Isolation of neurosphere-like bodies from an adult patient with refractory temporal lobe epilepsy

Isolamento de corpos neurosfera-like de paciente adulto com epilepsia do lobo temporal refratária

LETTER

Isolation of neurosphere-like bodies from an adult patient with refractory temporal lobe epilepsy

Isolamento de corpos neurosfera-like de paciente adulto com epilepsia do lobo temporal refratária

Ricardo Luiz Azevedo-Pereira^I; Emiliano Medei^I; Rosália Mendez-Otero^I; Jorge Paes Barreto Marcondes de Souza^II; Soniza Vieira Alves-Leon^II

^IInstitute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro RJ, Brazil

^IIUniversity Hospital Clementino Fraga Filho, Neurology and Neurosurgery Department, Federal University of Rio de Janeiro, Rio de Janeiro RJ, Brazil

^{Correspondence}

Epilepsy affects approximately 0.8% of the world population and is most common among those with chronic neurological diseases¹. Some patients are refractory to antiepileptic drugs and they develop refractory epilepsy². This kind of epilepsy presents a higher frequency of sudden death³; surgical treatment is indicated and has shown significantly higher efficacy compared to antiepileptic drug treatment⁴.

The surgical treatment of the patients creates the possibility of isolating neural stem cells (NSCs) in vivo. These cells are present in the subventricular zone (SVZ), around the lateral ventricles and in the sub-granular layer in the hippocampus (SGZ), and they give rise to neurons and glial cells during adulthood^5-6. The isolation of these cells could lead to a future source of autologous transplants for neurodegenerative diseases, as well as to the storage for future research for basic science studies of NSCs. However, only few studies have described the isolation of NSCs from human brain in vivo^7-9, and no such study has been performed in Brazil.

Here, we showed for the first time in Brazil the possibility of isolating neural progenitor cells from a woman with refractory epilepsy during interventional surgery.

CASE

A 50-year-old woman with a 15 year history of epilepsy wasreported. The surgery was carried out after the concordance of electrophysiological clinical analysis, MRI and ictal single photon emission computed tomography (SPECT) findings that have demonstrated an ictal onset zone in the right temporal lobe, atrophy and loss of volume in the right hippocampus (Fig 1A, B) and increased perfusion in the same side (Fig 1C).

The patient was subjected to an anterior temporal lobectomy and amigdalo-hippocampectomy, and in approaching the temporal horn of the lateral ventricle, subventricular sampling was done for tissue culture. The protocol was approved by the Ethical Committee from the Hospital Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, and the patient gave written informed consent.

The tissue was immediately transferred to DMEM-F12 at 4ºC. The pia mater and associated blood vessels were removed, and the tissue was dissociated with papain following the manufacturer's recommended procedures (Worthington Biochemical Corp). After dissociation, the single cells were cultured in neurosphere medium¹. The cells were cultured in 10-cm non-adherent culture plates. The culture was supplemented with 20 ng/mL epidermal growth factor (EGF), 10ng/mL basic fibroblast growth factor (bFGF) and B27 supplement twice a week. Additional neurosphere medium was administered once every week.

After 14 days in vitro, we observed, at least, 26 cellular aggregates similar to neurospheres. These neurosphere-like bodies had different diameters, ranging between 57 μm and 290 μm (Fig 2A). Individual neurospheres (n=10) were then transferred to glass coverslips previously incubated with 10 μg/mL poly-L-lysine followed by 20 μg/mL laminin. The growth factors were removed from the culture medium and the neurosphere-like aggregates adhered to the coverslips and showed different patterns of differentiation in the following days. In one pattern, lamellipodia-like projections were observed soon after plating (Fig 2B, 1 day in vitro) and in the following days, on top of these, many slender filopodia-like projections were present (Fig 2C, 30 days in vitro). The cells were fixed with 4% paraformaldehyde at 37ºC. The coverslips were incubated with normal goat serum followed by primary and secondary antibodies for immunocytochemistry. The lamellipodia-like processes were positive for glial fibrillary acidic protein (GFAP), suggesting differentiation into astrocytes and/or radial glial cells from the neurosphere-like bodies. In addition, we showed that on top of these processes, several β-III tubulin positive processes were present, suggesting neuronal differentiation (Fig 2D). A different pattern was observed in other neurospheres, in which only a few projections were present emanating from the neurospheres (Fig 2E, 5 days in vitro). One of the neurospheres detached from the substrate, leaving behind 3 attached cells that were used for the recording experiments (Fig 2F, 17 days in vitro). After recording, the coverslip with the cells was fixed and immunocytochemistry for GFAP and β-III tubulin was performed. In Figure 2G, it is possible to notice neurites (β-III tubulin positive), some of them closely associated with glial processes (GFAP positive).

