Many groups use newborn mouse hippocampal cell preparations to analyse differentiation and signaling processes. These cultures are usually kept cultivated for 2 to 4 weeks in vitro before use. Do cells prepared like that match in vivo cell types in form differentiation state and function? What about the influence of hormones produced in vivo but not provided in vitro? Processes that change daily may also be different.
Here is another question. Does the 'immortalized HT22 cell line work well? I think a coculture with mouse astrocytes would better approximate in vivo conditions.
We have characterized the in vitro hippocampal cells differentiation in terms of numbers of dendritic processes number, order of dendritic branches (primary, secondary, tertiary) and expression of several markers of neuronal identity. In rats, we consider a 3DIV (days of in vitro differentiation) culture similar to a hippocampus of an 18-days embryo. In contrast, a culture of >21DIV resembles the neuronal anatomy of an adult individual hippocampal neuron. We have seen that some proteins that are not expressed in adult neurons (e.g. Ezh2) are also not expressed in 21DIV neurons. It could be a good approach of couse understanding the obvious differences.
neuron differentiation in vitro is not the same as in vivo, but still is a good model to study some properties and events. As you noted for hormones, and other signaling gating molecules (e.g. chemotactic cues, trophic factors); in vitro development also lacks cell-cell and cell-ECM contacts present in the intact tissue which have been proven to affect neuron morphogenesis. Despite these differences, cultured neurons in vitro develop normally (they reach mature stage 5) and have been shown to be functionally normal (synapse formation, electrophysiological records, etc). Main overall differences are that neurogenesis and neuron migration present in vivo are largely absent in vitro. However, under both models one can see neuron differentiation events.
To Terence: HT22 are no good model for hippocampal neurons and it doesn`t get better with astrocytes present. We did co-cultures of HT22 and mouse astrocytes and saw NeuN-positive cells (HT22 were never NeuN-positive in our hands), but we saw them in mouse astrocyte control cultures in the same number. We conclude the NeuN-positive cells are a "contamination" of astrocyte culture with primary neurons...
Erik, maybe HT22 cells are not a great fit, but coculture of astrocytes and various staged primary culture could bring about a more in vivo phenotype. For example astrocytes are known to secrete apoE, that can be neuroprotective.
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