specific cell functions. We have examined two pure NPs, 3-acetoxy-7,8-dihydroxyserrulat-14-en-19-oic acid (RAD288) and 3,7,8-trihydroxyserrulat-14-en-19-oic acid (RAD289) isolated from the Australian plant Eremophila microtheca. We determined that RAD288 and RAD289 stimulated the viability and proliferation of OECs in two-dimensional cultures and increased cell viability in three-dimensional spheroids. Both compounds also enhanced OEC-mediated phagocytosis of neural debris. However, only RAD288 stimulated migration of OECs, demonstrating that key structural changes to the compound can dramatically affect the resultant cellular action. In addition, cell-type specific action is highlighted by the result that neither compound stimulated the viability of Schwann cells which are a closely-related glial cell type. Therefore, these small molecules may have high potential for selective activation of specific therapeutically-useful activities of OECs for transplantation therapies to repair the nervous system.

Schwann cell lamellipodia regulate cell-cell interactions and phagocytosis

&NA; Lamellipodia in Schwann cells (SCs) are crucial for myelination, but their other biological functions remain largely uncharacterised. Two types of lamellipodia exist in SCs: axial lamellipodia at the outermost edge of the cell processes, and radial lamellipodia appearing peripherally along the entire cell. We have previously shown that radial lamellipodia on olfactory glia (olfactory ensheathing cells; OECs) promote cell‐cell adhesion, contact‐mediated migration and phagocytosis. Here we have investigated whether lamellipodia in SCs have similar roles. Using live‐cell imaging, we show that the radial lamellipodia in SCs are highly motile, appear at multiple cellular sites and rapidly move in a wave‐like manner. We found that axial and radial lamellipodia had strikingly different roles and are regulated by different intracellular pathways. Axial lamellipodia initiated interactions with other SCs and with neurons by contacting radial lamellipodia on SCs, and budding neurites/axons. Most SC‐SC interactions resulted in repulsion, and, lamellipodial activity (unlike in OECs) did not promote contact‐mediated migration. We show that lamellipodia are crucial for SC‐mediated phagocytosis of both axonal debris and bacteria, and demonstrated that inhibition of lamellipodial activity by blocking the Rho/Rac pathways also inhibits phagocytosis. We also show that heregulin, which induces SC differentiation and maturation, alters lamellipodial behaviour but does not affect phagocytic activity. Overall, the results show that SC lamellipodia are important for cell interactions and phagocytosis. Graphical abstract Figure. No caption available. HighlightsSchwann cells have motile lamellipodia that regulate cell‐cell contact.Lamellipodia are important for phagocytic activity.Schwann cell lamellipodia are different to those in olfactory ensheathing cells in that radial lamellipodia do not drive contact‐mediated migration.

Linckosides enhance proliferation and induce morphological changes in human olfactory ensheathing cells

Linckosides are members of the steroid glycoside family isolated from the starfish Linckia laevigata. These natural compounds have notable neuritogenic activity and synergistic effects on NGF-induced neuronal differentiation of PC12 cells. Neurogenic factors or molecules that are able to mimic their activities are known to be involved in the survival, proliferation and migration of neurons and glial cells; however how glial cells respond to specific neurogenic molecules such as linckosides has not been investigated. This study aimed to examine the effect of three different linckosides (linckoside A, B and granulatoside A) on the morphological properties, proliferation and migration of human olfactory ensheathing cells (hOECs). The proliferation rate after all the treatments was higher than control as detected by MTS assay. Additionally, hOECs displayed dramatic morphological changes characterized by a higher number of processes after linckoside treatment. Interestingly changes in microtubule organization and expression levels of some early neuronal markers (GAP43 and βIII-tubulin) were also observed. An increase in the phosphorylation of ERK 1/2 after addition of the compounds suggests that this pathway may be involved in the linckoside-mediated effects particularly those related to morphological changes. These results are the first description of the stimulating effects of linckosides on hOECs and raise the potential for this natural compound or its derivatives to be used to regulate and enhance the therapeutic properties of OECs, particularly for cell transplantation therapies.

Transplantation of Olfactory Ensheathing Cells in Spinal Cord Injury

Spinal cord injury is characterized by massive cellular and axonal loss, a neurotoxic environment, inhibitory molecules and physical barriers that hamper nerve regeneration and reconnection leading to chronic paralysis. Transplantation of different types of cells is one of the strategies being examined in order to restore the lost cell populations and to re-establish a permissive environment for nerve regeneration. The mammalian olfactory system is one of the few zones in the body where neurogenesis occurs during the lifetime of the organism, with olfactory neurons being replaced daily with their axons elongating from the peripheral nervous system into the central nervous system to re-establish functional connections. The regenerative ability of this system is largely attributed to the presence of a unique group of cells called olfactory ensheathing cells (OECs). OECs have emerged as an encouraging cell candidate for transplantation therapies to repair the injured spinal cord with multiple animal models showing significant functional improvements and several human trials establishing that the procedure is safe and feasible. Even though the results are promising with some animal models showing remarkable restoration of function, the variability amongst studies in terms of outcome assessments, cell purity, cell culture and transplantation protocols make it difficult to reach firm conclusions about the effectiveness of OEC transplant therapy to treat the injured spinal cord. These variations need to be addressed in order to achieve a more realistic understanding of how the benefits of OEC transplantation enhance the therapeutic outcomes.