Research on Stem Cells

Project location: Italy
Project start date: March 2005 - Project end date: March 2008
Project number: 2005-01
Beneficiary: ATENA Onlus

- Repair of  Central Nervous System (CNS) lesions through cell replacement therapy by using  NSC induced towards  neuronal phenotypes

- Isolation of cancer stem cells from human brain tumors

(above) Giulio Maira M.D.Professor of Neurosurgery, Chief of Department of Neurosurgery Policlinico A. Gemelli Università Cattolica School of Medicine Rome

A new line of research on human brain cancer stem cells has recently been opened. The identification of brain tumor stem cells provides a powerful tool to investigate tumorigenesis and to develop innovative therapies. 

The Nando Peretti Foundation is financing the following projects:

 

 

A. Repair of  Central Nervous System (CNS) lesions through cell replacement therapy by using  NSC induced towards  neuronal phenotypes. 

 

B. Isolation of cancer stem cells from human brain tumors

 

A. Our preliminary results on transplantation of NSC to the SC in adult rats are encouraging on that the grafted cells survived and integrated in the injured SC tissue. However, differentiation of grafted NSC was restricted exclusively to the astrocytic phenotype (Pallini et al, submitted to the Journal of  Neurosurgery). Our current research project is directed to engineer NSC to express neurotrophin (NT) genes for a nerve regeneration therapy. The finding that in vivo direct delivery of Brain-Derived Neurotrophic Factor (BDNF) promote survival and/or differentiation of NSC and increase the number of the newly generated neurons, solicited us to transduce NSC to constitutively express BDNF and/or Nerve Growth Factor (NGF) by a lentivirus-mediated system. Hopefully, the neurogenesis will be induced by engrafted NSC, which will secrete neurotrophins locally, and in addition will promote axonal regeneration from the resident NSC. We chose lentiviral vectors to transduce NSC to express neurotrophins because this system offer these advantages: 1)  ability to infect non-mitotic cells as neurons,  2) high efficiency of  expression of the therapeutic gene(s), 3) gene(s) delivery sustained for long lasting time, 4)  simultaneous expression of up to three genes (Naldini L at al.;Pencea V. et al.; Benraiss A et al; Philips MF et al; Andsberg G et al; Kim DH et al.). 

 

B. In the last few months, we were able to isolate and to characterize cancer stem cells from human brain tumors of different phenotypes. These cells exhibit marked capacity of proliferation, self-renewal and differentiation. These features were highest from the most aggressive  clinical samples of glioblastomas and medulloblastomas compared with low-grade gliomas.

Operational procedures and methodology

A. NSC are isolated from the whole cortex  neonatal CD1mice and embryonic Wistar rats and from the subventricular zone (SVZ) of adult animals. NSC will be engineered with an advanced version of lentivirus, which allows expressing simultaneously and coordinately up to three genes.  In vitro NSC maintenance occurs in presence of basic Fibroblast Growth Factor (bFGF) and Epidermal Growth Factor (EGF) which allow self-renewing and extensive proliferation of NSC.

We intend to construct lentiviral vector bearing NT genes combined with their NT receptors or alternatively combination of NTs genes. To begin we will express simultaneously, under the same cytomegalovirus (CMV) promoter, BDNF or NGF followed by a fluorescent protein, named Enhanced Green Fluorescent Protein (EGFP), which result in fluorescent vector that can be easily  visualized inside the NSC in vitro and in vivo using a fluorescence microscopy. Whereas the first gene is translated in a more efficient manner, instead of translation initiation of the second and third gene rely on internal ribosome entry site of poliovirus (IRES) which has been optimized for maximum translational enhancement (Mitta B et al.).

On the basis of scientific reports (Lu P et al; Blesch A et al; FM Bareyre and ME Schwab) we expect that the cascade of events elicited by the NT itself or by the autocrine loop generated by co-expression of receptors and NTs simultaneously, would induce neurogenesis into the site of lesion, and consequently an increase of motor and sensory axon growth in adult CNS. 

Animal model. 

A. Adult female Wistar rats will be operated on using microsurgical procedures under aseptic conditions. After a T-8 spinal cord complete transection a suspension of neurospheres, timely engineered, will be grafted at the transection site.

A functional evaluation (foot print and walking tracks analysis and  Basso, Beattie, Bresnahan-BBB test) will be done at one, two and three months after the lesion.

At three months after the lesion, the animals will be re-anesthetized and immobilized in a stereotactic frame. After a bilateral frontoparietal craniectomy, the sensorimotor cortex of each side will be injected with 0.5 to 0.6 µl of an 8% solution of lectin-conjugated HRP tracer (wheat germ agglutinin), through a Hamilton microsyringe mounted on the stereotactic unit,  to label the cortico-spinal tract axons. Forty-eight hours after injection of HRP, the rats will be anesthetized and transcardially perfused with saline, followed by 2.5% buffered gluteraldehyde. The spinal cord will be then removed and processed using tetramethylbenzidine. 

B. Cancer stem cells will be isolated from different human brain tumors. Surgical specimens will be obtained fom different areas of the same tumor (core, periphery). The specimens will be dissociated and cultured in neural stem cell medium. Assays for tumorisphere formation, differentiation and immunostaining of cells from tumor spheres will be performed. We will investigate whether these cells, after transplantation in nude mice, may act as tumor founding cells or may develop tumors. The histological, cytological and architectural features of these tumors will be determined. 

Anticipated achievements and outcomes of the project

A. We have submitted to the  Journal of Neurosurgery a manuscript entitled "Transplantation of Neural Stem Cells to the Injured Spinal Cord. Experimental results in Rodents animal Models". In this paper, NSCs were homografted onto the injured spinal cord to assess their potential to survival and differentiate following traumatic injury. The results can be so summarized,  NSCs a) survive as long as 9 months after grafting b) migrate from the grafting site with a tropism towards the lesion epicenter, c) either remain undifferentiated or differentiated into the astrocytic phenotype without neuronal or oligodendrocytic differentiation, d) come close in relation with the tip of severed descending axonal paths including the Cortical Spinal Tract (CST) axons, and finally e) partly improve motor behavior on a foot-print analysis of ambulation. One main conclusion of this paper was that the use of genetically modified NSCs expressing trophic factors appears an attracting  tool in SC transplantation research to enhance neuronal differentiation.

B. The identification and availability of tumor stem cell lines represent an invaluable tool not only to study the basic mechanisms regulating cell proliferation  and differentiation in malignant gliomas, but also to develop novel approach to hinder their malignant growth. One first achievement will be to establish a relationship, if any, between tumorsphere formation and aggressive biological behaviour in specific cases. In fact, the amount of cancer stem cells in gliomas is supposed to be related to the infiltrative growth of the tumors. However, recent observations raises the hypothesis that these cells, which have been shown to migrate mostly at the periphery of gliomas, might represent an attempt of the host brain to counteract tumor growth.