This work was supported by grants from Ministerio&#160;de Ciencia e Innovaci&#243;n and Agencia Estatal de Investigaci&#243;n (MCIN/AEI) (PID2019-110045GB-I00; PID2022-142734OB-I00 and EUR2023-143479) to SRF. EJV (FPU20/00795) and DSL (FPU22/03797) are funded by the Spanish Formacio&#769;n de Profesorado Universitario fellowship program (FPU). LLC (PRE2020-094137) and JDM (PREP2022-000680) are funded by the Spanish Formacio&#769;n de Personal Investigador (FPI) fellowship program. CMM (CIACIF/2022/366) is funded by Generalitat Valenciana. MIL (CPI-22-481) is funded by Programa INVESTIGO fellowship (Next Generation EU). Open Access funding provided by the Ministerio de Ciencia e Innovacio&#769;n.

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

<ol>
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The authors declare no competing financial interests.

Due to the lack of definitive markers for identifying the NSC population in vivo, the analysis of NSCs has been primarily based on observing the behavior of cells isolated from neurogenic niches in ex vivo conditions. Pioneering work by Reynolds and Weiss laid the groundwork by establishing precise culture conditions, enabling the isolation and expansion of individual cells from young adult (2 months old) mouse SVZ tissue under non-adhesive conditions. These cells are usually propagated in a serum-free medium containing EGF and bFGF, conditions that entirely prevent differentiation into neurons and glia while promoting proliferation. Indeed, under these culture conditions9, most cells die during the first days in culture, but a small subset begins dividing and primarily forms floating neurospheres9. Enzymatic dissociation and subculture of these cell aggregates facilitate culture propagation, demonstrating the self-renewal capacity of these cultures.
Notably, neurosphere cultures show expansion potential with the addition of EGF only, while NSCs grown in a medium containing only bFGF do not show long-term cell proliferation30. Both pieces of evidence point out EGF as the primary NSC self-renewal mitogen. Nevertheless, the presence of both EGF and bFGF in the culture medium improves the self-renewal capacity of NSCs31, as well as contributes to balancing the differentiation potential into astrocytes, neurons, and oligodendrocytes32,33,34,35. Moreover, the use of a defined mixture of hormones and factors instead of commercial alternatives to supplement the medium ensures the high quality and reproducibility of NSC cultures. Under these conditions, mouse NSC cultures can persist as stable cell lines without undergoing immortalization. Nevertheless, a concern in neurosphere cultures is the potential of proliferative cell populations to undergo genetic transformations, bypassing cell cycle regulatory mechanisms and leading to an immortalized phenotype. Therefore, although neurosphere cultures are highly expandable, cultures beyond 10 in vitro passages are usually discarded for studies, as they may undergo replicative aging, and cells with abnormal chromosomal content or irregular growth dynamics might emerge. Moreover, the use of long-term established neurosphere cultures must be continuously monitored. Neurosphere cultures also offer the opportunity to examine their characteristics and potential in a controlled environment, providing a more precise and adjustable setting that can be modulated and monitored more accurately than in vivo. Through clonogenic or population analyses in vitro, it is possible to quantify the self-renewal and proliferation capacities of these cells, facilitating the identification of the underlying mechanisms governing these properties.
Nevertheless, despite the large list of advantages of working with NSCs in vitro, the nature of this culturing protocol and the delicacy of NSCs constitute a challenge in the field. For example, the number of primary neurospheres generated during a typical dissection can vary significantly based on the skill and precision of the experimenter. The primary neurosphere results illustrate this variability in the number of primary neurospheres obtained from the SVZ of 2-month-old mice, ranging between 500 and 3000 neurospheres. Various factors may contribute to this variability. First, the precision of the dissection minimizes unwanted parenchymal tissue, which inhibits primary sphere formation. Second, generating small pieces of SVZ tissue allows for efficient enzymatic digestion and trituration, thereby reducing cell loss. This highlights the need for sufficient prior practice of the dissection protocol and a fine-tuned development of tissue processing during the establishment of the neurosphere cultures.
