Int J Stem Cells.  2019 Jul;12(2):279-290. 10.15283/ijsc18042.

FGF-17 from Hypoxic Human Wharton's Jelly-Derived Mesenchymal Stem Cells Is Responsible for Maintenance of Cell Proliferation at Late Passages

• Han KH
• Kim MH
• Jeong GJ
• Kim AK
• Chang JW
• Kim DI

• KMID: 2465898
• DOI: http://doi.org/10.15283/ijsc18042

Abstract

BACKGROUND AND OBJECTIVES
Although it is well known that hypoxic culture conditions enhance proliferation of human mesenchymal stem cells, the exact mechanism is not fully understood. In this study, we investigated the effect of fibroblast growth factor (FGF)-17 from hypoxic human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs) on cell proliferation at late passages.
METHODS AND RESULTS
hWJ-MSCs were cultured in α-MEM medium supplemented with 10% fetal bovine serum (FBS) in normoxic (21% O₂) and hypoxic (1% O₂) conditions. Protein antibody array was performed to analyze secretory proteins in conditioned medium from normoxic and hypoxic hWJ-MSCs at passage 10. Cell proliferation of hypoxic hWJ-MSCs was increased compared with normoxic hWJ-MSCs from passage 7 to 10, and expression of secretory FGF-17 was highly increased in conditioned medium from hypoxic hWJ-MSCs at passage 10. Knockdown of FGF-17 in hypoxic and normoxic hWJ-MSCs decreased cell proliferation, whereas treatment of hypoxic and normoxic hWJ-MSCs with recombinant protein FGF-17 increased their proliferation. Signal transduction of FGF-17 in hypoxic and normoxic hWJ-MSCs involved the ERK1/2 pathway. Cell phenotypes were not changed under either condition. Differentiation-related genes adiponectin, Runx2, and chondroadherin were downregulated in normoxic hWJ-MSCs treated with rFGF-17, and upregulated by siFGF-17. Expression of alkaline phosphatase (ALP), Runx2, and chondroadherin was upregulated in hypoxic hWJ-MSCs, and this effect was rescued by transfection with siFGF-17. Only chondroadherin was upregulated in hypoxic hWJ-MSCs with rFGF-17.
CONCLUSIONS
In hypoxic culture conditions, FGF-17 from hypoxic hWJ-MSCs contributes to the maintenance of high proliferation at late passages through the ERK1/2 pathway.

Keyword

FGF-17; Hypoxic culture; Mesenchymal stem cells; Proliferation

MeSH Terms

Adiponectin
Alkaline Phosphatase
Cell Proliferation*
Culture Media, Conditioned
Fibroblast Growth Factors
Humans*
Mesenchymal Stromal Cells*
Phenotype
Signal Transduction
Transfection
Adiponectin
Alkaline Phosphatase
Culture Media, Conditioned
Fibroblast Growth Factors

Figure

• 

  Fig. 1 Enhanced proliferation of hypoxic hWJ-MSCs and levels of secretory proteins at late passages. (A) Cell numbers of normoxic and hypoxic hWJ-MSCs from passage 6 to 10. Results are expressed as the mean±SEM from three independent experiments (n=3, *p<0.05, t-test). (B) Scatterplot of protein expression levels between normoxic and hypoxic hWJ-MSCs at passage 10. The red line above represents upregulated proteins (>2 fold), and the green line below represents downregulated proteins (>2 fold) in conditioned medium from hypoxic hWJ-MSCs compared with normoxic hWJ-MSCs. (C) Clustering of upregulated proteins and downregulated proteins in conditioned medium from hypoxic hWJ-MSCs compared with normoxic hWJ-MSCs at passage 10 (fold change >2.0). Results were from a single sample analysis. (D) Absolute amount of FGF-17 in CM from normoxic hWJ-MSCs and hypoxic hWJ-MSCs (1:3 diluted CM) (n=3, **p<0.01, t-test). (E) Expression of FGFR-1, −2, −3 and −4 in normoxic and hypoxic hWJ-MSCs at passage 10. Nor-MSC, normoxic mesenchymal stem cells; Hypo-MSC, hypoxic mesenchymal stem cells.

• 

  Fig. 2 FGF-17 of hypoxic hWJ-MSCs had a role in the increase of cell proliferation. Hypoxic hWJ-MSCs were transfected with siFGF-17 at passage 10 for 48 h. (A) Expression of FGF-17. (B) Images of cells. (C) Cell number and (D) BrdU staining. (E) PCNA and Ki67 expression. Control, negative control siRNA; siFGF-17, siRNA targeting FGF-17. Hypoxic hWJ-MSCs were treated with rFGF-17 at passage 10 for 48 h, (F) Images of cells. (G) Cell number and (H) Cell viability (500 ng/ml of rFGF-17 treatment). (I) Cell viability of hypoxic hWJ-MSCs with blocking antibody of FGF-17. Control, untreated hypoxic hWJ-MSCs; +rFGF-17, hypoxic hWJ-MSCs treated with rFGF-17; +FGF-17 Ab, hypoxic hWJ-MSCs with blocking antibody of FGF-17. Results are expressed as the mean±SEM from three independent experiments (n=3, *p<0.05, **p<0.01, t-test).

