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Survival and Neurogenesis-Promoting Effects of the Co-Overexpression of BCLXL and BDNF Genes on Wharton's Jelly-Derived Mesenchymal Stem Cells - PubMed

  • ️Sat Jan 01 2022

Survival and Neurogenesis-Promoting Effects of the Co-Overexpression of BCLXL and BDNF Genes on Wharton's Jelly-Derived Mesenchymal Stem Cells

Paulina Borkowska et al. Life (Basel). 2022.

Abstract

The main problem with using MSC (mesenchymal stem cells) to treat the deficient diseases of the central nervous system is the low cell survival rate after the transplant procedure and their low ability to spontaneously differentiate into functional neurons. The aim of this study was to investigate the effects of genetically modifying MSC. A co-overexpression of two genes was performed: BCLXL was supposed to increase the resistance of the cells to the toxic agents and BDNF was supposed to direct cells into the neuronal differentiation pathway. As a result, it was possible to obtain the functional overexpression of the BCLXL and BDNF genes. These cells had an increased resistance to apoptosis-inducing toxicants (staurosporine, doxorubicin and H2O2). At the same time, the genes of the neuronal pathway (CHAT, TPH1) were overexpressed. The genetically modified MSC increased the survival rate under toxic conditions, which increased the chance of surviving a transplant procedure. The obtained cells can be treated as neural cell progenitors, which makes them a universal material that can be used in various disease models. The production of neurotransmitters suggests that cells transplanted into the brain and subjected to the additional influence of the brain's microenvironment, will be able to form synapses and become functional neurons.

Keywords: BCLXL; BDNF; co-overexpression; mesenchymal stem cells; neurogenesis; survival.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1

Overview of the general idea of the experiment. The diagram shows the experimental procedures that were performed on the FV group (Figure 2C+2D) in which there was a functional overexpression of the Bcl-XL and BDNF proteins. An analogous procedure was performed for the control group (C) and the EV group that had been transduced with empty vector backbones (Figure 2A+2B).

Figure 2
Figure 2

The simplified lentiviral vectors maps that were used for the overexpression. The sequence of the Bcl-XL gene enriched with the Kozak sequence was cloned into the plasmid (A) that contained the green fluorescence protein (EGFP) (C). The sequence of the BDNF gene enriched with the Kozak sequence was cloned into the plasmid (B) that contained the red fluorescence protein (tdTomato) (D). In the further parts of this work, plasmids A and B, which were used synergistically for the transduction, the abbreviations EV—empty vectors, and plasmids C and D under abbreviation FV—full vectors are used.

Figure 3
Figure 3

Characterization of an in vitro WJ-MSC culture. (A) Characteristic WJ-MSC fibroblast-like morphology. The WJ-MSC were cultured in a complete medium as is described in Materials and Methods. Changes in the cell morphology after a 12-day differentiation procedure in the control—C (B), empty vectors—EV (C) and full vectors—FV (D) groups. Scale bar = 50 µm.

Figure 4
Figure 4

Immunophenotypic analysis for the WJ-MSC using flow cytometry. Isotype controls (A,C,E) were used for gating. Almost all of the WJ-MSC cells were negative for CD34, CD11b, CD19, CD45 and HLA-DR (B) and positive for CD90 (D) and CD73 (F). Data shown are representative of at least three independent experiments.

Figure 5
Figure 5

Characterization of an in vitro WJ-MSC culture. (A) Characteristic WJ-MSC fibroblast-like morphology. The WJ-MSC were cultured in a complete medium as described in Materials and Methods. (B) The control cultured in standard medium (DMEM/F12 with 0.5% FBS and a 1% Antibiotic Antimycotic Solution) equally with adipogenic and/or osteogenic differentiation (C,D). WJ-MSC were cultured for three weeks in either an adipogenic (C) or osteogenic (D) differentiation medium. Lipid vacuoles were stained with Oil Red O (C) and matrix mineralization was determined using alkaline phosphatase staining (D). Scale bar = 50 µm.

Figure 6
Figure 6

Percentage of positive transduced cells over time (A). Quantitative analysis of the Bcl-XL (B) and BDNF (C) protein production over time. At each time point, the experiment was performed for all three groups: cells overexpressing the genes BCLXL and BDNF (full vectors—FV); empty vector transduced cells—(EV); control (C). In (A), there were no positively transduced cells in C group. In (C), BDNF protein level of EV group is the same as the C group (0 ng/mL all the time). The points represent the mean value (n = 4; two independent experiments). Statistically significant * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 two-way ANOVA test followed by a post hoc Tuckey’s test. Detailed statistical analysis is available in Supplementary Materials section.

Figure 7
Figure 7

Cell survival analysis. Transduction with a single vector compared with a synergistic transduction of the empty vectors (EV: iG2 + iT2) or full vectors (FV: Bcl-XL + BDNF). The WST-1 was measured 24 h after the addition of staurosporine; two-way ANOVA test followed by a post hoc Tuckey’s test (A). Cell survival was analyzed after various death inducers were used. The WST-1 was measured 24 h after the addition of each agent; two-way ANOVA test followed by a post hoc Tuckey’s test. (B). An examination of cell resistance to a toxic agent and an analysis of its type of death. The measurement was conducted 12 h after the addition 1 µM of staurosporine; two-way ANOVA test followed by a post hoc Tuckey’s test. (C). Apoptosis induction was done one day after transduction ended for (A) and after 14 days for (B,C). The points represent the mean value ± SD (n = 12; three independent experiments). Statistically significant * p < 0.05; ** p < 0.01; *** p < 0.001 or **** p <0.0001.

Figure 8
Figure 8

qRT-PCR analysis for the neuronal markers is presented as the fold change (2−ΔΔCT) in the level of their expression, which was normalized to the RPS17 reference gene; * p < 0.05, ** p < 0.01 or **** p < 0.0001 (A). The number of CHAT and TH positive cells. The calculations were based on a flow cytometry analysis. Points represent the mean value ± SD (n = 6; three independent experiments). Statistically significant * p < 0.05; two-way ANOVA test followed by a post hoc Tuckey’s test (B). Acetylcholine and dopamine release after depolarization by the neuronal-differentiated WJ-MSC. Points represent the mean value (n = 6; three independent experiments). Statistically significant * p < 0.05; two-way ANOVA test followed by a post hoc Tuckey’s test (C). Flow cytometric analysis for the expression of neural specific proteins: CHAT (choline acetyltransferase) and TH (tyrosine hydroxylase) after 12 days of differentiation. Percentage values represent the mean value (n = 6; three independent experiments) (D). Control group (C), the group that had been transduced with empty vectors (EV), the group of cells that had an overproduction of the Bcl-XL and BDNF proteins (FV) and in FV that had additionally been supplemented with bFGF or resveratrol.

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