nucleofection of neurosphere cultures obtained from murine subventricular zone-derived neural stem cells

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

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.

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%

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.

nucleofection-neurosphere-cultures-obtained-from-murine

Research

JoVE Journal

Neuroscience

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.

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 ...

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.