Changes in the secretome of tri-dimensional spheroid-cultured human mesenchymal stem cells in vitro by interleukin-1 priming

Elena Redondo-Castro, Catriona Cunningham, Jonjo Miller, Helena Brown, Stuart M Allan, Emmanuel Pinteaux*

*Corresponding author for this work

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Background
Mesenchymal stem cells (MSCs) are one of the most promising candidates for the treatment of major neurological disorders. Desirable therapeutic properties of MSCs include reparative and regenerative potential but, despite their proven safety, the efficacy of MSCs remains controversial. Therefore, it is essential to optimise culture protocols to enhance the therapeutic potential of the MSC secretome. Here we aimed to: assess the increase in secretion of cytokines that may induce repair, regeneration, or immunomodulation when cultured in three dimensions; study the effect of interleukin (IL)-1 priming on two- (2D) and three-dimensional (3D) cultures of MSC; and evaluate the potential use of the modified secretome using microglial-MSC co-cultures.

Methods
We established a 3D spheroid culture of human MSCs, and compared the secretome in 2D and 3D cultures under primed (IL-1) and unprimed conditions. BV2 microglial cells were stimulated with lipopolysaccharide (LPS) and treated with spheroid conditioned media (CM) or were co-cultured with whole spheroids. Concentrations of secreted cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Protein arrays were used to further evaluate the effect of IL-1 priming in 2D and 3D cultures.

Results
3D culture of MSCs significantly increased secretion of the IL-1 receptor antagonist (IL-1Ra), vascular endothelial growth factor (VEGF), and granulocyte-colony stimulating factor (G-CSF) compared with 2D culture, despite priming treatments with IL-1 being more effective in 2D than in 3D. The addition of CM of 3D-MSCs reduced LPS-induced tumour necrosis factor (TNF)-α secretion from BV2 cells, while the 3D spheroid co-cultured with the BV2 cells induced an increase in IL-6, but had no effect on TNF-α release. Protein arrays indicated that priming treatments trigger a more potent immune profile which is necessary to orchestrate an effective tissue repair. This effect was lost in 3D, partly because of the overexpression of IL-6.

Conclusions
Increased secretion of anti-inflammatory markers occurs when MSCs are cultured in 3D, but this specific secretome did not translate into anti-inflammatory effects on LPS-treated BV2 cells in co-culture. These data highlight the importance of optimising priming treatments and culture conditions to maximise the therapeutic potential of MSC spheroids.
Original languageEnglish
Article number11
JournalStem cell research & therapy
Volume9
DOIs
Publication statusPublished - 17 Jan 2018

Fingerprint

Stem cells
Mesenchymal Stromal Cells
Interleukin-1
Lipopolysaccharides
Conditioned Culture Medium
Interleukin-6
Repair
Anti-Inflammatory Agents
Tumor Necrosis Factor-alpha
Protein Array Analysis
Cytokines
Immunosorbents
Interleukin-1 Receptors
Coculture Techniques
Granulocyte Colony-Stimulating Factor
Therapeutics
Vascular Endothelial Growth Factor A
Assays
Proteins
Tissue

Keywords

  • spheroid
  • 3D culture
  • mesenchymal stem cell
  • inflammation
  • BV2 cells
  • microglia

Cite this

Changes in the secretome of tri-dimensional spheroid-cultured human mesenchymal stem cells in vitro by interleukin-1 priming. / Redondo-Castro, Elena; Cunningham, Catriona; Miller, Jonjo; Brown, Helena; Allan, Stuart M; Pinteaux, Emmanuel.

In: Stem cell research & therapy, Vol. 9, 11, 17.01.2018.

