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

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.

FIG. 1.

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.

FIG. 2.

Proliferative response of cortical Nestin-positive cells to extracts and the role of FGF2. (A) Proliferation in response to various concentrations of extract (E0) and CM from MSC. (B) Effect of FGF2 neutralization on cell proliferation stimulated by SB623-derived E0 at either 15% or 5% (equivalent to 0.2 and 0.07 ng/mL FGF2, respectively). *P<0.05; **P<0.01. Neutralizing anti-FGF2 (clone bFM1) or isotype control (mouse IgG1) were added at 2 μg/mL. (C, D) Immunostaining of rat cortical cells stimulated or not with 20% SB623-derived extract. After 7 days, cells cultures were labeled with 5-bromo-2-deoxyuridine (BRDU) and stained for either BRDU and Doublecortin (Dcx) (C) or for BRDU and Nestin (Nes) (D). We concluded that cells predominantly labeled with BRDU and increased in numbers in extract-treated cultures were nestin-positive cells.

FIG. 3.

Proliferative response of human umbilical vein endothelial cells (HUVEC) to extracts and the role of FGF2. (A) HUVEC proliferation in response to various dilutions of either MSC extract (E0), CM, or neurobasal medium after 2 day-culturing. (B) Proliferative responses of HUVEC to 15% SB623-extract (E0) (final FGF2 concentration 0.2 ng/mL), rVEGF (10 ng/mL), and rFGF2 (1 ng/mL), and to FGF2-neutralization in E0. bFM1 and IgG1 are anti-FGF2 neutralizing antibody and mouse isotype control antibody, respectively, both at 2 μg/mL. *P<0.05. No statistically significant difference between E0 alone and E0+IgG1.

FIG. 4.

Neuropoietic activity of extracts in comparison to the activity of dead and alive cells. Neuropoiesis assay was performed with rat cortical cells. Rat cells were plated into cell-derived extracellular matrix-coated wells and incubated with SB623-derived samples. The samples were produced as following: the SB623 cell suspension was aliquoted; one aliquot was used as live cells (A, alive), another was used after freeze/thaw (D, dead), and another was cleared after freeze/thaw by centrifugation (E, extract). Numbers of human cells or their equivalent per well are indicated on X-axis. After culturing, relative expression of genes was accessed using quantitative reverse transcriptase-polymerase chain reaction: rat Nestin (A), rat glial fibrillary acidic protein (GFAP) (B), and human glyceraldehyde 3 phosphate dehydrogenase (GAP) (C) expression levels were analyzed from the same wells. In another experiment (D, E), cell suspensions of live and dead cells were mixed in indicated proportions and their effect on rat GFAP expression was compared to effects of live or dead cells alone. *P<0.005. (E) Human GAP expression was analyzed in same wells as in (D) to confirm relative numbers of live human cells.

FIG. 5.

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.

FIG. 6.

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.