Skip to main content
Log in

Pipette-based Method to Study Embryoid Body Formation Derived from Mouse and Human Pluripotent Stem Cells Partially Recapitulating Early Embryonic Development Under Simulated Microgravity Conditions

  • ORIGINAL ARTICLE
  • Published:
Microgravity Science and Technology Aims and scope Submit manuscript

Abstract

The in vitro differentiation of pluripotent stem cells partially recapitulates early in vivo embryonic development. More recently, embryonic development under the influence of microgravity has become a primary focus of space life sciences. In order to integrate the technique of pluripotent stem cell differentiation with simulated microgravity approaches, the 2-D clinostat compatible pipette-based method was experimentally investigated and adapted for investigating stem cell differentiation processes under simulated microgravity conditions. In order to keep residual accelerations as low as possible during clinorotation, while also guaranteeing enough material for further analysis, stem cells were exposed in 1-mL pipettes with a diameter of 3.5 mm. The differentiation of mouse and human pluripotent stem cells inside the pipettes resulted in the formation of embryoid bodies at normal gravity (1 g) after 24 h and 3 days. Differentiation of the mouse pluripotent stem cells on a 2-D pipette-clinostat for 3 days also resulted in the formation of embryoid bodies. Interestingly, the expression of myosin heavy chain was downregulated when cultivation was continued for an additional 7 days at normal gravity. This paper describes the techniques for culturing and differentiation of pluripotent stem cells and exposure to simulated microgravity during culturing or differentiation on a 2-D pipette clinostat. The implementation of these methodologies along with -omics technologies will contribute to understand the mechanisms regulating how microgravity influences early embryonic development.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Briegleb, W.: Some qualitative and quantitative aspects of the fast-rotating clinostat as a research tool. ASGSB Bull 5(2), 23–30 (1992)

    Google Scholar 

  • Caiozzo, V.J., Haddad, F., Baker, M.J., Herrick, R.E., Prietto, N., Baldwin, K.M.: Microgravity-induced transformations of myosin isoforms and contractile properties of skeletal muscle. J. Appl. Physiol. 81(1), 123–132 (1996)

    Google Scholar 

  • Cogoli, M.: The fast rotating clinostat: a history of its use in gravitational biology and a comparison of ground-based and flight experiment results. ASGSB Bull 5(2), 59–67 (1992)

    Google Scholar 

  • Colleoni, S., Galli, C., Gaspar, J.A., Meganathan, K., Jagtap, S., Hescheler, J., Sachinidis, A., Lazzari, G.: Development of a neural teratogenicity test based on human embryonic stem cells: response to retinoic acid exposure. Toxicol. Sci. 124(2), 370–377 (2011). doi:10.1093/toxsci/kfr245

    Article  Google Scholar 

  • Crawford-Young, S.J.: Effects of microgravity on cell cytoskeleton and embryogenesis. Int. J. Dev. Biol. 50 (2-3), 183–191 (2006). doi:10.1387/ijdb.052077sc

    Article  Google Scholar 

  • Doss, M.X., Winkler, J., Chen, S., Hippler-Altenburg, R., Sotiriadou, I., Halbach, M., Pfannkuche, K., Liang, H., Schulz, H., Hummel, O., Hubner, N., Rottscheidt, R., Hescheler, J., Sachinidis, A.: Global transcriptome analysis of murine embryonic stem cell-derived cardiomyocytes. Genome Biol. 8(4), R56 (2007). doi:10.1186/gb-2007-8-4-r56

    Article  Google Scholar 

  • Eiermann, P., Kopp, S., Hauslage, J., Hemmersbach, R., Gerzer, R., Ivanova, K.: Adaptation of a 2-D clinostat for simulated microgravity experiments with adherent cells. Microgravity Sci. Technol. 25, 153–159 (2013)

  • Fitts, R.H., Desplanches, D., Romatowski, J.G., Widrick, J.J.: Spaceflight effects on single skeletal muscle fiber function in the rhesus monkey. Am. J. Physiol. Regul. Integr. Comp. Physiol. 279(5), R1546–1557 (2000)

    Google Scholar 

  • Gabut, M., Samavarchi-Tehrani, P., Wang, X., Slobodeniuc, V., O’Hanlon, D., Sung, H.K., Alvarez, M., Talukder, S., Pan, Q., Mazzoni, E.O., Nedelec, S., Wichterle, H., Woltjen, K., Hughes, T.R., Zandstra, P.W., Nagy, A., Wrana, J.L., Blencowe, B.J.: An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming. Cell 147(1), 132–146 (2011). doi:doi:10.1016/j.cell.2011.08.023

