USING IPSC FOR UNDERSTANDING CNS PHYSIOLOGY….….24

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neurogenesis. These observations strongly suggest a neurogenesis-associated oxidative stress, which is not deleterious to the cells but rather regulates self-renewal and proliferative properties! of neural precursor cells⁵³.! In vitro neurodevelopmental models using neuroblastoma cell line have highlighted ROS as a key factor for neuronal differentiation and decreasing ROS generation restrains neural differentiation⁵⁴.

Taken together, these recent observations reveal the important role of ROS in neural differentiation and even more precisely at the early stage of neural differentiation when the cells exhibit higher proliferation rate. The exact source of these regulatory ROS and their targets remain to be identified.

1.3.1.2. USING IPSC FOR UNDERSTANDING CNS PHYSIOLOGY

As stated earlier, a main focus of stem cell biology is to generate human cellular systems to recapitulate specific diseases in vitro. However, in order to successfully mimic the in vivo condition it is essential to understand normal tissue physiology, such as for example the metabolic environmental factors, which are necessary for normal neurogenesis and neural differentiation. The studies described above have shown that stem cells can be used to study not only disease specific pathological aspects of oxidative stress-mediated metabolic changes but also how low oxygen and certain level of ROS generation represent physiological factors associated with neurogenesis.

! Identification of cellular environmental factors to improve neuronal differentiation and maintenance of neural stem/progenitor cells in vitro is key for human stem cells biology. To this extent, several studies were designed to modulate levels of O2 and ROS in stem cells culture and assess their neural differentiation.

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Stacpoole and colleagues, have studied differentiation culture of neural precursor cells derived from human embryonic stem cells using either 3% O2 (hypoxia) or 20% O2

(hyperoxia). This study revealed an enhanced survival rate of NPCs in hypoxic conditions in comparison to 20% environmental oxygen. In addition, the neural differentiation condition with 3% O2 resulted in a twofold increase of motor neuron precursors defined by the marker OLIG2 (encoding oligodendrocyte lineage transcription factor 2). These finding represent a step towards recapitulation of appropriate environmental factors impacting neural differentiation, and thus it may represent a significant advancement in disease modeling and cell-based therapies⁵⁵.

Additionally, the group of Le Belle and colleagues described an in vitro neural differentiation study in which NPCs derived from human embryonic stem cells were cultured under different concentration of exogenously ROS (H2O2). They defined two NPCs populations with low level of ROS (ROS ^lo) and high level of ROS (ROS ^hi) in which they demonstrate a higher NPCs proliferation and self-renewal rate in ROS ^hi in comparison to ROS ^lo 46.

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1.4. CANCER STEM CELLS

Cancer is normally initiated by a mutation in regulatory mechanisms that control cell division and proliferation, signal transduction pathway and DNA damage (oncogenic mutations). Under normal conditions, cells with abnormal mutations are eliminated from the cell replication cycle, however mutations may accumulate at some low incidence and initiate cancer. Malignant tumors contain cells with functional heterogeneity with different proliferation and differentiation abilities. A defined subset of cancer cells display stem cell characteristics including self-renewal and differentiation into all types of cells found within cancers. These cells are called cancer stem cells (CSC). They are characterized by self-renewal and differentiation into malignant progenitors. These unique properties allow the preservation of cancer stem cell pool within the tumor and causes malignant spreading of the tumor, invasion into surrounding healthy tissue and new tumor site formation. For this reason, various therapeutic approaches hold hope for cancer treatment by targeting proliferative cancer stem cells⁵⁶.

Cancer stem cells have been reported in most of the human tumors and can be identified and isolated from the patient biopsy by using approaches such as fluorescence activated cell sorting (FACS) for specific surface markers^57,58 and can be cultured in vitro.

The isolation of cancer stem cells and in vitro culture for various cancers, including brain tumors, opened novel perspectives in the field of cancer biology and related mechanism.

During the 80s, the group of Mina Bissell was one of the first to underline the importance of developing three-dimensional based models in vitro in order to more closely recapitulate the multicellular physiological tumor environment. This was defined as “tumor engineering”⁵⁹. Her rationale was that “cancer is not a disease of single cells but rather a problem of the organs". Accordingly, three-dimensional-based in vitro models have been

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developed to study a variety of solid tumors, including glioblastomas. This type of models provides important insights into tumor biology such as cell-cell interaction, cellular differentiation and tissue re-organization, synthesis of extracellular matrix (ECM) and cell-matrix interaction⁶⁰. These are the so called “multicellular tumor spheroids” which consist of a three-dimensional arrangement of mixed tumor cells and extracellular matrix. Once cancer stem cells are grown above a certain size, a characteristic cellular layer structure is formed due to gradient supply of oxygen and nutrients. This results in a characteristic cytoarchitecture which is composed of an outer layer of proliferating cells, a middle layer of quiescent viable cells and a center of dead necrotic cells (Figure 5)⁶¹.

This method to recapitulate the tumor in vitro has been extensively used for diverse tumors, including glioblastoma, which is discussed in the next section.

Figure 5. Multicellular spheroid growth. Tumor stem cells can be grown in culture due to the high proliferation rate. Cell growth in spheroids results in formation of different layers due to gradient supply of necessary factors for cell proliferation such as oxygen (O₂) and nutrient. This results in an outer layer of proliferation cell, a middle layer of viable cells and eventually a necrotic center.

O₂ and nutrients CO2 and waste

quiescent viable cells proliferating cells

necrotic cells Cancer stem cell

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