Another limitation of these cultures lies in the fact that neurospheres can comprise both NSCs and progenitor cells, making it challenging to distinguish between these two populations within primary cultures. While various markers like GFAP, Nestin, Musashi, and SOX2 have been reported to be expressed by NSCs8, none of these have been exclusively associated with NSCs. Emerging FACS techniques based on cell surface antigen expression have enabled the isolation of NSCs and their progeny. These studies have demonstrated that transit-amplifying progenitors are unable to form neurospheres upon passage25,36. Thus, while the relationship between SVZ cells and neurosphere-initiating cells requires further refinement18,19,20,21,22,23, the ability of neurospheres to be serially passaged over an extended period may reflect the presence of NSCs in the cultures.
This neurosphere culture system has served as a robust model for investigating the impact of signaling pathways and gene expression in maintaining NSC self-renewal capacity in vitro15,29. One approach to explore these aspects involves transfecting NSCs to either overexpress or knock down specific genes. This can be accomplished through various techniques, including viral and non-viral methods. While viral vectors often achieve high gene transfer efficiency, they have important limitations, such as the high safety requirements and time-consuming vector production26. Conversely, classical transfection methods like lipofection and electroporation achieve very low transfection rates, making them unfeasible for hard-to-transfect cells. The nucleofection technology offers a user-friendly approach by combining cell type-specific nucleofection solutions with unique electrical parameters for each cell type37,38. This ensures DNA transfer directly into the cell nucleus39, allowing the DNA incorporation in a cell cycle-independent manner. Consequently, nucleofection emerges as a viable technique for transfecting difficult-to-transfect cells such as mouse NSCs28,40.
One limitation of this method is that a minimum of 2 x 106&#160;cells is recommended and necessary for each nucleofection, although in optimal conditions, a lower number of cells can be used per single nucleofection (i.e., 5 &#215; 105 cells). Another drawback of the method is that the quality and concentration of DNA used for nucleofection significantly influence gene transfer efficiency. Utilizing endotoxin-free prepared DNA is highly recommended to prevent elevated cell mortality due to endotoxin presence. Additionally, using more than 6 &#181;g of total DNA for nucleofection can substantially reduce both gene transfer efficiency and cell viability. Finally, electric pulse administration is also critical for the survival of nucleofected cells.
In this work, we exemplified the manipulation of Snrpn gene expression by employing a strategy involving the downregulation of its expression using episomal plasmids. These plasmids are not integrated into the genome of the cells, and as NSCs keep proliferating in vitro, the introduced DNA will be subsequently diluted along cell division and, thus, lose the effect of genetic manipulation. Therefore, this strategy is valuable to study the effect of an acute alteration in a short period or to birthdate cells in vitro. Some alternatives to evaluate a more prolonged effect of gene perturbation are available. For example, an integrative system such as the transposon-based piggyBAC could be used. This system consists of introducing the desired coding sequence contained in the plasmid flanked by transposable sequences and co-nucleofect the cells with a plasmid containing the sequence for the enzyme transposase41. Alternatively, transposons or the CRISPR/Cas systems could be used. 
Further development of transfecting technologies will be an important step towards high throughput assays to assess the role of different genes on adult neural stem cell physiology. In combination with increasingly sophisticated purification and expansion methods, these studies will enable the understanding of the in vitro biology of adult NSCs and the comparison of the biological differences between floating neurospheres and NSCs in vivo.

