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. 2024 Aug 6:18:1449063.
doi: 10.3389/fncel.2024.1449063. eCollection 2024.

Neuroprotective and anti-inflammatory properties of proteins secreted by glial progenitor cells derived from human iPSCs

Diana I Salikhova  1   2 , Margarita O Shedenkova  1   2 , Anastasya K Sudina  1   2 , Ekaterina V Belousova  1   2 , Irina A Krasilnikova  3 , Anastasya A Nekrasova  3 , Zlata A Nefedova  4 , Daniil A Frolov  5 , Timur Kh Fatkhudinov  1 , Andrey V Makarov  1 , Alexander M Surin  6 , Kirill V Savostyanov  3 , Dmitry V Goldshtein  1   2 , Zanda V Bakaeva  3   4
Affiliations

Neuroprotective and anti-inflammatory properties of proteins secreted by glial progenitor cells derived from human iPSCs

Diana I Salikhova et al. Front Cell Neurosci. .

Abstract

Currently, stem cells technology is an effective tool in regenerative medicine. Cell therapy is based on the use of stem/progenitor cells to repair or replace damaged tissues or organs. This approach can be used to treat various diseases, such as cardiovascular, neurological diseases, and injuries of various origins. The mechanisms of cell therapy therapeutic action are based on the integration of the graft into the damaged tissue (replacement effect) and the ability of cells to secrete biologically active molecules such as cytokines, growth factors and other signaling molecules that promote regeneration (paracrine effect). However, cell transplantation has a number of limitations due to cell transportation complexity and immune rejection. A potentially more effective therapy is using only paracrine factors released by stem cells. Secreted factors can positively affect the damaged tissue: promote forming new blood vessels, stimulate cell proliferation, and reduce inflammation and apoptosis. In this work, we have studied the anti-inflammatory and neuroprotective effects of proteins with a molecular weight below 100 kDa secreted by glial progenitor cells obtained from human induced pluripotent stem cells. Proteins secreted by glial progenitor cells exerted anti-inflammatory effects in a primary glial culture model of LPS-induced inflammation by reducing nitric oxide (NO) production through inhibition of inducible NO synthase (iNOS). At the same time, added secreted proteins neutralized the effect of glutamate, increasing the number of viable neurons to control values. This effect is a result of decreased level of intracellular calcium, which, at elevated concentrations, triggers apoptotic death of neurons. In addition, secreted proteins reduce mitochondrial depolarization caused by glutamate excitotoxicity and help maintain higher NADH levels. This therapy can be successfully introduced into clinical practice after additional preclinical studies, increasing the effectiveness of rehabilitation of patients with neurological diseases.

