ADULT HUMAN BRAIN MENINGES HOST NEURAL STEM/PRECURSOR CELLS THAT CAN BE IN VITRO EXPANDED AND SHOW MULTIPOTENT NEURAL DIFFERENTIATION POTENTIAL IN 2D AND 3D CULTURE

During brain and spinal cord development majority of the cell types are produced by the multipotent, self-renewing and proliferating neural stem/progenitor cells (NSCs) through the processes like neurogenesis and gliogenesis [1]. In the developed mammalian brain these NSCs have been found to be restricted to limited regions called neurogenic niches [2]. These neurogenic niches provide microenvironments for the NSCs survival, proliferation and neural differentiation. However, in adult human brain the presence of active NSC niches and the generation of new neurons after birth is still debatable [3]. Recent data highlighted that meninges, the protective coverings of the brain and spinal cord, are a neurogenic niche. NSCs have been detected in the developing and adult brain and spinal cord meninges both in physiological [4-7] and pathological conditions [8-11]. These NSCs have been described to migrate from meninges to the brain parenchyma and generate cortical neurons during perinatal stage [6]. However, majority of the data regarding the presence of NSCs in the meninges comes from the studies in mice and rats and it is not known whether NSCs exist in the human brain meninges. In this thesis work, we study the presence of NSCs in fetal and adult human brain meninges and assessed NSC properties in vitro in 2D and 3D culture. To perform this, we first set up the methodologies for brain meninges cell isolation, and characterization of NSC in vitro properties, differentiation in 2D and 3D culture and transplantation in vivo using the murine models. We observed that isolated cells from the adult mice brain meninges exhibited in vitro the stem cell properties including proliferation, self-renewal, and migration. In vitro cultured mice NSCs derived from adult brain meninges (mM-NSCs) expressed typical NSCs markers at gene and protein levels. Furthermore, mM-NSCs have neural differentiation potential and can be differentiated in 2D culture into neurons and oligodendrocytes. Similarly, we found mM-NSCs show self-assembly properties and multipotent differentiation potential into neurons, astrocytes, and oligodendrocytes in 3D culture. Moreover, neurons in 3D neural organoids expressed pre- and post-synaptic protein markers and showed spontaneous and synchronized calcium fluctuations. Additionally, we found that mM-NSCs survive and differentiate following in vivo transplantation into the adult mouse hippocampus. In human, we observed for the first time that cells expressing NSC markers are present in the developing human brain meninges and that cells expressing these NSC markers persist in the adult human brain meninges. We successfully isolated and culture NSCs from the adult human brain meninges (hM-NSCs). In vitro culture hM-NSCs exhibit typical NSC properties and express NSC markers. We were able to differentiate hM-NSCs into mature electrically functional neurons. We also observed that human neurons derived from differentiated hM-NSCs in vitro matured with their own timings and improved their electrical properties with age. Moreover, we found that hM-NSCs show self-assembly properties and generate 3D neural organoids in which they showed multipotent differentiation potential. Whole transcriptomic analysis showed that hM-NSCs were closely related to IPSCs-derived NSCs with respect to the upregulation of neural stem/progenitor markers and gene ontologies. Overall, our data suggest that NSC are present in the adult human brain meninges, can be isolated, and show multipotent differentiation potential in 2D and 3D cultures. Our findings highlight the presence of NSCs in the adult human brain meninges and they could further help understanding human brain development and cellular plasticity. The presence of a somatic NSC pool in the meninges of the adult human brain may provide a new source of adult NSCs which may be potentially used in regenerative medicine for brain repair in neurodegenerative diseases.