Neural stem cells (NSCs) are multipotent stem cells resident in the brain. These cells possess the ability to self-renew and differentiate into the three neural lineages: astrocytes, oligodendrocytes, and neurons1. Consequently, NSCs play a crucial role in adult neurogenesis in mammals, a process where new neurons are generated in the brain2. NSCs predominantly reside in two regions within the adult brain termed neurogenic niches: the subventricular zone (SVZ) along the walls of the lateral ventricles and the subgranular zone (SGZ) within the dentate gyrus of the hippocampus3,4. NSCs (also known as B cells) in the SVZ, the most active neurogenic niche in the adult mouse, self-renew and produce transit-amplifying progenitors (TAPs or C cells) that subsequently differentiate into neuroblasts (A cells). These neuroblasts migrate through the rostral migratory stream (RMS) to the olfactory bulbs (OB), where they undergo full differentiation into interneurons, integrating into the pre-existing circuitry3,4,5,6.
Understanding the intricate interplay of molecular cues and signals that regulate NSCs within these niches is important to harness their potential for therapeutic applications. For that purpose, various methods have been developed to study this cell population, ranging from selective primary culture of NSCs to cell selection using surface markers7,8,9,10. The present manuscript details the isolation and culturing of SVZ NSCs in vitro using a serum-free selective medium containing both mitogens: basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). This medium facilitates cell proliferation and maintains the stemness of NSCs obtained from the SVZ of adult mouse&#160;brains forming in vitro clonal, three-dimensional, non-adherent aggregates known as neurospheres9. Neurosphere cultures serve as a controlled platform for manipulating and studying the molecular mechanisms and factors affecting NSC proliferation, self-renewal, differentiation, and survival11. Notably, the number of primary neurospheres formed in the culture allows an estimation of the number of NSCs present in the SVZ in vivo, making it a powerful tool for studying the effects of different conditions on the adult NSC pool12,13. Furthermore, once the primary culture is established, NSCs can generate new aggregates (secondary neurospheres) upon passaging through symmetric divisions under proliferation conditions14. Thus, low-density seeding of secondary neurosphere cells (clonal assay) can be utilized to assess the self-renewal rate of these cultures4,15,16,17.
Despite the potential of neurospheres in uncovering the mechanisms governing NSC regulation, some researchers question the validity of in vitro findings, arguing that the artificial conditions in which cells grow might not faithfully replicate the intricate in vivo microenvironment of neurogenic niches18,19,20,21,22. Another controversial point revolves around the observed heterogeneity in neurospheres. Nonetheless, this variability is believed to mirror the symmetric and asymmetric divisions of the NSCs that naturally occur in vivo23,24. Furthermore, recent validation has supported the utilization of NSC cultures to predict mechanisms operating within the SVZ neurogenic niche in vivo, and several studies have demonstrated that NSCs cultured in vitro accurately maintain the transcriptomic profile observed in vivo11,25.
Therefore, neurosphere cultures not only serve as a method to explore NSC proliferation and differentiation abilities, but also offer a system to study the influence of genes governing NSC biology. A pivotal technique for investigating gene function in NSCs is gene expression perturbation. siRNAs or genes delivered through plasmids can be transfected into cell cultures, resulting in knockdown or upregulation of the target gene. This versatile approach significantly reduces the time and cost compared to establishing cultures using conditional knockout mice, presenting a promising avenue for unraveling the genetic bases of neurogenesis and exploring therapeutic prospects. Altering the expression of specific genes in NSCs enables the modulation of their behavior, influencing crucial biological processes such as proliferation, differentiation, and migration. However, the prospect of transfecting NSCs, particularly within mouse neurospheres, presents notable challenges. The three-dimensional structure of neurospheres compromises the efficiency of transfection, often resulting in low rates of successful exogenous nucleic acid delivery, which limits the extent of genetic manipulation26,27. Additionally, transfection procedures can detrimentally affect cell viability and functionality27. In this context, we present the nucleofection system as a method to mitigate cell damage, achieving a high survival rate and ensuring higher efficacy in gene delivery assays used to perturb NSC cultures.
