scholarly journals Creation and characterisation of 11β hydroxysteroid dehydrogenase type 1 transgenic mesenchymal stem cells for use in models of acute respiratory distress syndrome

2019 ◽  
Author(s):  
RAHUL Y. MAHIDA ◽  
Zhengqiang Yuan ◽  
Krishna K. Kolluri ◽  
Sebastian T. Lugg ◽  
Aaron Scott ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Respiratory Distress Syndrome ◽  
Stem Cell ◽  
Transgene Expression ◽  
Distress Syndrome ◽  
Lentiviral Vector ◽  
Transfection Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Alveolar Space

Abstract Background: Human bone marrow mesenchymal stem cell (MSC) administration reduces inflammation in pre-clinical models of lung injury, however clinical efficacy in patients with acute respiratory distress syndrome (ARDS) has not been shown. Upregulation of the glucocorticoid activating enzyme, 11β hydroxysteroid dehydrogenase type 1 (HSD-1) within the alveolar space elevates local anti-inflammatory cortisol levels, and promotes alveolar macrophage efferocytosis. Administration of HSD-1 transgenic MSCs (tMSCs) that overexpress HSD-1 may enhance local cortisol activation. This combined cellular and gene therapy may be more effective at reducing inflammation in ARDS than cellular therapy alone. Methods: Molecular cloning was used to create a recombinant lentiviral vector containing the HSD-1 and green fluorescent protein (GFP) transgenes. MSCs were transfected using this lentivirus, then transfection efficiency was assessed using flow-cytometry. HSD-1 transgene expression was assessed using immunofluorescence and western blotting. Thin layer chromatography was used to assess HSD-1 function in tMSCs. Bi-lineage differentiation and flow-cytometry were used to determine whether tMSCs maintained a stem cell phenotype. Results: A recombinant lentiviral vector was created containing the HSD-1 and GFP transgenes under the control of a tetracycline promoter. MSCs were successfully transfected, with a transfection efficiency of 91.1%. HSD-1 transgene expression was confirmed by immunofluorescence and western blot. Functional HSD-1 activity was evident within tMSCs, with predominant reductase cortisol activation, following treatment with the transcriptional activator doxycycline. HSD-1 reductase activity was maintained for 72 hours after doxycycline was removed from tMSCs. HSD-1 tMSCs maintained the capacity for osteogenic and adipogenic differentiation, and maintained expression of MSC surface markers. Conclusions: We successfully transduced tMSCs to express the HSD-1 transgene that functions predominantly as a reductase. Administration of HSD-1 tMSCs to in vivo models of ARDS may attenuate inflammation through activation of cortisol in the alveolar space.

scholarly journals Strategies to Enhance Mesenchymal Stem Cell-Based Therapies for Acute Respiratory Distress Syndrome

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Jibin Han ◽  
Yanmin Li ◽  
Yuanyuan Li
Keyword(s):  
Acute Respiratory Distress Syndrome ◽  
Stem Cell ◽  
Respiratory Distress Syndrome ◽  
Respiratory Distress ◽  
Distress Syndrome ◽  
Therapeutic Potential ◽  
Large Body ◽  
Acute Onset ◽  
Trophic Factors ◽  
Preclinical Models

Acute respiratory distress syndrome (ARDS) is a multifaced disease characterized by the acute onset of hypoxemia, worsened pulmonary compliance, and noncardiogenic pulmonary edema. Despite over five decades of research, specific treatments for established ARDS are still lacking. MSC-based therapies have the advantage of targeting nearly all pathophysiological components of ARDS by means of a variety of secreted trophic factors, exerting anti-inflammatory, antioxidative, immunomodulatory, antiapoptotic, and proangiogenic effects, resulting in significant structural and functional recovery following ARDS in various preclinical models. However, the therapeutic efficacy of transplanted MSCs is limited by their poor engraftment and low survival rate in the injured tissues, major barriers to clinical translation. Accordingly, several strategies have been explored to improve MSC retention in the lung and enhance the innate properties of MSCs in preclinical models of ARDS. To provide a comprehensive and updated view, we summarize a large body of experimental evidence for a variety of strategies directed towards strengthening the therapeutic potential of MSCs in ARDS.

Co-Epression of Two Transgenes in Lentiviral Vector Using Independent Transcriptional Units.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5254-5254
Author(s):  
Friedrich G. Schuening ◽  
Michail M. Zaboikin ◽  
Narasimhachar Srinivasakumar ◽  
Tatiana N. Zaboikina
Keyword(s):  
Flow Cytometry ◽  
Transgene Expression ◽  
Dna Methyltransferase ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Egfp Expression ◽  
Drug Resistant ◽  
Gfp Expression ◽  
Ic50 Values ◽  
Hiv 1

Abstract There are several gene therapy approaches, which require transfer and co-expression of two transgenes within one target cell. To this end, we have created and tested two-gene expression HIV-1 based vectors, which encode enhanced green fluorescent protein (EGFP) and P144K mutant of canine O6-methylguanine-DNA-methyltransferase (MGMT) transgenes under either the phosphoglycerate kinase gene promoter (PGKp), or the elongation factor 1 alpha promoter (EF1a_p). Eight different configurations of the two transgene expression cassettes were created and tested within the same lentiviral backbone (see Figure). Individual VSV-G pseudotyped vectors stocks were produced and used for infection of canine thyroid adenocarcinoma (CTAC) cells at low multiplicity of infection (MOI = 0.1) to ensure 1 copy of proviral vector per transduced cell. The cells were harvested one week later and an aliquot was assayed for EGFP expression by flow cytometry. Another portion was subjected to selection with O6-benzylguanine (BG, 40 m M for 18 hrs) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 100 m M for 2 hrs) and kept in culture for additional two weeks to eliminate cells expressing insufficient MGMT. The percentage of GFP positive cells, prior to selection with BG and BCNU, ranged between 0.1 % to 6.6% for the dual-transgene expression cassette encoding HIV-1 vectors. Following selection with BG and BCNU, the percentage of GFP positive cells increased for all vectors with the exception of vector #2 PGEM. Two of the vectors (#1 PMEG and #8 EGMP) demonstrated over 80% GFP positivity after selection. The results of flow cytometry after selection were corroborated by fluorescence microscopy of individual BCNU-resistant colonies. GFP expression was readily detected in drug-resistant colonies transduced with vectors #1 PMEG or #8 EGMP. Weaker GFP expression was detected in drug resistant cells transduced with vectors #3 PMGE, #5 EMPG or #6 EGPM. No significant GFP expression was observed in drug-resistant colonies transduced with vectors #2 PGEM, #4 PGME or #7 EMGP. Drug resistance to BCNU (IC50 values) provided by each of the vectors, was also determined. The data showed that the IC50 values for #1 PMEG and #8 EGMP vectors were 2.4-fold and 4.4-fold, respectively, higher than for mock transduced control cells. The above results indicate that coordinated co-expression of two transgenes using independent expression cassettes is promoter, position and orientation dependent. The data also indicate that two potentially useful vectors (#1 PMEG and #8 EGMP) have been identified for evaluation, ex vivo and in vivo, in the canine model for co-expression of two transgenes.

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