02501nas a2200253   4500000000100000000000100001008004100002260001500043653001400058653002400072653001400096653001800110653001600128653001600144653002100160653002300181100002000204700002400224245013200248856010400380490000700484520174200491022001402233       2024                            d  c2024-05-0910aMicroglia10aSpecies differences10aastrocyte10aheterogeneity10ahuman brain10amouse brain10aoligodendrocytes10aprotein processing1 aTyler J. Wenzel1 aDarrell D. Mousseau00aBrain organoids engineered to give rise to glia and neural networks after 90 days in culture exhibit human-specific proteoforms  uhttps://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2024.1383688/full0 v183 a<p>Human brain organoids are emerging as translationally relevant models for the study of human brain health and disease. However, it remains to be shown whether human-specific protein processing is conserved in human brain organoids. Herein, we demonstrate that cell fate and composition of unguided brain organoids are dictated by culture conditions during embryoid body formation, and that culture conditions at this stage can be optimized to result in the presence of glia-associated proteins and neural network activity as early as three-months <italic>in vitro.</italic> Under these optimized conditions, unguided brain organoids generated from induced pluripotent stem cells (iPSCs) derived from male–female siblings are similar in growth rate, size, and total protein content, and exhibit minimal batch-to-batch variability in cell composition and metabolism. A comparison of neuronal, microglial, and macroglial (astrocyte and oligodendrocyte) markers reveals that profiles in these brain organoids are more similar to autopsied human cortical and cerebellar profiles than to those in mouse cortical samples, providing the first demonstration that human-specific protein processing is largely conserved in unguided brain organoids. Thus, our organoid protocol provides four major cell types that appear to process proteins in a manner very similar to the human brain, and they do so in half the time required by other protocols. This unique copy of the human brain and basic characteristics lay the foundation for future studies aiming to investigate human brain-specific protein patterning (e.g., isoforms, splice variants) as well as modulate glial and neuronal processes in an <italic>in situ</italic>-like environment.</p>  a1662-5102