This manuscript aims to illustrate the procedure for isolating, expanding, and nucleofecting NSCs from the adult SVZ neurogenic niche to perturb genes using the neurosphere culture system. This method surpasses the effectiveness of traditional transfection protocols, presenting significantly higher survival rates and improved gene delivery efficiency among the targeted cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m pore-filter bottles (250 mL)</td>
			<td>VWR</td>
			<td>514-0330P</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m pore-filter bottles (500 mL)</td>
			<td>VWR</td>
			<td>514-0332P</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>LabClinics</td>
			<td>PLC30012</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Fisher</td>
			<td>10738771</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>LabClinics</td>
			<td>PLC30024</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>BioWest</td>
			<td>L0180-100</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well plate</td>
			<td>ThermoFisher</td>
			<td>150687</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>LabClinics</td>
			<td>PLC30006</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Labclinics</td>
			<td>PLC30096</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase solution</td>
			<td>Sigma</td>
			<td>A6964-100ML</td>
			<td>Referred as enzymatic solution</td>
		</tr>
		<tr>
			<td>Amaxa Mouse Neural Stem Cell Nucleofector Kit</td>
			<td>Lonza</td>
			<td>VPG-1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Amaxa Nucleofector Iib</td>
			<td>Lonza</td>
			<td>10700807</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic/Antimitotic (A/A)</td>
			<td>Sigma</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-t-Transferrine</td>
			<td>Sigma</td>
			<td>T2252-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Fibroblast Growth Factor (bFGF)</td>
			<td>Sigma</td>
			<td>F0291</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Sigma</td>
			<td>203350</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>B4287-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 40 &#956;m</td>
			<td>LabClinics</td>
			<td>PLC93040</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxynucleotide triphosphate (dNTPs)</td>
			<td>NZYTech</td>
			<td>MB08701</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Saline Buffer (DPBS)</td>
			<td>Gibco</td>
			<td>14080-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) F12 1:1</td>
			<td>Gibco</td>
			<td>11320-074</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli DH5&#945; Competent Cells</td>
			<td>ThermoFisher</td>
			<td>EC0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Earles&#39;s Balanced Salt Solution (EBSS)</td>
			<td>Gibco</td>
			<td>24010-043</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor - Human recombinant (EGF)</td>
			<td>Gibco</td>
			<td>53003-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>E-6511</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11274-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors Sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G-7021</td>
			<td></td>
		</tr>
		<tr>
			<td>GoTaq G2 Flexi DNA Polymerase</td>
			<td>Promega</td>
			<td>M7805</td>
			<td>Kit includes reagents for PCR</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H-3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Insuline</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar (Lennox)</td>
			<td>LabKem</td>
			<td>AGLB-00P-500</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth (Lennox)</td>
			<td>LabKem</td>
			<td>LBBR-00P-500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C-8277</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Blaubrand</td>
			<td>BR718620</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Labbox</td>
			<td>WATR-00A-10K</td>
			<td></td>
		</tr>
		<tr>
			<td>NZYMaxiprep Endotoxin Free Kit</td>
			<td>NZYTech</td>
			<td>MB39901</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain Lyophilized</td>
			<td>Worthington</td>
			<td>LS003119</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P-6149</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P-7505</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10316-14</td>
			<td></td>
		</tr>
		<tr>
			<td>shSCRAMBLE</td>
			<td>Mission (Sigma)</td>
			<td>SHC003</td>
			<td></td>
		</tr>
		<tr>
			<td>shSNRPN</td>
			<td>Mission (Sigma)</td>
			<td>TRCN0000109285</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>Sigma</td>
			<td>S-9133</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Fine Science Tools</td>
			<td>10090-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile PES Syringe Filters (0.22 &#956;m pore-filter)</td>
			<td>Epica</td>
			<td>SFPE-22E-050</td>
			<td></td>
		</tr>
		<tr>
			<td>T12.5 cm2 Flask</td>
			<td>Biofil</td>
			<td>TCF012025</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 cm2 Flask</td>
			<td>LabClinics</td>
			<td>PLC70025</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 cm2 Flask</td>
			<td>LabClinics</td>
			<td>PLC70075</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, expansion, and nucleofection of neural stem cells from adult murine subventricular zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

