ELIJAH SCHAFFER (GUEST, BLAZETV HOST): And these people are realizing what I've been noticing all along. The line between BlazeTV, between Fox News and Infowars, is getting blurred in the most healthy way possible
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SH-SY5Y neuroblast-like cells are a subclone of the parental neuroblastoma cell line SK-N-SH. The parental cell line was generated in 1970 from a bone marrow biopsy that contains both neuroblast-like and epithelial-like cells15. SH-SY5Y cells have a stable karyotype consisting of 47 chromosomes, and can be differentiated from a neuroblast-like state into mature human neurons through a variety of different mechanisms including the use of RA, phorbol esters, and specific neurotrophins such as brain-derived neurotrophic factor (BDNF). Prior evidence suggests that the use of different methods can select for specific neuron subtypes such as adrenergic, cholinergic, and dopaminergic neurons16,17. This latter aspect makes SH-SY5Y cells useful for a multitude of neurobiology experiments.
Several studies have noted important differences between SH-SY5Y cells in their undifferentiated and differentiated states. When SH-SY5Y cells are undifferentiated, they rapidly proliferate and appear to be non-polarized, with very few, short processes. They often grow in clumps and express markers indicative of immature neurons18,19. When differentiated, these cells extend long, branched processes, decrease in proliferation, and in some cases polarize2,18. Fully differentiated SH-SY5Y cells have been previously demonstrated to express a variety of different markers of mature neurons including growth-associated protein (GAP-43), neuronal nuclei (NeuN), synaptophysin (SYN), synaptic vesicle protein II (SV2), neuron specific enolase (NSE) and microtubule associated protein (MAP)2,16,17,20, and to lack expression of glial markers such as glial fibrillary acidic protein (GFAP)4. In further support that differentiated SH-SY5Y cells represent a homogeneous neuronal population, removal of BDNF results in cellular apoptosis4. This suggests that survival of differentiated SH-SY5Y cells is dependent on trophic factors, similar to mature neurons.
Use of SH-SY5Y cells has increased since the subclone was established in 19783. Some examples of their use include investigating Parkinson's disease17, Alzheimer's disease21, and the pathogenesis of viral infection including poliovirus22, enterovirus 71 (EV71)23,24, varicella-zoster virus (VZV)1, human cytomegalovirus25, and herpes simplex virus (HSV)2,26. It is important to note that several studies using SH-SY5Y cells have used these cells in their undifferentiated form, especially in the field of neurovirology27-36. The difference in the observed phenotype of undifferentiated versus differentiated SH-SY5Y cells raises the question of whether the observed progression of infection would be different in mature differentiated neurons. For example, differentiated SH-SY5Y cells have a higher efficiency of HSV-1 uptake versus undifferentiated, proliferating SH-SY5Y cells, which may be due to a lack of surface receptors that bind HSV and modulate entry on undifferentiated SH-SY5Y cells2. It is therefore critical that when designing an experiment focused on testing neurons in vitro, SH-SY5Y cells should be differentiated in order to obtain the most accurate results for translation and comparison to in vivo models.
SH-SY5Y cells demonstrate a variety of different phenotypes during the course of differentiation and it is important to be able to identify healthy neurons from those that are stressed. On differentiation day 1, prior to the introduction of Differentiation Media #1, cells have a flat, retracted phenotype with short, stubby processes (Figure 4A). Following 5 days of serum deprivation, SH-SY5Y cells begin to develop longer projections and demonstrate a more neuronal phenotype (Figure 4B). Passaging is a harsh process for SH-SY5Y cells, and on days immediately following passaging, cells appear unhealthy. This is evidenced by cell body clumping and the presence of fewer, shorter processes. (See before images: Figures 4C and 4E, and after images: Figures 4D and 4F). Approximately 48 hr following a split, cells appear to recover, and fully mature, differentiated neurons are obtained at Day 18 (Figure 2B). This is evidenced by a reduction in cell body clumping, and extension of numerous thin, branched neuritic processes that often connect to neighboring cells.
