In vitro osteogenic differentiation of mesenchymal stem cells

Abstract

The use of adipose-derived stromal/stem cells (ASCs) continues to increase in the field of regenerative medicine and other clinical applications. Adipose tissue can be collected in a less invasive procedure, when compared to bone marrow aspirations, from patients undergoing cosmetic liposuction or abdominoplasty procedures either as an aspirate or as intact tissue. Adipose tissue is seen as medical waste that would otherwise been discarded. ASCs are seen as the one of the most promising stem cell populations for tissue regeneration as they can be harvested with relative ease, can yield large quantities, grow under standard cell culture conditions, differentiate into multiple lineages, and secrete various cytokines.

One of the clinical applications of ASCs is their role in enhancement of bone regeneration. To achieve osteogenic differentiation of ASCs, the ASCs are exposed to a differentiation cocktail containing β-glycerophosphate, ascorbate-2-phosphate and dexamethasone. Currently there is no consensus regarding the most optimal osteogenic differentiation medium for in vitro osteogenic differentiation of mesenchymal stromal/stem cells (MSCs), and the concentrations of the stimulating factors vary amongst published osteogenic induction media. It is for this reason we tested 3 previously published osteogenic differentiation media with varying concentrations of β-glycerophosphate, ascorbate-2-phosphate and dexamethasone. The success of the different differentiation cocktails was assessed using two osteogenic assays namely Alizarin Red S (ARS) and alkaline phosphatase (ALP) which were used to quantify the amount of calcified product and ALP enzyme activity respectively. Of the three differentiation media, one differentiation medium that produced the best osteogenic differentiation was chosen for further downstream testing.

Foetal bovine serum (FBS) has been the gold standard for medium supplementation when expanding ASCs ex vivo despite the many disadvantages associated with its use, such as batch-to-batch variability, the presence of xenogenic proteins, possibility of zoonotic disease transmission, and ethical concerns regarding animal welfare to name a few. Furthermore, for ASCs to be used in a clinical setting, they need to comply with Good Manufacturing Practice (GMP) guidelines, which strongly advise against the use of FBS in clinical-grade cell therapy products. For this reason, researchers and clinicians continue to seek optimal and GMP compliant alternatives to FBS. Human alternatives to FBS will not only overcome FBS-associated disadvantages, but also more accurately mimic the environmental niche thus making the cell therapy product more physiologically compatible and consequently more reliable when applied clinically.

The current study explored the use of two human alternatives to FBS namely platelet-rich plasma (PRP) and pooled human platelet lysate (pHPL) for use in osteogenic differentiation of ASCs in vitro. ASCs were expanded and differentiated in medium supplemented with either FBS, PRP or pHPL. Again, the two osteogenic assays, ARS and ALP were used to determine the success of osteogenic differentiation. To examine the kinetics of osteogenic gene expression, RNA was isolated at 4 time points during the differentiation period (days 0, 7, 14 and 21) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed. We found that both human alternatives were superior to FBS supplemented medium in terms of the amount of calcified bone product produced and the rate at which osteogenesis occurred. RT-qPCR data revealed that cells grown in FBS take longer to switch from a proliferative to an osteogenic differentiation state. Results revealed that PRP and pHPL are effective substitutes to FBS when differentiating ASCs into osteoblasts, and that this can effectively be assessed in the therapeutic setting.

The aim of this study is to optimize and standardize protocols to differentiate MSCs into osteoblasts.