Limits...
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

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.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4499789&req=5

f4: 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.

Mentions: The neuropoiesis assay [23] quantifies growth and differentiation of rat embryonic cortical cells promoted by small numbers of human MSC in direct cocultures. This sensitive assay was used to (i) assess the activity of extracts and (ii) compare the activity of extracts to the activity of live and dead cells. For these experiments, a working suspension of either MSC or SB623 in NB was divided into three aliquots. One aliquot was left intact; cells were alive (denoted “A”). Two aliquots were frozen and then thawed, which made them suspensions of dead cells (denoted “D”). One of these two aliquots was then cleared by centrifugation, becoming the extract (denoted “E”). All three aliquots were plated at identical dilutions, which corresponded to 500, 250, and 125 of live MSC or SB623 cells/well. Cortical cells (5,000 cells/well) were added to all wells. After culturing, expression of rat nestin, rat GFAP, and human GAP were determined (Fig. 4A–C, correspondingly). It should be noted that extracts in this experiment had 40 times lower strength than our standard extract preparations: indeed, an extract from 500 cells in 100 μL culture medium corresponded to 2.5% dilution of E0 prepared as described in the section “Materials and Methods” (106 cells/5 mL medium).


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: 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.
Mentions: The neuropoiesis assay [23] quantifies growth and differentiation of rat embryonic cortical cells promoted by small numbers of human MSC in direct cocultures. This sensitive assay was used to (i) assess the activity of extracts and (ii) compare the activity of extracts to the activity of live and dead cells. For these experiments, a working suspension of either MSC or SB623 in NB was divided into three aliquots. One aliquot was left intact; cells were alive (denoted “A”). Two aliquots were frozen and then thawed, which made them suspensions of dead cells (denoted “D”). One of these two aliquots was then cleared by centrifugation, becoming the extract (denoted “E”). All three aliquots were plated at identical dilutions, which corresponded to 500, 250, and 125 of live MSC or SB623 cells/well. Cortical cells (5,000 cells/well) were added to all wells. After culturing, expression of rat nestin, rat GFAP, and human GAP were determined (Fig. 4A–C, correspondingly). It should be noted that extracts in this experiment had 40 times lower strength than our standard extract preparations: indeed, an extract from 500 cells in 100 μL culture medium corresponded to 2.5% dilution of E0 prepared as described in the section “Materials and Methods” (106 cells/5 mL medium).

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