RHD-specific microRNA for regulation of the DEL blood group: integration of computational and experimental approaches.

OBJECTIVE: The discovery of specific microRNAs (miRNA) mediates a better understanding of molecular mechanisms, diagnosis and prognosis of complex phenotypes. Synthesis of the RhD blood group involves multiple factors causing variation in the expression of RHD antigens. The mechanism underlying the extremely weak expression of RHD antigen associated with the RHD variant called DEL (D-elute) is incompletely understood. Down-regulation of gene expression through miRNA is a guide to the potential involvement of miRNAs in the DEL blood group. In order to determine the association of miRNAs and Rh-DEL blood donors with DEL variant, we investigated the expression level RHD-specific miRNA. METHODS: Blood samples were serologically tested for RhD blood group determination. DNA was analysed using SSP-PCR for the Asian-type DEL allele (RHD 1227 G>A). Bioinformatics analyses were applied for prediction of candidate RHD-specific miRNA. The RHD-specific miRNA expression level was quantitated using a real-time-qPCR approach. The miRNA expression levels of various RhD blood groups were compared and statistically analysed. RESULTS: The bioinformatics tools (n = 3) for prediction of miRNA targeting on RHD identified miR-98 as the miRNA potentially specific for the 3' UTR of RHD. The relative expression levels of miR-98 among D-positive (n = 50), D-negative (n = 49) and DEL (n = 63) subjects showed no statistically significant differences (P-values = 0.58). CONCLUSION: This is the first attempt to determine whether miR-98 is involved in RHD expression using computational and experimental approaches. Further investigations are necessary to fully characterize the miRNA genetics in DEL blood group regulation.

Re-using blood products as an alternative supplement in the optimisation of clinical-grade adipose-derived mesenchymal stem cell culture.

OBJECTIVES: Adipose-derived mesenchymal stem cells (ADMSCs) are a promising strategy for orthopaedic applications, particularly in bone repair. Ex vivo expansion of ADMSCs is required to obtain sufficient cell numbers. Xenogenic supplements should be avoided in order to minimise the risk of infections and immunological reactions. Human platelet lysate and human plasma may be an excellent material source for ADMSC expansion. In the present study, use of blood products after their recommended transfusion date to prepare human platelet lysate (HPL) and human plasma (Hplasma) was evaluated for in vitro culture expansion and osteogenesis of ADMSCs. METHODS: Human ADMSCs were cultured in medium supplemented with HPL, Hplasma and a combination of HPL and Hplasma (HPL+Hplasma). Characteristics of these ADMSCs, including osteogenesis, were evaluated in comparison with those cultured in fetal bovine serum (FBS). RESULTS: HPL and HPL+Hplasma had a significantly greater growth-promoting effect than FBS, while Hplasma exhibited a similar growth-promoting effect to that of FBS. ADMSCs cultured in HPL and/or Hplasma generated more colony-forming unit fibroblasts (CFU-F) than those cultured in FBS. After long-term culture, ADMSCs cultured in HPL and/or Hplasma showed reduced cellular senescence, retained typical cell phenotypes, and retained differentiation capacities into osteogenic and adipogenic lineages. CONCLUSION: HPL and Hplasma prepared from blood products after their recommended transfusion date can be used as an alternative and effective source for large-scale ex vivo expansion of ADMSCs.Cite this article: J. Phetfong, T. Tawonsawatruk, K. Seenprachawong, A. Srisarin, C. Isarankura-Na-Ayudhya, A. Supokawej. Re-using blood products as an alternative supplement in the optimisation of clinical-grade adipose-derived mesenchymal stem cell culture. Bone Joint Res 2017;6:414-422. DOI: 10.1302/2046-3758.67.BJR-2016-0342.R1.

Lipid-membrane affinity of chimeric metal-binding green fluorescent protein.

The Green Fluorescent Protein (GFP) is a useful marker to trace the expression of cellular proteins. However, little is known about changes in protein interaction properties after fusion to GFP. In this study, we present evidence for a binding affinity of chimeric cadmium-binding green fluorescent proteins to lipid membrane. This affinity has been observed in both cellular membranes and artificial lipid monolayers and bilayers. At the cellular level, the presence of Cd-binding peptide promoted the association of the chimeric GFP onto the lipid membrane, which declined the fluorescence emission of the engineered cells. Binding affinity to lipid membranes was further investigated using artificial lipid bilayers and monolayers. Small amounts of the chimeric GFP were found to incorporate into the lipid vesicles due to the high surface pressure of bilayer lipids. At low interfacial pressure of the lipid monolayer, incorporation of the chimeric Cd-binding GFP onto the lipid monolayer was revealed. From the measured lipid isotherms, we conclude that Cd-binding GFP mediates an increase in membrane fluidity and an expansion of the surface area of the lipid film. This evidence was strongly supported by epifluorescence microscopy, showing that the chimeric Cd-binding GFP preferentially binds to fluid-phase areas and defect parts of the lipid monolayer. All these findings demonstrate the hydrophobicity of the GFP constructs is mainly influenced by the fusion partner. Thus, the example of a metal-binding unit used here shines new light on the biophysical properties of GFP constructs.