Qualitative and Quantitative Comparison of the Proteome of Erythroid Cells Differentiated from Human iPSCs and Adult Erythroid Cells by Multiplex TMT Labelling and NanoLC-MS/MS

Trakarnsanga, Kongtana; Wilson, Marieangela C.; Griffiths, Rebecca E.; Toye, Ashley M.; Carpenter, Lee; Heesom, Kate J; Parsons, Stephen H.; Anstee, David J.; Frayne, Jan

doi:10.1371/journal.pone.0100874

Cited by 53 publications

(67 citation statements)

References 40 publications

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“…Similar variation between lines has been previously reported for RPE 16,21,45 and other cell types derived from iPSC. [45][46][47][48][49] The SCAC method secreted higher levels of PEDF in iPSC-3-RPE compared to iPSC-1-RPE. The directed method showed superior mature RPE transcript expression and PEDF secretion in iPSC-3-RPE.…”

Section: Discussionmentioning

confidence: 95%

Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods

Leach

Croze

et al. 2016

Journal of Ocular Pharmacology and Therapeutics

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Purpose: The application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic, with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells, but previous studies have shown variability in iPSC propensity to differentiate into RPE. Methods: This study provides a comparison between 2 different methods for RPE differentiation: (1) a commonly used spontaneous continuously adherent culture (SCAC) protocol and (2) a more rapid, directed differentiation using growth factors. Integration-free iPSC lines were differentiated to RPE, which were characterized with respect to global gene expression, expression of RPE markers, and cellular function. Results: We found that all 5 iPSC lines (iPSC-1, iPSC-2, iPSC-3, iPSC-4, and iPSC-12) generated RPE using the directed differentiation protocol; however, 2 of the 5 iPSC lines (iPSC-4 and iPSC-12) did not yield RPE using the SCAC method. Both methods can yield bona fide RPE that expresses signature RPE genes and carry out RPE functions, and are similar, but not identical to fetal RPE. No differences between methods were detected in transcript levels, protein localization, or functional analyses between iPSC-1-RPE, iPSC-2-RPE, and iPSC-3-RPE. Directed iPSC-3-RPE showed enhanced transcript levels of RPE65 compared to directed iPSC-2-RPE and increased BEST1 expression and pigment epithelium-derived factor (PEDF) secretion compared to directed iPSC-1-RPE. In addition, SCAC iPSC-3-RPE secreted more PEDF than SCAC iPSC-1-RPE. Conclusions: The directed protocol is a more reliable method for differentiating RPE from various pluripotent sources and some iPSC lines are more amenable to RPE differentiation.

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Section: Discussionmentioning

confidence: 95%

Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods

Leach

Croze

et al. 2016

Journal of Ocular Pharmacology and Therapeutics

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“…[18][19][20] In addition, where sufficient sample was available, individual patient samples were analyzed alongside the pooled control samples ( Figure 1B). Aliquots of 100 mg of 10 samples per experiment were digested with trypsin (2.5 mg trypsin per 100 mg of protein; 37°C, overnight), labeled with TMT reagents according to the manufacturer's protocol (Thermo Fisher Scientific, Loughborough, United Kingdom) and the labeled samples were pooled.…”

Section: Proteomic Analysis: Tmt Labeling and High Ph Reversed-phase mentioning

confidence: 99%

Quantitative proteomics of plasma vesicles identify novel biomarkers for hemoglobin E/β-thalassemic patients

