The Role of Melanotransferrin (CD228) in the regulation of the differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells (hBM-MSC)

Abstract

Melanotransferrin (CD228), firstly reported as a melanoma-associated antigen, is a membrane-bound glycoprotein of an iron-binding transferrin homolog. CD228 was found to be expressed significantly higher in human bone marrow-derived mesenchymal stem cells (hBM-MSC) than in human embryonic fibroblasts (FB) by RT-PCR, western blotting and flow cytometry. The expression of CD228 declined in aged hBM-MSC as osteogenesis-related genes did. We examined a possible role for CD228 in the regulation of osteogenesis and adipogenesis of hBM-MSC. Surprisingly, siRNA-mediated CD228 knockdown increased the expression of the transcription factor DLX5 and enhanced osteogenesis of hBM-MSC evidenced by an increased expression of the runt-related transcription factor 2 (RUNX2), osterix (Osx), and osteocalcin (OC), as well as higher alkaline phosphatase (ALP) activity and extracellular calcium deposition. Interestingly, hBM-MSC transfected with CD228 siRNA also showed an increase in intracellular lipid level during adipogenesis, indicated by oil red O staining of differentiated adipocytes. Overall, our study unveils CD228 as a cell surface molecule expressed by young hBM-MSC, but not by FB. It also provides evidence to suggest a role for CD228 as a negative regulator of osteogenesis and of lipid accumulation during adipogenesis in hBM-MSC in vitro.

Competitive Hybridization of a Microarray Identifies CMKLR1 as an Up-Regulated Gene in Human Bone Marrow-Derived Mesenchymal Stem Cells Compared to Human Embryonic Fibroblasts

“…The hBMSCs (ScienCell Research Laboratories, Carlsbad, CA, USA) were grown in α-MEM (Life Tech., Carlsbad, CA, USA) supplemented with 16.5% fetal bovine serum (FBS, Lonza, Basel, Switzerland), 100 units/mL penicillin and 100 µg/mL streptomycin (HyClone, Logan, UT, USA). Multipotency and expression of MSC-specific CD markers proposed by International Society for Cellular Therapy (ISCT ® ) were previously confirmed for the hBMSC employed in this experiment [19,33]. Human embryonic fibroblasts (hEF) [19,34] were grown in Dulbecco's modified Eagle's medium (DMEM) (Life Tech) supplemented with 10% FBS, L-glutamine (Life Tech), 100 units/mL penicillin and 100 µg/mL streptomycin.…”

“…Multipotency and expression of MSC-specific CD markers proposed by International Society for Cellular Therapy (ISCT ® ) were previously confirmed for the hBMSC employed in this experiment [19,33]. Human embryonic fibroblasts (hEF) [19,34] were grown in Dulbecco's modified Eagle's medium (DMEM) (Life Tech) supplemented with 10% FBS, L-glutamine (Life Tech), 100 units/mL penicillin and 100 µg/mL streptomycin. Both hBMSC and hEF were incubated at 37 • C in a humidified 5% CO 2 incubator.…”

“…CD106 and CD146 are found to be highly expressed in the MSC, while CD10 and CD26 expression is high in the fibroblast in comparison with the other cells [18]. Recently, CD200 and CD228 have been found to be differentially expressed between the two cells [19]. Once the expression pattern of these new markers is confirmed to be consistent in various MSC preparations, they could be included in an MSC-specific surface marker panel.…”

“…hBMSC younger than passage eight was plated at 4 × 10 3 cells/well in a 96-well plate or 4 × 10 5 cells in a 60-mm dish one day prior to treatment of osteoblast differentiation medium (ODM). ODM consisted of 100 nM dexamethasone, 50 µM ascobate-2-phosphate and 10 mM β-glycerophosphate in 10% FBS-supplemented α-MEM [19]. ODM with or without 100 ng/mL RARRES2 was refreshed every 3-4 days up to 14 days.…”

“…The hBMSCs were seeded at 4 × 10 3 cells/well in a 96-well plate one day prior to treatment of ODM with or without 100 ng/mL RARRES2 for cell viability and alkaline phosphatase assays. Cell viability was measured on day 4 of ODM treatment by MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, Sigma Aldrich) as described in Huynh et al [19]. MTT/well (100 µL 0.5 mg/mL in 10% FBS-supplemented α-MEM) was incubated for 3 h at 37 • C. An equal amount of formazan solubilizer (10% SDS in 0.01 N HCl) was added and further incubated at 37 • C overnight.…”