Research output: Contribution to journalArticle

Redondo-Castro, Elena ; Cunningham, Catriona ; Miller, Jonjo ; Brown, Helena ; Allan, Stuart M ; Pinteaux, Emmanuel. / Changes in the secretome of tri-dimensional spheroid-cultured human mesenchymal stem cells in vitro by interleukin-1 priming. In: Stem cell research & therapy. 2018 ; Vol. 9.
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title = "Changes in the secretome of tri-dimensional spheroid-cultured human mesenchymal stem cells in vitro by interleukin-1 priming",
abstract = "BackgroundMesenchymal stem cells (MSCs) are one of the most promising candidates for the treatment of major neurological disorders. Desirable therapeutic properties of MSCs include reparative and regenerative potential but, despite their proven safety, the efficacy of MSCs remains controversial. Therefore, it is essential to optimise culture protocols to enhance the therapeutic potential of the MSC secretome. Here we aimed to: assess the increase in secretion of cytokines that may induce repair, regeneration, or immunomodulation when cultured in three dimensions; study the effect of interleukin (IL)-1 priming on two- (2D) and three-dimensional (3D) cultures of MSC; and evaluate the potential use of the modified secretome using microglial-MSC co-cultures.MethodsWe established a 3D spheroid culture of human MSCs, and compared the secretome in 2D and 3D cultures under primed (IL-1) and unprimed conditions. BV2 microglial cells were stimulated with lipopolysaccharide (LPS) and treated with spheroid conditioned media (CM) or were co-cultured with whole spheroids. Concentrations of secreted cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Protein arrays were used to further evaluate the effect of IL-1 priming in 2D and 3D cultures.Results3D culture of MSCs significantly increased secretion of the IL-1 receptor antagonist (IL-1Ra), vascular endothelial growth factor (VEGF), and granulocyte-colony stimulating factor (G-CSF) compared with 2D culture, despite priming treatments with IL-1 being more effective in 2D than in 3D. The addition of CM of 3D-MSCs reduced LPS-induced tumour necrosis factor (TNF)-α secretion from BV2 cells, while the 3D spheroid co-cultured with the BV2 cells induced an increase in IL-6, but had no effect on TNF-α release. Protein arrays indicated that priming treatments trigger a more potent immune profile which is necessary to orchestrate an effective tissue repair. This effect was lost in 3D, partly because of the overexpression of IL-6.ConclusionsIncreased secretion of anti-inflammatory markers occurs when MSCs are cultured in 3D, but this specific secretome did not translate into anti-inflammatory effects on LPS-treated BV2 cells in co-culture. These data highlight the importance of optimising priming treatments and culture conditions to maximise the therapeutic potential of MSC spheroids.",
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author = "Elena Redondo-Castro and Catriona Cunningham and Jonjo Miller and Helena Brown and Allan, {Stuart M} and Emmanuel Pinteaux",
note = "Funding The work was supported with funds from the Stroke Association and from EPSRC, MRC Centre for Doctoral Training in Regenerative Medicine studentship grant EP/L014904/1, and the Manchester Regenerative Medicine Network (MaRM). Availability of data and materials The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.",
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T1 - Changes in the secretome of tri-dimensional spheroid-cultured human mesenchymal stem cells in vitro by interleukin-1 priming

AU - Redondo-Castro, Elena

AU - Cunningham, Catriona

AU - Miller, Jonjo

AU - Brown, Helena

AU - Allan, Stuart M

AU - Pinteaux, Emmanuel

N1 - Funding The work was supported with funds from the Stroke Association and from EPSRC, MRC Centre for Doctoral Training in Regenerative Medicine studentship grant EP/L014904/1, and the Manchester Regenerative Medicine Network (MaRM). Availability of data and materials The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

