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 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.
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 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.
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 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 × 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 µg 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.
The authors have nothing to disclose.
This work was supported by grants from Ministerio de Ciencia e Innovación and Agencia Estatal de Investigación (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 Formación de Profesorado Universitario fellowship program (FPU). LLC (PRE2020-094137) and JDM (PREP2022-000680) are funded by the Spanish Formación 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 Innovación.
0.22 μm pore-filter bottles (250 mL) | VWR | 514-0330P | |
0.22 μm pore-filter bottles (500 mL) | VWR | 514-0332P | |
12-well plate | LabClinics | PLC30012 | |
15 mL tube | Fisher | 10738771 | |
24-well plate | LabClinics | PLC30024 | |
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) | BioWest | L0180-100 | |
4′,6-diamidino-2-phenylindole (DAPI) | Sigma | D9542 | |
48-well plate | ThermoFisher | 150687 | |
6-well plate | LabClinics | PLC30006 | |
96-well plate | Labclinics | PLC30096 | |
Accutase solution | Sigma | A6964-100ML | Referred as enzymatic solution |
Amaxa Mouse Neural Stem Cell Nucleofector Kit | Lonza | VPG-1004 | |
Amaxa Nucleofector Iib | Lonza | 10700807 | |
Antibiotic/Antimitotic (A/A) | Sigma | A5955 | |
Apo-t-Transferrine | Sigma | T2252-1G | |
Basic Fibroblast Growth Factor (bFGF) | Sigma | F0291 | |
Blasticidin | Sigma | 203350 | |
Bovine Serum Albumin (BSA) | Sigma | B4287-25MG | |
Cell strainer 40 μm | LabClinics | PLC93040 | |
Deoxynucleotide triphosphate (dNTPs) | NZYTech | MB08701 | |
Dimethyl sulfoxide (DMSO) | Sigma | D8418 | |
Dulbecco’s Phosphate Saline Buffer (DPBS) | Gibco | 14080-055 | |
Dulbecco's Modified Eagle's Medium (DMEM) F12 1:1 | Gibco | 11320-074 | |
E. coli DH5α Competent Cells | ThermoFisher | EC0112 | |
Earles's Balanced Salt Solution (EBSS) | Gibco | 24010-043 | |
Epidermal Growth Factor – Human recombinant (EGF) | Gibco | 53003-018 | |
Ethylenediaminetetraacetic acid (EDTA) | Sigma | E-6511 | |
Fine Forceps | Fine Science Tools | 11274-20 | |
Fine Scissors Sharp | Fine Science Tools | 14060-09 | |
Glucose | Sigma | G-7021 | |
GoTaq G2 Flexi DNA Polymerase | Promega | M7805 | Kit includes reagents for PCR |
Heparin | Sigma | H-3149 | |
Insuline | Sigma | I6634 | |
LB agar (Lennox) | LabKem | AGLB-00P-500 | |
LB broth (Lennox) | LabKem | LBBR-00P-500 | |
L-Cysteine | Sigma | C-8277 | |
L-Glutamine | Gibco | 25030-024 | |
Neubauer chamber | Blaubrand | BR718620 | |
Nuclease free water | Labbox | WATR-00A-10K | |
NZYMaxiprep Endotoxin Free Kit | NZYTech | MB39901 | |
Papain Lyophilized | Worthington | LS003119 | |
Progesterone | Sigma | P-6149 | |
Putrescine | Sigma | P-7505 | |
Scalpel | Fine Science Tools | 10316-14 | |
shSCRAMBLE | Mission (Sigma) | SHC003 | |
shSNRPN | Mission (Sigma) | TRCN0000109285 | |
Sodium Selenite | Sigma | S-9133 | |
Spatula | Fine Science Tools | 10090-17 | |
Sterile PES Syringe Filters (0.22 μm pore-filter) | Epica | SFPE-22E-050 | |
T12.5 cm2 Flask | Biofil | TCF012025 | |
T25 cm2 Flask | LabClinics | PLC70025 | |
T75 cm2 Flask | LabClinics | PLC70075 | |
Tweezers | Fine Science Tools | 91150-20 |
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