et al. 2018

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Key Points• Chaperones, antioxidants, ironsequestering proteins, and cathepsin S exhibited increased abundance in thalassemic EVs.• Haptoglobin and hemopexin are reduced in thalassemic patients' EVs, reflecting hemolysis. These could be used as clinical biomarkers.Hemoglobin E (HbE)/b-thalassemia has a wide spectrum of clinical manifestations that cannot be explained purely by its genetic background. Circulating extracellular vesicles (EVs) are one factor that likely contributes to disease severity. This study has explored the differences in protein composition and quantity between EVs from HbE/b-thalassemic patients and healthy individuals. We used tandem mass tag labeling mass spectrometry to analyze the EV proteins isolated from the plasma of 15 patients compared with the controls.To reduce biological variation between individuals, the EV proteins isolated from randomly assigned groups of 5 HbE/b-thalassemic patients were pooled and compared with 5 pooled age-and sex-matched controls in 3 separate experiments. Alpha hemoglobin-stabilizing protein had the highest fold increase. Catalase, superoxide dismutase, T-complex proteins, heat shock proteins, transferrin receptor, ferritin, and cathepsin S were also upregulated in thalassemic circulating EVs. Importantly, haptoglobin and hemopexin were consistently reduced in patients' EVs across all data sets, in keeping with the existing hemolysis that occurs in thalassemia. The proteomic data analysis of EV samples isolated from 6 individual HbE/b-thalassemic patients and western blotting results corroborated these findings. In conclusion, we have successfully identified consistent alterations of protein quantity between EVs from HbE/b-thalassemic and healthy individuals. This work highlights haptoglobin, hemopexin, and cathepsin S as potential clinically relevant biomarkers for levels of hemolysis and inflammation. Monitoring of these plasma proteins could help in the clinical management of thalassemia.

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“…Protein ratios represent the median of the raw measured peptide ratios for each protein. These methods are described in Trakarnsanga et al (5).The mass spectrometry (MS) proteomics data have been deposited to the ProteomeXchange Consortium (17) via the PRIDE partner repository with the dataset identifier PXD003276.…”

Section: Isolation Of Adult and Cord Blood Rbcs And Cd34mentioning

confidence: 99%

Comparison of the Proteome of Adult and Cord Erythroid Cells, and Changes in the Proteome Following Reticulocyte Maturation

Wilson

Trakarnsanga

Heesom

et al. 2016

Molecular & Cellular Proteomics

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105

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Cord blood stem cells are an attractive starting source for the production of red blood cells in vitro for therapy because of additional expansion potential compared with adult peripheral blood progenitors and cord blood banks usually being more representative of national populations than blood donors. Consequently, it is important to establish how similar cord RBCs are to adult cells. In this study, we used multiplex tandem mass tag labeling combined with nano-LC-MS/MS to compare the proteome of adult and cord RBCs and reticulocytes. 2838 unique proteins were identified, providing the most comprehensive compendium of RBC proteins to date. Using stringent criteria, 1674 proteins were quantified, and only a small number differed in amount between adult and cord RBC. We focused on proteins critical for RBC function. Of these, only the expected differences in globin subunits, along with higher levels of carbonic anhydrase 1 and 2 and aquaporin-1 in adult RBCs would be expected to have a phenotypic effect since they are associated with the differences in gaseous exchange between adults and neonates. Since the RBC and reticulocyte samples used were autologous, we catalogue the change in proteome following reticulocyte maturation. The majority of proteins (>60% of the 1671 quantified) reduced in abundance between 2-and 100-fold following maturation. However, ϳ5% were at a higher level in RBCs, localized almost exclusively to cell membranes, in keeping with the known clearance of intracellular recycling pools during reticulocyte maturation. Overall, these data suggest that, with respect to the proteome, there is no barrier to the use of cord progenitors for the in vitro generation of RBCs for transfusion to adults other than the expression of fetal, not adult, hemoglobin. Molecular & Cellular Proteomics

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Qualitative and Quantitative Comparison of the Proteome of Erythroid Cells Differentiated from Human iPSCs and Adult Erythroid Cells by Multiplex TMT Labelling and NanoLC-MS/MS

Cited by 53 publications

References 40 publications

Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods

Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods

Quantitative proteomics of plasma vesicles identify novel biomarkers for hemoglobin E/β-thalassemic patients

Comparison of the Proteome of Adult and Cord Erythroid Cells, and Changes in the Proteome Following Reticulocyte Maturation

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