An Approach to Measuring Protein Turnover in Human Induced Pluripotent Stem Cell Organoids by Mass Spectrometry

Abstract

Patient-derived organoids from induced pluripotent stem cells have emerged as a model for studying human diseases beyond conventional two-dimensional (2D) cell culture. Briefly, these three-dimensional organoids are highly complex, capable of self-organizing, recapitulate cellular architecture, and have the potential to model diseases in complex organs, such as the brain. For example, the hallmark of Parkinson's disease - proteostatic dysfunction leading to the selective death of neurons in the substantia nigra - present a subtle distinction in cell type specificity that is simply lost in 2D cell culture models. As such, the development of robust methods to study global proteostasis and protein turnover in organoids will remain a critical need as organoid models evolve. To solve this problem, we have designed a workflow to extract proteins from organoids and measure global protein turnover using mass spectrometry and stable isotope labeling using amino acids in cell culture (SILAC). This allowed us to measure the turnover rates of 844 proteins and compare protein turnover to previously reported data in primary cell cultures and in vivo models. Taken together, this method will facilitate the study of proteostasis in organoid models of human disease and will provide an analytical and statistical framework to measure protein turnover in organoids of all cell types.

Physiological Electric Field: A Potential Construction Regulator of Human Brain Organoids

“…Until now, following this construction principle, brain organoids constructed in the laboratory could be categorized into two groups: (1) brain organoids that have been pre-patterned, which could be cultured by using multiple patterning factors to guide cells that are differentiated into specific neural tissue formations [ 29 , 30 , 31 ]; (2) un-patterned brain organoids that contain spontaneously differentiated neuronal cells. Their formation relies on ESC/PSC’s own morphogenesis; therefore, they encompass a variety of morphological structures with a mixture of features seen in developing brain subareas, such as the forebrain, midbrain, hindbrain, retina, and choroid plexus [ 8 , 12 , 32 , 33 , 34 ].…”