|Year : 2023 | Volume
| Issue : 2 | Page : 108-118
Proteome architecture of human-induced pluripotent stem cell-derived three-dimensional organoids as a tool for early diagnosis of neuronal disorders
R Negi1, A Srivastava2, AK Srivastava3, Abhishek Pandeya3, P Vatsa1, UA Ansari1, AB Pant1
1 Developmental Toxicology Division, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research, Lucknow; Academy of Scientific and Innovative Research, Ghaziabad, Uttar Pradesh, India
2 Department of Biochemistry, University of Lucknow, Lucknow, India
3 Developmental Toxicology Division, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research, Lucknow, India
|Date of Submission||26-Jan-2023|
|Date of Decision||25-Mar-2023|
|Date of Acceptance||04-May-2023|
|Date of Web Publication||03-Jun-2023|
A B Pant
Developmental Toxicology Division, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research, Lucknow - 226 001, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
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
|How to cite this article:|
Negi R, Srivastava A, Srivastava A K, Pandeya A, Vatsa P, Ansari U A, Pant A B. Proteome architecture of human-induced pluripotent stem cell-derived three-dimensional organoids as a tool for early diagnosis of neuronal disorders. Indian J Pharmacol 2023;55:108-18
|How to cite this URL:|
Negi R, Srivastava A, Srivastava A K, Pandeya A, Vatsa P, Ansari U A, Pant A B. Proteome architecture of human-induced pluripotent stem cell-derived three-dimensional organoids as a tool for early diagnosis of neuronal disorders. Indian J Pharmacol [serial online] 2023 [cited 2023 Sep 22];55:108-18. Available from: https://www.ijp-online.com/text.asp?2023/55/2/108/378032
| » Introduction|| |
Creating disease models from induced pluripotent stem cell (iPSC) lines originating from people with neurodegenerative diseases (NDDs), researchers have gained a more profound knowledge of the genetic variations and cellular phenotypes that characterize the illness state. Neurons generated from iPSCs taken from patients with Alzheimer's or Down syndrome showed characteristic pathologies associated with those diseases, including amyloid β and Tau clumps and neurofibrillary tangles. All of the primary familial Parkinson's disease-linked cytopathies (Lewy bodies' accumulation, mitochondrial dysfunction, and neurites degeneration) were observed in dopaminergic neurons successfully generated from iPSCs with mutations of PD-associated genes (SNCA, PINK1 PARKIN, AND LRRK2). Recent years have also seen the development of cellular models for rare NDDs like amyotrophic lateral sclerosis (ALS).
The three-dimensional (3D) design of organoids derived from iPSCs improves our understanding of the role of various cellular factors/entities in the etiology, development, and diagnosis of NDDs. The change from a two-dimensional to a 3D cellular model system has been validated by discovering proteomic similarities between iPSC-derived 3D neuro-spheroids and postmortem brain tissue obtained from Alzheimer's patients. These 3D models are crucial because they help us learn more about neurodegenerative disorders and pinpoint druggable targets shared amongst different patients and those unique to them in cases where genetics plays a crucial role in disease progression.
The present work was designed to determine whether a 3D organoid model derived from human iPSC (hiPSC) might help investigate ALS, a rare neurodegenerative illness. Researchers studying NDDs such as Parkinson's and Alzheimer's have previously constructed brain organoids using specific cocktails of numerous culture conditions along with well-defined chemicals and growth factors. However, very few 3D organoid investigations have been conducted on ALS. This study attempts to characterize the differences in proteome between organoids derived from TDP-43-mutated hiPSCs and those derived from normal hiPSCs. This research has helped to shed light on the crucial functional biomolecules/mechanisms involved in disease progression in the ALS syndrome caused by mutations in the TDP-43 gene. In addition, this study will identify potent and selective proteome targets for effective draggability for future therapeutic interventions.
| » Materials and Methods|| |
Culture and characterization of human-induced pluripotent stem cells
hiPSCs cell line was procured from Gibco™. The hiPSCs were grown following the protocol of Beers et al. protocol. After every fifth passage, healthy colonies with >80% confluence were characterized for normal morphology, stemness, and potency marker expression. The purified and characterized hiPSCs population was used to differentiate into organoids for all experiments. Cells were characterized for pluripotency markers SOX2 and OCT4.
