Morphological Analysis of Human Induced Pluripotent Stem Cells During Induced Differentiation and Reverse Programming

Abstract

The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report, human mesenchymal cells (hMSCs) generated from the human ES cell line H9, were reprogrammed back to induced pluripotent state using Oct-4, Sox2, Nanog, and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes, lipid droplets, glycogen, scarce reticulum) and nuclear levels (features of nuclear plasticity, presence of euchromatin, reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal–epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming.

Quantitative volumetric Raman imaging of three dimensional cell cultures

“…DNA was visualized using the O–P–O band (789 cm −1 ) while the CH 2 symmetric stretching highlighted areas rich in lipid structures (2,857 cm −1 ). The 3D reconstruction of hiPSCs emulates the dense morphology of human pluripotent stem cell colonies26. hiPSC-derived CMs were ∼3 μm in height, which matches previous reports of their particularly flat nature27.…”

5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts

“…5-aza-CR increased plasticity was also accompanied by autophagic phenomena, that have been reported to play a critical role in the maintenance of adult stem cells40, possibly reducing senescence via the active removal of oxigen reactive species41. Interestingly the presence of autophagic vacuoles have been previously reported in ultrastructural analysis of hiPSC42. Furthermore, post 5-aza-CR cell nuclei were characterized by sparse chromatin and appeared devoid of compact formations.…”

High-throughput physical phenotyping of cell differentiation

“…For example, upregulation of Pax6 or PKC-a during differentiation may stabilize the cytoskeleton 44,45 . Furthermore, loosely organized heterochromatin and/or abundant euchromatin modifications have been observed in pluripotent cells compared to that observed in differentiated cells [46][47][48][49][50][51] . These changes to cytoskeletal and nuclear structure may contribute to the decrease in deformability and overall change in physical phenotype observed in RPEs compared to that in iPSCs (Figure 2).…”