Inducing neuronal characteristics of HT22 cells through differentiation with pro-neural factors

Abstract

We are interested in using an easily cultured immortalized neuronal cell line as a replacement for primary cultures. HT22 cells are an SV40 transformed, immortalized mouse hippocampal neuron cell line, that have been used under the assumption that they will function similar to primary culture neurons. However, this assumption may be inaccurate since HT22 cells show low expression of plasmids containing neuron specific promoters. We hypothesized that differentiation of HT22 cells with removal of sera and addition of pro neural factors, would improve their neuronal characteristics. We characterize the neuronal qualities of HT22 cells grown with and without sera and after switching to a neuron supporting media containing either N2 or B27 supplements. These supplements are consistently used in the culturing of primary neurons and the differentiation of immortalized neurons. Additionally, the effects of nerve growth factor (NGF) and retinoic acid (RA) to further induce and support neuronal characteristics is being tested. NGF has been shown to induce differentiation and structural changes in HT22 cells into neuronal cells, and RA has been shown to regulate a late stage neuronal differentiation promoter, CaMKIIα. HT22 cell differentiation is characterized by multiple metrics. Cell cycle progression analyses are performed using propidium iodide (PI) staining and measured using flow cytometry, as differentiated HT22 cells are mitotically arrested and would be prevented from entering S phase. Cell death assays are also performed, using a combination of PI and Annexin V staining to determine if post differentiation HT22 cells die by apoptosis, unlike pre-differentiation HT22 cells. Post differentiation, HT22 cell CaMKIIα expression levels are determined using green fluorescent protein (GFP) as a reporter gene and measured by confocal imaging. We have also characterized the membrane properties and expression of ion channels post-differentiation using electrophysiology. Results indicate differentiation with a combination of N2 supplement, RA, and NGF has the greatest improvement of HT22 cell neuronal characteristics compared to undifferentiated HT22 cells. An increase in apoptosis rates has been observed in HT22 cells differentiated with B27 supplement alone, but rates of apoptosis seem to decrease when differentiated with B27 supplement, NGF, and RA. Using this model, we hope to characterize gene expression from viruses and plasmids containing neuronal promoters. Funding Sources: Murchison Summer Research Fellowship, McNair Scholars Program, Biology Summer Research Fellowship, Trinity University Start-Up Funds, The Brain and Behavior Research Foundation NARSAD Young Investigator Award, and the Mary E. Groff Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.