Generation of mutated human-induced pluripotent stem cells (TDP-43 mutation)
The mutation in hiPSCs was accomplished using CRISPR/Cas-9 technology and predesigned gRNA in accordance with the protocol established in our laboratory.
Human-induced pluripotent stem cell derived organoids
hiPSCs (Normal and TDP-43 mutated) colonies were grown in a Matrigel basement matrix coated 60 mm culture dish, and when they reached 80% confluence, they were parted into small squares on the dish surface with a sterile needle. These colonies were gently lifted and transferred into ultra-low-attachment 6-well plates containing Essential 8 medium supplemented with ROCK inhibitor Y-27632 (day 0) for the first 24 h. For neural induction, dorsomorphin (10 μM) and SB-431542 (10 μM) were added to the DMEM/F12 medium containing 20% KSR, non-essential amino acid (1:100), Glutamax (1:100) for the first 5 days. The floating spheroids were transferred to a serum-free neurobasal medium (NBM) containing B-27, bFGF (20 ng/ml), and EGF (20 ng/ml) till day 24 days. From day 25, bFGF and EGF in the NBM were replaced with BDNF (20 ng/ml) and NT3 (20 ng/ml) to promote the differentiation of neural progenitors into neurons. These similar culture conditions were maintained till day 32.
Characterization of human organoids
Following the similar protocol as mentioned earlier for characterization of hiPSCs, the organoids were characterized for neural progenitor markers (Pax-6 and Nestin), neuronal markers (MAP-2 and Tuj-1), and glial markers (Sβ100).
Sample preparation and digestion
Organoids from normal and TDP-43-mutated hiPSCs were homogenized in liquid chromatography–mass spectrometry (LC-MS) specific protein extraction buffer, and samples were processed further as per the protocol described earlier by our group.
Liquid chromatography-mass spectrometry
Proteomic analyses was carried out using high-resolution MS following the protocol established in our laboratory.
In silico analysis
The Database for Annotation, Visualization, and Integrated Discovery (DAVID) bioinformatics resource and the PANTHER™ (Protein Analysis through Evolutionary Relationships) online analysis tools were used for the bioinformatics analysis. The former bioinformatics tool was used to identify the Reactome Pathways and the KEGG pathways. The latter, the PANTHER™ platform, was used for the gene ontology and pathway analysis of deregulated proteins. The details such as procurement of the materials used in the study, and procedures followed have been provided in the materials and methods section of supplementary document.
| » Results|| |
Culture and characterization of human-induced pluripotent stem cells
The regular culturing and passaging of the hiPSCs revealed that cells cryopreserved up to 10 passages had a revival rate of over 80% with satisfactory morphology and characteristics [Figure 1]a. The immunocytochemistry showed the expression of pluripotency markers OCT-4 and SOX-2 [Figure 1]b. The expression of these markers in the cells revived from cryopreserved vials from various passage numbers has also confirmed the applicability of these cryopreserved cells in further experimentations.
|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|
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Human-induced pluripotent stem cell-derived organoid formation and their characterization
hiPSC-derived organoids were produced using the protocol as mentioned earlier in the material and method section [depicted in [Figure 2]a. The hiPSC colonies differentiated into embryoid bodies (EBs) in approximately 6 days. These EBs then further grew and had a characteristic and defined translucent outer rim of newly generated neuro-ectoderm and, at later stages, had a distinct neuroepithelial layer between days 6 and 25. Post this, at the end of 32 days, we obtained a fully developed organoid under specific culture conditions. The phase contrast images of various developmental stages of hiPSC-derived organoids are represented in [Figure 2]b. The developed organoid' size limitation was due to the unavailability of a defined circulatory system, oxygen, and nutrient diffusion, creating a core with dead cells or fluid. The mature organoid at day 32 manifested neural progenitor markers (PAX6) and Nestin), neuronal markers (MAP2 and Tuj-1), and astrocyte marker (Sβ100) [Figure 2]c.