  • Gaspar, J.A., Doss, M.X., Hengstler, J.G., Cadenas, C., Hescheler, J., Sachinidis, A.: Unique metabolic features of stem cells, cardiomyocytes, and their progenitors. Circ. Res. 114(8), 1346–1360 (2014). doi:10.1161/CIRCRESAHA.113.302021

    Article  Google Scholar 

  • Gaspar, J.A., Doss, M.X., Winkler, J., Wagh, V., Hescheler, J., Kolde, R., Vilo, J., Schulz, H., Sachinidis, A.: Gene expression signatures defining fundamental biological processes in pluripotent, early, and late differentiated embryonic stem cells. Stem. Cells Dev. 21(13), 2471–2484 (2012). doi:10.1089/scd.2011.0637

    Article  Google Scholar 

  • Gualandris-Parisot, L., Husson, D., Bautz, A., Durand, D., Kan, P., Aimar, C., Membre, H., Duprat, A.M., Dournon, C.: Effects of space environment on embryonic growth up to hatching of salamander eggs fertilized and developed during orbital flights. Biol. Sci. Space 16(1), 3–11 (2002)

    Article  Google Scholar 

  • Harrison, B.C., Allen, D.L., Girten, B., Stodieck, L.S., Kostenuik, P.J., Bateman, T.A., Morony, S., Lacey, D., Leinwand, L.A.: Skeletal muscle adaptations to microgravity exposure in the mouse. J. Appl. Physiol. 95(6), 2462–2470 (2003). doi:10.1152/japplphysiol.00603.2003

    Article  Google Scholar 

  • Hemmersbach, R., von der Wiesche, M., Seibt, D.: Ground-based experimental platforms in gravitational biology and human physiology. Signal Transduct. 6(6), 381–387 (2006). doi:10.1002/sita.200600105

    Article  Google Scholar 

  • Herranz, R., Anken, R., Boonstra, J., Braun, M., Christianen, P.C, de Geest, M., Hauslage, J., Hilbig, R., Hill, R.J., Lebert, M., Medina, F.J., Vagt, N., Ullrich, O., van Loon, J.J., Hemmersbach, R.: Ground-based facilities for simulation of microgravity: organism-specific recommendations for their use, and recommended terminology. Astrobiology 13(1), 1–17 (2013). doi:10.1089/ast.2012.0876

    Article  Google Scholar 

  • Higashibata, A., Szewczyk, N.J., Conley, C.A., Imamizo-Sato, M., Higashitani, A., Ishioka, N.: Decreased expression of myogenic transcription factors and myosin heavy chains in Caenorhabditis elegans muscles developed during spaceflight. J. Exp. Biol. 209(Pt 16), 3209–3218 (2006). doi:10.1242/jeb.02365

    Article  Google Scholar 

  • Kojima, Y., Sasaki, S., Kubota, Y., Ikeuchi, T., Hayashi, Y., Kohri, K.: Effects of simulated microgravity on mammalian fertilization and preimplantation embryonic development in vitro. Fertil. Steril. 74(6), 1142–1147 (2000). doi:10.1016/S0015-0282(00)01583-1

    Article  Google Scholar 

  • Krug, A.K., Kolde, R., Gaspar, J.A., Rempel, E., Balmer, N.V., Meganathan, K., Vojnits, K., Baquie, M., Waldmann, T., Ensenat-Waser, R., Jagtap, S., Evans, R.M., Julien, S., Peterson, H., Zagoura, D., Kadereit, S., Gerhard, D., Sotiriadou, I., Heke, M., Natarajan, K., Henry, M., Winkler, J., Marchan, R., Stoppini, L., Bosgra, S., Westerhout, J., Verwei, M., Vilo, J., Kortenkamp, A., Hescheler, J., Hothorn, L., Bremer, S., van Thriel, C., Krause, K.H., Hengstler, J.G., Rahnenfuhrer, J., Leist, M., Sachinidis, A.: Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach. Arch. Toxicol. 87(1), 123–143 (2013). doi:10.1007/s00204-012-0967-3

    Article  Google Scholar 

  • Livak, K.J., Schmittgen, T.D.: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Methods (San Diego, Calif.) 25(4), 402–408 (2001). doi:10.1006/meth.2001.1262

  • Meganathan, K., Jagtap, S., Wagh, V., Winkler, J., Gaspar, J.A., Hildebrand, D., Trusch, M., Lehmann, K., Hescheler, J., Schluter, H., Sachinidis, A.: Identification of thalidomide-specific transcriptomics and proteomics signatures during differentiation of human embryonic stem cells. PLoS One 7(8), e44228 (2012). doi:10.1371/journal.pone.0044228

