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PRDX6 Promotes the Differentiation of Human Mesenchymal Stem (Stromal) Cells to Insulin-Producing Cells.

ABSTRACT: Mesenchymal stem cells (MSCs) can be differentiated in vitro to form insulin-producing cells (IPCs). However, the proportion of induced cells is modest. Extracts from injured pancreata of rodents promoted this differentiation, and three upregulated proteins were identified in these extracts. The aim of this study was to evaluate the potential benefits of adding these proteins to the differentiation medium alone or in combination. Our results indicate that the proportion of IPCs among the protein(s)-supplemented samples was significantly higher than that in the samples with no added proteins. The yield from samples supplemented with PRDX6 alone was 4-fold higher than that from samples without added protein. These findings were also supported by the results of fluorophotometry. Gene expression profiles revealed higher levels among protein-supplemented samples. Significantly higher levels of GGT, SST, Glut-2, and MafB expression were noted among PRDX6-treated samples. There was a stepwise increase in the release of insulin and c-peptide, as a function of increasing glucose concentrations, indicating that the differentiated cells were glucose sensitive and insulin responsive. PRDX6 exerts its beneficial effects as a result of its biological antioxidant properties. Considering its ease of use as a single protein, PRDX6 is now routinely used in our differentiation protocols.

SUBMITTER: Gabr MM

PROVIDER: S-EPMC6995490 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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PRDX6 Promotes the Differentiation of Human Mesenchymal Stem (Stromal) Cells to Insulin-Producing Cells.

Gabr Mahmoud M MM Zakaria Mahmoud M MM Refaie Ayman F AF Khater Sherry M SM Ashamallah Sylvia A SA Rashed Sahar A SA Fouad Ali M AM Ismail Amani M AM Ghoneim Mohamed A MA

BioMed research international 20200121

Mesenchymal stem cells (MSCs) can be differentiated in vitro to form insulin-producing cells (IPCs). However, the proportion of induced cells is modest. Extracts from injured pancreata of rodents promoted this differentiation, and three upregulated proteins were identified in these extracts. The aim of this study was to evaluate the potential benefits of adding these proteins to the differentiation medium alone or in combination. Our results indicate that the proportion of IPCs among the protein ...[more]

PMID: 32051828

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Project description:Mesenchymal stromal cells (MSCs) are an attractive option for cell therapy for type 1 diabetes mellitus (DM). These cells can be obtained from many sources, but bone marrow and adipose tissue are the most studied. MSCs have distinct advantages since they are nonteratogenic, nonimmunogenic and have immunomodulatory functions. Insulin-producing cells (IPCs) can be generated from MSCs by gene transfection, gene editing or directed differentiation. For directed differentiation, MSCs are usually cultured in a glucose-rich medium with various growth and activation factors. The resulting IPCs can control chemically-induced diabetes in immune-deficient mice. These findings are comparable to those obtained from pluripotent cells. PD-L1 and PD-L2 expression by MSCs is upregulated under inflammatory conditions. Immunomodulation occurs due to the interaction between these ligands and PD-1 receptors on T lymphocytes. If this function is maintained after differentiation, life-long immunosuppression or encapsulation could be avoided. In the clinical setting, two sites can be used for transplantation of IPCs: the subcutaneous tissue and the omentum. A 2-stage procedure is required for the former and a laparoscopic procedure for the latter. For either site, cells should be transplanted within a scaffold, preferably one from fibrin. Several questions remain unanswered. Will the transplanted cells be affected by the antibodies involved in the pathogenesis of type 1 DM? What is the functional longevity of these cells following their transplantation? These issues have to be addressed before clinical translation is attempted. Graphical Abstract Bone marrow MSCs are isolated from the long bone of SD rats. Then they are expanded and through directed differentiation insulin-producing cells are formed. The differentiated cells are loaded onto a collagen scaffold. If one-stage transplantation is planned, a drug delivery system must be incorporated to ensure immediate oxygenation, promote vascularization and provide some growth factors. Some mechanisms involved in the immunomodulatory function of MSCs. These are implemented either by cell to cell contact or by the release of soluble factors. Collectively, these pathways results in an increase in T-regulatory cells.

Project description:BackgroundThe purpose of this study was to investigate allogenic immune responses following the transplantation of insulin-producing cells (IPCs) differentiated from human adipose tissue-derived stem cells (hAT-MSCs) into humanized mice.MethodshAT-MSCs were isolated from liposuction aspirates obtained from HLA-A2-negative healthy donors. These cells were expanded and differentiated into IPCs. HLA-A2-positive humanized mice (NOG-EXL) were divided into 4 groups: diabetic mice transplanted with IPCs, diabetic but nontransplanted mice, nondiabetic mice transplanted with IPCs and normal untreated mice. Three million differentiated cells were transplanted under the renal capsule. Animals were followed-up to determine their weight, glucose levels (2-h postprandial), and human and mouse insulin levels. The mice were euthanized 6-8 weeks posttransplant. The kidneys were explanted for immunohistochemical studies. Blood, spleen and bone marrow samples were obtained to determine the proportion of immune cell subsets (CD4+, CD8+, CD16+, CD19+ and CD69+), and the expression levels of HLA-ABC and HLA-DR.ResultsFollowing STZ induction, blood glucose levels increased sharply and were then normalized within 2 weeks after cell transplantation. In these animals, human insulin levels were measurable while mouse insulin levels were negligible throughout the observation period. Immunostaining of cell-bearing kidneys revealed sparse CD45+ cells. Immunolabeling and flow cytometry of blood, bone marrow and splenic samples obtained from the 3 groups of animals did not reveal a significant difference in the proportions of immune cell subsets or in the expression levels of HLA-ABC and HLA-DR.ConclusionTransplantation of IPCs derived from allogenic hAT-MSCs into humanized mice was followed by a muted allogenic immune response that did not interfere with the functionality of the engrafted cells. Our findings suggest that such allogenic cells could offer an opportunity for cell therapy for insulin-dependent diabetes without immunosuppression, encapsulation or gene manipulations.