|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|
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Proteome enrichment of human-induced pluripotent stem cell-derived organoids
We used label-free shotgun proteomics to provide greater protein coverage for normal and mutated TDP-43 organoids, as depicted in [Figure 3]. The analysis of nLC-HRMS data filtered with at least two unique peptides and a 1% false discovery rate (FDR) per protein showed 872 proteins in the normal organoid and 904 proteins in mutated organoids.The list of the proteins according to their arbitrary abundance units of the normal and mutated organoid, are provided in [Supplementary Table 1] and [Supplementary Table 2], respectively. The bioinformatics tool DAVID used to analyze the proteomic data has provided functional annotation and enriched biological themes in GO in terms of significantly enhanced pathways in both experimental groups. The significantly (p < 0.05) identified KEGG pathways are shown in [Figure 4]a and [Figure 4]b of normal and mutated organoids, respectively. The associated detailed list is enclosed as [Supplementary Table 3]. The major enriched pathways of identified proteins from normal and mutated organoids were related to neurodegenerative disorders, proteasomes, and hypoxia-inducible factor 1 (HIF-1) signaling pathways. Reactome pathways analysis by the DAVID platform also reveals the proteins involved in various critical biological processes such as axon guidance, nervous system development pathways, and cellular stress response. The detailed list of the Reactome pathways associated with the identified proteins (p < 0.05) from both normal and mutated organoids are provided in [Supplementary Table 4] and [Supplementary Table 5], respectively. The biological processes dictated by the identified proteins, including those related to metabolic and cellular processes (cytoplasmic translation, DNA replication) and the proteasome catabolic process, etc., in both normal and mutated organoids at day 32 are enlisted in the [Supplementary Table 6] and [Supplementary Table 7], respectively. The identified proteins play a role in structural and cytoplasmic integrity (cytoplasm, cell membrane, cytoskeleton, and nucleoplasm) and cellular connection and communications (extracellular exosomes, axonal outgrowth cone, postsynaptic density, cell-cell adherent junction, and focal adhesion proteins) in normal and mutated organoid are also listed in [Supplementary Table 8] and [Supplementary Table 9].
|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|
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|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|
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Differential proteomic analysis of TDP-43 mutated versus normal organoids
When the global proteomic profiling of both TDP-43 mutated and normal organoids was compared, we found 1208 proteins (with at least two unique peptides and 1% FDR). A list of all differentially regulated proteins is provided in [Table 1]. Comparative analysis of mutated organoid versus normal organoid showed 82 proteins upregulated and 44 proteins downregulated with a significance p < 0.05, presented in the volcano plot [Figure 5]a. Gene ontology of deregulated proteins was performed for pathway enrichment analysis by the PANTHER™ platform. The results of PANTHER™ illustrate that these proteins are involved in clusters such as axon guidance, Alzheimer's disease-presenilin pathway, and Huntington's disease pathway [Figure 5]b.
|Table 1: A list of differentially regulated proteins in human-induced pluripotent stem cell-derived TDP-43-mutated and normal organoids|
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|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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| » Discussion|| |
TDP-43 or Transactive response DNA Binding Protein is a DNA/RNA-binding protein a mutation that causes TDP-43 cytoplasmic inclusions in motor neurons and has been reported in 90% of cases of sporadic ALS. Cerebral organoids have emerged as an unparallel and reliable functional model for studying the rare human neurological disease pathophysiology in-depth and allowing us to explore the paracrine role of cells in the 3D milieu for disease progression. The amalgamation of high throughput proteomic approaches with the 3D organoid-based disease model could be a sheer interphase to predict the critical player protein molecules and associated mechanisms in the etiology of the disease. Therefore, our efforts were directed toward creating a 3D-organoid derived from a TDP-43-mutated hiPSCs to circumvent ethical constraints in accessing neural tissue from ALS patients and to provide it as an alternative for studying hallmark changes in the disease. Our differential proteomic results showed the deregulation of a total of 126 proteins (44 upregulated and 82 downregulated). We have found 39 altered proteins (25 upregulated and 14 downregulated) in the TDP-43-mutated organoids previously reported in two independent human tissue-based proteomic studies [Supplementary Table 10]., Nonetheless, we found several inconsistencies in the expression of a few proteins reported in these two studies. This could be attributed to the fact that we are at the naive stage of our model, with no stratification of the defined brain region. The consistency in our proteomic findings with previously reported reflects the overlapping molecular similarity of our generated diseased model with human neural tissue toward imitating human diseased condition. Furthermore, our comparative data of TDP-43-mutated organoids have revealed significant changes in numerous proteins that regulate a wide range of normal cellular functions, including cellular clearance system (Ubiquitin–Proteasome Pathway and autophagy), cellular translation and transcription machinery, cytoskeletal and axonal integrity, cellular migration and differentiation, mitochondrial, and cellular energetics.
