Quantitative analysis of glycans, related genes, and proteins in two human bone marrow stromal cell lines using an integrated strategy

Li, Xiang; Li, Dongliang; Pang, Xingchen; Yang, Ganglong; Deeg, H. Joachim; Guan, Feng

doi:10.1016/j.exphem.2015.04.009

Cited by 8 publications

(6 citation statements)

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“…Based on our in vitro observations, we intravenously co-injected HS5 cells and MDS marrow-derived hematopoietic cells into Nod.cg-Prkdc scid Il2rg tm1wjll (NSG) mice, and observed that differential stromal contact had structural and functional effects on proliferation of MDS precursor cells [40]. Also, we used glycomics (lectin microarray) and proteomics (SILAC method) approaches to compare two bone marrow stromal cell lines, HS5 and HS27a, and observed differential expression of several glycans and proteins [18, 41]. These and many similar findings indicate that microenvironmental factors play key roles in both normal and pathological hematopoiesis, and that hematopoiesis is modulated by bidirectional signals between hematopoietic cells and the microenvironment.…”

Section: Discussionmentioning

confidence: 99%

Screening differentially expressed proteins from co-cultured hematopoietic cells and bone marrow-derived stromal cells by quantitative proteomics (SILAC) method

Wang

et al. 2019

Clin Proteom

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Background Bone marrow stromal cells protect hematopoietic cells and provide drug resistance by delivering bunch of variable proteins. Thus, alterations of protein expression are typically associated with cell–cell signal transduction and regulation of cellular functions. Methods Co-culture models of bone marrow stromal cells and hematopoietic cells are often used in studies of their crosstalk. Studies of altered protein expression initiated by stromal cell/hematopoietic cell interactions are an important new trend in microenvironmental research. There has been no report to date of global quantitative proteomics analysis of crosstalk between hematopoietic cells and stromal cells. In this study, we analyzed quantitative proteomes in a co-culture system of stromal HS5 cells and hematopoietic KG1a cells, and simultaneously tracked differentially expressed proteins in two types of cells before and after co-culture by stable isotope labeling by amino acids in cell culture (SILAC) method. Results We have shown that in co-cultured KG1a, 40 proteins (including CKAP4, LMNA, and SERPINB2) were upregulated and 64 proteins (including CD44, CD99, and NCAM1) were downregulated relative to KG1a alone. We utilized IPA analysis to discover that the NOD-like receptor signaling pathway was upregulated, whereas platelet activation was downregulated in co-cultured KG1a cells. Furthermore, 95 proteins (including LCP1, ARHGAP4, and UNCX) were upregulated and 209 proteins (including CAPG, FLNC, and MAP4) were downregulated in co-cultured HS5 relative to HS5 alone. The tight junction pathway was downregulated and glycolysis/gluconeogenesis pathway was dysfunctional in co-cultured HS5. Most importantly, the significantly differentially expressed proteins can also be confirmed using different co-cultured cell lines. Conclusion Altogether, we recommend such quantitative proteomics approach for the studies of the hematopoietic–stroma cross-talk, differentially expressed proteins and related signaling pathways identification. The differentially expressed proteins identified from this current SILAC method will provide a useful basis for ongoing studies of crosstalk between stromal cells and hematopoietic cells in co-culture systems. All these result suggested our ongoing studies can focus on the mechanisms underlying CKAP4 increase and CD44 decrease in co-cultured hematopoietic cells, and the increase of LCP1 and decrease of CAPG in co-cultured stromal cell. The proteomic profiles from the KG1a/stromal cell co-culture system give new molecular insights into the roles of these cells in MDS pathophysiology and related bone disease. Electronic supplementary material The online version of this article (10.1186/s12014-019-9249-x) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Screening differentially expressed proteins from co-cultured hematopoietic cells and bone marrow-derived stromal cells by quantitative proteomics (SILAC) method

Wang

et al. 2019

Clin Proteom

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“…Similar enrichment of genes of glycan biosynthesis pathway is suggestive of their crucial role in cell adhesion, differentiation, cell survival, and signal transduction processes. 34 Several studies have shown that tolerance (induced, operational, immunologic, or "prope") was accomplished through activation and differentiation of B cell subtypes and regulatory T cells. 11,[35][36][37][38][39][40][41][42] However, these studies were mostly limited to analysis of gene expression profiles in the PBMCs.…”

Section: Discussionmentioning

confidence: 99%

Intragraft Molecular Pathways Associated with Tolerance Induction in Renal Transplantation

