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Stem Cell Reviews and Reports

, Volume 10, Issue 1, pp 1–15 | Cite as

Evidence for self-maintaining pluripotent murine stem cells in embryoid bodies

  • Wael A. Attia
  • Osama M. Abd El Aziz
  • Dimitry Spitkovsky
  • John A. Gaspar
  • Peter Dröge
  • Frank Suhr
  • Davood Sabour
  • Johannes Winkler
  • Kesavan Meganathan
  • Smita Jagtap
  • Markus Khalil
  • Jürgen Hescheler
  • Konrad Brockmeier
  • Agapios Sachinidis
  • Kurt PfannkucheEmail author
Article

Abstract

Pluripotent stem cells have great potential for regenerative medicine; however, their clinical use is associated with a risk of tumor formation. We utilized pluripotent cells expressing green fluorescent protein and puromycin resistance under control of the Oct4 promoter to study the persistence of potential pluripotent cells under embryoid body (EB) culture conditions, which are commonly used to obtain organotypic cells. We found that i.) OCT4-expressing cells dramatically decrease during the first week of differentiation, ii.) the number of OCT4-expressing cells recovers from day 7 on, iii.) the OCT4-expressing cells are similar to embryonic stem cells grown in the presence of leukemia inhibitory factor LIF but express several markers associated with germ cell formation, such as DAZL and STRA-8 and iv.) the persistence of potentially pluripotent cells is independent of supportive cells in EBs. Finally, OCT4-expressing cells, isolated from EBs after 2-month of culture, were further maintained under feeder-free conditions in absence of LIF and continued to express OCT4 in 95 % of the population for at least 36 days. These findings point to an alternative state of stable OCT4 expression. In the frame of the landscape model of differentiation two attractors of pluripotency might be defined based on their different characteristics.

Keywords

OCT4 Pluripotency Self-renewal Stem cells Embryoid bodies Landscape model 

Notes

Acknowledgments

We thank Professor Austin Smith (Institute for Stem Cell Biology University of Cambridge, UK) for providing the Oct4-GFP-ires-PAC ES cell line and Christoph Göttlinger for help with the FACS sorter. Thanks to Manoj Gupta and Daniel Derichsweiler for support with flow cytometry and Moritz Haustein for statistical analysis. This project was supported by grants from the German Academic Exchange Service (DAAD) to Wael A Attia and Osama Abd El Aziz and the European Union funded RAMSES project to KB (7th framework program), by grants of the Imhoff Foundation (Imhoff Stiftung) and the Maria Pesch Foundation (Maria Pesch Stiftung) to KP and by grants of the National Medical Research Council and the Ministry of Education of Singapore (PD).

Author Disclosure Statement

No conflicts of interests were declared.

Supplementary material

12015_2013_9472_Fig7_ESM.gif (169 kb)
Supplemental Figure 1

Induction of differentiation by retinoic acid decreases retention of Oct4+ cells. Embryoid bodies were treated with 1 μM retinoic acid from day 4 to day 8 of differentiation. After 3 weeks of differentiation the persistence of Oct4+ cells as indicated by activation of the Oct4-reporter transgene (GFP+ cells) was largely decreased as compared to control. (GIF 169 kb)

12015_2013_9472_MOESM1_ESM.eps (1.4 mb)
High resolution image (EPS 1417 kb)
12015_2013_9472_Fig8_ESM.gif (90 kb)
Supplemental Figure 2

Expression of GFP in puromycin-selected EBs (Panels A–C). Embryoid bodies were formed from Oct4-GFP-IRES-PAC ES cells and cultured till day 7 under suspension culture conditions. Beginning at day 7, puromycin was added to the culture medium. Panels A and B show the expression of GFP in the puromycin treated EBs after 7 days of selection (independent experiments). Panel C shows the expression of GFP in EBs that were selected with puromycin for 14 days. Green lines illustrate the distribution of GFP+ cells, black lines show the expression of GFP in Oct4-GFP-IRES-PAC ES-cells for comparison. Panel D: Growth of GFP+ cell-clusters under permanent selection with puromycin. Panel E–F: Chromosome spreads of metaphase chromosomes from ES cells (E) and GFP+ cells isolated after 22 days from the EB. (GIF 89 kb)

12015_2013_9472_MOESM2_ESM.eps (5.5 mb)
High resolution image (EPS 5638 kb)
12015_2013_9472_Fig9_ESM.gif (172 kb)
Supplemental Figure 3

Comparison of FACS-sorted GFP+ cells from day 16 embryoid bodies with starting ES cells by gene expression analysis. Expression of marker genes for primordial germ cells (A) and epiblast stem cells (B) were compared between FACS-sorted cells and ES cells. (GIF 172 kb)

12015_2013_9472_MOESM3_ESM.eps (5.7 mb)
High resolution image (EPS 5857 kb)
12015_2013_9472_MOESM4_ESM.pptx (675 kb)
Supplemental Figure 4 Heatmap of gene arra expression data (PPTX 675 kb)
12015_2013_9472_MOESM5_ESM.xls (686 kb)
Supplemental raw data The supplemental raw data table lists up- and down-regulated genes as well as gene ontology analysis in GFP+ cells sorted from day 16 EBs in comparison to GFP+ ES cells. (XLS 685 kb)
Supplementary video 1

(MPG 3565 kb)

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Wael A. Attia
    • 1
    • 2
  • Osama M. Abd El Aziz
    • 1
    • 2
  • Dimitry Spitkovsky
    • 3
  • John A. Gaspar
    • 3
  • Peter Dröge
    • 4
  • Frank Suhr
    • 5
    • 6
  • Davood Sabour
    • 3
  • Johannes Winkler
    • 3
  • Kesavan Meganathan
    • 3
  • Smita Jagtap
    • 3
  • Markus Khalil
    • 2
  • Jürgen Hescheler
    • 3
  • Konrad Brockmeier
    • 2
  • Agapios Sachinidis
    • 3
  • Kurt Pfannkuche
    • 2
    • 3
    • 7
    Email author
  1. 1.Cairo UniversityCairoEgypt
  2. 2.Paediatric CardiologyUniversity of CologneCologneGermany
  3. 3.Center of Physiology and Pathophysiology, Institute for NeurophysiologyUniversity of CologneCologneGermany
  4. 4.Nanyang Technological UniversitySingaporeSingapore
  5. 5.Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport MedicineGerman Sport University CologneCologneGermany
  6. 6.The German Research Center of Elite SportGerman Sport University CologneCologneGermany
  7. 7.Institute for Neurophysiology & Department for Paediatric CardiologyUniversity Clinic of CologneCologneGermany

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