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Extracellular membrane vesicles from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning.

Kilpinen L, Impola U, Sankkila L, Ritamo I, Aatonen M, Kilpinen S, Tuimala J, Valmu L, Levijoki J, Finckenberg P, Siljander P, Kankuri E, Mervaala E, Laitinen S - J Extracell Vesicles (2013)

Bottom Line: Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells.Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI.We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.

View Article: PubMed Central - PubMed

Affiliation: Finnish Red Cross Blood Service, Helsinki, Finland.

ABSTRACT

Background: Mesenchymal stromal cells (MSC) are shown to have a great therapeutic potential in many immunological disorders. Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells. Umbilical cord blood is an attractive but still less studied source of MSCs. We investigated the production of extracellular membrane vesicles (MVs) from human umbilical cord blood derived MSCs (hUCBMSC) in the presence (MVstim) or absence (MVctrl) of inflammatory stimulus.

Methods: hUCBMSCs were cultured in serum free media with or without IFN-γ and MVs were collected from conditioned media by ultracentrifugation. The protein content of MVs were analyzed by mass spectrometry. Hypoxia induced acute kidney injury rat model was used to analyze the in vivo therapeutic potential of MVs and T-cell proliferation and induction of regulatory T cells were analyzed by co-culture assays.

Results: Both MVstim and MVctrl showed similar T-cell modulation activity in vitro, but only MVctrls were able to protect rat kidneys from reperfusion injury in vivo. To clarify this difference in functionality we made a comparative mass spectrometric analysis of the MV protein contents. The IFN-γ stimulation induced dramatic changes in the protein content of the MVs. Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI. We further discovered that differently produced MV pools contained specific Rab proteins suggesting that same cells, depending on external signals, produce vesicles originating from different intracellular locations.

Conclusions: We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.

No MeSH data available.


Related in: MedlinePlus

(A) Scanning electron microscopic (FEI Quanta 250 FEG SEM) pictures of MVs produced by serum deprivation at magnification of 60,000× (i) and 90,000× (ii). Negative stainings (FEI Tecnai 12 TEM) of the same sample at magnification of 30,000×(iii). (B) Scanning electron microscopic pictures of MVs produced by IFN-γ stimulation at magnification of 60,000× (i) and 90,000× (ii). (C) Representative nanoparticle tracking analysis (NTA) profiles of MVctrl and MVstim. (D) Size distribution of MV ctrl and MVstim measured by NTA. Results are mean±SEM of 3 independent experiments. (E) The effect of MVs or hUCBMSCs on Treg induction after 7 days of MV or MSC cultured with allogeneic PBMCs. Representative flowcytometric analysis of CD25+-FOXP3+ Tregs are shown for CD4+ gated T lymphocytes. (F) The effect of MV and MSCs on T-cell proliferation analyzed by CFSE labelling of PBMCs and activation of T-cells with monoclonal CD3 antibody.
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Figure 0001: (A) Scanning electron microscopic (FEI Quanta 250 FEG SEM) pictures of MVs produced by serum deprivation at magnification of 60,000× (i) and 90,000× (ii). Negative stainings (FEI Tecnai 12 TEM) of the same sample at magnification of 30,000×(iii). (B) Scanning electron microscopic pictures of MVs produced by IFN-γ stimulation at magnification of 60,000× (i) and 90,000× (ii). (C) Representative nanoparticle tracking analysis (NTA) profiles of MVctrl and MVstim. (D) Size distribution of MV ctrl and MVstim measured by NTA. Results are mean±SEM of 3 independent experiments. (E) The effect of MVs or hUCBMSCs on Treg induction after 7 days of MV or MSC cultured with allogeneic PBMCs. Representative flowcytometric analysis of CD25+-FOXP3+ Tregs are shown for CD4+ gated T lymphocytes. (F) The effect of MV and MSCs on T-cell proliferation analyzed by CFSE labelling of PBMCs and activation of T-cells with monoclonal CD3 antibody.

Mentions: hUCBMSCs were induced to produce MVs by culturing them in serum-free conditions with (MVstim) or without (MVctrl) IFN-γ-stimulation for 24–48 hours. Cell viability in both culture conditions was always >95% (n=10). Electron microscopy analysis of the vesicles showed variation in size, with the smallest being around 20 nm and the largest >500 nm (Fig. 1A and B). NTA confirmed the wide size range seen by electron microscopy analysis (Fig. 1C). Also, the presence of very small vesicles (<50 nm) was confirmed. There was no significant difference in the size distribution between IFN-γ stimulated and control conditions (Fig. 1D). Based on the analysis of protein amount, IFN-γ stimulus gave only slightly better MV yield (data not shown). The protein yield was on average 16 µg of protein in a vesicle fraction for every 10×106 cells after 24-h production. When hUCBMSCs or MVs derived from hUCBMSCs were co-cultured with PBMCs for 7 days, we were able to show that MVs alone as well as MSCs were able to induce the formation of T-cells with Treg phenotype (CD4+ CD25+ FOXP3+). The percentage of Tregs was increased from 3.3% to 4.5% when PBMCs were co-cultured with MSCs (Fig. 1E). However, we were not able to see any remarkable differences in Treg induction when PBMCs were co-cultured with either MVctrl (4.5%) or MVstim (4.0%).