To probe the electrical activity of the differentiated cells, depolarizing pulses were applied from -90 to +60 mV in 10 mV steps (holding potential = -70 mV). With this approach, an outward rectifier current was observed typical of glial cells (Fig 2H).

DISCUSSION

Mammalian NSCs have been isolated from the SVZ around the lateral ventricle and the SGZ^5,6. These cells are self-renewable as cell aggregates called neurospheres, and they can give rise to all the cells of the nervous system. These neural progenitor cells can generate neurons and astrocytes, but they have lower plasticity than neural stem cells¹⁰. Here, we isolated neural progenitor cells which formed neurosphere-like bodies that gave rise to β-III tubulin-positive neurons and GFAP-positive astrocytes. After differentiation, electrical properties were recorded from these cells showing functionality in vitro. In our study, the cells did not differentiate into oligondendrocytes and were not self-renewable (data not shown). Therefore, they are not neural stem cells.

Up to now, few studies have reported the isolation of human neural stem/progenitor cells in vivo^7-9. Although we did not obtain NSCs, we isolated neural progenitor cells from the SVZ for the first time in Brazil. These cells were isolated after being sampled by means of surgical procedure of an adult patient with refractory epilepsy, and these results can be reproduced by other groups.

Methods of isolation and characterization of progenitor/stem cells have been reported in order to clarify their basic biological mechanisms. Additionally, these cells can be differentiated in vitro into all cells of the nervous system, which could be useful in the screening of drugs and, in the future, these cells could be expanded in culture as a source for cellular transplants for neurological diseases.

ACKNOWLEDGEMENTS - We would like to thank Felipe Marins for technical support and Andrew McUsic for English review.

Received 1June 2009

Received in final form 14 October 2009

Accepted 29 October 2009

• 1. Pitkänenand A, Sutula TP. Is epilepsy a progressive disorder? Lancet Neurol 2002;1:173-181.
• 2. Rouvel-Tallec A. New antiepileptic drugs. Rev Med Interne 2009;30: 335-339.
• 3. Tomson T, Nashef L, Ryvlin P. Sudden unexpected death in epilepsy: current knowledge and future directions. Lancet Neurol 2008;7:1021-1031.
• 4. Wiebe S, Warren T, Girvin JP, Eliaszw M. A randomized, controlled trial of surgery dor temporal lobe epilepsy. N Eng J Med 2001;342:311-318.
• 5. Doetsch F, Caille I, Lim DA, et al. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 1999;97:703-716.
• 6. Johansson CB, Momma S, Clarke DL, et al. Identification of a neural stem cell in the adult mammalian central nervous system. Cell 1999;96,25-34.
• 7. Sanai N, Tramontin AD, Quinones-Hinojosa A, et al. Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration. Nature 2004;427:740-744.
• 8. Moe MC, Varghese M, Danilov AI, et al. Multipotent progenitor cells from the adult human brain: neurophysiological differentiation to mature neurons. Brain 2005;128:2189-2199.
• 9. Moe MC, Westerlund U, Varghese M, et al. Development of neuronal networks from single stem cells harvested from the adult human brain. Neurosurgery 2005;56:1182-1190.
• 10. Kukekov VG, Laywell ED, Suslov O, et al. Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain. Exp Neurol 1999; 156,333-344.

Publication Dates

• Publication in this collection
  06 Jan 2011
• Date of issue
  Dec 2010