Biology subjects

Mohamed, N.-V.

Publications and source records attributed to Mohamed, N.-V..

3 recordsLinked to original sources

bioRxiv2021

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

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 Parkinsons 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.

neuroscience↗

bioRxiv2021

Microfabricated disk technology: rapid scale up in midbrain organoid generation

By providing a three-dimensional in vitro culture system with key features of the substantia nigra region in the brain, 3D neuronal organoids derived from human induced pluripotent stem cells (iPSCs) provide living neuronal tissue resembling the midbrain region of the brain. However, a major limitation of conventional brain organoid culture is that it is often labor-intensive, requiring highly specialized personnel for moderate throughput. Additionally, the methods published for long-term cultures require time-consuming maintenance to generate brain organoids in large numbers. With the increasing need for human midbrain organoids (hMOs) to better understand and model Parkinsons disease (PD) in a dish, there is a need to implement new workflows and methods to both generate and maintain hMOs, while minimizing batch to batch variation. In this study, we developed a method with microfabricated disks to scale up the generation of hMOs. This opens up the possibility to generate larger numbers of hMOs, in a manner that minimizes the amount of labor required, while decreasing variability and maintaining the viability of these hMOs over time. Taken together, producing hMOs in this manner opens up the potential for these to be used to further PD studies.

neuroscience↗

bioRxiv2021

Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy

SNCA, the first gene associated with Parkinsons disease, encodes the -synuclein (-syn) protein, the predominant component within pathological inclusions termed Lewy bodies (LBs). The presence of LBs is one of the classical hallmarks found in the brain of patients with Parkinsons disease, and LBs have also been observed in patients with other synucleinopathies. However, the study of -syn pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use 3D midbrain organoids (hMOs), differentiated from human induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These hMOs recapitulate key features of -syn pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication hMOs express elevated levels of -syn and exhibit an age-dependent increase in -syn aggregation, manifested by the presence of both oligomeric and phosphorylated forms of -syn. These phosphorylated -syn aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, hMOs from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinsons disease and provide a powerful system to study the pathogenesis of synucleinopathies.

neuroscience↗