Extracellular membrane vesicles from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning.

Kilpinen L, Impola U, Sankkila L, Ritamo I, Aatonen M, Kilpinen S, Tuimala J, Valmu L, Levijoki J, Finckenberg P, Siljander P, Kankuri E, Mervaala E, Laitinen S - J Extracell Vesicles (2013)

(A) Scanning electron microscopic (FEI Quanta 250 FEG SEM) pictures of MVs produced by serum deprivation at magnification of 60,000× (i) and 90,000× (ii). Negative stainings (FEI Tecnai 12 TEM) of the same sample at magnification of 30,000×(iii). (B) Scanning electron microscopic pictures of MVs produced by IFN-γ stimulation at magnification of 60,000× (i) and 90,000× (ii). (C) Representative nanoparticle tracking analysis (NTA) profiles of MVctrl and MVstim. (D) Size distribution of MV ctrl and MVstim measured by NTA. Results are mean±SEM of 3 independent experiments. (E) The effect of MVs or hUCBMSCs on Treg induction after 7 days of MV or MSC cultured with allogeneic PBMCs. Representative flowcytometric analysis of CD25+-FOXP3+ Tregs are shown for CD4+ gated T lymphocytes. (F) The effect of MV and MSCs on T-cell proliferation analyzed by CFSE labelling of PBMCs and activation of T-cells with monoclonal CD3 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3860334&req=5

Figure 0001: (A) Scanning electron microscopic (FEI Quanta 250 FEG SEM) pictures of MVs produced by serum deprivation at magnification of 60,000× (i) and 90,000× (ii). Negative stainings (FEI Tecnai 12 TEM) of the same sample at magnification of 30,000×(iii). (B) Scanning electron microscopic pictures of MVs produced by IFN-γ stimulation at magnification of 60,000× (i) and 90,000× (ii). (C) Representative nanoparticle tracking analysis (NTA) profiles of MVctrl and MVstim. (D) Size distribution of MV ctrl and MVstim measured by NTA. Results are mean±SEM of 3 independent experiments. (E) The effect of MVs or hUCBMSCs on Treg induction after 7 days of MV or MSC cultured with allogeneic PBMCs. Representative flowcytometric analysis of CD25+-FOXP3+ Tregs are shown for CD4+ gated T lymphocytes. (F) The effect of MV and MSCs on T-cell proliferation analyzed by CFSE labelling of PBMCs and activation of T-cells with monoclonal CD3 antibody.
Mentions: hUCBMSCs were induced to produce MVs by culturing them in serum-free conditions with (MVstim) or without (MVctrl) IFN-γ-stimulation for 24–48 hours. Cell viability in both culture conditions was always >95% (n=10). Electron microscopy analysis of the vesicles showed variation in size, with the smallest being around 20 nm and the largest >500 nm (Fig. 1A and B). NTA confirmed the wide size range seen by electron microscopy analysis (Fig. 1C). Also, the presence of very small vesicles (<50 nm) was confirmed. There was no significant difference in the size distribution between IFN-γ stimulated and control conditions (Fig. 1D). Based on the analysis of protein amount, IFN-γ stimulus gave only slightly better MV yield (data not shown). The protein yield was on average 16 µg of protein in a vesicle fraction for every 10×106 cells after 24-h production. When hUCBMSCs or MVs derived from hUCBMSCs were co-cultured with PBMCs for 7 days, we were able to show that MVs alone as well as MSCs were able to induce the formation of T-cells with Treg phenotype (CD4+ CD25+ FOXP3+). The percentage of Tregs was increased from 3.3% to 4.5% when PBMCs were co-cultured with MSCs (Fig. 1E). However, we were not able to see any remarkable differences in Treg induction when PBMCs were co-cultured with either MVctrl (4.5%) or MVstim (4.0%).

Bottom Line: Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells.Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI.We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.

View Article: PubMed Central - PubMed

Affiliation: Finnish Red Cross Blood Service, Helsinki, Finland.

ABSTRACT

Background: Mesenchymal stromal cells (MSC) are shown to have a great therapeutic potential in many immunological disorders. Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells. Umbilical cord blood is an attractive but still less studied source of MSCs. We investigated the production of extracellular membrane vesicles (MVs) from human umbilical cord blood derived MSCs (hUCBMSC) in the presence (MVstim) or absence (MVctrl) of inflammatory stimulus.

Methods: hUCBMSCs were cultured in serum free media with or without IFN-γ and MVs were collected from conditioned media by ultracentrifugation. The protein content of MVs were analyzed by mass spectrometry. Hypoxia induced acute kidney injury rat model was used to analyze the in vivo therapeutic potential of MVs and T-cell proliferation and induction of regulatory T cells were analyzed by co-culture assays.

Results: Both MVstim and MVctrl showed similar T-cell modulation activity in vitro, but only MVctrls were able to protect rat kidneys from reperfusion injury in vivo. To clarify this difference in functionality we made a comparative mass spectrometric analysis of the MV protein contents. The IFN-γ stimulation induced dramatic changes in the protein content of the MVs. Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI. We further discovered that differently produced MV pools contained specific Rab proteins suggesting that same cells, depending on external signals, produce vesicles originating from different intracellular locations.

Conclusions: We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.

No MeSH data available.


Related in: MedlinePlus