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

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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><tbody><tr><td>0.22 &amp;mu;m pore-filter bottles (250 mL)</td><td>VWR</td><td>514-0330P</td><td></td></tr><tr><td>0.22 &amp;mu;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&amp;prime;,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 &amp;mu;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&amp;rsquo;s Phosphate Saline Buffer (DPBS)</td><td>Gibco</td><td>14080-055</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM) F12 1:1</td><td>Gibco</td><td>11320-074</td><td></td></tr><tr><td>E. coli DH5&amp;alpha; Competent Cells</td><td>ThermoFisher</td><td>EC0112</td><td></td></tr><tr><td>Earles&#039;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 &amp;mu;m pore-filter)</td><td>Epica</td><td>SFPE-22E-050</td><td></td></tr><tr><td>T12.5 cm&lt;sup&gt;2&lt;/sup&gt; Flask</td><td>Biofil</td><td>TCF012025</td><td></td></tr><tr><td>T25 cm&lt;sup&gt;2&lt;/sup&gt; Flask</td><td>LabClinics</td><td>PLC70025</td><td></td></tr><tr><td>T75 cm&lt;sup&gt;2&lt;/sup&gt; 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.

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/66651/66651fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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, 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 ...

isolation-expansion-nucleofection-neural-stem-cells-from-adult-murine

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.2K Views.  Universitat de València. Ons onderzoek is gericht op het bestuderen van hoe de neurale stamcelpopulatie wordt gereguleerd in de hersenen van volwassen zoogdieren. Het begrijpen van de intrinsieke en extrinsieke moleculaire mechanismen die neurale stamcellen binnen de neurogene niches reguleren, is belangrijk om de biologie te begrijpen en om toekomstige potentiële therapeutische toepassingen te ontwikkelen. Om het gedrag van neurale stamcellen te bestuderen, worden zowel in vivo als...

Video: Auteur in de schijnwerpers: Verbeterde nucleofectie voor zeer efficiënte genafgifte in neurale stamcelculturen afkomstig van de subventriculaire zone van muizen

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Developmental Biology

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

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

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 5-8. 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 10-14, 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 neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

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

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Isolation and Culturing of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Once dissected, transfer the mouse brain to cold PBS buffer, and rinse it twice to remove excess blood. Next, place the brain in the brain matrix on ice, and make coronal cuts to obtain one-millimeter-thick slices. Subsequently, transfer the posterior brain slices containing SCZ regions to cold PBS.
Under a dissecting microscope with low magnification, microdissect the SCZ from the white matter area of the cortex and the hippocampus with a bent 30-gauge needle. Then, remove the cortex regions above the SCZ, and place the dissected SCZ into cold PBS in a 35-millimeter Petri dish on ice.
The region of subcallosal zone can be dissected precisely from the whole tissue by using brain matrix and bent 30-gauge needle.
Following that, remove the PBS, and then, immediately chop the dissected tissues into small pieces. Resuspend them with one milliliter of digestion buffer. Afterward, transfer the sample to a 15-milliliter tube containing two milliliters of digestion buffer. Then, incubate it for 30 minutes in a 37 degrees Celsius water bath.
In this step, tap the tube mildly to dissociate the digested tissue, and then centrifuge the tube at 145 times g for five minutes. After five minutes, discard the supernatant, resuspend the digested tissue with one milliliter of pre-warmed and two medium to wash out the digestion buffer, and gently pipette the sample solution a maximum of five times.
Next, centrifuge the sample again at 145 times g for five minutes. After discarding the supernatant, resuspend the cell pellet in one milliliter of N2 medium. Place one milliliter of N2 medium in a non-coated six-well plate, and add one milliliter of the suspended cells to make a final volume of two milliliters.
Next, add 20 nanograms per milliliter EGF and 20 nanograms per milliliter bFGF into each well. Gently shake the six-well culture dish by hand to mix the added growth factors with the plated cells. Then, keep the six-well plate in a 37 degrees Celsius and 5% carbon dioxide incubator.
Add 20 nanograms per milliliter EGF and 20 nanograms per milliliter bFGF to each well every day for eight days. Every third day, add 200 microliters of N2 media to maintain approximately two milliliters of the medium. Here is an image of the neurospheres after eight days.
In this procedure, gather the neurospheres, and transfer them into a new 15-millimeter conical tube. Centrifuge the tube at 145 times g for five minutes. Subsequently, discard the supernatant, and resuspend the neurospheres with 0.5 milliliters of dissociation buffer. Incubate them with 0.5 milliliters of dissociation buffer for five minutes in a 37 degrees Celsius water bath to dissociate the neurospheres into single cells.
Gently pipette the sample solution up and down less than five times, and centrifuge the tube at 145 times g for five minutes. Subsequently, discard the supernatant, and resuspend the neurospheres with one milliliter of N2 medium. After coating a six-well plate with PLL laminin, plate the cells at 2.5 x 105 cells per milliliter with two milliliters of N2 medium per well. Maintain the SCZ adult neural stem cells with a daily treatment of growth factors for five days before passaging them.

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

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