Brain pericytes in culture display diverse morphological and functional phenotypes.

Pericytes play several important functions in the neurovascular unit including contractile control of capillaries, maintenance of the BBB, regulation of angiogenesis, and neuroinflammation. There exists a continuum of pericyte subtypes along the vascular tree which exhibit both morphological and transcriptomic differences. While different functions have been associated with the pericyte subtypes in vivo, numerous recent publications have used a primary human brain vascular pericytes (HBVP) cell line where this pericyte heterogeneity has not been considered. Here, we used primary HBVP cultures, high-definition imaging, cell motility tracking, and immunocytochemistry to characterise morphology, protein expression, and contractile behaviour to determine whether heterogeneity of pericytes also exists in cultures. We identified five distinct morphological subtypes that were defined using both qualitative criteria and quantitative shape analysis. The proportion of each subtype present within the culture changed as passage number increased, but pericytes did not change morphological subtype over short time periods. The rate and extent of cellular and membrane motility differed across the subtypes. Immunocytochemistry revealed differential expression of alpha-smooth muscle actin (αSMA) across subtypes. αSMA is essential for cell contractility, and consequently, only subtypes with high αSMA expression contracted in response to physiological vasoconstrictors endothelin-1 (ET1) and noradrenaline (NA). We conclude that there are distinct morphological subtypes in HBVP culture, which display different behaviours. This has significance for the use of HBVP when modelling pericyte physiology in vitro where relevance to in vivo pericyte subtypes along the vascular tree must be considered.