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. 2023 Mar-Apr;55(2):108-118.
doi: 10.4103/ijp.ijp_56_23.

Proteome architecture of human-induced pluripotent stem cell-derived three-dimensional organoids as a tool for early diagnosis of neuronal disorders

R Negi  1 , A Srivastava  2 , A K Srivastava  3 , Abhishek Pandeya  3 , P Vatsa  1 , U A Ansari  1 , A B Pant  1
Affiliations

Proteome architecture of human-induced pluripotent stem cell-derived three-dimensional organoids as a tool for early diagnosis of neuronal disorders

R Negi et al. Indian J Pharmacol. 2023 Mar-Apr.

Abstract

Background and objectives: Induced pluripotent stem cells (iPSCs) derived three-dimensional (3D) model for rare neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) is emerging as a novel alternative to human diseased tissue to explore the disease etiology and potential drug discovery. In the interest of the same, we have generated a TDP-43-mutated human iPSCs (hiPSCs) derived 3D organoid model of ALS disease. The high-resolution mass spectrometry (MS)-based proteomic approach is used to explore the differential mechanism under disease conditions and the suitability of a 3D model to study the disease.

Materials and methods: The hiPSCs cell line was procured from a commercial source, grown, and characterized following standard protocols. The mutation in hiPSCs was accomplished using CRISPR/Cas-9 technology and predesigned gRNA. The two groups of organoids were produced by normal and mutated hiPSCs and subjected to the whole proteomic profiling by high-resolution MS in two biological replicates with three technical replicas of each.

Results: The proteomic analysis of normal and mutated organoids revealed the proteins associated with pathways of neurodegenerative disorders, proteasomes, autophagy, and hypoxia-inducible factor-1 signaling. Differential proteomic analysis revealed that the mutation in TDP-43 gene caused proteomic deregulation, which impaired protein quality mechanisms. Furthermore, this impairment may contribute to the generation of stress conditions that may ultimately lead to the development of ALS pathology.

Conclusion: The developed 3D model represents the majority of candidate proteins and associated biological mechanisms altered in ALS disease. The study also offers novel protein targets that may uncloud the precise disease pathological mechanism and be considered for future diagnostic and therapeutic purposes for various neurodegenerative disorders.

Keywords: Amyotrophic lateral sclerosis; brain organoids; human-induced pluripotent stem cells; neurodegenerative disorders; proteome profiling.

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Conflict of interest statement

None

Figures

Figure 1
Figure 1
(a) Phase contrast images of cultured hiPSCs. (b) Immunofluorescence staining with OCT-4, SOX-2 in hiPSCs cells. Nuclei are stained with DAPI represented as blue color and expression of OCT-4 and SOX-2 represented in green and red color, respectively. The original magnification of images is ×400. hiPSCs = Human-induced pluripotent stem cells
Figure 2
Figure 2
(a) Schematic representation of formation of organoid from hiPSCs. (b) The phase contrast images of various developmental stages of hiPSC derived organoids (c) Immunofluorescence staining of 32-day organoid manifesting neural progenitor markers (PAX6 [green] and Nestin [red]), neuronal markers (MAP2 [red] and Tuj-1 [green]), and astrocyte marker (Sβ100 [green]). Nuclei are stained with DAPI (blue). The original magnification of images is ×400. hiPSCs = Human-induced pluripotent stem cells
Figure 3
Figure 3
Overview of proteomic profiling of normal and mutated (TDP-43) organoid. Experimental proteomics workflow, i.e., from generation to collection of organoids and their processing for proteomic analysis. After sample preparation (extraction and digestion), peptides underwent a reverse phage fractionation and on-line detection using orbitrap high resolution MS/MS acquisition. The acquired peptides were identified, and quantified using the software Protein Discoverer 2.4. This was followed by functional annotation of protein groups by in silico analyses
Figure 4
Figure 4
Heat map showing associated KEGG pathways (P < 0.05) of identified proteins in (a) normal organoid (b) TDP-43-mutated organoid using DAVID bioinformatic tool
Figure 5
Figure 5
(a) Volcano plot of data from label-free quantification of proteins identified with at least two unique peptides (1208 proteins) in TDP-43 mutated versus normal organoid. Red and blue dots represent up- and down-regulated proteins, respectively, with a log2 fold change ±1 and -log10 P > 1.3 (b) Pathway enrichment analyses of up- and down-regulated proteins. The 22 most significantly enriched pathways in the proteomic data of TDP-43 mutated versus normal organoid were identified by PANTHER™. Enriched terms are colored and P-value cut-off was taken <0.05
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