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In Vitro Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line.

ABSTRACT: There are very few model systems which demonstrate hematopoiesis in vitro. Previously, we described unique splenic stromal cell lines which support the in vitro development of hematopoietic cells and particularly myeloid cells. Here, the 5G3 spleen stromal cell line has been investigated for capacity to support the differentiation of hematopoietic cells from progenitors in vitro. Initially, 5G3 was shown to express markers of mesenchymal but not endothelial or hematopoietic cells and to resemble perivascular reticular cells in the bone marrow through gene expression. In particular, 5G3 resembles CXCL12-abundant reticular cells or perivascular reticular cells, which are important niche elements for hematopoiesis in the bone marrow. To analyse the hematopoietic support function of 5G3, specific signaling pathway inhibitors were tested for the ability to regulate cell production in vitro in cocultures of stroma overlaid with bone marrow-derived hematopoietic stem/progenitor cells. These studies identified an important role for Wnt and Notch pathways as well as tyrosine kinase receptors like c-KIT and PDGFR. Cell production in stromal cocultures constitutes hematopoiesis, since signaling pathways provided by splenic stroma reflect those which support hematopoiesis in the bone marrow.

SUBMITTER: Lim HK

PROVIDER: S-EPMC6323497 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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In Vitro Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line.

Lim Hong Kiat HK Periasamy Pravin P O'Neill Helen C HC

Stem cells international 20181225

There are very few model systems which demonstrate hematopoiesis in vitro. Previously, we described unique splenic stromal cell lines which support the in vitro development of hematopoietic cells and particularly myeloid cells. Here, the 5G3 spleen stromal cell line has been investigated for capacity to support the differentiation of hematopoietic cells from progenitors in vitro. Initially, 5G3 was shown to express markers of mesenchymal but not endothelial or hematopoietic ...[more]

PMID: 30675170

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Project description:Type 1 diabetes mellitus (T1DM) is caused by the selective destruction of insulin-producing ?-cells. This process is mediated by cells of the immune system through release of nitric oxide, free radicals and pro-inflammatory cytokines, which induce a complex network of intracellular signalling cascades, eventually affecting the expression of genes involved in ?-cell survival.The aim of our study was to investigate possible mechanisms of resistance to cytokine-induced ?-cell death. To this purpose, we created a cytokine-resistant ?-cell line (?-TC3R) by chronically treating the ?-TC3 murine insulinoma cell line with IL-1? + IFN-?. ?-TC3R cells exhibited higher proliferation rate and resistance to cytokine-mediated cell death in comparison to the parental line. Interestingly, they maintained expression of ?-cell specific markers, such as PDX1, NKX6.1, GLUT2 and insulin. The analysis of the secretory function showed that ?-TC3R cells have impaired glucose-induced c-peptide release, which however was only moderately reduced after incubation with KCl and tolbutamide. Gene expression analysis showed that ?-TC3R cells were characterized by downregulation of IL-1? and IFN-? receptors and upregulation of SOCS3, the classical negative regulator of cytokines signaling. Comparative proteomic analysis showed specific upregulation of 35 proteins, mainly involved in cell death, stress response and folding. Among them, SUMO4, a negative feedback regulator in NF-kB and JAK/STAT signaling pathways, resulted hyper-expressed. Silencing of SUMO4 was able to restore sensitivity to cytokine-induced cell death in ?-TC3R cells, suggesting it may play a key role in acquired cytokine resistance by blocking JAK/STAT and NF-kB lethal signaling.In conclusion, our study represents the first extensive proteomic characterization of a murine cytokine-resistant ?-cell line, which might represent a useful tool for studying the mechanisms involved in resistance to cytokine-mediated ?-cell death. This knowledge may be of potential benefit for patients with T1DM. In particular, SUMO4 could be used as a therapeutical target.

Dataset Information

In Vitro Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line.

Publications

<i>In Vitro</i> Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line.

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