Braun, F.

Durante, M.

Hessel, P.

Luft, S.

Ritter, S.

Scholz, M.

Schroeder, I. S.

GSI Repository

Cardiac differentiation of human embryonic stem cells as a measure for radiation risk in early embryogenesis*   I. Schroeder1, S. Luft 1, F. Braun2, P. Hessel1, M. Scholz1, M. Durante1,3, and S. Ritter#1 1GSI, Darmstadt, 2Hochschule Darmstadt, 3Technische Universität Darmstadt, Germany  The early embryo is particularly vulnerable to ionizing radiation responding to exposure with prenatal death, or-gan malformation or other detrimental ramifications [1]. Thus, a thorough assessment of the radiation risk is man-datory in situations of inevitable or unintended exposure of the conceptus in utero. However, our current under-standing of radiation induced effects is predominantly based on animal studies or radiation accidents [2] ham-pering an in-depth evaluation of the underlying mecha-nisms. Human embryonic stem (hES) cells, derived from the inner cell mass of the blastocyst during embryogene-sis, present a valuable tool to examine radiation effects on the early embryo as they can differentiate into all cells of the body mimicking human development in vitro [3]. Of special interest is the impact of diagnostic or therapeutic irradiation on the formation of the heart as one of the ear-liest organogenic processes taking place from week 2-7 in humans (for concise review see [4]). We established a hES cell-based cardiac differentiation protocol, which modulates the Wnt-signaling pathway that is crucial for in vivo cardiogenesis [5]. In an insulin-free culture it leads to the stepwise generation of mesoderm (by inducing Wnt via CHIR99021), cardiac mesoderm (by inhibiting Wnt via IWP2), cardiovascular progenitors and beating cardi-omyocytes recapitulating all stages of the developing heart within 15 days (Figure 1). Using quantitative PCR, specific markers could be detected at every differentiation stage. In contrast to hES cells and spontaneously differen-tiating cells, in hES cells subjected to directed differentia-tion according to Lian et al. [5] the appearance of early cardiomyocytes (d8-d15) was marked by beating clusters and the expression of early cardiomyocyte markers such as alpha myosin heavy chain (aMHC, Figure 2). Further culture led to the synchronization of cardiomyocyte clus-ters with increased beating rates and an elevated expres-sion of genes specific for mature cardiomyocytes. Suc-cessful cardiac development was also evidenced post-transcriptionally by increased levels of cardiac mi-croRNAs. Currently, techniques to determine the beat-rate and other electrophysiological parameters in cardio-myocytes derived from non-irradiated or irradiated hES cells are established. Preliminary data suggest that the cardiac differentiation of hES cells surviving the exposure to 1 Gy X-rays is accelerated leading to premature ex-pression of cardiac markers when compared to unexposed cells. Such acceleration may render the cells unsuscepti-ble to subsequent positional and temporal patterning ex-plaining radiation induced effects such as the above men-tioned organ malformations or prenatal death. Currently, the underlying mechanisms are studied in more detail.        References [1] C.H. McCollough et al., Radiographics 27 (2007) 909. [2] ICRP Publication 90, Ann. ICRP 33 (2003). [3] T. Vazin and W.J. Freed, Restor Neurol Neurosci 28 (2010) 589. [4] S.M. Evans et al., Circ Res 107 (2010) 1428. [5] X. Lian et al., Nat Protoc 8 (2013) 162.     ___________________________________________ * Work supported by BMBF (02NUK025A). #  s.ritter@gsi.de Figure 1. Differentiation scheme according to Lian et al. Differentiation promoting factors are depicted in blue and green, inhibiting factors are shown in red, d: day. Figure 2. aMHC mRNA expression in cells subjected to spontaneous or directed differentiation according to Lian et al., d: days. GSI SCIENTIFIC REPORT 2014 APPA-HEALTH-39DOI:10.15120/GR-2015-1-APPA-HEALTH-39 333

Cardiac differentiation of human embryonic stem cells as a measure for radiation risk in early embryogenesis

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Cardiac differentiation of human embryonic stem cells as a measure for radiation risk in early embryogenesis

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