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.

Peer Review #1 of "Trace elements during primordial plexiform network formation in human cerebral organoids (v0.2)"

“…While greater concentrations of Zn in adult brain are toxic to neural cells and also cause axonal degeneration in mice (Chuah, Tennent & Jacobs, 1995), Zn modulates stem cell proliferation and neuronal differentiation during neurogenesis (Gao et al, 2009;Levenson & Morris, 2011;Morris & Levenson, 2013). In accordance, previous data from our group have demonstrated increased Zn during neural differentiation of human pluripotent stem cells (Cardoso et al, 2011). This may highlight the importance of Zn for neural stem cells commitment and justify why the levels found in cerebral organoids are subtly higher than those described in adult brain (Fig 3F and S3 Table) (Dexter et al, 1991;Rajan et al, 1997;Rahil-Khazen et al, 2002).…”

Trace elements during primordial plexiform network formation in human cerebral organoids

“…While higher concentrations of zinc in adult brain are toxic to neural cells and also cause axonal degeneration in mice (47), zinc modulates the proliferation of stem cells and neuronal differentiation during neurogenesis (31)(32)(33). In accordance, previous data from our group have demonstrated increased zinc during neural differentiation of human pluripotent stem cells (48), which may highlight the importance of zinc for the commitment of neural stem cells and justify why levels found in cerebral organoids are subtly higher than those described in the adult brain (Table 2) (9,49,50).…”

Synchrotron radiation X‐ray micro‐fluorescence: Protocol to study mesenchymal stem cells

“…μ‐XRF analysis gives the possibility of determining simultaneously major, minor, and trace elements in cells (multi‐elemental analysis), in a non‐destructive way without the necessity to perform any pre‐treatment of the samples, in contrast with the conventional electron microscopy technique (Cardoso et al, ; James et al, ). Glutaraldehyde alone or in combination with other components such as formaldehyde has been used for specimen preservation to analyze by conventional microscopy (Arro et al, ; Boyde and Franc, ; Boyde and Maconnachie, ; Brunk et al, ).…”

“…Also, no change or precipitation of elements was observed when samples were in contact with paraformaldehyde. Contradicting Cardoso et al (), in the presented study it was not possible to observe cells fixed or preserved in PBS solution. We worked with undifferentiated cells that were smaller than the differentiated cells used by Cardoso et al When dry, the smaller cells were hidden on the back of the crystals formed by precipitation.…”

“…Although the μ‐XRF has been used for years, its general application to cell biology has only been generally appreciated in the last decade and showed to be a very useful tool, particularly for the identification of cellular phenotypes (Banas et al, ; Ortega et al, ). Many research in medical sciences have shown the successful use of this technique to characterize various types of human cells and tissues because it allows the investigator to obtain images (multi‐elemental mapping) of samples in the micrometer scale, enabling chemical determinations of heterogeneous samples (Cardoso et al, ; Chonanant et al, ). In addition, the spectroscopic approach is more straightforward, non‐destructive, and requires less sample preparation compared with the conventional methodologies (Cardoso et al, ; Pérez et al, ).…”