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Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells.

Kinzebach S, Dietz L, Klüter H, Thierse HJ, Bieback K - BMC Cell Biol. (2013)

Bottom Line: Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP.Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS.The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Service Baden-Württemberg, Friedrich-Ebert-Str, 107, Mannheim, Hessen D-68167, Germany. thierse@immunoproteomics.de.

ABSTRACT

Background: Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay.

Results: Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation.In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system.

Conclusions: The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion.

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Influence of selected cytokines on MSC proliferation; receptor expression profiles and concentration. (A) MSC were seeded in 5% pHPL, tPRP and FBS, respectively, and stimulated with for 3 days with 50 ng/ml HGF, 10 ng/ml IGF-1 or 25 ng/ml bFGF. Cell counts were acquired with the CellTiter-Glo assay and then normalized to the unstimulated control to derive relative cell count values. (B)% positivity of IGF, FGF and HGF receptor expression of BM- and LA-MSC (donors 1–3, respectively) in pHPL, tPRP and FBS assessed by flow cytometry. (C – E): IGF, FGF and HGF concentrations were determined by ELISA in pHPL and tPRP supplemented medium (medium, 6 different batches); medium stored for 24 h (control medium) and conditioned by MSC (CM) (each n = 3). Symbols indicate statistically significant diffences between: * stimulation; + supplements; # MSC sources; (one symbol p < 0.05; two symbols p < 0.01).
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Figure 4: Influence of selected cytokines on MSC proliferation; receptor expression profiles and concentration. (A) MSC were seeded in 5% pHPL, tPRP and FBS, respectively, and stimulated with for 3 days with 50 ng/ml HGF, 10 ng/ml IGF-1 or 25 ng/ml bFGF. Cell counts were acquired with the CellTiter-Glo assay and then normalized to the unstimulated control to derive relative cell count values. (B)% positivity of IGF, FGF and HGF receptor expression of BM- and LA-MSC (donors 1–3, respectively) in pHPL, tPRP and FBS assessed by flow cytometry. (C – E): IGF, FGF and HGF concentrations were determined by ELISA in pHPL and tPRP supplemented medium (medium, 6 different batches); medium stored for 24 h (control medium) and conditioned by MSC (CM) (each n = 3). Symbols indicate statistically significant diffences between: * stimulation; + supplements; # MSC sources; (one symbol p < 0.05; two symbols p < 0.01).

Mentions: Cytokines were not identified within the current 2D DIGE approach probably due to their often low concentration in plasma. Therefore, within a second set of experiments selected cytokines, known to foster MSC proliferation in FBS [40-42], were added to the different supplements and MSC proliferation was measured. Here we a) tested the effect of adding different concentrations of cytokines, b) evaluated growth factor receptor expression and c) the concentration of cytokines in the culture medium/conditionened medium. We decided to use 50 ng/ml HGF, 10 ng/ml IGF-1 and 25 ng/ml bFGF [35,40,41]. Cytokines failed to induce a proliferative response in MSC cultured with 5% platelet derivatives (Figure 4A), whereas control cells in 5% FBS responded adequately. bFGF inhibited BM-MSC proliferation in the human supplements. Comparable findings have been observed by Cheon et al. testing the effects of EGF, FGF and ITS (insulin, transferring, selenium) on AT-MSC proliferation in FBS or human serum. In both settings, the combination of ITS and growth factors induced proliferation, whereas FGF on its own was highly effective in FBS only [43]. Addition of EGF, bFGF and PDGFbb to 3% platelet poor plasma, however, reached a proliferation rate comparable to FBS [44], advising a fine tuning in multifactorial design studies.


Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells.

Kinzebach S, Dietz L, Klüter H, Thierse HJ, Bieback K - BMC Cell Biol. (2013)

Influence of selected cytokines on MSC proliferation; receptor expression profiles and concentration. (A) MSC were seeded in 5% pHPL, tPRP and FBS, respectively, and stimulated with for 3 days with 50 ng/ml HGF, 10 ng/ml IGF-1 or 25 ng/ml bFGF. Cell counts were acquired with the CellTiter-Glo assay and then normalized to the unstimulated control to derive relative cell count values. (B)% positivity of IGF, FGF and HGF receptor expression of BM- and LA-MSC (donors 1–3, respectively) in pHPL, tPRP and FBS assessed by flow cytometry. (C – E): IGF, FGF and HGF concentrations were determined by ELISA in pHPL and tPRP supplemented medium (medium, 6 different batches); medium stored for 24 h (control medium) and conditioned by MSC (CM) (each n = 3). Symbols indicate statistically significant diffences between: * stimulation; + supplements; # MSC sources; (one symbol p < 0.05; two symbols p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231358&req=5

Figure 4: Influence of selected cytokines on MSC proliferation; receptor expression profiles and concentration. (A) MSC were seeded in 5% pHPL, tPRP and FBS, respectively, and stimulated with for 3 days with 50 ng/ml HGF, 10 ng/ml IGF-1 or 25 ng/ml bFGF. Cell counts were acquired with the CellTiter-Glo assay and then normalized to the unstimulated control to derive relative cell count values. (B)% positivity of IGF, FGF and HGF receptor expression of BM- and LA-MSC (donors 1–3, respectively) in pHPL, tPRP and FBS assessed by flow cytometry. (C – E): IGF, FGF and HGF concentrations were determined by ELISA in pHPL and tPRP supplemented medium (medium, 6 different batches); medium stored for 24 h (control medium) and conditioned by MSC (CM) (each n = 3). Symbols indicate statistically significant diffences between: * stimulation; + supplements; # MSC sources; (one symbol p < 0.05; two symbols p < 0.01).
Mentions: Cytokines were not identified within the current 2D DIGE approach probably due to their often low concentration in plasma. Therefore, within a second set of experiments selected cytokines, known to foster MSC proliferation in FBS [40-42], were added to the different supplements and MSC proliferation was measured. Here we a) tested the effect of adding different concentrations of cytokines, b) evaluated growth factor receptor expression and c) the concentration of cytokines in the culture medium/conditionened medium. We decided to use 50 ng/ml HGF, 10 ng/ml IGF-1 and 25 ng/ml bFGF [35,40,41]. Cytokines failed to induce a proliferative response in MSC cultured with 5% platelet derivatives (Figure 4A), whereas control cells in 5% FBS responded adequately. bFGF inhibited BM-MSC proliferation in the human supplements. Comparable findings have been observed by Cheon et al. testing the effects of EGF, FGF and ITS (insulin, transferring, selenium) on AT-MSC proliferation in FBS or human serum. In both settings, the combination of ITS and growth factors induced proliferation, whereas FGF on its own was highly effective in FBS only [43]. Addition of EGF, bFGF and PDGFbb to 3% platelet poor plasma, however, reached a proliferation rate comparable to FBS [44], advising a fine tuning in multifactorial design studies.

Bottom Line: Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP.Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS.The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Service Baden-Württemberg, Friedrich-Ebert-Str, 107, Mannheim, Hessen D-68167, Germany. thierse@immunoproteomics.de.

ABSTRACT

Background: Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay.

Results: Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation.In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system.

Conclusions: The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion.

Show MeSH
Related in: MedlinePlus