Author Spotlight: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Our research is focused on studying how the neural stem cell population is regulated in the adult mammalian brain. Understanding the intrinsic and extrinsic molecular mechanisms that regulate neural stem cells within the neurogenic niches is important to comprehend the biology and to develop future potential therapeutic applications. To study neural stem cell behavior, both in vivo and in vitro approaches are widely used.In vitro neural stem cell cultures offer controlled environments and the possibility to easily manipulate and monitor this population of cells. Techniques for gene expression preservation in vitro are a versatile approach to study molecular mechanisms governing cell biology. However, an efficient and reproducible method for overexpressing and knocking down candidate genes in neural stem cells is still challenging in the field.Traditional transfection methods for gene delivery have proven effective in central nervous system cells. Moreover, these methods affect cell viability and functionality. Thus, refinement of alternative approaches is crucial to manipulate gene expression in neural stem cells.With this protocol, we present an improved nucleofection system that achieves high efficiency of gene delivery in neural stem cell cultures from the adult murine subventricular zone, along with survival rates higher than 80%

Optimal culture conditions enable isolation and expansion of adult SVZ-derived NSCs in vitro 
Cultures of NSCs derived from the adult SVZ have served as a valuable in vitro method for investigating the molecular mechanisms and niche signals regulating NSCs within their specific microenvironments. The neurosphere assay outlined in this manuscript was employed to examine the NSC count within the adult SVZ. SVZ tissue was isolated from the brains of 3-month-old mice, dissociated, and cultured in complete NSC medium supplemented with both EGF and bFGF or each separately. After 10 days in vitro (DIV), the total count of primary spheres formed under these three distinct culture conditions was quantified using phase contrast microscopy (Figure 2A,B). Remarkably, our findings demonstrate that the presence of EGF led to maximal primary sphere formation, which is evident from the reduced count of primary spheres observed in cultures with only bFGF (Figure 2A,B). To evaluate the self-renewal capacity of NSCs under varied medium conditions, cells were subcultured and plated at low density (5 cells/&#181;L) in media supplemented with the aforementioned combinations of mitogens. Secondary spheres quantification revealed that SVZ NSCs need at least EGF to efficiently self-renew and that the combination of both EGF and bFGF ameliorates the self-renewing capacity of cells (Figure 2B). Moreover, for a detailed analysis of growth dynamics across diverse media conditions, the number of cells seeded and obtained throughout 7 passages was recorded. Growth curves obtained from different media conditions confirmed that cultures supplemented with EGF alone or in combination with bFGF exhibited improved growth dynamics compared to cultures solely supplemented with bFGF (Figure 2C). Collectively, these findings substantiate that concurrent use of both EGF and bFGF enhances NSC culturing yield in vitro.
In order to investigate the NSC count within the SVZ across different postnatal ages and evaluate the impact of mice aging on neurosphere culture efficiency, SVZ tissue from mice aged 1 month up to 12 months was dissected. Notably, our findings revealed a significant decline in the number of primary spheres with increasing age, showcasing maximum efficiency in sphere count at around 2 to 4 months of age (Figure 2D). Additionally, to evaluate the self-renewal capacity of NSCs during subculturing, NSCs from mice aged 2 to 4 months were subcultured and seeded at clonal density (5 cells/&#181;L) in complete medium. Quantification&#160;of the number of secondary spheres throughout subsequent passages indicated a considerable decrease in culture efficiency over passages (Figure 2E). Based on all these observations, conducting self-renewal assays during early passages is recommended to further optimize NSC culture conditions.
Nucleofection is a highly efficient technique for manipulating gene expression in adult NSCs 
Given that NSCs are not easily transfectable, to manipulate gene expression, here we present a protocol of nucleofection with a higher rate of successful gene delivery without the need to use viral transduction. Following dissection and culturing of individual SVZs from 2 to 4-month-old mice, NSCs were nucleofected with a GFP-carrying plasmid as described (Figure 3A). Detection of GFP-positive cells 2 days post-nucleofection revealed an efficiency ranging from 30% to 50%, consistent with previous literature28. To specifically isolate successfully nucleofected NSCs, cells were sorted by FACS based on GFP fluorescence intensity 3 to 5 days post-nucleofection (Figure 3A-C). Approximately 40% of pre-FACS analyzed cells exhibited high GFP fluorescence levels and were subsequently selected by sorting (Figure 3C). Reseeding of sorted GFP+ NSCs showed that all cells were GFP positive, validating the nucleofection-based cell isolation method (Figure 3D). Notably, nucleofected NSCs maintained viability through subsequent passages, confirming nucleofection feasibility for adult NSCs. These results emphasize the effectiveness of combining nucleofection and FACS to establish a pure culture of modified NSCs.
