A novel fully validated LC–MS/MS method for quantification of pyridoxal-5′-phosphate concentrations in samples of human whole blood

Quantification of pyridoxal-5´-phosphate (PLP) in biological samples is challenging due to the presence of endogenous PLP in matrices used for preparation of calibrators and quality control samples (QCs). Hence, we have developed an LC-MS/MS method for accurate and precise measurement of the concentrations of PLP in samples (20 µL) of human whole blood that addresses this issue by using a surrogate matrix and minimizing the matrix effect. We used a surrogate matrix comprising 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for making calibrators, QCs and the concentrations were adjusted to include the endogenous PLP concentrations in the surrogate matrix according to the method of standard addition. PLP was separated from the other components of the sample matrix using protein precipitation with trichloroacetic acid 10% w/v. After centrifugation, supernatant were injected directly into the LC-MS/MS system. Calibration curves were linear and recovery was > 92%. QCs were accurate, precise, stable for four freeze-thaw cycles, and following storage at room temperature for 17h or at -80 °C for 3 months. There was no significant matrix effect using 9 different individual human blood samples. Our novel LC-MS/MS method has satisfied all of the criteria specified in the 2012 EMEA guideline on bioanalytical method validation.

Human mesenchymal stem cells retain multilineage differentiation capacity including neural marker expression after extended in vitro expansion

The suitability of human mesenchymal stem cells (hMSCs) in regenerative medicine relies on retention of their proliferative expansion potential in conjunction with the ability to differentiate toward multiple lineages. Successful utilisation of these cells in clinical applications linked to tissue regeneration requires consideration of biomarker expression, time in culture and donor age, as well as their ability to differentiate towards mesenchymal (bone, cartilage, fat) or non-mesenchymal (e.g., neural) lineages. To identify potential therapeutic suitability we examined hMSCs after extended expansion including morphological changes, potency (stemness) and multilineage potential. Commercially available hMSC populations were expanded in vitro for > 20 passages, equating to > 60 days and > 50 population doublings. Distinct growth phases (A-C) were observed during serial passaging and cells were characterised for stemness and lineage markers at representative stages (Phase A: P+5, approximately 13 days in culture; Phase B: P+7, approximately 20 days in culture; and Phase C: P+13, approximately 43 days in culture). Cell surface markers, stem cell markers and lineage-specific markers were characterised by FACS, ICC and Q-PCR revealing MSCs maintained their multilineage potential, including neural lineages throughout expansion. Co-expression of multiple lineage markers along with continued CD45 expression in MSCs did not affect completion of osteogenic and adipogenic specification or the formation of neurospheres. Improved standardised isolation and characterisation of MSCs may facilitate the identification of biomarkers to improve therapeutic efficacy to ensure increased reproducibility and routine production of MSCs for therapeutic applications including neural repair.

Cell surface heparan sulfate proteoglycans as novel markers of human neural stem cell fate determination

Multipotent neural stem cells (NSCs) provide a model to investigate neurogenesis and develop mechanisms of cell transplantation. In order to define improved markers of stemness and lineage specificity, we examined self-renewal and multi-lineage markers during long-term expansion and under neuronal and astrocyte differentiating conditions in human ESC-derived NSCs (hNSC H9 cells). In addition, with proteoglycans ubiquitous to the neural niche, we also examined heparan sulfate proteoglycans (HSPGs) and their regulatory enzymes. Our results demonstrate that hNSC H9 cells maintain self-renewal and multipotent capacity during extended culture and express HS biosynthesis enzymes and several HSPG core protein syndecans (SDCs) and glypicans (GPCs) at a high level. In addition, hNSC H9 cells exhibit high neuronal and a restricted glial differentiative potential with lineage differentiation significantly increasing expression of many HS biosynthesis enzymes. Furthermore, neuronal differentiation of the cells upregulated SDC4, GPC1, GPC2, GPC3 and GPC6 expression with increased GPC4 expression observed under astrocyte culture conditions. Finally, downregulation of selected HSPG core proteins altered hNSC H9 cell lineage potential. These findings demonstrate an involvement for HSPGs in mediating hNSC maintenance and lineage commitment and their potential use as novel markers of hNSC and neural cell lineage specification.