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Cell Injury-Induced Release of Fibroblast Growth Factor 2: Relevance to Intracerebral Mesenchymal Stromal Cell Transplantations.

Aizman I, Vinodkumar D, McGrogan M, Bates D - Stem Cells Dev. (2015)

Bottom Line: We found that fibroblast growth factor (FGF)2 and FGF1, but not vascular endothelial growth factor and monocyte chemoattractant protein 1 levels were high in extracts despite being low in conditioned media.However, suspensions of killed cells strongly potentiated the astrogenic effects of live MSC.We therefore propose that both dying and surviving grafted MSC contribute to tissue regeneration.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Research, SanBio, Inc. , Mountain View, California.

ABSTRACT
Beneficial effects of intracerebral transplantation of mesenchymal stromal cells (MSC) and their derivatives are believed to be mediated mostly by factors produced by engrafted cells. However, the mesenchymal cell engraftment rate is low, and the majority of grafted cells disappear within a short post-transplantation period. Here, we hypothesize that dying transplanted cells can affect surrounding tissues by releasing their active intracellular components. To elucidate the type, amounts, and potency of these putative intracellular factors, freeze/thaw extracts of MSC or their derivatives were tested in enzyme-linked immunosorbent assays and bioassays. We found that fibroblast growth factor (FGF)2 and FGF1, but not vascular endothelial growth factor and monocyte chemoattractant protein 1 levels were high in extracts despite being low in conditioned media. Extracts induced concentration-dependent proliferation of rat cortical neural progenitor cells and human umbilical vein endothelial cells; these proliferative responses were specifically blocked by FGF2-neutralizing antibody. In the neuropoiesis assay with rat cortical cells, both MSC extracts and killed cells induced expression of nestin, but not astrocyte differentiation. However, suspensions of killed cells strongly potentiated the astrogenic effects of live MSC. In transplantation-relevant MSC injury models (peripheral blood cell-mediated cytotoxicity and high cell density plating), MSC death coincided with the release of intracellular FGF2. The data showed that MSC contain a major depot of active FGF2 that is released upon cell injury and is capable of acutely stimulating neuropoiesis and angiogenesis. We therefore propose that both dying and surviving grafted MSC contribute to tissue regeneration.

No MeSH data available.


Related in: MedlinePlus

FGF2 release by MSC and SB623 in high cell density/hypoxia/insufficient nutrients cultures. MSC or SB623 were plated in round 96-well plates at 1×106 cells/mL, 350 μL/well, and tightly closed to prevent gas exchange. At the indicated time points, the well contents were collected. FGF2 (A) and LDH activity (B) were measured in both culture medium (CM) and cells (intracellular) after releasing intracellular contents by freeze/thaw treatment. The results are expressed per million cells.
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f6: FGF2 release by MSC and SB623 in high cell density/hypoxia/insufficient nutrients cultures. MSC or SB623 were plated in round 96-well plates at 1×106 cells/mL, 350 μL/well, and tightly closed to prevent gas exchange. At the indicated time points, the well contents were collected. FGF2 (A) and LDH activity (B) were measured in both culture medium (CM) and cells (intracellular) after releasing intracellular contents by freeze/thaw treatment. The results are expressed per million cells.

Mentions: When MSC or their derivatives are transplanted into a peri-infarct zone, they are deposited at high density into a hostile environment with limited diffusion of oxygen and nutrients. To model this environment in vitro, MSCs or SB623 cells were plated at very high density in round-bottom wells of 96-well plates in NB/B27/GLX and incubated sealed to prevent gas-exchange. The media became rapidly acidic indicating a hypoxic environment. Cells tended to adhere homotypically and very few cells were able to attach to the bottom of the well. Of note, even after 5 days of culturing in this harsh environment, these cultures still contained a few living cells that were able to attach, grow, and proliferate, if replated under normal growth conditions (data not shown). The contents of the wells were harvested at several time points, centrifuged to separate CM from cells and debris, and the pellets were freeze/thawed to release the intracellular contents from surviving cells. Measurement of FGF2 and LDH in medium (“CM”) and after mechanical damage (“intracellular”) showed that while intracellular FGF2 content quickly dropped within 20 h of initial plating, levels of released FGF2 (in CM) were quite steady between 4 h and 2 days; with substantially higher FGF2 levels released from SB623, than from MSC (around 2 vs. 0.5 ng/1 million cells, respectively) (Fig. 6). A decrease in the level of FGF2 in the CM levels was detected on day 5. The LDH pattern was different to that of FGF2: intracellular LDH activity remained high longer than did FGF2. This result indicated that dying cells release high levels of intracellular FGF2 when placed in harsh culture conditions, while surviving cells reduce their intracellular FGF2 levels. It appeared that FGF2 released into the media remained stable for some time despite these conditions. We also noticed that SB623 typically survived better than MSCs in these harsh conditions as judged by higher levels of intracellular LDH at later time points.


Cell Injury-Induced Release of Fibroblast Growth Factor 2: Relevance to Intracerebral Mesenchymal Stromal Cell Transplantations.

Aizman I, Vinodkumar D, McGrogan M, Bates D - Stem Cells Dev. (2015)

FGF2 release by MSC and SB623 in high cell density/hypoxia/insufficient nutrients cultures. MSC or SB623 were plated in round 96-well plates at 1×106 cells/mL, 350 μL/well, and tightly closed to prevent gas exchange. At the indicated time points, the well contents were collected. FGF2 (A) and LDH activity (B) were measured in both culture medium (CM) and cells (intracellular) after releasing intracellular contents by freeze/thaw treatment. The results are expressed per million cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4499789&req=5

f6: FGF2 release by MSC and SB623 in high cell density/hypoxia/insufficient nutrients cultures. MSC or SB623 were plated in round 96-well plates at 1×106 cells/mL, 350 μL/well, and tightly closed to prevent gas exchange. At the indicated time points, the well contents were collected. FGF2 (A) and LDH activity (B) were measured in both culture medium (CM) and cells (intracellular) after releasing intracellular contents by freeze/thaw treatment. The results are expressed per million cells.
Mentions: When MSC or their derivatives are transplanted into a peri-infarct zone, they are deposited at high density into a hostile environment with limited diffusion of oxygen and nutrients. To model this environment in vitro, MSCs or SB623 cells were plated at very high density in round-bottom wells of 96-well plates in NB/B27/GLX and incubated sealed to prevent gas-exchange. The media became rapidly acidic indicating a hypoxic environment. Cells tended to adhere homotypically and very few cells were able to attach to the bottom of the well. Of note, even after 5 days of culturing in this harsh environment, these cultures still contained a few living cells that were able to attach, grow, and proliferate, if replated under normal growth conditions (data not shown). The contents of the wells were harvested at several time points, centrifuged to separate CM from cells and debris, and the pellets were freeze/thawed to release the intracellular contents from surviving cells. Measurement of FGF2 and LDH in medium (“CM”) and after mechanical damage (“intracellular”) showed that while intracellular FGF2 content quickly dropped within 20 h of initial plating, levels of released FGF2 (in CM) were quite steady between 4 h and 2 days; with substantially higher FGF2 levels released from SB623, than from MSC (around 2 vs. 0.5 ng/1 million cells, respectively) (Fig. 6). A decrease in the level of FGF2 in the CM levels was detected on day 5. The LDH pattern was different to that of FGF2: intracellular LDH activity remained high longer than did FGF2. This result indicated that dying cells release high levels of intracellular FGF2 when placed in harsh culture conditions, while surviving cells reduce their intracellular FGF2 levels. It appeared that FGF2 released into the media remained stable for some time despite these conditions. We also noticed that SB623 typically survived better than MSCs in these harsh conditions as judged by higher levels of intracellular LDH at later time points.

Bottom Line: We found that fibroblast growth factor (FGF)2 and FGF1, but not vascular endothelial growth factor and monocyte chemoattractant protein 1 levels were high in extracts despite being low in conditioned media.However, suspensions of killed cells strongly potentiated the astrogenic effects of live MSC.We therefore propose that both dying and surviving grafted MSC contribute to tissue regeneration.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Research, SanBio, Inc. , Mountain View, California.

ABSTRACT
Beneficial effects of intracerebral transplantation of mesenchymal stromal cells (MSC) and their derivatives are believed to be mediated mostly by factors produced by engrafted cells. However, the mesenchymal cell engraftment rate is low, and the majority of grafted cells disappear within a short post-transplantation period. Here, we hypothesize that dying transplanted cells can affect surrounding tissues by releasing their active intracellular components. To elucidate the type, amounts, and potency of these putative intracellular factors, freeze/thaw extracts of MSC or their derivatives were tested in enzyme-linked immunosorbent assays and bioassays. We found that fibroblast growth factor (FGF)2 and FGF1, but not vascular endothelial growth factor and monocyte chemoattractant protein 1 levels were high in extracts despite being low in conditioned media. Extracts induced concentration-dependent proliferation of rat cortical neural progenitor cells and human umbilical vein endothelial cells; these proliferative responses were specifically blocked by FGF2-neutralizing antibody. In the neuropoiesis assay with rat cortical cells, both MSC extracts and killed cells induced expression of nestin, but not astrocyte differentiation. However, suspensions of killed cells strongly potentiated the astrogenic effects of live MSC. In transplantation-relevant MSC injury models (peripheral blood cell-mediated cytotoxicity and high cell density plating), MSC death coincided with the release of intracellular FGF2. The data showed that MSC contain a major depot of active FGF2 that is released upon cell injury and is capable of acutely stimulating neuropoiesis and angiogenesis. We therefore propose that both dying and surviving grafted MSC contribute to tissue regeneration.

No MeSH data available.


Related in: MedlinePlus