Abstract
Pluripotent stem cells (PSCs) have various degrees of pluripotency, which necessitates selection of PSCs with high pluripotency before their application to regenerative medicine. However, the quality control processes for PSCs are costly and time-consuming, and it is essential to develop inexpensive and less laborious selection methods for translation of PSCs into clinical applications. Here we developed an imaging system, termed Phase Distribution (PD) imaging system, which visualizes subcellular structures quantitatively in unstained and unlabeled cells. The PD image and its derived PD index reflected the mitochondrial content, enabling quantitative evaluation of the degrees of somatic cell reprogramming and PSC differentiation. Moreover, the PD index allowed unbiased grouping of PSC colonies into those with high or low pluripotency without the aid of invasive methods. Finally, the PD imaging system produced three-dimensional images of PSC colonies, providing further criteria to evaluate pluripotency of PSCs. Thus, the PD imaging system may be utilized for screening of live PSCs with potentially high pluripotency prior to more rigorous quality control processes.
Highlights
Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells, have variations in their capacity to differentiate[1]
The refraction and structure phases from two RM-differential interference contrast (DIC) images were compounded into compound refraction and structure phases, respectively (Supplementary Fig. S2, Step 3), which were recomposed into a single image (Supplementary Fig. S2, Step 4)
The two retardation modulated differential interference contrast (RM-DIC) images obtained from different directions are decomposed, deconvoluted, and compounded before integrating into a single image, which is detailed enough to highlight organelles including nucleus, nucleoli, and mitochondria within a live cell (Fig. 1)
Summary
Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have variations in their capacity to differentiate[1]. More recent studies reported label-free and non-invasive approaches, some of which are combined with computational data processing, to evaluate pluripotency of PSCs8–10 These methods typically utilize the morphological features of cells and colonies but not of subcellular structures due to the limited resolving power of microscopy. If observed in a non-invasive manner, morphological changes of subcellular structures such as mitochondria may serve as a useful marker to evaluate the pluripotency of PSCs. Non-invasive visualization of subcellular structures has been enabled by recent development of differential interference contrast (DIC) microscope combined with retardation modulation[19,20] and two switchable orthogonal shear directions[21,22,23] such as an orientation-independent differential interference contrast (OI-DIC) microscopy[24,25,26,27,28]. We reported a similar technique termed retardation modulated differential interference contrast (RM-DIC) microscopy, which allows three-dimensional (3D) measurement of the microstructures of phase objects[29,30,31,32]
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