Expansion of Embryonic and Adult Neural Stem Cells by <em>In Utero</em> Electroporation or Viral Stereotaxic Injection

Abstract

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1 . Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2 . However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4 . Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes [5][6][7][8] . By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9 . Both approaches, whose basic principles were already described by other video protocols [10][11][12][13][14] , were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15 . These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult ne...

SARA regulates neuronal migration during neocortical development through L1 trafficking

“…IUE procedures were performed on E13.5 mouse brains as previously described (Li et al, 2011;Artegiani et al, 2012). Briefly, the embryonic ventricles were injected with a mixture of plasmids (2 µg/µl), Fast Green dye and Tris-EDTA buffer.…”

Lentiviruses allow widespread and conditional manipulation of gene expression in the developing mouse brain

“…Primers (supplementary material Table S1) were designed to express the GFP in frame with the nls, and stop codons were added to terminate translation in both the recombined and not-recombined construct. Viral preparations, as previously described (Artegiani et al, 2011;Artegiani et al, 2012), were further optimized to increase viral concentration. GFP nls or GFP lox vectors together with pCD/NL-BH (Mochizuki et al, 1998) and pczVSV-G (Pietschmann et al, 1999) plasmids (coding for HIV-1 Gag/Pol and VSV-G, respectively) were co-transfected (5 µg each) in 293T cells (5×10 6 in a 10 cm dish, total of 45 dishes).…”

“…1 μl of viral suspension mixed with 0.05% Fast Green was injected through the uterine walls into the embryonic lateral ventricle as described for in utero electroporation (Artegiani et al, 2012;Lange et al, 2009). Brains were collected at E15.5, E18.5 and postnatal day (P) 2 and 10.…”

Modified Bone Marrow Stromal Cells Therapy for Central Nervous System Disorders

“…Stem cells are a kind of cells characterized of species diversity, selfreplication and renewal ability, high proliferation potential and multiple differentiation potential [1][2][3][4]. Compared with other types of stem cell transplantation, treatment using Bone Marrow Stromal Cells (BMSCs) has more advantage, which has rich resources, easy to access, multiple potential, ethical limits, subculture amplification etc.…”

“…This may be because of the neural disorder is more complex, involving more cell types, more complicated cerebral blood circulation and micro environment [2]. We have to face more clinical hurdles including (1) limited engraftment, survival, and proliferation; (2) immunogenicity with allogenic transplantation; (3) poor differentiation, maturation, and integration [34].…”