• 

  Fig. 3 FGF-17 of normoxic hWJ-MSCs had a role in the increase of cell proliferation. Normoxic hWJ-MSCs were transfected with siFGF-17 at passage 7 for 48 h. (A) Expression of FGF-17. (B) Images of cells. (C) Cell number and (D) BrdU staining. Control, negative control siRNA; siFGF-17, siRNA targeting FGF-17. Normoxic hWJ-MSCs were treated with rFGF-17 at passage 7 for 48 h. (E) Images of cells. (F) Cell numbers. (G) Cell viability (500 ng/ml of rFGF-17 treatment). (H) PCNA and Ki67 expression. Control, untreated normoxic hWJ-MSCs; +rFGF-17, normoxic hWJ-MSCs treated with rFGF-17. Results are expressed as the mean±SEM from three independent experiments (n=3, *p<0.05, **p<0.01, t-test).

• 

  Fig. 4 Effects of FGF-17 on signal transduction in hypoxic and normoxic hWJ-MSCs. (A) Expression of proteins p21, p27, p53, phospho-AKT, phospho-STAT3, phospho-ERK, ERK and GAPDH in hypoxic hWJ-MSCs transfected with siFGF-17. (B) Relative expression of phospho-ERK in hypoxic hWJ-MSCs transfected with siFGF-17 (n=3, *p<0.05, t-test). Control, hypoxic hWJ-MSC only; siFGF-17, hypoxic hWJ-MSCs with siFGF-17. (C) Expression of proteins p21, p27, p53, phospho-AKT, phospho-STAT3, phospho-ERK, ERK and GAPDH in hypoxic hWJ-MSCs treated with 500 ng/ml of rFGF-17. (D) Relative expression of phospho-ERK in hypoxic hWJ-MSCs treated with 500 ng/ml of rFGF-17 (n=3, *p<0.05, t-test). Control, hypoxic hWJ-MSC only; +rFGF-17, hypoxic hWJ-MSCs with rFGF-17. (E) Expression of proteins p21, p27, p53, phospho-AKT, phospho-STAT3, phospho-ERK, ERK and GAPDH in normoxic hWJ-MSCs transfected with siFGF-17. (F) Relative expression of phospho-ERK in normoxic hWJ-MSCs transfected with siFGF-17 (n=3, *p<0.05, t-test). Control, normoxic hWJ-MSC only; siFGF-17, normoxic hWJ-MSCs with siFGF-17. (G) Expression of proteins p21, p27, p53, phospho-AKT, phospho-STAT3, phospho-ERK, ERK and GAPDH in normoxic hWJ-MSCs treated with 500 ng/ml of rFGF-17. (H) Relative expression of phospho-ERK in normoxic hWJ-MSCs treated with 500 ng/ml of rFGF-17 (n=3, **p<0.01, t-test). Control, normoxic hWJ-MSC only; +rFGF-17, normoxic hWJ-MSCs with rFGF-17. Nor-MSC, normoxic mesenchymal stem cells; Hypo-MSC, hypoxic mesenchymal stem cells.

• 

  Fig. 5 Effects of FGF-17 on phenotypic characterization of hypoxic and normoxic hWJ-MSCs. (A, B) Negative phenotype markers (CD31, CD34, CD45, and CD144) and positive phenotype markers (CD44, CD73, CD90, and CD105) were analyzed in hypoxic hWJ-MSCs treated with siFGF-17 or rFGF-17 with specific antibody staining and flow cytometry. The gray bar corresponds to the isotype negative control, the dotted line corresponds to hypoxic hWJ-MSCs only, the strong black line indicates hypoxic hWJ-MSCs transfected with siFGF-17 or treated with 500 ng/ml of rFGF-17. (C, D) The same phenotype markers were analyzed in normoxic hWJ-MSCs treated with siFGF-17 or rFGF-17 with specific antibody staining and flow cytometry. The gray bar corresponds to the isotype negative control, the dotted line corresponds to normoxic hWJ-MSCs only, and the strong black line indicates normoxic hWJ-MSCs transfected with siFGF-17 or treated with 500 ng/ml of rFGF-17. (E) Adipogenesis-related gene adiponectin, osteogenesis-related genes ALP and Runx2, and chondrogenesis genes chondroadherin and SOX9 were analyzed in hypoxic hWJ-MSCs transfected with siFGF-17 by QRT-PCR assay. Results are expressed as the mean±SEM from three independent experiments (n=3, *p<0.05; Nor-MSC vs. Hypo-MSC, †p<0.05; Hypo-MSC vs. Hypo-MSC+ siFGF-17, t-test). (F) The same differentiation-related genes were analyzed in hypoxic hWJ-MSCs treated with 500 ng/ml of rFGF-17. Results are expressed as the mean±SEM from three independent experiments (n=3, *p<0.05; Nor-MSC vs. Hypo-MSC, †p<0.05; Hypo-MSC vs. Hypo-MSC+rFGF-17, t-test). (G) The same differentiation-related genes were analyzed in normoxic hWJ-MSCs transfected with siFGF-17 by QRT-PCR assay. Results are expressed as the mean±SEM from three independent experiments (n=3, *p<0.05; Nor-MSC vs. Nor-MSC+ siFGF-17, t-test). (H) The same differentiation-related genes were analyzed in normoxic hWJ-MSCs treated with 500 ng/ml of rFGF-17. Results are expressed as the mean±SEM from three independent experiments (n=3, *p<0.05; Nor-MSC vs. Nor-MSC+rFGF-17, t-test). Nor-MSC, normoxic mesenchymal stem cells; Hypo-MSC, hypoxic mesenchymal stem cells.

Reference

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