Noninvasive measurement of alkaline phosphatase activity in embryoid bodies and coculture spheroids with scanning electrochemical microscopy.
Alkaline phosphatase (ALP) is an enzyme commonly used as an undifferentiated marker of embryonic stem cells (ESCs). Although noninvasive ALP detection has long been desired for stem cell research and in cell transplantation therapy, little progress has been made in developing such techniques. In this study, we propose a noninvasive evaluation method for detecting ALP activity in mouse embryoid bodies (mEBs) using scanning electrochemical microscopy (SECM). SECM has several advantages, including being noninvasive, nonlabeled, quantitative, and highly sensitive. First, we found that SECM-based ALP evaluation permits the comparison of ALP activity among mEBs of different sizes by monitoring the p-aminophenol (PAP) production rate in aqueous solution containing p-aminophenylphosphate (PAPP) normal to the surface area of each sample. Second, coculture spheroids, consisting of mEB and MCF-7 cells for the core and the concentric outer layer, respectively, were prepared as model samples showing heterogeneous ALP activities. The overall PAP production rate dramatically declined in the presence of the MCF-7 cell outer layer, which blocked the mass transfer of PAPP to inner mEB. This result indicated that the SECM response mainly originated from ALP located at the surface of the cellular aggregate, including mEBs and coculture spheroids. Third, taking advantage of the noninvasive nature of SECM, we examined the relevance of ALP activity and cardiomyocyte differentiation. Collectively, these results suggested that noninvasive SECM-based ALP activity normalized by the sample surface enables the selection of EBs with a higher potential to differentiate into cardiomyocytes, which can contribute toward various types of stem cell research.
Graduate School of Environmental Studies, Tohoku University , Sendai 980-8579, Japan.
Toshiharu
T
Arai
Taku
T
Nishijo
Yoshiharu
Y
Matsumae
Yuanshu
Y
Zhou
Kosuke
K
Ino
Hitoshi
H
Shiku
Tomokazu
T
Matsue
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation; D018920:Coculture Techniques; D058732:Embryoid Bodies / Q000166:cytology / Q000201:enzymology*; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D061986:MCF-7 Cells; D051379:Mice; D064347:Microscopy, Electrochemical, Scanning; D032383:Myocytes, Cardiac / Q000166:cytology; D018874:Spheroids, Cellular / Q000166:cytology*
D000469:Alkaline Phosphatase