Protein aggregation is common among almost all neurodegenerative disorders, including ALS. Although these protein aggregations manifest in the late stages of diseases, the trigger of their accumulation is primarily devoted to impairment in the cellular clearance mechanism. Impaired ubiquitin-proteasome pathways and autophagic function play a role in clearing aggregated forms of TDP-43 and are linked to ALS. Using a hiPSC-derived in vitro ALS model system, our previous study has also shown that astrocyte-mediated hyperactivated autophagy imparts neurotoxicity and promotes motor neuron degeneration in the case of Cu/ZnSOD1 L29R linked ALS disease. We found an increased expression of the LAMP2 protein, an important regulator of autophagy, in the TDP-43-mutated organoid compared to the normal organoid, which is consistent with our previous findings.
We have also found altered expression of proteins associated with chaperons (AHSA1) ubiquitin-proteasome system (PSMA5, PSMC6, COPS2, and CUL3) and endoplasmic reticulum (ER) proteostasis system (TSG101, MLEC, RAB8A, SRP9, VPS26A, ASNA1, NBAS, and AAGAB). This indicates that TDP-43 mutation might disrupt the protein quality control system in addition to autophagy. This may further promote the accumulation of toxic misfolded proteins inside the cells. It has been reported that depletion of TSG101 (an essential subunit of endosomal sorting complexes required for transports) may cause the improper formation of multivesicular bodies (MVBs) in the cell. This malformation of MVBs showed elevated levels of autophagy but a decrease in autophagic degradation, which could cause the accumulation of TDP-43 aggregates in ALS. Malectin (MLEC) expression is induced on ER stress to prevent the secretion of defective proteins and maintain ER quality control machinery. The decreased expression of MLEC in our proteomic findings could explain the failure of other quality control mechanisms inside the TDP-43-mutated organoids.
Our proteomic findings have demonstrated a broader collection of the proteins regulating neural cell differentiation and migration via cell cycle progression and a mechanistic balance between transcription and translation. The impairment in cellular replicative capacity and basal mechanisms for cell growth propels the cellular system toward senescence and untimely degeneration in TDP-43-induced proteinopathies and accelerates ALS pathological manifestations. TAF15, CDKN1B, CDC42, RPS6, and NELFE are some of the proteins reported to be altered in different models of ALS disease and defined their role in ALS-related pathologies.,, We have found significant variations in the levels of a variety of critical regulatory proteins of the cell cycle (CDKN1B, CDKN2AIP, CDK1, CDC42), DNA replication (CRIP2, H1F0, HDAC1, RECQL, PPIL1), transcriptional regulation (TCF25, TCEAL3, PUF60, NHP2 L1, CSTF2, SRRM1, PPP1R8) and translational control of protein synthesis (TCOF1, TAF15, RPL11, RPS6, EIF3F, NELFE, THUMPD1, YBX3), which suggests that TDP-43 mutation disrupts the standard cellular machinery required for cell proliferation, migration, and differentiation, and this malfunctioning may lead to neuronal degeneration. It was recently reported on how the perturbations in cytoskeletal architecture in ALS conditions affect vital processes essential for defining the wholesome environment inside the cell. The compromises in to-integrity may account for impaired axonal structure and transport, mitochondrial disruption, dysfunctional neuronal growth cones, synapses, and neuronal cargo trafficking in ALS disease. The decreased expression and distribution of microtubule-associated protein MAP2 from the cytoplasm to the membrane in the spinal cord of SOD1G37R mice were reported to be an early event in motor neuron degeneration before the onset of clinical symptoms. Gelsolin (GSN) and alpha-internecine (INA) proteins are indispensable for actin and intermediate filament assembly, and their expression in ALS disease demands a further investigation for their role in ALS pathology., Our proteomic findings encompass the altered levels of these cytostructural proteins, such as CEP170, MAP2, GSN, NCAM1, INA, CACYBP, FLNB, and CORO7, which indicate the damage of neuronal cytostructure in TDP-43-mutated organoids. It has also been shown that the structural protein KIF1A is required for the axonal transport of vesicles carrying synaptic vesicle precursor proteins and