Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2017 Nov 28;8(1):271.
doi: 10.1186/s13287-017-0720-1.

Two Sides of the Same Coin? Unraveling Subtle Differences Between Human Embryonic and Induced Pluripotent Stem Cells by Raman Spectroscopy

Free PMC article
Comparative Study

Two Sides of the Same Coin? Unraveling Subtle Differences Between Human Embryonic and Induced Pluripotent Stem Cells by Raman Spectroscopy

Elvira Parrotta et al. Stem Cell Res Ther. .
Free PMC article


Background: Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial.

Methods: Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays.

Results: Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm-1, which is enriched in human induced pluripotent stem cells.

Conclusions: Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.

Keywords: Human embryonic stem cells; Human induced pluripotent stem cells; Multivariate analysis; Raman imaging.

Conflict of interest statement

Author information

Not applicable.

Ethics approval and consent to participate

A skin biopsy was collected from a healthy individual after obtaining written informed consent from the donor. The study was approved by the Ethics Committee of the Magna Graecia University and the Azienda Ospedaliero-Universitaria “Mater Domini”.

Consent for publication

All of the authors have read and approved the manuscript for publication.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Fig. 1
Fig. 1
Raman imaging of typical human ESCs and iPSCs. Color-reconstituted Raman images of human embryonic stem cells (hESCs, upper left panel) and human induced pluripotent stem cells (hiPSCs) (upper right panel). White scale bar = 5 μm. Small insets show corresponding bright-field images recorded after Raman scanning. Raman peak at 785 cm–1 (DNA/RNA bases) mapped in red, 1670 cm–1 (proteins) in blue, 2850 cm–1 (lipids) in green, and a combination of 748 and 1585 cm–1 (cytochrome C) in magenta. hiPSCs exhibit a much higher level of DNA/RNA bases in well-defined regions of the cell. Curves in the lower panel are average spectra of hESCs (top curve) and hiPSCs (bottom curve), where the peaks used for the color-reconstituted images are indicated with the corresponding color
Fig. 2
Fig. 2
Principal component (PC) curves as biochemical indicators. a Loading curves of the first three PCs calculated by PCA. Since PCA is performed on the overall dataset of all probed cells, the computed PCs are the same throughout all of the measured spectra, and their characteristic bands indicate sensitive biochemical features (see text for details concerning peak assignment). While the PC1 curve only resembles the global average spectrum, the PC2 and PC3 curves account for the significant biochemical differences between the different cells, as well as different regions of the same cell. Scatter plot of PC3 vs PC2 scores for hESCs (blue dots) and hiPSCs (red dots) from Fig. 1. Each dot corresponds to one spectrum (pixel) of Raman mapping. Blue and red dots closely overlap, except for the top-left part of the graph which corresponds to positive PC3 and negative PC2 scores (b). Loading curves of (a) support that this PC3–PC2 region can be assigned to DNA/RNA bases, the typical frequencies of which are exhibited as positive bands in the PC3 curve (mainly 785, 1098, 1484, and 1575 cm–1), and as a sharp negative band (785 cm–1) for the PC2 curve. hESC human embryonic stem cell, hiPSC human induced pluripotent stem cell
Fig. 3
Fig. 3
Semiquantitative comparison of Raman images by cluster analysis. KCA performed on the PCA results for Raman assignment of different cellular regions. Top row reports results for three typical hESCs, while second row reports results for three typical hiPSCs (scale bar = 5 μm). For the KCA calculation, six clusters were imposed within the cells (see text for further details), and the red cluster is only evident in hiPSCs (upper panel). Lower graph shows average Raman spectra of each cluster, where the curves have the same color as the corresponding cluster. The red curve exhibits all of the major peaks ascribed to DNA/RNA bases, and consequently the red regions inside the cells are assigned to DNA/RNA compartments. The absence of red clusters inside hESCs does not mean that DNA/RNA bases are missing therein, only that their expression is much lower than the DNA/RNA abundance in the red regions of hiPSCs
Fig. 4
Fig. 4
Quantification of nucleic acid levels. DNA and RNA extracted from both pluripotent stem cell lines were quantified with a NanoDrop 2000 UV-Vis spectrophotometer (a) and with ethidium bromide staining on agarose gel electrophoresis (b). Error bars indicate mean ± SEM. Statistical comparison between hiPSCs and hESCs by paired Student’s t test (*p < 0.05). hESC human embryonic stem cell, hiPSC human induced pluripotent stem cell
Fig. 5
Fig. 5
Cell cycle and proliferation rate analysis. a Flow cytometric analysis of hESCs and hiPSCs stained with CFSE and cultured for 2 h (T0) and 4 days (T4) after staining. b Proliferation rate of hiPSCs quantified and compared to that of hESCs. Quantitative data expressed as mean ± SD of three independent experiments. Statistical comparison for each generation by paired Student’s t test (**p < 0.01, ***p < 0.001). c Cell cycle progression analysis of hiPSCs and hESCs. Cells were stained with propidium iodide (PI) and analyzed by fluorescence-activated cell sorting. Data shown as mean ± SD from three independent experiments. d Statistical comparison between hiPSCs and hESCs for each phase of the cell cycle by paired Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001). e Quantitative real-time PCR (qRT-PCR) analysis of the cell-cycle-associated proteins CCNA2, CCNB1, CCND1, and CCNE1 in hESCs and hiPSCs. All expression values normalized to GAPDH and relative to hESCs. Data represent the mean ± SD from three independent experiments. CFSE 5,6-carboxyfluorescein diacetate succinimidyl ester, hESC human embryonic stem cell, hiPSC human induced pluripotent stem cell
Fig. 6
Fig. 6
Karyotype analysis and fluorescence-based quantification of mitochondria. a Representative image and karyotype of an M-Fish stained hESC (left) and an M-Fish stained hiPSC (right), confirming that both cell lines have normal karyotypes. b Mitochondrial staining using the MitoTracker Green FM of hESC and hiPSCs. Magnification × 20. c Representative graphs of mean fluorescence intensity values in a single cell colony and average intensity ± SD in two cell lines. hESC human embryonic stem cell, hiPSC human induced pluripotent stem cell

Similar articles

See all similar articles

Cited by 4 articles


    1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–7. doi: 10.1126/science.282.5391.1145. - DOI - PubMed
    1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–72. doi: 10.1016/j.cell.2007.11.019. - DOI - PubMed
    1. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318:1917–20. doi: 10.1126/science.1151526. - DOI - PubMed
    1. Wobus AM, Boheler KR. Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev. 2005;85:635–78. doi: 10.1152/physrev.00054.2003. - DOI - PubMed
    1. Hanna J, Wernig M, Markoulaki S, Sun CW, Meissner A, Cassady JP, Beard C, Brambrink T, Wu LC, Townes TM, Jaenisch R. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science. 2007;318:1920–3. doi: 10.1126/science.1152092. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources