Dataset Information

Secretome analysis of human bone marrow derived mesenchymal stromal cells

ABSTRACT: As an essential cellular component of the bone marrow (BM) microenvironment mesenchymal stromal cells (MSC) play a pivotal role for the physiological regulation of hematopoiesis, in particular through the secretion of cytokines and chemokines. Mass spectrometry (MS) facilitates the identification and quantification of a large amount of secreted proteins (secretome), but can be hampered by the false-positive identification of contaminating proteins released from dead cells or derived from cell medium. To reduce the likelihood of contaminations we applied an approach combining secretome and proteome analysis to characterize the physiological secretome of BM derived human MSC. Our analysis revealed a secretome consisting of 315 proteins. Pathway analyses of these proteins revealed a high abundance of proteins related to cell growth and/or maintenance, signal transduction and cell communication thereby representing key biological functions of BM derived MSC on protein level. Within the MSC secretome we identified several cytokines and growth factors such as VEGFC, TGF-β1, TGF-β2 and GDF6 which are known to be involved in the physiological regulation of hematopoiesis. By comparing the peptide patterns of secretomes and cell lysates 17 proteins were identified as candidates for proteolytic processing. Taken together, our combined MS work-flow reduced the likelihood of contaminations and enabled us to carve out a specific overview about the composition of the secretome from human BM derived MSC. This methodological approach and the specific secretome signature of BM derived MSC may serve as basis foffuture comparative analyses of the interplay of MSC and HSPC in patients with hematological malignancies.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Mesenchymal Cell, Bone Marrow

SUBMITTER: Falk Baberg

LAB HEAD: Prof. Kai Stühler

PROVIDER: PXD011643 | Pride | 2019-02-12

REPOSITORIES: Pride

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Secretome analysis of human bone marrow derived mesenchymal stromal cells.

Baberg Falk F Geyh Stefanie S Waldera-Lupa Daniel D Stefanski Anja A Zilkens Christoph C Haas Rainer R Schroeder Thomas T Stühler Kai K

Biochimica et biophysica acta. Proteins and proteomics 20190201 4

As an essential cellular component of the bone marrow (BM) microenvironment mesenchymal stromal cells (MSC) play a pivotal role for the physiological regulation of hematopoiesis, in particular through the secretion of cytokines and chemokines. Mass spectrometry (MS) facilitates the identification and quantification of a large amount of secreted proteins (secretome), but can be hampered by the false-positive identification of contaminating proteins released from dead cells or derived from cell me ...[more]

PMID: 30716505

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Project description:We have previously reported that the deficiency of p53 alone or in combination with Rb (Rb-/- p53-/-) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53-/- and Rb-/-p53-/- MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53-/- and Rb-/-p53-/- ASCs. In addition, gene expression profiling revealed a link between p53- or Rb-p53-deficient BM-MSCs and ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem a crucial factor in the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless of the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development. To analyse whether the BM-MSC and Fat-MSC (ASC) differentiation stage may define the sarcoma phenotype, RbloxP/loxPp53loxP/loxP BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage and both Rb and p53 were excised with adenoviral vectors expressing the Cre-recombinase gene (Ad-CMV-Cre) at different stages (day 0 and 10) along osteogenic differentiation. NSG mice were inoculated subcutaneously with 5M-CM-^W10^6 mutant cells. Animals were killed when tumors reached 1 cm3 or 150 days after infusion. Some of the obtained tumors were mechanically disaggregated to establish ex vivo MSC-transformed cell lines. Gene expression analysis was performed using: WT BM-MSCs and ASCs, Rb-/-p53-/- BM-MSCs and ASCs previously differentiated to the osteogenic lineage for 10 days and a tumor cell line derived from p53-/-Rb-/- BM-MSC differentiated to the osteogenic lineage for 10 days.