As an example of gene modulation in adult NSCs, gene attenuation via short hairpin (sh) RNA nucleofection was employed. Specifically, a shRNA targeting the Small nuclear ribonucleoprotein polypeptide N (Snrpn) gene, carrying a CAG-GFP reporter (shSNRPN) was nucleofected in NSC cultures derived from 2-month-old mice. Downregulation of Snrpn was confirmed by qPCR and immunocytochemistry in cells nucleofected with the shSNRPN plasmid but not for the control shSCRAMBLE plasmid (Figure 3E,F)29. To elucidate the effects of Snrpn downregulation on NSC self-renewal capacity, a low-density assay was performed in nucleofected cells (Figure 3F,G). Quantification of secondary neurospheres in nucleofected cultures revealed an increased neurosphere formation capacity upon Snrpn downregulation (Figure 3G). This assay underscores the nucleofection capacity to manipulate gene expression in adult NSCs and, more specifically, identifies the role of Snrpn in maintaining stemness in adult NSCs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66651/66651fig01.jpg" /> 
Figure 1: Detailed description of SVZ dissection. (A) After removing the mouse brain, the entire brain is transferred with DPBS to a silicon pad for dissection. (B) OB and cerebellum are removed from the brain using a scalpel. (C) The brain is divided into two hemispheres to proceed with dissection separately. (D) The brain is opened along the line of the corpus callosum, separating the cortex and striatum from the hippocampus, septum, and diencephalon, thereby exposing the lateral ventricles. (E-F)&#160;The hippocampus, septum, and diencephalon are removed following the ventral limit of the ventricles. (G-I)&#160;The tissue beyond the rostral and caudal ends of the SVZ and the cortex is removed following the corpus callosum. (J-K)&#160;The tissue is tilted so that the SVZ faces sideways, allowing the removal of the striatal tissue beneath the SVZ. (L) A thin piece of tissue containing the SVZ is obtained. Black dashed lines indicate the cutting site. Black dotted lines indicate the location of the SVZ at each step. Abbreviations: OB = olfactory bulb; CB = cerebellum. Scale bar in A-H: 5 mm; in I-L: 3 mm. <a href="https://www.jove.com/files/ftp_upload/66651/66651fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66651/66651fig02.jpg" /> 
Figure 2: Both mitogens EGF and bFGF are necessary for an optimal culture and expansion of NSCs in vitro. (A) Phase-contrast microscopy images of primary and secondary neurosphere cultures in NSC medium supplemented with both EGF and bFGF or each mitogen separately. (B) Number of primary spheres obtained per mouse and per well based on the mitogen composition of the culture medium: no mitogens (gray, n=9), EGF+bFGF (blue, n=33), only EGF (green, n=20), or only bFGF (pink, n=19; left panel). Number of secondary neurospheres formed at low density (5 cells/&#956;L) per well based on the mitogen composition of the culture medium): no mitogens (gray, n=11), bFGF (pink, n=18), EGF (green, n=18) or EGF+bFGF (blue, n=37; right panel). The Kruskal-Wallis test with Dunn&#39;s post hoc analysis was used.&#160;(C) Growth curves showing the total number of cells formed after 8 passages in distinct neurosphere cultures according to the mitogens present in the culture medium: bFGF (pink, n=5), EGF (green, n=5) or EGF+bFGF (blue, n=10). Linear regression analysis was used. (D) The number of primary neurospheres derived from individual mice dissected and disaggregated from animals at various postnatal development stages. Linear regression analysis was used. n indicated in brackets. (E) The number of secondary neurospheres formed at low density (5 cells/&#181;L) through subsequent passages in vitro to assess their self-renewal capacity.&#160;The Kruskal-Wallis test was used, and p values are included: *: p&#60;0.05; **: p&#60;0.01; ***: p&#60;0.001; ****: p&#60;0.0001. n.s: non-significant. Error bars represent the SEM. Scale bar in A is 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66651/66651fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66651/66651fig03.jpg" /> 
Figure 3: Schematic of the nucleofection process and FACS-based cell selection of nucleofected cells. (A) Overview of the nucleofection process and FACS-based cell selection of nucleofected cells. 1) Plasmid DNA solution is combined with the nucleofection solution and added to the cells. This mixture is then transferred to a cuvette for nucleofection. 2) The cuvette containing DNA and cells is inserted in the nucleofector, where an electric shock is applied. For neurosphere cultures isolated from the adult SVZ, we recommend using the A-031 NSC program. 3) Following nucleofection, cells are transferred to a flask containing complete medium supplemented with EGF and bFGF. 4) Cells are sorted by FACS based on the expression of the reporter contained in the plasmid DNA. 5) Sorted cells undergo further culture for subsequent experiments.(B) FACS-based selection strategy and gating for nucleofected NSCs. First, living cells are selected based on high FSC-A and high SSC-A (left graph), and then cell doublets and triplets are eliminated by analyzing FSC-A versus FSC-H parameters (right graph). (C)&#160;FACS gating for nucleofected cells based on their GFP expression (SSC-A versus FITC-A fluorescence). (D) Phase-contrast microscopy and GFP fluorescence images of nucleofected NSCs before (n=3) and after (n=4) FACS selection (left panel). The percentage of nucleofected GFP+ cells is depicted before and after FACS selection (right panel). t-test was used. (E) Quantitative PCR (qPCR) analysis of Snrpn expression in proliferating NSCs nucleofected with Snrpn-specific shRNA (n=4). Nucleofection using a shRNA SCRAMBLE served as the control (n=4). t-test was used. (F) Immunocytochemistry images displaying SNRPN (in red) in NSCs 7 days after the shRNA nucleofection (outer panels). DAPI was utilized to counterstain DNA. Representative images depicting neurospheres derived from NSCs under shSNRPN and shSCRAMBLE conditions (middle panels). (G) Quantification of the number of neurospheres formed in NSC cultures after nucleofection with shSNRPN (n=8) or shSCRAMBLE (n=8). t-test was used. p values are included: *: p&#60;0.05; **: p&#60;0.01; ***: p&#60;0.001; ****: p&#60;0.0001. n.s: non-significant. Error bars represent the SEM. Scale bar in D, 10 &#181;m; in F, 100 &#181;m (high magnification images in F, 7 &#181;m). <a href="https://www.jove.com/files/ftp_upload/66651/66651fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...