Factors that are essential to obtaining reproducible and viable neuronal cultures include using heat-inactivated FBS, minimizing trypsin incubation time, and gentle trituration. Importantly, this protocol details the use of four different media formulations with various concentrations of hiFBS, thereby creating a gentle transition into a serum-starved state for the cells. When mature SH-SY5Y cells are healthy, they demonstrate numerous projections that connect to surrounding neurons (Figure 5A). It should be noted that a distinctive epithelial phenotype will overtake the neuronal cultures if they are mishandled during the differentiation process (Figure 5B). Additionally, if neurons begin to die, neurites will retract, cell bodies will begin to clump together and round up, and debris from neurite degeneration will start to accumulate at and around cell processes. This process is akin to what is demonstrated in Figures 5C and 5D. While Figure 5C shows a more natural progression of cell death following nutrient and environmental deprivation, Figure 5D shows differentiated SH-SY5Y neurons 4 hr following infection with an HSV-1 strain, KOS, at a multiplicity of infection (MOI) of 106 PFU. This protocol has been thoroughly optimized in the lab and yields reproducible, homogeneous populations of neurons, which are essential for downstream analyses and experimentation.
Figure 1: Timetable of differentiation procedure. The differentiation process consists of 11 steps spread out over the course of an 18 day period. On the first day of the differentiation protocol (day 0), between 25,000 and 100,000 cells are plated onto uncoated 35 mm dishes. On days 1, 3, and 5, old media is removed and Differentiation Media #1 is applied. On day 7, cells are split 1:1 onto uncoated 35mm dishes in Differentiation Media #1. On day 8, the media is changed to Differentiation Media #2, and on day 10, cells are again split 1:1, but this time onto ECM-coated 35 mm dishes in Differentiation Media #2. On days 11, 14, and 17, old media is removed and Differentiation Media #3 is applied. On day 18, differentiated neurons are ready to use for downstream applications.
Figure 2: Morphological appearance of undifferentiated and differentiated SH-SY5Y cells. (A) Undifferentiated SH-SY5Y cells have a flat phenotype with few projections while (B) differentiated SH-SY5Y neurons demonstrate extensive and elongated neuritic projections. Images were obtained in phase at 20X magnification using an inverted epifluorescence microscope.
Figure 3: Markers of neuronal differentiation. Immunofluorescence illuminates neuronal features of fully differentiated SH-SY5Y cells. (A) Anti-SMI31 (green) stains the phosphorylated neurofilament H in the extensive network of neurites. (B) Anti-MAP2 (red) labels microtubule-associated protein 2, revealing the neuronal soma and proximal portion of neurites. Corresponding phase image for each immunofluorescence panel is shown to the right. Images were obtained at 10X magnification using an inverted epifluorescence microscope. Scale bar, 100 μm.
Figure 4: Intermediate steps of the differentiation protocol. (A) Day 1 of differentiation. Cells maintain a retracted phenotype with short projections. (B) Day 5 of differentiation. Cells have been exposed to 5 days of serum deprivation (Differentiation Media #1). The surviving cells begin to elongate and form longer processes that connect with neighboring cells. (C) Day 7 of differentiation prior to split. Cells demonstrate high numbers of long processes, with fewer numbers of cells demonstrating an epithelial-like phenotype. (D) Day 8 of differentiation, one day after the first passage. Following splitting, cell bodies form clumps and processes appear short as a result of the passaging procedure. (E) Day 10 of differentiation, prior to split onto ECM-coated plates. Cells demonstrate longer processes that make connections with nearby cells. Cell body clustering is also evident. (F) Day 11 of differentiation, one day following second split. Cells are stressed following the second passage and many neurons are ultimately lost. However, the remaining population is viable, homogenous, and neuronal in phenotype. Cell bodies produce larger clusters and processes begin to emit from the base of the clusters.
Figure 5: Epithelial overgrowth and neuronal death - two alternative outcomes of the differentiation process. (A) Healthy, mature SH-SY5Y neurons demonstrate diffuse axonal projections connecting to neighboring cells. (B) In some instances, cells with a more epithelial-like phenotype overtake maintenance cultures. This over-population of epithelial-like cells may be due to infrequent passaging of maintenance cultures. These cultures should be discarded, as the epithelial-like cells will continue to outnumber neurons. Arrows denote epithelial-like cells. (C) When SH-SY5Y cells are unhealthy and begin to die, cell bodies round up and processes degrade, generating a significant amount of debris. (D) Noticeable accumulation of cellular debris and retraction of neuronal processes is evident in mature, differentiated neurons, 4 hr following infection with a strain of herpes simplex virus 1 (HSV-1) KOS at a multiplicity of infection (MOI) of 106 PFU. 59ce067264