A Novel Approach for Determining the Critical Quality Attributes of Mesenchymal Stem Cells by Specifying Cell Population With Replication Potential.

We introduce a novel approach to determine the critical quality attributes (CQAs) of mesenchymal stem cells (MSCs) expected to exert immunosuppressive effects. MSCs retained homeostatic replication potentials, such as sustainable growth and consistent cell morphology as a population, in early passages, but lost them in late passages. Characteristic surface markers of MSCs (ie, CD73, CD90, and CD105) were no longer expressed at 2 weeks after subcutaneous transplantation into NOG mice when MSCs from late passages were transplanted, but not when MSCs from early passages were transplanted, suggesting that the biological effects of the MSCs differed according to the timing of cell harvesting and highlighting the importance of specifying MSCs that retained homeostatic features to define the CQAs. The homeostatic features of MSCs related to the balance of the redox system, nutrient requirements, and mitochondrial function were also observed until a certain passage. Therefore, we could define the CQAs of MSCs related to manufacturing by selecting process parameters (PPs) underlying the homeostatic features of MSCs and measuring these PPs quantitatively to specify the cell population with homeostatic features by limiting the passage number. The validity of the PPs stipulated in our pilot study was verified using an SKG murine arthritis model, and critical PPs (CPPs) were then selected among the PPs. Thus, CQAs related to manufacturing in the developmental phase could be defined by the CPPs in this manner, and the concept of CQAs could be refined continuously toward commercial manufacturing.

Improving the differentiation potential of pluripotent stem cells by optimizing culture conditions.

Embryoid cells and induced pluripotent stem cells (iPSCs) are pluripotent stem cells (PSCs). They retain differentiation and self-renewal potential. However, the differentiation potential of PSCs can be changed by the culture medium. PSCs retain their differentiation potential when cultured with medium that supports the glycolytic pathway, showing high expression of chromodomain-helicase-DNA-binding protein 7 (CHD7), but lose their differentiation potential with medium that supports mitochondrial function, showing reduced levels of CHD7. Labeling cells by their copy number variant profile revealed that genetically different PSC populations can be cultured by medium selection. Another factor that defines the self-renewal potential of PSCs is culture condition. PSCs form colonies as they grow, and spontaneous differentiation inevitably occurs along the rim of these colonies in areas that lack cell-to-cell contact; because of this, undifferentiated cell populations would diminish if differentiated cells are not removed properly. Seeding cells on a less potent cell-binding material may minimize the inclusion of differentiated cells, exploiting the reduced adhesive properties of differentiated cells. Culturing cells with medium that supports the glycolytic pathway, using CHD7 as a biomarker for differentiation potential, and culturing cells on less sticky material can improve the differentiation potential of already established PSC clones.

Development and evaluation of a simultaneous and efficient quantification strategy for final prostanoid metabolites in urine.

Prostanoids (PNs) play critical roles in various physiological and pathological processes. Therefore, it is important to understand the alternation of PN expression profiles. However, a simultaneous and efficient quantification system for final PN metabolites in urine has not yet been established. Here, we developed and evaluated a novel method to quantify all final PN metabolites. By purification using a reverse phase solid phase extraction (SPE) column, the matrix effects against the final PGD2, PGE2, and PGF2α metabolites were low, and their accuracies were nearly 100%. The matrix effects against the final PGI2 and TXA2 metabolites were high using reverse phase SPE column purification alone. By applying a tandem SPE method that combined reverse phase and ion exchange SPE columns, the matrix effects decreased so that the accuracy was nearly 100%. To validate the reliability of the method, each final metabolite was quantified from mouse urine to which the PNs (PGD2, PGE2, and PGI2) were intravenously administered. As a result, the amounts of PN metabolites were correlated with those of the PNs administered to the blood in a dose-dependent manner. To validate the method using human samples, the urinary metabolites of Crohn's disease (CD, a PN-related disease) patients and healthy individuals were quantified. All five metabolites were successfully quantified. Only final PGE2 metabolite levels were significantly higher in CD patients than those in healthy individuals, so that the urinary metabolite profiles of CD patients is determined. In conclusion, we developed a novel method to quantify all final PN metabolites simultaneously and efficiently and demonstrated the practicality of the method using human CD patient samples.