neurotrophic factors. It is proposed as the risk-conferring gene for ALS. Another report also suggests the role of CLPTM1 in GABAergic neurotransmission in NDD. The role of GAP43 in maintaining the neurites integrity and synaptic plasticity in degenerating anterior horn presynaptic terminal of neurons in ALS patients is suggested to be an early symptom of the disease. We observed lower expression of structural proteins, which are crucial for axon formation (GAP43, EFNB1, PLXNB2), neurotransmission (CLPTM1, SYAP1), axonal transport (KIF1A), and synaptic integrity (GALNT2) in TDP-43-mutated organoid compared to normal organoid, provides us a clue for their role in TDP-43 mutation-driven synaptic dysfunction in ALS condition. The extracellular matrix (ECM) linked proteins (collagen and integrins) are found to trigger several signaling mechanisms that play a critical role in cell-cell communications, cell proliferation, migration, and synaptic transmission in normal cells. Apart, the ECM proteins (AGRN, FBLN1, VANGL2) are also crucial for the maintenance of motor neurons and neuromuscular junction (NMJ) in ALS patient cells and animal models of neuromuscular degeneration., Our data also represents the differential expression of ECM proteins (COL1A1, COL1A2, FBLN1, FN1, AGRN, ITGA5, FNDC3A, VANGL2) in TDP-43-mutated organoids compared to normal organoids. This provides the candidate proteins that may contribute toward the degeneration of synapse and NMJ induced by TDP-43 mutation. However, the precise role of these proteins warrants further in-depth analysis. The FGFBP proteins play an essential role in the maturation of presynaptic junctions in developing NMJs in association with neuronal agrin (AGRN) protein. The observed downregulation in signaling protein FGFBP3 in the current study suggests that its loss may hamper the binding of other FGF ligands to activate FGF-signaling at developing NMJ for the formation of synaptic clusters.
Because motor neurons are rapid-acting and work in association with the muscular system, they demand a proper energy supply for uninterrupted functioning. The studies on ALS disease have explored the role of mitochondrial dysfunction, inefficient energy production, and altered redox status in the etiology of the disease., A proteomic study has reported that deregulation of mitochondrial and metabolic impairment (AKR1A1, LDHA, ALDOA, PYCR1, ATP5F1C, PCCB) may be the key triggering factors in ALS-related disease phenotype. Similarly, our proteomic study has also observed the deviation in expression of mitochondrial resident proteins (COX1, ECHS1, PYCR1, ATP5F1C, PCCB, GLDC, NDUFS2), and energy-producing glycolysis and gluconeogenesis pathway proteins (SUCLG1, ALDOA, AKR1A1, OXCT1, LDHA, TIGAR GFPT1) in TDP-43-mutated organoids compared to normal organoids. These changes indicate that TDP-43 mutation creates a compromised energy state in cells where mitochondria and other energy mechanisms are not efficiently supplying the chain of ATPs to all the physiological processes and, in turn, drive the neuronal cells into a stressed degenerative condition. The higher levels of these molecular alterations are evident in the microenvironment within and around the cells. In the current study, the changes in protein related to pro-and anti-inflammatory response (PTGR1, CTPS1) and apoptosis (MAGED1) indicate that TDP-43 mutation inside the cells has activated the stress-responsive mechanisms that propel the neuronal system toward cell death.
During the development of the iPSC-derived 3D model of ALS, the major challenge we faced was the limitations in oxygen and nutrient availability at the core of the organoid. Because apical to basal surface diffusion is the only oxygen and nutrient supply mode in our organoid culture conditions, a hypoxic and anoxic region was manifested in the developed organoids. In the bioinformatics analysis of our data, we have obtained a list of proteins involved in HIF-1 pathways [Supplementary Table 11] in the normal and mutated organoids. The abundance of these proteins was found higher in TDP-43-mutated organoids, which could be driven by TDP-43 mutation. In the current study, higher levels of NQO1 observed in TDP-43-mutated organoid is a clear sign of activation of NQO1-mediated HIF-1α expression, which further led to the indication of HIF-1 α mediated transcriptional activation of hypoxia genes such as LDHA at the core of developed organoids.