Gallon

Mathew

Bontha

et al. 2017

JASN

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The modern immunosuppression regimen has greatly improved short-term allograft outcomes but not long-term allograft survival. Complications associated with immunosuppression, specifically nephrotoxicity and infection risk, significantly affect graft and patient survival. Inducing and understanding pathways underlying clinical tolerance after transplantation are, therefore, necessary. We previously showed full donor chimerism and immunosuppression withdrawal in highly mismatched allograft recipients using a bioengineered stem cell product (FCRx). Here, we evaluated the gene expression and microRNA expression profiles in renal biopsy samples from tolerance-induced FCRx recipients, paired donor organs before implant, and subjects under standard immunosuppression (SIS) without rejection and with acute rejection. Unlike allograft samples showing acute rejection, samples from FCRx recipients did not show upregulation of T cell- and B cell-mediated rejection pathways. Gene expression pathways differed slightly between FCRx samples and the paired preimplantation donor organ samples, but most of the functional gene networks overlapped. Notably, compared with SIS samples, FCRx samples showed upregulation of genes involved in pathways, like B cell receptor signaling. Additionally, prediction analysis showed inhibition of proinflammatory regulators and activation of anti-inflammatory pathways in FCRx samples. Furthermore, integrative analyses (microRNA and gene expression profiling from the same biopsy sample) identified the induction of regulators with demonstrated roles in the downregulation of inflammatory pathways and maintenance of tissue homeostasis in tolerance-induced FCRx samples compared with SIS samples. This pilot study highlights the utility of molecular intragraft evaluation of pathways related to FCRx-induced tolerance and the use of integrative analyses for identifying upstream regulators of the affected downstream molecular pathways.

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“…Human marrow stromal cell lines HS27a and HS5 have been used in studies of MDS as well as various types of cancer. We previously characterized glycan expression levels in these two cell lines using an integrated strategy combining gene microarray, proteomic, and lectin microarray analyses [33]. However, integrated analysis using differential genomic and proteomic techniques has not yet been performed.…”

Section: Discussionmentioning

confidence: 99%

Quantitative proteomic analysis and comparison of two bone marrow stromal cell lines using the SILAC method

Wan

Zhang

et al. 2016

Experimental Hematology

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Two human bone marrow stromal cell lines, HS5 and HS27a, co-cultured with myeloid cells, have frequently been used in studies of cross talk between cells in the bone marrow microenvironment and hematopoietic cells. Altered expression of proteins is typically associated with cell-cell signal transduction and regulation of cellular functions. Many studies have focused on key proteins that contribute to functional differences in cell co-culture models, but global quantitative proteome analysis of HS5 and HS27a has not been performed. We employed the stable isotope labeling by amino acids in cell culture (SILAC) method using two stable isotopes each of arginine and lysine to label proteins in the two cell lines. Labeled proteins were analyzed by 2-D ultrahigh-resolution liquid chromatography- LTQ/Orbitrap mass spectrometry. Among 4,213 unique identified and annotated proteins in the cell lines, 1,462 were detected in two independent experiments. Of these, 69 exhibited significant upregulation and 48 significant downregulation (>95% confidence) in HS27a relative to HS5 cells. Gene ontology term and pathway analysis indicated that the differentially regulated proteins were involved in cellular movement, cell-to-cell signaling and interaction, and hematologic system development and function. A total of 55 items were identified in both genomic and proteomic databases. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed on 7 proteins randomly selected from 28 differentially expressed proteins that were identified in both databases and were involved in the top networks/pathways. We observed a decrease in apoptosis in co-cultured KG1a cells when integrin αV was inhibited in HS27a cells, which suggested the functional role of integrin αV in the co-culture system. The integrated genomic/proteomic approach described here, and the identified proteins, will provide a useful basis for further elucidation of molecular mechanisms in the bone marrow microenvironment and for ongoing studies of cross talk among stromal cells and myeloma cells in co-culture systems.

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Quantitative analysis of glycans, related genes, and proteins in two human bone marrow stromal cell lines using an integrated strategy

Cited by 8 publications

References 54 publications

Screening differentially expressed proteins from co-cultured hematopoietic cells and bone marrow-derived stromal cells by quantitative proteomics (SILAC) method

Screening differentially expressed proteins from co-cultured hematopoietic cells and bone marrow-derived stromal cells by quantitative proteomics (SILAC) method

Intragraft Molecular Pathways Associated with Tolerance Induction in Renal Transplantation

Quantitative proteomic analysis and comparison of two bone marrow stromal cell lines using the SILAC method

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