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Research

JoVE Journal

Neuroscience

Murine Brain Extraction and Subventricular Zone (SVZ) Dissection for Isolation of Neural Stem Cells

To begin the extraction of the brain from the properly euthanized mouse, after separating the head from the rest of the body, expose the brain by initially cutting the skull along the sagittal suture. With the help of fine tweezers, remove the bones. Be careful not to damage the brain tissue beneath the skull during this step.Using a spatula, carefully extract the brain from the skull. Place it into a 12-well plate containing cold Dulbecco's Phosphate Buffered Saline, or DPBS. Next, transfer the entire brain onto the silicon pad, where the dissection will take place.Use a scalpel to remove the olfactory bulb located at the rostral end of the brain, and the cerebellum situated in the coddle part while retaining the central portion of the brain containing both lateral ventricles. Then divide the brain along the longitudinal fissure into two hemispheres before proceeding with the dissection of both hemispheres separately. Working with one hemisphere, reorient it so that the medial area faces upward.Open the brain along the line of the corpus callosum, separating the cortex and striatum from the hippocampus, septum, and diencephalon, thereby exposing the lateral ventricles. Remove the hippocampus, septum, and encephalon, following the ventral limit of the ventricles. Then remove the tissue beyond the rostral and coddle ends of the subventricular zone or SVZ, and the cortex, following the corpus callosum.Tilt the tissue so that the SVZ faces sideways. Remove the striatal tissue beneath the SVZ to obtain a thin piece of tissue containing the neural stem cells. Store both dissected SVZs from the mouse in a 12-well plate containing cold DPBS till tissue processing.