In summary, the current proteomic assessment of the developed 3D model of ALS derived from TPD-43-mutated hiPSCs showed the majority of hallmark proteins and processes already reported elsewhere, contributing a weighty segment in the origination and progression of the disease. Our study has provided numerous novel candidate proteins as a part of reported biological processes that could produce a new segment of investigation in the diagnosis and therapeutics against ALS. hiPSC-derived 3D model developed in the present study could be a decent replacement for human-diseased neuronal tissue to study the intricacies of disease mechanisms, however a further validation will be required to strengthen the system.
The authors are grateful to Mrs. Deepshikha Srivastava, Technical Officer, Central Instrumentation Facility, CSIR-Indian Institute of Toxicology Research, Lucknow, India, for extending the operational support for LC-MS/MS during proteomics studies.
Financial support and sponsorship
Authors are grateful to the Indian Council of Medical Research (ICMR), New Delhi, India, for providing the financial support through an extramural research grant (Grant Sanction No. 5/4-5/3/9/DHR/Neuro/2021- NCD-1).
Conflicts of interest
There are no conflicts of interest.
| » Supplementary File|| |
Materials and Methods
Reagents and Consumables: Dithiothreitol (DTT) (Cat# 10708984001), Iodoacetamide (Cat# I1149), LiChrosolv®, liquid chromatography–mass spectrometry (LC-MS) grade water, for MS and Protease Inhibitor Cocktail (Cat# P8340) were purchased from Merck. Human-induced pluripotent stem cell (hiPSC) cell line (Episomal hiPSCs, Cat#A18945) was commercially purchased from Gibco™. All medium and reagents including Essential 8 medium (Cat# A1517001), DMEM/F12 medium (Cat# 12500062), Neurobasal™ Medium (Cat# 21103049), N2 supplement (Cat# 17502048), B27 Supplement (Cat# 17504044), StemFlex™ Medium (Cat # A3349401), StemPro Accutase™ (Cat# A1110501), KnockOut™ SR (Cat# 10828010), 4-well chamber slides (Cat # 177399) and D-PBS (Cat# 21600010), GlutaMAX™ (Cat# 35050061), UltraPure Urea™ (Cat# 15505035), Pierce™ Detergent Removal Spin Columns (Cat# 87776), Pierce™ C18 Spin Columns (Cat# 89870), were procured from Thermo Fisher Scientific, USA. Normocin™ (Cat # ant-nr-1) was procured from InvivoGen. Trypsin/Lys-C Mix, LCMS grade (Cat# V5073) was purchased from Promega Corporation. Sodium bicarbonate (Cat# 1681049) and 100X Non-Essential Amino Acid (Cat# 1681049) were procured from MP Biomedicals. Several growth factors like Recombinant Human FGF-basic 154 a.a (Cat# 100-18B), Human BDNF (Cat# 45002), Human NT-3 (Cat #AF-45003), and Human EGF (AF-10015) were procured from PeproTech Inc. ROCK inhibitor-Y-27632 (Cat# 1254), SB431542 (Cat# 1614), and Dorsomorphin (Cat# 3093) were procured from Tocris Bioscience, UK. Corning® Matrigel® Basement Membrane Matrix, LDEV-free (Cat# 354234), Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free, (Cat# 356230), ultra-low attachment 6-well plates (Cat# 3271) were obtained from Corning®. All primary antibodies were procured from Abcam and secondary antibodies were purchased from Thermo Fisher Scientific, USA.
