ABSTRACT
Background: Bone marrow mesenchymal stem cells [BM-MSCs] have emerged as a potential therapy for various inflammatory diseases. Because of some limitations, several recent studies have suggested the use of embryonic stem cell-derived MSCs [ESC-MSCs] as an alternative for BM-MSCs. Some of the therapeutic effects of the ESCMSCs are related to the secretion of a broad array of cytokines and growth factors, known as secretome.Harnessing this secretome for therapeutic applications requires the optimization of production of secretary molecules. It has been shown that aggregation of MSCs into 3D spheroids, as a preconditioning strategy, can enhance immunomodulatory potential of such cells. In this study, we investigated the effect of secretome derived from human ESC-MSCs [hESC-MSCs] spheroids on secretion of IL-1Beta, IL-10, and tumor necrosis factor Alpha [TNF-Alpha] from lipopolysaccharide [LPS]-induced peripheral blood mononuclear cells [PBMCs]
Methods: In the present study, after immunophenotyping and considering mesodermal differentiation of hESC-MSCs, the cells were nonadherently grown to prepare 3D aggregates, and then conditioned medium or secretome was extracted from the cultures. Afterwards, the anti-inflammatory effects of the secretome were assessed in an in vitro model of inflammation
Results: Results from this study showed that aggregate-prepared secretome from hESC-MSCs was able to significantly decrease the secretion of TNF-Alpha [301.7 +/- 5.906, p < 0.0001] and IL-1 Alpha [485.2 +/- 48.38, p < 0.001] from LPS-induced PBMCs as the indicators of inflammation, in comparison with adherent culture-prepared secretome [TNF-Alpha : 166.6 +/- 8.04, IL-1Beta: 125.2 +/- 2.73]
Conclusion: Our study indicated that cell aggregation can be an appropriate strategy to increase immunomodulatory characteristics of hESC-MSCs
ABSTRACT
Background: Mesenchymal stem cells [MSCs] are important candidates for MSC-based cellular therapy. Current paradigm states that MSCs support local progenitor cells in damaged tissue through paracrine signaling. Therefore, the study of paracrine effects and secretome of MSCs could lead to the appreciation of mechanisms and molecules associated with the therapeutic effects of these cells. This study analyzed anti-inflammatory and immune-modulatory effects of MSC secretomes derived from embryonic stem cells [ESCs] and bone marrow cells after hypoxia and normoxia preconditioning
Methods: ESCs differentiated into MSCs and characterized by flow cytometry as well as by differentiation into adipocytes and osteoblasts. The experimental groups were consisted of individual groups of ESC-MSCs and BM-MSCs [bone marrow-derived mesenchymal stromal cells], which were preconditioned with either hypoxia or normoxia for 24, 48 and 72 h. After collecting the cell-free medium from each treatment, secretomes were concentrated by centrifugal filters. Using a peripheral blood mononuclear cell [PBMC] assay and ELISA, IL-10 concentration in PBMCs was evaluated after their incubation with different secretomes from preconditioned and non-preconditioned MSCs
Results: A significant difference was observed between ESC-MSC normoxia and ESC-MSC hypoxia in IL-10 concentration, and normoxia secretomes increased IL-10 secretion from PBMCs. Moreover, the strongest IL-10 secretion from PBMCs could be detected after the stimulation by ESC-MSC conditioned secretomes, but not BM-MSC conditioned medium
Conclusions: Human hypoxia preconditioned ESC-MSC secretome indicated stronger immune-modulatory effects compared to BMMSC conditioned medium. It could be suggested that induced MSCs confer less immune-modulatory effects but produce more inflammatory molecules such as tumor necrosis factor alpha, which needs further investigation
Subject(s)
Humans , Human Embryonic Stem Cells , Culture Media, Conditioned/pharmacology , Cell Hypoxia/physiology , Blood Cells , Leukocytes, Mononuclear/physiology , Interleukin-10/metabolismABSTRACT
Background: As a drug target and an antigenic agent, HIV-1 protease [HIV-1 PR] is at the center of attention for designing anti-AIDS inhibitors and diagnostic tests. In previous studies, the production of the recombinant protease has been faced with several difficulties; therefore, the aims of this study were the easy production, purification of the soluble form of protease in E. coli and investigation of its immunoreactivity
Methods: Protease coding region was isolated from the serum of an infected individual, amplified by RT-PCR and cloned into PTZ57R using TA-cloning. Protease coding frame was isolated by PCR and cloned in pET102/D. TOPO expression vector and cloned protease was expressed in Escherichia coli [E. coli] BL21. Produced recombinant protein was purified by affinity Ni-NTA column and protein concentration was checked by BCA protein assay kit. Subsequently, immunoreactivity of recombinant protease [rPR] was assayed by Western blotting and ELISA
Results: Cloning of the HIV protease by TOPO cloning system in pET102/D.TOPO was confirmed with PCR and sequencing. The concentration range of purified recombinant protein was 85 to 100 micro g/ml. Immunogenicity of rPR was confirmed by Western blotting and ELISA
Conclusion: Soluble production of recombinant HIV-1 protease [HIV-1 rPR] was performed successfully. This recombinant protein disclosed 86% specificity and 90% sensitivity in immunoassay tests