Isolation and Seeding of Murine Subventricular Zone-Derived Neural Stem Cells (NSCs) for Neurosphere Formation

To isolate neuro stem cells or NSC's from the dissected neuron subventricular zones or SVZ's, cut each SVZ into four to five small pieces to facilitate the disaggregation of the tissue. Using a sterile, plastic pasteur pipette, transfer the chopped SVZ's to a 15 milliliter centrifuge tube while ensuring no fragments are left in the plate. Let the tissue sediment at the bottom of the tube before removing the remaining DPBS.Add 500 microliters of filtered containing enzymatic mix for the two SVZ's per brain, and incubate the tube in a water bath at 37 degrees Celsius for 30 minutes. After incubation, add five milliliters of control medium pre-warned at 37 degrees celsius to each sample. Centrifuge the sample at 300 G for five minutes.Carefully remove the supernatant using a micro pipette or a vacuum pump. Using a fire polished pasture glass pipette, add one milliliter of control medium to the pellet. Mechanically disaggregate the pellet carefully by pipetting up and down 10 to 20 times until a homogeneous cell suspension is obtained.Then, add four milliliters of control medium, and mix by inversion to wash the cells. After centrifuging at 300 G for five minutes, homogenously re-suspend the resulting pellet in one milliliter of complete medium by pipetting up and down 10 times. After diluting one part of an aliquot of the cell suspension with one part of trypan blue, count the cells using a neubauer chamber under the microscope.Ensure cellular disaggregation at the single cell level before seeding the cells equally in eight wells of a 48 well plate in a final volume of 500 microliters of complete medium. Incubate the cells for five to seven days to let the primary neurospheres form.

Expansion of Neurospheres Obtained from Murine Subventricular Zone-Derived Neural Stem Cells

To perform passage of neurospheres obtained from neuro stem cells isolated from neuron subventricular zone, collect the medium containing the neurospheres from the multi-well plates and transfer them to a 15 milliliter centrifuge tube. Centrifuge the neurospheres for five minutes at 300 G.After removing the supernatant, add 200 microliters of enzymatic solution to the pellet and gently tap the bottom of the tube to dislodge it. Incubate for 10 minutes at room temperature to facilitate the neurosphere dissociation.Then add one milliliter of control medium to halt the enzymatic reaction. Mechanically dissociate neurospheres with a P 1000 micro pipette by pipetting up and down 10 to 20 times. Add an additional volume of four milliliters of control medium and mix by inversion to wash the cells.Centrifuge the cells for five minutes at 300 G before removing the supernatant. Add one milliliter of complete medium and resuspend the pellet to homogenize the cellular suspension by pipetting up and down 10 times. After diluting one part of an aliquot of the cell suspension with one part of trypan blue, count the cell suspension using a Neubauer chamber.To expand the culture, seed cells at a density of 10, 000 cells per square centimeter using complete medium in an appropriate culture plate or flask.

Nucleofection of Neurosphere Cultures Obtained from Murine Subventricular Zone-Derived Neural Stem Cells

Using the recommended kit, proceed to prepare 95 microliters of the nucleofection solution in a 1.5 milliliter tube for each desired nucleofection condition. Also prepare a T25 flask filled with four milliliters of pre-warmed complete medium per condition and keep it in the incubator until use. To perform nucleofection, centrifuge the desired number of cells for five minutes at 300 G before removing the supernatant.Add one milliliter of DPBS and re-suspend the pellet homogenously by pipetting up and down 10 times. After repeating the centrifugation, re-suspend the pellet in 95 microliters of nucleofection solution. Combine the 95 microliters of cells with a pre-mixed solution containing five microliters of each plasmid selected for nucleofection.After transferring this solution to a cuvette, place it in the nucleofector device to nucleofect the cells with the plasmids using the optimized neural stem cell program of the nucleofector system. After completing the electroporation, using the Pasteur pipette provided in the kit, carefully introduce warm complete medium into the cuvette. Transfer the contents of the cuvette to the previously prepared T25 flask containing pre-warmed complete medium.Incubate the nucleofected cells in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide for three to five days and visualize the nucleofected neurospheres.

806 Views.  Universitat de València.  Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

Summary