PY - 2018/1/17

Y1 - 2018/1/17

N2 - BackgroundMesenchymal stem cells (MSCs) are one of the most promising candidates for the treatment of major neurological disorders. Desirable therapeutic properties of MSCs include reparative and regenerative potential but, despite their proven safety, the efficacy of MSCs remains controversial. Therefore, it is essential to optimise culture protocols to enhance the therapeutic potential of the MSC secretome. Here we aimed to: assess the increase in secretion of cytokines that may induce repair, regeneration, or immunomodulation when cultured in three dimensions; study the effect of interleukin (IL)-1 priming on two- (2D) and three-dimensional (3D) cultures of MSC; and evaluate the potential use of the modified secretome using microglial-MSC co-cultures.MethodsWe established a 3D spheroid culture of human MSCs, and compared the secretome in 2D and 3D cultures under primed (IL-1) and unprimed conditions. BV2 microglial cells were stimulated with lipopolysaccharide (LPS) and treated with spheroid conditioned media (CM) or were co-cultured with whole spheroids. Concentrations of secreted cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Protein arrays were used to further evaluate the effect of IL-1 priming in 2D and 3D cultures.Results3D culture of MSCs significantly increased secretion of the IL-1 receptor antagonist (IL-1Ra), vascular endothelial growth factor (VEGF), and granulocyte-colony stimulating factor (G-CSF) compared with 2D culture, despite priming treatments with IL-1 being more effective in 2D than in 3D. The addition of CM of 3D-MSCs reduced LPS-induced tumour necrosis factor (TNF)-α secretion from BV2 cells, while the 3D spheroid co-cultured with the BV2 cells induced an increase in IL-6, but had no effect on TNF-α release. Protein arrays indicated that priming treatments trigger a more potent immune profile which is necessary to orchestrate an effective tissue repair. This effect was lost in 3D, partly because of the overexpression of IL-6.ConclusionsIncreased secretion of anti-inflammatory markers occurs when MSCs are cultured in 3D, but this specific secretome did not translate into anti-inflammatory effects on LPS-treated BV2 cells in co-culture. These data highlight the importance of optimising priming treatments and culture conditions to maximise the therapeutic potential of MSC spheroids.

AB - BackgroundMesenchymal stem cells (MSCs) are one of the most promising candidates for the treatment of major neurological disorders. Desirable therapeutic properties of MSCs include reparative and regenerative potential but, despite their proven safety, the efficacy of MSCs remains controversial. Therefore, it is essential to optimise culture protocols to enhance the therapeutic potential of the MSC secretome. Here we aimed to: assess the increase in secretion of cytokines that may induce repair, regeneration, or immunomodulation when cultured in three dimensions; study the effect of interleukin (IL)-1 priming on two- (2D) and three-dimensional (3D) cultures of MSC; and evaluate the potential use of the modified secretome using microglial-MSC co-cultures.MethodsWe established a 3D spheroid culture of human MSCs, and compared the secretome in 2D and 3D cultures under primed (IL-1) and unprimed conditions. BV2 microglial cells were stimulated with lipopolysaccharide (LPS) and treated with spheroid conditioned media (CM) or were co-cultured with whole spheroids. Concentrations of secreted cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Protein arrays were used to further evaluate the effect of IL-1 priming in 2D and 3D cultures.Results3D culture of MSCs significantly increased secretion of the IL-1 receptor antagonist (IL-1Ra), vascular endothelial growth factor (VEGF), and granulocyte-colony stimulating factor (G-CSF) compared with 2D culture, despite priming treatments with IL-1 being more effective in 2D than in 3D. The addition of CM of 3D-MSCs reduced LPS-induced tumour necrosis factor (TNF)-α secretion from BV2 cells, while the 3D spheroid co-cultured with the BV2 cells induced an increase in IL-6, but had no effect on TNF-α release. Protein arrays indicated that priming treatments trigger a more potent immune profile which is necessary to orchestrate an effective tissue repair. This effect was lost in 3D, partly because of the overexpression of IL-6.ConclusionsIncreased secretion of anti-inflammatory markers occurs when MSCs are cultured in 3D, but this specific secretome did not translate into anti-inflammatory effects on LPS-treated BV2 cells in co-culture. These data highlight the importance of optimising priming treatments and culture conditions to maximise the therapeutic potential of MSC spheroids.

KW - spheroid

KW - 3D culture

KW - mesenchymal stem cell

KW - inflammation

KW - BV2 cells

KW - microglia

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U2 - 10.1186/s13287-017-0753-5

DO - 10.1186/s13287-017-0753-5

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C2 - 29343288

VL - 9

JO - Stem cell research & therapy

JF - Stem cell research & therapy

SN - 1757-6512

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