Culture and characterization of human-induced pluripotent stem cell (hiPSC): hiPSCs cell line was procured from Gibco™. The hiPSCs were grown according to the Beers, Gulbranson, et al. protocol.,, In brief, hiPSCs cells were seeded in a 60 mm tissue culture dish coated with Corning® Matrigel® Basement Membrane Matrix, LDEV-free, in pre-warmed Essential 8 medium (E8 medium). After reaching 80% confluence, cells were routinely passaged and cryopreserved in a split ratio of 1:3 to 1:4 and placed in a humidified incubator with 5% CO2 at 37°C. After every fifth passage, healthy colonies with >80% confluence were characterized for normal morphology, stemness, and potency marker expression. The purified and characterized hiPSCs population was used to differentiate into organoids for all experiments. HiPSCs were seeded onto a Matrigel basement matrix coated 4-well chamber slide with E8 medium for immunocytochemistry. After 24 h, cells were fixed using 4% paraformaldehyde (PFA) for 20 min at room temperature (RT), followed by permeabilization with 0.5% Triton X-100 in 1X phosphate buffer saline (PBS) for 10–15 min on a rocker shaker at RT. Following a wash with 1X PBS, cells were kept in a blocking solution containing 0.2% Triton X-100 in 0.1% bovine serum albumin for 1 h at RT. After blocking, cells were incubated overnight at 4°C with primary antibodies for pluripotency markers SOX2 and OCT4 (1:200 dilution). The next day the cells were washed with 1X PBS and incubated with secondary antibodies (1:2500 dilution) labeled with Alexa fluor-488 and -1595 for 2 h at 25°C. After a final wash with 1X PBS, nuclear stain 4, 6-diamidino-2-phenylindole (DAPI) was added to the cells, and images were taken under a confocal fluorescent microscope (Carl Zeiss LSM 880).
Generation of Mutated hiPSCs (TDP-43 mutation): The mutation in hiPSCs was accomplished using CRISPR/Cas-9 technology and predesigned gRNA in accordance with the protocol established in our laboratory. In a nutshell, cells were transfected using an electroporation transfection system (Invitrogen™ Neon™ Transfection System MPK5000) to deliver TDP-43-specific predesigned guide RNA as per the manufacturer's protocol. Following electroporation with the TDP-43 IVTgRNA 5'-ACATCCGATTTAATAGTGTT-3', hiPSCs were plated in Matrigel basement matrix coated 12-well plates in StemFlex medium containing ROCK inhibitor (10 μM) and kept in a humidified incubator at 37°C with 5% CO2. The characterization of cells for transfection efficacy was carried out in accordance with the protocol outlined in our previous studies.
HiPSC-derived Organoids: hiPSCs (Normal and TDP-43 mutated) colonies were grown in a Matrigel basement matrix coated 60 mm culture dish, and when they reached 80% confluence, they were parted into small squares on the dish surface with a sterile needle. These colonies were gently lifted and transferred into ultra-low-attachment 6-well plates containing Essential 8 medium supplemented with ROCK inhibitor Y-27632 (day 0) for the first 24 h. For neural induction, Dorsomorphin (10 μM) and SB-431542 (10 μM) were added to the DMEM/F12 medium containing 20% KSR, non-essential amino acid (1:100), Glutamax (1:100) for the first 5 days. The floating spheroids were transferred to a serum-free neurobasal medium (NBM) containing B-27, bFGF (20 ng/ml), and EGF (20 ng/ml) till day 24 days. From day 25, bFGF and EGF in the NBM were replaced with BDNF (20 ng/ml) and NT3 (20 ng/ml) to promote the differentiation of neural progenitors into neurons. These similar culture conditions were maintained till day 32.
Characterization of human organoids: Following the same steps mentioned for the characterization of hiPSCs, 32-day organoids from ultra-low-attachment 6-well plates were transferred to 4-well chamber slides and fixed with 4% PFA for 20 min at RT. After the blocking process was complete, the organoids were incubated with primary antibodies (1:200 dilutions) for neural progenitor markers (Pax-6 and Nestin), neuronal markers (MAP-2 and Tuj-1), and glial markers (Sβ100) for overnight at 4°C. The organoids were then washed with 1X PBS and incubated with secondary antibodies labelled with Alexa fluor-488 and -595 (1:2500 dilution) for 2 h at RT. After 1X PBS wash, the cells were stained with the 4, 6-diamidino-2-phenylindole (DAPI), and fluorescence microscopy images were captured using a confocal fluorescent microscope (Carl Zeiss LSM 880).