    Article  Google Scholar 

  • Meganathan, K., Sotiriadou, I., Natarajan, K., Hescheler, J., Sachinidis, A.: Signaling molecules, transcription growth factors and other regulators revealed from in-vivo and in-vitro models for the regulation of cardiac development. Int. J. Cardiol. 183, 117–128 (2015). doi:DOI10.1016/j.ijcard.2015.01.049

  • Ronca, A.E.: Studies toward birth and early mammalian development in space. Adv. Space Res. 32(8), 1483–1490 (2003). doi:10.1016/S0273-1177(03)90385-1

    Article  Google Scholar 

  • Ronca, A.E., Alberts, J.R.: Effects of prenatal spaceflight on vestibular responses in neonatal rats. J. Appl. Physiol. 89(6), 2318–2324 (2000)

    Google Scholar 

  • Shinde, V., Klima, S., Sureshkumar, P.S., Meganathan, K., Jagtap, S., Rempel, E., Rahnenführer, J., Hengstler, J.G., Waldmann, T., Hescheler, J., Leist, M., Sachinidis, A.: Human pluripotent stem cell based developmental toxicity assays for chemical safety screening and systems biology data generation. e52333 (2015a). doi:10.3791/52333

  • Shinde, V., Stöber, R., Nemade, H., Sotiriadou, I., Hescheler, J., Hengstler, J., Sachinidis, A.: Transcriptomics of hepatocytes treated with toxicants for investigating molecular mechanisms underlying hepatotoxicity. In: Vinken, M., Rogiers, V. (eds.) Protocols in In Vitro Hepatocyte Research, vol. 1250. Methods in Molecular Biology, pp. 225-240. Springer, New York (2015b)

  • Thiel, C.S., Hauschild, S., Tauber, S., Paulsen, K., Raig, C., Raem, A., Biskup, J., Gutewort, A., Hurlimann, E., Unverdorben, F., Buttron, I., Lauber, B., Philpot, C., Lier, H., Engelmann, F., Layer, L.E., Ullrich, O.: Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity. Biomed. Res. Int. (2015). doi:10.1155/2015/363575

    Google Scholar 

  • Trouillas, M., Saucourt, C., Guillotin, B., Gauthereau, X., Ding, L., Buchholz, F., Doss, M.X., Sachinidis, A., Hescheler, J., Hummel, O., Huebner, N., Kolde, R., Vilo, J., Schulz, H., Boeuf, H.: Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives. BMC Genomics. 10, 73 (2009). doi:10.1186/1471-2164-10-73

    Article  Google Scholar 

  • Wagh, V., Doss, M.X., Sabour, D., Niemann, R., Meganathan, K., Jagtap, S., Gaspar, J.A., Ardestani, M.A., Papadopoulos, S., Gajewski, M., Winkler, J., Hescheler, J., Sachinidis, A.: Fam40b is required for lineage commitment of murine embryonic stem cells. Cell Death Dis. 5, e1320 (2014). doi:10.1038/cddis.2014.273

    Article  Google Scholar 

  • Wagh, V., Jagtap, S., Meganathan, K., Potta, S.P., Winkler, J., Hescheler, J., Sachinidis, A.: Effect of chemopreventive agents on differentiation of mouse embryonic stem cells. Front. Biosci. (Elite Ed) 4, 156–168 (2012)

    Article  Google Scholar 

  • Wakayama, S., Kawahara, Y., Li, C., Yamagata, K., Yuge, L., Wakayama, T.: Detrimental effects of microgravity on mouse preimplantation development in vitro. PLoS One 4(8), e6753 (2009). doi:10.1371/journal.pone.0006753

    Article  Google Scholar 

  • Wang, Y., An, L., Jiang, Y., Hang, H.: Effects of simulated microgravity on embryonic stem cells. PLoS One 6(12), e29214 (2011). doi:10.1371/journal.pone.0029214

    Article  Google Scholar 

Download references

Acknowledgments

This work was funded by the European Space Agency, Noordwijk, Netherlands in the frame of the GBF Programm (4000110259 ESA-CORA-GBF)

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Agapios Sachinidis.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shinde, V., Brungs, S., Hescheler, J. et al. Pipette-based Method to Study Embryoid Body Formation Derived from Mouse and Human Pluripotent Stem Cells Partially Recapitulating Early Embryonic Development Under Simulated Microgravity Conditions. Microgravity Sci. Technol. 28, 287–295 (2016). https://doi.org/10.1007/s12217-015-9469-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12217-015-9469-2

Keywords

Navigation