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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

Fibroblast growth factor (FGF)2 content of mesenchymal stromal cells (MSC) and SB623 cell extracts and conditioned media (CM). Total amounts of FGF2 in extracts (A) or CM (B) obtained from 1 million cells were calculated based on FGF2-enzyme-linked immunosorbent assay (ELISA) (A) or high sensitivity (HS)-FGF2-ELISA (B). (A) *P<0.05; No statistically significant difference in (B). (C) Lactate dehydrogenase (LDH) activity released from 1 million cells into extracts prepared from cryopreserved cells (E0) or from cells that were cultured to produce CM (E1). No statistically significant difference between MSC and SB623. Bars represent the average across seven cell lots. Error bars represent standard deviation.
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f1: Fibroblast growth factor (FGF)2 content of mesenchymal stromal cells (MSC) and SB623 cell extracts and conditioned media (CM). Total amounts of FGF2 in extracts (A) or CM (B) obtained from 1 million cells were calculated based on FGF2-enzyme-linked immunosorbent assay (ELISA) (A) or high sensitivity (HS)-FGF2-ELISA (B). (A) *P<0.05; No statistically significant difference in (B). (C) Lactate dehydrogenase (LDH) activity released from 1 million cells into extracts prepared from cryopreserved cells (E0) or from cells that were cultured to produce CM (E1). No statistically significant difference between MSC and SB623. Bars represent the average across seven cell lots. Error bars represent standard deviation.

Mentions: The release of FGF2 from SB623 and their parental MSC by either secretion or mechanical cell rupture was measured. Figure 1 represents averaged results from cell lots obtained from seven donors. Cryopreserved MSC and SB623 were thawed, washed, and subjected to one freeze/thaw cycle. The released cell contents were extracted (E0 extracts); and the amount of FGF2 was quantified (Fig. 1A). On average, 3.9 and 7.2 ng of FGF2 were released from 106 MSC and SB623, respectively. One freeze/thaw cycle was sufficient to kill all the cells (as tested with Trypan blue and cell plating), while each additional freeze/thaw cycle decreased FGF2 concentration by about 20% (not shown). CM conditioned by the same number of either MSC or SB623 cells contained only∼0.02 ng of FGF2 (Fig. 1B). To control for potential differences in cell metabolic activity, LDH was measured in E0 and also in cell extracts obtained after the production of CM by plated MSC and SB623 (E1) (Fig. 1C). LDH activity differed between E0 and E1 (0.3 vs. 0.13 U/106 cells, respectively), but not between MSC and SB623, indicating that metabolic activity dropped in starving cells compared with cells ruptured after cryopreservation, but was similar between MSC and SB623. The comparison between FGF2 contents of CM and E0 indicated that FGF2 was predominantly intracellular, while very little was secreted. This difference appeared to be true for FGF1, but not for VEGF and MCP1 (Table 1).


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)

Fibroblast growth factor (FGF)2 content of mesenchymal stromal cells (MSC) and SB623 cell extracts and conditioned media (CM). Total amounts of FGF2 in extracts (A) or CM (B) obtained from 1 million cells were calculated based on FGF2-enzyme-linked immunosorbent assay (ELISA) (A) or high sensitivity (HS)-FGF2-ELISA (B). (A) *P<0.05; No statistically significant difference in (B). (C) Lactate dehydrogenase (LDH) activity released from 1 million cells into extracts prepared from cryopreserved cells (E0) or from cells that were cultured to produce CM (E1). No statistically significant difference between MSC and SB623. Bars represent the average across seven cell lots. Error bars represent standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Fibroblast growth factor (FGF)2 content of mesenchymal stromal cells (MSC) and SB623 cell extracts and conditioned media (CM). Total amounts of FGF2 in extracts (A) or CM (B) obtained from 1 million cells were calculated based on FGF2-enzyme-linked immunosorbent assay (ELISA) (A) or high sensitivity (HS)-FGF2-ELISA (B). (A) *P<0.05; No statistically significant difference in (B). (C) Lactate dehydrogenase (LDH) activity released from 1 million cells into extracts prepared from cryopreserved cells (E0) or from cells that were cultured to produce CM (E1). No statistically significant difference between MSC and SB623. Bars represent the average across seven cell lots. Error bars represent standard deviation.
Mentions: The release of FGF2 from SB623 and their parental MSC by either secretion or mechanical cell rupture was measured. Figure 1 represents averaged results from cell lots obtained from seven donors. Cryopreserved MSC and SB623 were thawed, washed, and subjected to one freeze/thaw cycle. The released cell contents were extracted (E0 extracts); and the amount of FGF2 was quantified (Fig. 1A). On average, 3.9 and 7.2 ng of FGF2 were released from 106 MSC and SB623, respectively. One freeze/thaw cycle was sufficient to kill all the cells (as tested with Trypan blue and cell plating), while each additional freeze/thaw cycle decreased FGF2 concentration by about 20% (not shown). CM conditioned by the same number of either MSC or SB623 cells contained only∼0.02 ng of FGF2 (Fig. 1B). To control for potential differences in cell metabolic activity, LDH was measured in E0 and also in cell extracts obtained after the production of CM by plated MSC and SB623 (E1) (Fig. 1C). LDH activity differed between E0 and E1 (0.3 vs. 0.13 U/106 cells, respectively), but not between MSC and SB623, indicating that metabolic activity dropped in starving cells compared with cells ruptured after cryopreservation, but was similar between MSC and SB623. The comparison between FGF2 contents of CM and E0 indicated that FGF2 was predominantly intracellular, while very little was secreted. This difference appeared to be true for FGF1, but not for VEGF and MCP1 (Table 1).

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