Project description:We have previously reported that the deficiency of p53 alone or in combination with Rb (Rb-/- p53-/-) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53-/- and Rb-/-p53-/- MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53-/- and Rb-/-p53-/- ASCs. In addition, gene expression profiling revealed a link between p53- or Rb-p53-deficient BM-MSCs and ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem a crucial factor in the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless of the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development. BM-MSC and ASC cultures were established from 3 mouse strains: (a) WT, (b) p53loxP/loxP and (c) p53loxP/loxP RbloxP/loxP. The corresponding mutant cells were generated by excision of the LoxP-flanked sequences by infection of all MSC cultures with adenoviral vectors expressing the Cre-recombinase gene (Ad-CMV-Cre). NSG mice were inoculated subcutaneously with 5×10^6 mutant cells. Animals were killed when tumors reached 1 cm3 or 150 days after infusion. Some of the obtained tumors were mechanically disaggregated to establish ex vivo MSC-transformed cell lines. Gene expression analysis was performed using BM-MSCs and ASCs from all genotypes, as well as tumor cell lines derived from p53-/- and p53-/-Rb-/- BM-MSC and ASC cultures.

Project description:Human SARS-CoV-2 and avian IBV are highly contagious and deadly coronaviruses causing devastating respiratory diseases in human and chicken, respectively. Lacking effective therapies exacerbates the disease associated with SARS-CoV-2 and IBV infections. Thus, novel therapeutic agents are in demand for controlling viral transmission and disease progression. Mesenchymal stem cells secreted factors (secretome) proven to be safe and highly efficient alternative to stem cells in mesenchymal stem cell-based therapy (MSCT). The most favorable advantage of secretome is the bypass of the side effects of MSCT that include but not limited to tumorigenesis, immune rejection, and infection. In this study, we aimed to investigate the antiviral activities of human Wharton’s jelly mesenchymal stem cells secretome against SARS-CoV-2 and IBV infection. The half-maximal inhibitory concentrations (IC50), and cytotoxic concentration (CC50) values of hWJ-MSC secretome (hWJ-MSC-S) were determined in Vero-E6 cells. Virucidal, anti-adsorption, and anti-replication mechanisms of hWJ-MSC-S against SARS-CoV-2 and IBV infection were evaluated in vitro. In Ovo anti-IBV activity of hWJ-MSC-S was estimated using Specific pathogen-free (SPF) embryonated chicken eggs (ECEs). The antiviral efficacy of hWJ-MSC-S was determined in terms of reduction in viral infection. Protein composition of hWJ-MSC-S was analyzed by mass spectrometry analysis, and gene ontology of secretome proteins was evaluated. Our data showed hWJ-MSC-S to significantly inhibits infection of human SARS-CoV-2 and avian IBV with very low cytotoxicity and embryotoxicity profile. CC50 value of hWJ-MSC-S in Vero-E6 was 39200 µg/ml. IC50, SI values of hWJ-MSC secretome against SARS-CoV-2 were 166.6 µg/ml, and 235.29, respectively. Similarly, IC50, SI values of hWJ-MSC-S against IBV were 439.9 µg/ml, and 89.11, respectively. hWJ-MSC-S reduced PFU/ml of SARS-CoV-2 and IBV by > 90%. Antiviral mechanistic studies demonstrated that hWJ-MSC-S had >95% and >90% virucidal effects on SARS-CoV-2 and IBV infections, respectively. The anti-replication effect was approximately 87.33% against SARS-CoV-2 infection, and >90% against IBV infection. In ovo model, hWJ-MSC-S significantly inhibited IBV infection. At a concentration of 1000 µg/ml, hWJ-MSC-S reduced IBV titre from 763000 ± 32638 to 38.333 ± 6.0093 EID50/ml (>99% inhibition). Similarly, hWJ-MSC-S reduced IBV titer from 763000 ± 32638 to 1896 ± 926.1 EID50/ml (>97% inhibition) at a concentration of 500 µg/ml. In addition, 250 µg/ml concentration of hWJ-MSC-S reduce IBV titer from 763000 ± 32638 to 20380 ± 2885 EID50/ml (>96% inhibition). LC/MS-MS analysis revealed that biological processes of hWJ-MSC-S proteins were mostly related to Immunomodulatory effect. Collectively, our results not only uncovered the antiviral potency of hWJ-MSC-S against SARS-CoV-2 and IBV, but also described the mechanism by which hWJ-MSC-S inhibit viral infection. These findings could be utilized in future pre-clinical and clinical studies to develop an effective therapeutic against human COVID-19 and avian IB respiratory diseases using hWJ-MSC-S.

Dataset Information

Secretome analysis of human bone marrow derived mesenchymal stromal cells

Publications

Secretome analysis of human bone marrow derived mesenchymal stromal cells.

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