Keywords: LPS-induced inflammation; glial progenitor cells; glutamate excitotoxicity; human induced pluripotent stem cells; secreted proteins.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Characterization of cultured cortical neurons and assessment of dose-dependent effects of SP-GPCs in a glutamate excitotoxicity model. (A) Morphology of the resulting culture of cortical neurons. Phase contrast microscopy. (B) Immunophenotyping of neuroglial culture for astroglial GFAP (green) and neuronal β-III-tubulin (red) markers. Cell nucleus stained by DAPI (blue). Scale bar: 200 μm. (C) Determination of the viability of cortical neurons (MTT test); *p ≤ 0.05 compared to glutamate (Glu, 100 μM) (n = 5). Data were analyzed by one-way ANOVA with Holm-Šidak correction. Data are presented as means and standard deviations. SP-GPCs at concentrations from 5 to 45 μg/ml led to an increase in the number of viable neurons to control values. The addition of memantine (Mem, 100 μM) increased neuronal survival by 30 ± 13%.
FIGURE 2
FIGURE 2
Comparison of parameters of dynamic changes in calcium homeostasis when modeling glutamate excitotoxicity under control and with the addition of SP-GPCs (15 μg/ml) 24 h before the experiment. [Ca2+]i and ΔΨm measured using Fura2 and Rh123, respectively.(A) Average curves of [Ca2+]i changes in the control group (black line, n = 485) and the group with the addition of SP-GPCs (red line, n = 434). (B) Average curves of the dynamics of changes in ΔΨm in the control group and the group with the addition of SP-GPCs. Data are presented as means and standard deviations. (C,D) Analysis of changes in [Ca2+]i and ΔΨm by calculating the areas under the curves (AUC) in different periods using the example of one representative neuron. (E) Effect of SP-GPCs on AUC values during the period of Glu exposure (15 min) (AUCGlu), (F) during the period after removal of Glu in a nominally calcium-free buffer (20 min) (AUCEGTA), and (H) % recovery of the original level [Ca2+]i. (I) under the influence of protonophore FCCP (5 min) (AUCFCCP), (G) the degree of decrease in ΔΨm, expressed as the ratio AUCFCCP/AUCGlu, (J) the degree of recovery of ΔΨm, expressed as the ratio AUCFCCP/AUCEGTA. Data were analyzed using a nonparametric unpaired t test followed by the Mann–Whitney test and presented as medians and interquartile range. ****p ≤ 0.0001 compared to control.
FIGURE 3
FIGURE 3
Effect of SP-GPCs on the dynamics of [NADH]i changes under the influence of glutamate. Data are presented as means and standard deviations. (A) Average curves of changes in [NADH]i in the control group and the group with added SP-GPCs (n = 196 in the control group and 182 in the SP-GPCs group). (B) Decrease in [NADH]i during 15 min of exposure to glutamate, expressed in %, the initial level of [NADH]i is taken as 100%, and the level of [NADH]i under exposure to FCCP is taken as 0%. (C,D) change in [NADH]i and ΔΨm in representative neurons from the control and SP-GPCs supplemented groups, respectively. Data were analyzed using a nonparametric unpaired t test followed by the Mann–Whitney test and presented as medians and interquartile ranges. ***p ≤ 0.001 compared to control.
FIGURE 4
FIGURE 4
Characterization of mixed glial culture and assessment of the dose-dependent effects of SP-GPCs on the secretion of the inflammatory mediator NO. (A) Morphology of the resulting microglia culture with an admixture of astrocytes (10 DIV). Phase contrast microscopy. (B) Immunophenotyping of glial culture for astroglial GFAP (green) and microglial CD11b (red) markers. Cell nucleus stained by DAPI (blue). Scale bar: 200 μm. (C) relative NO secretion by astrocytes and microglia upon addition of SP-GPCs under physiological conditions, and (D) during LPS-dependent inflammation (Griess test). Data were analyzed by rank analysis of variance (ANOVA on ranks) followed by Dunn’s test and presented as medians and interquartile ranges, *p ≤ 0.05 compared with the LPS group, n = 5. In the model of LPS-dependent inflammation, the addition of SP-GPCs at concentrations of 15 and 45 μg/ml led to a decrease in NO secretion. NO values during incubation with SP-GPCs at a concentration of 5 μg/ml were comparable to those in the LPS group. As a positive control, dexamethasone (Dex; 0.6 μM) was supplemented.
FIGURE 5
FIGURE 5
Inducible NO synthase activity upon addition of LPS and SP-GPCs to a mixed glial culture. (A) immunocytochemical study of iNOS (green cells). Cell nuclei are stained with DAPI (blue). (B) quantification of iNOS-positive cells, n = 4. Data were analyzed by rank analysis of variance (ANOVA on ranks) followed by Dunn’s test and presented as medians and interquartile ranges, *p ≤ 0.05 compared with LPS. Scale bar 200 μm. Incubation of a mixed glial culture (12 DIV) with LPS contributed to an increase in the number of iNOS-positive cells compared to the control. The addition of SP-GPCs at concentrations of 5 to 45 μg/ml under physiological conditions did not result in an increase in the population of iNOS-immunopositive cells.
FIGURE 6
FIGURE 6
Inducible NO synthase activity upon addition of SP-GPCs to a mixed glial culture in a model of LPS-dependent inflammation. (A) immunocyto- chemical study of iNOS (green cells). Cell nuclei are stained with DAPI (blue). (B) quantification of iNOS-positive cells, n = 4. Data were analyzed by rank analysis of variance (ANOVA on ranks) followed by Dunn’s test and presented as medians and interquartile ranges, *p ≤ 0.05 compared with LPS. Scale bar 200 μm. In the model of LPS-dependent inflammation, the addition of SP-GPCs at concentrations of 15 and 45 μg/ml contributed to a decrease in iNOS-positive cells in a mixed glial culture (12 DIV). The addition of dexamethasone (Dex; 0.6 μM) during LPS-induced inflammation effectively reduced the number of iNOS-positive cells and led to comparable to control values.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of the article. This work financially supported by the Ministry of Science and Higher Education of the Russian Federation (Project No. KBK 075 0110 47 1 S7 24600 621) by “Development of New Drugs for Therapy of Neurological Diseases.”

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