Differentiation and Characterization of Brain-Specific Pericyte-like Cells from Induced Pluripotent Stem Cells (iPSCs): Comparative Protocol Analysis, Transcriptional Profiling, and Metabolic Studies

Pericytes (PCs) emerged as indispensable players in vascular homeostasis, profoundly impacting blood-brain barrier (BBB) formation and maintenance. Their multifaceted roles in vascular stability, permeability regulation, and their role in neuroinflammatory process underscore their significance in preserving the delicate balance required for optimal brain function. Dysfunction in PCs has been associated with various neurovascular disorders and neurodegenerative diseases. In this study, human induced pluripotent stem cells (iPSCs) were cultured and differentiated into brain-specific PC-like cells using two distinct protocols. Morphological changes, immunocytochemistry staining, and qPCR analysis were employed to characterize the phenotype, mRNA and protein expression profile of PC-like cells. Subsequently, to understand the response of differentiated PCs to an inflammatory environment, metabolic studies using Gas Chromatography-Mass Spectrometry (GC-MS) were conducted to elucidate their metabolic profile under inflammatory conditions induced by the bacterial membrane component lipopolysaccharide (LPS). The results revealed similar characteristics for PCs following the different differentiation protocols. One protocol demonstrated superior efficiency in generating brain PC-like cells compared to the other. Immunocytochemistry staining confirmed successful differentiation, while qPCR analysis provided further insights, indicating the presence of key PC markers. Metabolic analysis unveiled dynamic alterations in metabolite levels in response to LPS-induced inflammation, suggesting perturbations in metabolic pathways. This work highlights the importance of considering differentiation protocols and determining the most effective approach. Additionally, it offers valuable insights into the metabolic responses of PCs in the context of neuroinflammation. By elucidating these mechanisms, this study contributes to the common understanding of brain PC culture and lays a foundation for future metabolic studies of PCs under inflammatory conditions.