Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: state of the science.

Brain tumour stem cells (BTSCs) are a small population of cells that has self-renewal, transplantation, multidrug resistance and recurrence properties, thus remain novel therapeutic target for brain tumour. Recent studies have shown that peroxisome proliferator-activated receptor gamma (PPARγ) agonists induce growth arrest and apoptosis in glioblastoma cells, but their effects on BTSCs are largely unknown. In this study, we generated gliospheres with more than 50% CD133+ BTSC by culturing U87MG and T98G human glioblastoma cells with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). In vitro treatment with PPARγ agonist, 15-Deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2) or all-trans retinoic acid resulted in a reversible inhibition of gliosphere formation in culture. Peroxisome proliferator-activated receptor gamma agonists inhibited the proliferation and expansion of glioma and gliosphere cells in a dose-dependent manner. Peroxisome proliferator-activated receptor gamma agonists also induced cell cycle arrest and apoptosis in association with the inhibition of EGF/bFGF signalling through Tyk2-Stat3 pathway and expression of PPARγ in gliosphere cells. These findings demonstrate that PPARγ agonists regulate growth and expansion of BTSCs and extend their use to target BTSCs in the treatment of brain tumour.

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PPARγ agonists inhibit growth and expansion of CD133+ brain tumour stem cells

Self-renewing, multipotent neural progenitor cells (NPCs) reside in the adult mammalian spinal cord ependymal region. The current study characterized, in vitro, the native differentiation potential of spinal cord NPCs isolated from adult enhanced green fluorescence protein rats. Neurospheres were differentiated, immunocytochemistry (ICC) was performed, and the positive cells were counted as a percentage of Hoescht+ nuclei in 10 random fields. Oligodendrocytes constituted most of the NPC progeny (58.0% of differentiated cells; 23.4% in undifferentiated spheres). ICC and electron microscopy (EM) showed intense myelin production by neurospheres and progeny. The number of differentiated astrocytes was 18.0%, but only 2.8% in undifferentiated spheres. The number of differentiated neurons was 7.4%, but only 0.85% in undifferentiated spheres. The number of differentiated radial glia (RG) was 73.0% and in undifferentiated spheres 80.9%. EM showed an in vitro phagocytic capability of NPCs. The number of undifferentiated NPCs was 32.8% under differentiation conditions and 78.9% in undifferentiated spheres. Compared with ependymal region spheres, the spheres derived from the peripheral white matter of the spinal cord produced glial-restricted precursors. These findings indicate that adult rat spinal cord ependymal NPCs differentiate preferentially into oligodendrocytes and RG, which may support axonal regeneration in future trials of transplant therapy for spinal cord injury.

Oligodendrocytes and Radial Glia Derived From Adult Rat Spinal Cord Progenitors: Morphological and Immunocytochemical Characterization:

The epidermal growth factor receptor family consists of four related tyrosine kinases: the epidermal growth factor receptor (EGF-R or ErbB), ErbB2, ErbB3, and ErbB4. These receptors are capable of ext

Expression of the EGF receptor family members ErbB2, ErbB3, and ErbB4 in germinal zones of the developing brain and in neurosphere cultures containing CNS stem cells.

A comparison of proliferative capacity and passaging potential between neural stem and progenitor cells in adherent and neurosphere cultures

Genetically unmodified neural precursor cells (NPCs) derived from rate embryonic cortex were stimulated with Long-epidermal growth factor in vitro to form multicellular neurospheres. In our study, histology and transmission electron microscopy was for the first time utilized for analysis of three-dimensional neurospheres. Immunohistochemical phenotypization carried out on paraffin-embedded section revealed that most cells in the neurosphere expressed high levels of vimentin that is normally found in immature neural cells. Majority of glial cells expressed glial fibrillary acidic protein (GFAP) that is a specific marker for differentiated astrocytes. On the other hand, myelin basic protein (MBP), a marker of oligodendrocytes, was found only in a discrete subpopulation of glial cells. Similarly, electron microscopy recognized only rate myelin sheaths surrounding axons. Some cells displayed neuron-like morphology and were labelled with antibodies recognizing the neuronal antigens: 210 kDa neurofilaments (NF 210) and microtubule associated protein-2 (MAP-2). Surprisingly well-developed interneuronal synapses were found electronmicroscopically. The presence of synaptic vesicles was also confirmed by immunohistochemical detection of synaptophysin. Our results show that NPCs in neurospheres gradually differentiate into glial and neuronal cells.

Differentiation of epidermal growth factor-responsive neural precursor cells within neurospheres.

We demonstrated that proliferation of dissociated neural E14 and E19 rat precursor cells could be induced by Long-EGF. Dividing EGF-responsive neural progenitor cells stimulated by Long-EGF formed spherical multicellular clusters (neurospheres) which may reach macroscopical size. We have studied the inner organization of cells in semithin sections and revealed that the cell population within the neurosphere is not uniform. These are original findings as other researchers did not process neurospheres histologically. Cells located in a central area possessed neuron-like morphology whereas peripheral cells where differentiated to a lesser degree. Generally, we have observed several apoptotic cells or apoptotic bodies per section. Moreover, the central portion of the neurosphere contained degenerating cells that probably die from worsened nutrition conditions in the large neurosphere. After plating the neurosphere in serum-supplemented medium, cells began to migrate radially from the edge of the neurosphere and differentiate. The cells lying in the vicinity to the cluster mimicked radial glia whereas the cells located at the periphery of the colony took morphology of astroglial cells. These observations suggest EGF-responsive neural precursor cells retained their ability to produce both neuronal and glial phenotypes after prolonged cultivation period.

Cultivation of neural EGF-responsive precursor cells.

Our previous studies tend to document the temporary invasiveness of intraparenchymal fetal isografts. The present study exploits a model situation in which contacted host brain surface remains undamaged. We analyzed the behavior of transplant fragments that accidentally disseminated in cerebrospinal fluid pathways. The transplant seeding occurred in 7 cases out of 85 cases of healthy solid isografts taken from various parts of fetal brain. Fragments were found either as free (floating or simply lying close to the surface) or attached. Some attached fragments invasively penetrated the brain parenchyma. This speaks in favor of invasive properties of fetal neural transplants.

Nĕmecek S

Papers

Differentiation of epidermal growth factor-responsive neural precursor cells within neurospheres.

Cultivation of neural EGF-responsive precursor cells.

Cerebrospinal fluid dissemination of fetal neural isografts in brain of adult rats.