Project description:BACKGROUND:Human induced pluripotent stem cells (hiPSCs) represent a potentially unlimited source of pancreatic endocrine lineage cells. Although insulin-producing ? cells derived from hiPSCs have been successfully induced, much work remains to be done to achieve mature ? cells. Lysine-specific demethylase 1 (LSD1) plays an important role in the regulation of hiPSC self-renewal and differentiation. We propose a new strategy to acquire insulin-producing cells (IPCs) from hiPSCs by knocking down LSD1. METHODS:Knockdown of LSD1 in hiPSCs with five shRNA. Assessment of the effects of shRNA on hiPSC proliferation, cell cycle, and apoptosis. Using knockdown hiPSCs with 31.33% LSD1 activity, we achieved a four-step differentiation into IPCs and test its differentiation efficiency, morphology, and marker genes and proteins. We implanted the IPCs into the renal subcapsular of SCID-Beige diabetic mice to evaluate the hypoglycemic effect in vivo. We tested LSD1 and HDAC1 whether they are present in the CoREST complex through IP-WB, and analyzed LSD1, CoREST, HDAC1, H3K4me2/me3, and H3K27me3 protein expression before and after knockdown of LSD1. RESULTS:Differentiated hiPSCs were 38.32%?±?3.54% insulin-positive cells and released insulin/C-peptide in response to glucose stimulus in a manner comparable to adult human islets. Most of the IPCs co-expressed mature ? cell-specific markers. When transplanted under the left renal capsule of SCID-Beige diabetic mice, these IPCs reversed hyperglycemia, leading to a significant increase in the definitive endoderm cells. IP-WB results showed that LSD1, HDAC1, and CoREST formed a complex in hiPSCs. Chip-PCR results showed that LSD1, HDAC1, and CoREST were enriched in the same district during the SOX17 and FOXA2 promoter region. Inhibition of LSD1 would not affect the level of CoREST but decreased the HDAC1 expressions. The H3K4me2/me3 and H3K9act level of SOX17 and FOXA2 promoter region increased after inhibited of LSD1, and promoted transcriptional activation. The H3K4me2/me3 and H3K9act level of OCT4 and SOX2 promoter region decreased with the transcriptional repressed. CONCLUSIONS:LSD1 regulated histone methylation and acetylation in promoter regions of pluripotent or endodermal genes. Our results suggest a highly efficient approach to producing IPCs from hiPSCs.

Project description:BackgroundMesenchymal stem/stromal cells (MSCs) and macrophages are critical components in many tissue microenvironments, including that in adipose tissue. The close interaction between MSCs and macrophages modulates various adipose-related disease development. However, the effects of macrophages on the fate of MSCs remain largely elusive. We here studied the effect of macrophages on the adipogenic differentiation of MSCs.MethodsMacrophages were obtained from THP-1 cells treated with phorbol-12-myristate-13-acetate (PMA). The induced matured macrophages were then induced to undergo classically activated macrophage (M1) or alternatively activated macrophage (M2) polarization with Iipopolysaccharide (LPS)/interferon (IFN)-γ and interleukin (IL)-4/IL-13, respectively. The supernatants derived from macrophages under different conditions were applied to cultured human adipose tissue-derived mesenchymal stem/stromal cells (hADSCs) undergoing adipogenic differentiation. Adipogenic differentiation was evaluated by examining Oil Red O staining of lipid droplets and the expression of adipogenesis-related genes with real-time quantitative polymerase chain reaction (Q-PCR) and western blot analysis.ResultsThe adipogenic differentiation of hADSCs was impaired when treated with macrophage-derived supernatants, especially that from the M1-polarized macrophage (M1-sup). The inhibitory effect was found to be mediated by the inflammatory cytokines, mainly tumor necrosis factor-α (TNF-α) and IL-1β. Blocking TNF-α and IL-1β with neutralizing antibodies partially alleviated the inhibitory effect of M1-sup.ConclusionMacrophage-derived supernatants inhibited the adipogenic differentiation of hADSCs in vitro, irrespective of the polarization status (M0, M1 or M2 macrophages). M1-sup was more potent because of the higher expression of pro-inflammatory cytokines. Our findings shed new light on the interaction between hADSCs and macrophages and have implications in our understanding of disrupted adipose tissue homeostasis under inflammation.

Dataset Information

PRDX6 Promotes the Differentiation of Human Mesenchymal Stem (Stromal) Cells to Insulin-Producing Cells.

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PRDX6 Promotes the Differentiation of Human Mesenchymal Stem (Stromal) Cells to Insulin-Producing Cells.

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