Synchrotron radiation X-ray microfluorescence reveals polarized distribution of atomic elements during differentiation of pluripotent stem cells

Abstract

The mechanisms underlying pluripotency and differentiation in embryonic and reprogrammed stem cells are unclear. In this work, we characterized the pluripotent state towards neural differentiated state through analysis of trace elements distribution using the Synchrotron Radiation X-ray Fluorescence Spectroscopy. Naive and neural-stimulated embryoid bodies (EB) derived from embryonic and induced pluripotent stem (ES and iPS) cells were irradiated with a spatial resolution of 20 µm to make elemental maps and qualitative chemical analyses. Results show that these embryo-like aggregates exhibit self-organization at the atomic level. Metallic elements content rises and consistent elemental polarization pattern of P and S in both mouse and human pluripotent stem cells were observed, indicating that neural differentiation and elemental polarization are strongly correlated.

Figure 1. Sample assembly.

In order to fixate EB samples on ultralene film, an aluminum base was used and film was attached to it with regular tape. EBs were water rinsed and air dried on this assembled piece.

Figure 2. Beamline geometry.

A) XRF line set up at the LNLS. B) Beamline geometry (view from the top).

Figure 3. Atomic distribution within murine embryoid bodies.

Top line: 4 days old murine embryoid body; Middle line: 8 days old control murine embryoid body; Bottom line: 8 days old neural induced murine embryoid body. Atomic elements are shown on the top line above the heat maps. All derived from the murine embryonic stem cell line R1. Maps are representative of all samples. Bright field images scale bar: 25 µm.

Figure 4. P and S polarization is significant on 8 days old neural induced murine embryoid bodies.

Left and right intensity of P, S, Cu and Zn in R1 embryoid bodies. A) 4 days old group; B) 8 days old control group; C) 8 days old neural induced group. Left side is represented by black bars and right side is represented by grey bars, *p<0.05.

Figure 5. Murine embryoid bodies show similar levels of P, S, Cu and Zn at different stages of differentiation.

Measure of elemental content normalized per irradiated area, A) P; B) S; C) Cu; D) Zn, *p<0.05.

Figure 6. Atomic polarization is independent of cell density, proliferation and death.

A) Example of a H9 14 days old neural induced embryoid body divided in half for nuclei quantification; B) Symmetry measure: left and right percentage of nuclei for neural induced R1 (n = 5) and H9 (n = 4) embryoid bodies; C) Example of PH3 staining (red) on a murine embryoid body; D) Example of TUNEL staining (green) on an H9 embryoid body. All DAPI (blue).

Figure 7. Nestin expression is polarized after neural induction in human embryoid bodies.

Nestin staining of H9 embryoid bodies, A) 7 days old group; B) 14 days old control group; C and D) 14 days old neural induced group. E to H) DAPI staining; I to L) merge. The embryoid bodies depicted in this figure are corresponding examples of the total population. Scale bar 50 µm.

Figure 8. Beta-III tubulin staining is exclusive to 14 days old neural induced embryoid bodies.

Beta-III tubulin staining of H9 embryoid bodies (green), A) 14 days old control group; B) 14 days old neural induced group; C and D) DAPI staining (blue); E and F) merge. Scale bar 50 µm.

Figure 9. Atomic distribution within human embryoid bodies.

Top line: 7 days old H9 embryoid body; Middle line: 14 days old control H9 embryoid body; Bottom line: 14 days old neural induced H9 embryoid body. Elements are shown on the top line above the heat maps. Maps are representative of all samples. Bright field images scale bar: 50 µm.

Figure 10. Human pluripotent cells-derived embryoid bodies retain P and S polarization under neural stimulus.

Left and right intensity of P, S, Cu and Zn in H9 embryoid bodies. A) 7 days old group; B) 14 days old control group; C) 14 days old neural induced group. Black bars correspond to the left side, grey bars correspond to the right side of atomic maps, *p<0.05 and **p<0.01.

Figure 11. Human embryoid bodies show similar levels of P, S, Cu and Zn at different stages of differentiation.

Measure of elemental content normalized per irradiated area, A) P; B) S; C) Cu; D) Zn, *p<0.05; **p<0.01.

Figure 12. Human embryoid bodies and neurospheres present comparable amounts of metallic elements.

Embryoid bodies and neurospheres metals were normalized by phosphorus content. A) Cu/P ratio; B) Zn/P ratio. *p<0.05.

Figure 13. Phosphorus co-localizes with sulfur, but does not overlap with zinc in neural induced EBs.