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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

Peripheral blood mononuclear cells (PBMC)-mediated cytotoxic injury of MSC and SB623, and FGF2 release in cocultures. (A) MSC (M) or SB623 (S) were cocultured with 10- or 30-fold excess of PBMC (10×P or 30×P, correspondingly). Release of LDH and FGF2 into the culture medium by these cocultures or by separately plated mesenchymal cells or PBMC was measured. Calculations of specific LDH release (ie, specific cell lysis) and specific FGF2 release were done the same way. (B) FGF2 concentrations measured for calculating specific FGF2 release (in A) are shown.
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f5: Peripheral blood mononuclear cells (PBMC)-mediated cytotoxic injury of MSC and SB623, and FGF2 release in cocultures. (A) MSC (M) or SB623 (S) were cocultured with 10- or 30-fold excess of PBMC (10×P or 30×P, correspondingly). Release of LDH and FGF2 into the culture medium by these cocultures or by separately plated mesenchymal cells or PBMC was measured. Calculations of specific LDH release (ie, specific cell lysis) and specific FGF2 release were done the same way. (B) FGF2 concentrations measured for calculating specific FGF2 release (in A) are shown.

Mentions: When cells are injected into the brain, damage to small brain vessels may occur. This disruption of the vasculature may result in exposure of the implanted cells to local PBMC and subsequent cytotoxic effects. Preliminary experiments were conducted to establish in vitro conditions, in which PBMC cytotoxicity toward MSC could be observed. Although there was high degree of variability in the cytotoxic effect of PBMC with different MSC or SB623 lots, we observed that in all assays, cytotoxicity was stronger at 18 h than at 5 h post exposure and also greater when PBMCs were pre-cultured for 7 days, as opposed to 1 day. The presence of IL2 in PBMC cultures did not affect their cytotoxicity. Therefore, PBMC-mediated cell lysis and target cell release of FGF2 were assessed in 18 h-cocultures of PBMCs (pre-cultured for 7 days without IL2) and target cells (either MSC or SB623). An example of these data is shown in Fig. 5A. The specific cell lysis was directly dependent on PBMC:target cell ratios and varied from 30% to 90% at 30:1 PBMC/target cell ratio for different donors of MSC, SB623, and PBMC. Specific FGF2 release was expressed the same way as was the specific cell lysis. The percentage of specific FGF2 release was lower than that of LDH by about 2–2.5 times, but nevertheless correlated well with the specific cell lysis. Although in this experiment specific lysis in MSC-PBMC cocultures was higher than that in SB623-PBMC cocultures, more FGF2 was released in latter case, due to higher intracellular levels in SB623 (Fig. 5B). This together with higher spontaneous release of FGF2 from SB623 cells in the absence of PBMC lead to approximately equal specific FGF2 release for MSC- and SB623-PBMC cocultures. The result showed that substantial amounts of FGF2 can be released by both MSC and SB623 as a result of the cytotoxic effects of PBMC.


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)

Peripheral blood mononuclear cells (PBMC)-mediated cytotoxic injury of MSC and SB623, and FGF2 release in cocultures. (A) MSC (M) or SB623 (S) were cocultured with 10- or 30-fold excess of PBMC (10×P or 30×P, correspondingly). Release of LDH and FGF2 into the culture medium by these cocultures or by separately plated mesenchymal cells or PBMC was measured. Calculations of specific LDH release (ie, specific cell lysis) and specific FGF2 release were done the same way. (B) FGF2 concentrations measured for calculating specific FGF2 release (in A) are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Peripheral blood mononuclear cells (PBMC)-mediated cytotoxic injury of MSC and SB623, and FGF2 release in cocultures. (A) MSC (M) or SB623 (S) were cocultured with 10- or 30-fold excess of PBMC (10×P or 30×P, correspondingly). Release of LDH and FGF2 into the culture medium by these cocultures or by separately plated mesenchymal cells or PBMC was measured. Calculations of specific LDH release (ie, specific cell lysis) and specific FGF2 release were done the same way. (B) FGF2 concentrations measured for calculating specific FGF2 release (in A) are shown.
Mentions: When cells are injected into the brain, damage to small brain vessels may occur. This disruption of the vasculature may result in exposure of the implanted cells to local PBMC and subsequent cytotoxic effects. Preliminary experiments were conducted to establish in vitro conditions, in which PBMC cytotoxicity toward MSC could be observed. Although there was high degree of variability in the cytotoxic effect of PBMC with different MSC or SB623 lots, we observed that in all assays, cytotoxicity was stronger at 18 h than at 5 h post exposure and also greater when PBMCs were pre-cultured for 7 days, as opposed to 1 day. The presence of IL2 in PBMC cultures did not affect their cytotoxicity. Therefore, PBMC-mediated cell lysis and target cell release of FGF2 were assessed in 18 h-cocultures of PBMCs (pre-cultured for 7 days without IL2) and target cells (either MSC or SB623). An example of these data is shown in Fig. 5A. The specific cell lysis was directly dependent on PBMC:target cell ratios and varied from 30% to 90% at 30:1 PBMC/target cell ratio for different donors of MSC, SB623, and PBMC. Specific FGF2 release was expressed the same way as was the specific cell lysis. The percentage of specific FGF2 release was lower than that of LDH by about 2–2.5 times, but nevertheless correlated well with the specific cell lysis. Although in this experiment specific lysis in MSC-PBMC cocultures was higher than that in SB623-PBMC cocultures, more FGF2 was released in latter case, due to higher intracellular levels in SB623 (Fig. 5B). This together with higher spontaneous release of FGF2 from SB623 cells in the absence of PBMC lead to approximately equal specific FGF2 release for MSC- and SB623-PBMC cocultures. The result showed that substantial amounts of FGF2 can be released by both MSC and SB623 as a result of the cytotoxic effects of PBMC.

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