Sample Preparation and Digestion: The normal and mutated hiPSCs were used to form organoids under the specific aforementioned culture conditions. Organoids from normal and TDP-43-mutated hiPSCs were homogenized in LC-MS specific protein extraction buffer (100 mM Tris HCL, 0.15 M NaCl, 1 mM EDTA, 1% NP40, 0.15% sodium deoxycholate, 1 mM PMSF, 0.5 mM DTT, and 10 μl/mL protease inhibitor cocktail). The samples were prepared similarly as mentioned in our previous report. In brief, the quantified protein was subjected to in-solution protein digestion by adding the enzyme Trypsin/Lys-C and incubating overnight at 37°C. Formic acid to a final concentration of 0.5% was added to terminate protein digestion. Following enzyme digestion, the tryptic peptides were purified using Pierce C18 Spin Columns as per the manufacturer's protocol. The vacuum-dried samples were resuspended in 0.1% formic acid for further processing.
LC-MS: Proteomic analyses was carried out using cutting-edge, high-resolution MS. Peptides of both experimental groups were loaded onto reverse phase C 18 column connected with EASY-nLC 1200 system (Thermo Fisher Scientific, MA) that was linked online to a Q-Exactive mass spectrometer with a nano-electrospray ion source. The digested peptides (approximately 800 ng) were subjected to reverse-phase chromatography using two-column systems, the first being a nanoViper Trap Column (Acclaim™ PepMap™ 100 C18 HPLC Column, 75 μm × 2 cm, 3μm, 100 A, Thermo Fisher Scientific) and followed by separation on EASY-Spray™ analytical column (PepMap™ RSLC C18, 7 5μm × 50 cm, 2 μm, 100 A, Thermo Fisher Scientific). For reverse phase chromatography, two different solvents were used: solvent (A) contains 1% v/v acetonitrile and 0.1% v/v formic acid in the water, while solvent (B) contains 80% v/v acetonitrile and 0.1% v/v formic acid in water. Following the elution of the peptides in solvent A, a linear gradient of solvent B was used for 130 min at a flow rate of 0.3 μl/min. The gradient started at 5% B and increased linearly to 40% B in 120 min, then 10 min to 95% B. The electrospray ionization voltage of 2.2 KV was achieved, and the heated capillary temperature for MS was set at 3000C. Technical triplicates were run for each biological replicate.
Information-dependent acquisition (IDA)/data-dependent mode (DDA): The mass spectrometer data were acquired in DDA or IDA with ion mobility separation (high-definition data-independent MS), which significantly improved proteome coverage. In positive ion mode, samples were injected using nano-electrospray ionization through an EASY-Spray™ column equipped with an EASY-Spray™ ionization Source (Thermo Fisher Scientific). For internal calibration, the lock mass channel with (poly-dimethyl cyclosiloxane [PCM] ion [protonated [Si(CH3)2O]; m/z = 445.12003) was used. The scanning mode parameter in MS1 with a scanning range of 350 to 1700 m/z. The automatic gain control (AGC) for the number of ions and the maximum amount of time that the ions accumulated in the C-trap were 1 X 106 ions with a full injection time of 50 ms and a resolution of 70,000. The AGC control dd-MS2 parameter was 5 × 104 ions with a maximum inject time of 100 ms and a resolution of 17,500. To avoid sequencing identical peptides, dynamic exclusion was set to 40 s, and the top 15 most prevalent precursors were used to collect MS data. Total ion chromatogram was used to confirm the peptide sample's identity, and base peak spectra of the entire MS1 were generated. Proteome Discoverer version 2.4 was used to analyze the raw spectrum obtained from Xcalibur™ Software (Thermo Fisher Scientific). The analysis of raw data for protein identification and quantification was carried out using default algorithms and set parameters for ion detection and quantitation, and the false discovery rate was set at 1%. Other variable parameters include up to two missed trypsin cleavages, oxidation (M) as variable modifications, and carbamidomethylation (C) as fixed modifications. The unlabeled/label-free quantitative analysis was based on relative abundance expressed as log 2 values.
In silico analysis: The DAVID bioinformatics resource and the PANTHER™ (Protein Analysis through Evolutionary Relationships) online analysis tools were used for the bioinformatics analysis. The DAVID bioinformatics resource was used to identify the Reactome Pathways and the KEGG pathways. Later on, PANTHER™ platform, was used for the gene ontology and pathway analysis of deregulated proteins.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]