Alternative protocols to induce chondrogenic differentiation: transforming growth factor-β superfamily

Abstract

Mesenchymal stem cells (MSCs) are an accepted candidate for cell-based therapy of multiple diseases. The interest in MSCs and their possible application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently, like aggregation and transforming growth factor beta (TGFβ) delivery, hypoxia has been indicated as crucial for complete chondrogenesis. The aim of this study was to test different culture conditions for directing stem cell differentiation into the chondrogenic lineage in vitro by testing different TGFβ superfamily members into the culture media under normoxic conditions. All chondrogenic culture conditions used allowed the differentiation of bone marrow-MSCs (BM-MSCs) into chondrogenic lineage. Chondrogenic induction capacity depended on the growth factor added to the culture media. In particular, the chondrogenic culture condition that better induced chondrogenesis was the medium that included the combination of three growth factors: bone morphogenetic protein-2 (BMP-2), BMP-7 and TGFβ-3. In this culture media, differentiated cells showed the highest levels expression of two markers of chondrogenesis, SOX9 and COL2A1, compared to the control points (p < 0.05, two-tailed t test). In our experimental conditions, the combination of BMP-2, BMP-7 and TGFβ-3 was the most effective in promoting chondrogenesis of BM-MSCs. These results underline the importance of determining in each experimental design the best protocol for in vitro directing stem cell differentiation into the chondrogenic lineage.

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Abbreviations

AA:

Ascorbic acid

AGG:

Aggrecan

ALP:

Alkaline phosphatase

APM1:

Adiponectin

BM-MSCs:

Bone marrow-mesenchymal stem cells

BMP-2:

Bone morphogenetic protein-2

BMP-7:

Bone morphogenetic protein-7

cDNA:

Complementary deoxyribonucleic acid

COL10A1:

Collagen type XA1

COL1A1:

Collagen type IA1

COL2A1:

Collagen type IIA1

DMEM:

Dulbecco’s modified Eagle’s medium

FABP4:

Fatty acid-binding protein 4

FBS:

Fetal bovine serum

HE:

Hematoxylin–eosin

IGF-1:

Insulin-like growth factor-1

KO:

Knockout serum

LPL:

Lipoprotein lipase

MMP13:

Matrix metalloproteinase 13

MSCs:

Mesenchymal stem cells

MT:

Masson’s trichrome

MTG:

Monotioglycerol

OP:

Osteoprotegerin

OP-1:

Osteogenic protein 1

P/S:

Penicillin and streptomycin

PCR:

Polymerase chain reaction

qPCR:

Real-Time PCR

REL:

Relative expression levels

rHuBMP-2:

Recombinant human bone morphogenetic protein 2

RNA:

Ribonucleic acid

SaO:

Safranin O

SOX9:

Sex determining region Y-box 9

TB:

Toluidine blue

TBP:

TATA box binding protein

TGFβ:

Transforming growth factor beta

TGFβ-3:

Transforming growth factor beta-3

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Acknowledgments

This study was supported by grants: Servizo Galego de Saúde, Xunta de Galicia (PS07/84), Cátedra Bioiberica de la Universidade da Coruña and Instituto de Salud Carlos III CIBER BBN; Ministerio Ciencia e Innovacion PLE2009-0144; Fondo Investigacion Sanitaria-PI 08/2028 with participation of funds from FEDER (European Community), Tamara Hermida-Gómez is the beneficiary of a contract from Fondo de Investigación Sanitaria (2008), Spain. We would like to thank P.Filgueira and M.J.Sánchez for technical assistance.

Conflict of interest

The authors declare that no competing financial interest exists.

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Correspondence to Silvia Díaz-Prado or Francisco J. Blanco.

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Cicione, C., Muiños-López, E., Hermida-Gómez, T. et al. Alternative protocols to induce chondrogenic differentiation: transforming growth factor-β superfamily. Cell Tissue Bank 16, 195–207 (2015). https://doi.org/10.1007/s10561-014-9472-7

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Keywords

  • Cell therapy
  • Autologous chondrocyte implantation
  • Focal lesions
  • Hyaline articular cartilage