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Isolation, characterization and differentiation of cells expressing pluripotent/multipotent markers from adult human ovaries

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Abstract

Pluripotent stem cells are still generally accepted not to exist in adult human ovaries, although increasing studies confirm the presence of pluripotent/multipotent stem cells in adult mammalian ovaries, including those of humans. The aim of this study is to isolate, characterize and differentiate in vitro stem cells that originate from the adult human ovarian cortex and that express markers of pluripotency/multipotency. After enzymatic degradation of small ovarian cortex biopsies retrieved from 18 women, ovarian cell cultures were successfully established from 17 and the formation of cell colonies was observed. The presence of cells/colonies expressing some markers of pluripotency (alkaline phosphatase, surface antigen SSEA-4, OCT4, SOX-2, NANOG, LIN28, STELLA), germinal lineage (DDX4/VASA) and multipotency (M-CAM/CD146, Thy-1/CD90, STRO-1) was confirmed by various methods. Stem cells from the cultures, including small round SSEA-4-positive cells with diameters of up to 4 μm, showed a relatively high degree of plasticity. We were able to differentiate them in vitro into various types of somatic cells of all three germ layers. However, these cells did not form teratoma when injected into immunodeficient mice. Our results thus show that ovarian tissue is a potential source of stem cells with a pluripotent/multipotent character for safe application in regenerative medicine.

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Acknowledgments

The authors thank all patients who kindly donated their ovarian tissue for this research and are also grateful to Dr. Elvira Malicev and Prof. Primoz Rozman from the Blood Transfusion Center Ljubljana for flow-cytometry analyses and the FACS service, to Prof. Gregor Sersa from the Institute of Oncology Ljubljana for providing IGROV-1 and the melanoma cell line, to Prof. Rok Romih from the Institute of Cell Biology, Medical Faculty, University of Ljubljana for transmission electron microscopy of cell colonies, to Dr. Natasa Toplak from Omega for the TaqMan Protein Expression Assays, to Sabine Conrad, MTA from the University of Tübingen, Germany for technical assistance with the Fluidigm analyses, to Dr. Lenart Girandon from Educell for providing adipogenic induction medium, Oil Red O solution and silver nitrate solution and to all the other people and institutions supporting this research.

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Authors and Affiliations

  1. Reproductive Unit, Department of Obstetrics and Gynaecology, University Medical Centre Ljubljana, Slajmerjeva 3, 1000, Ljubljana, SI, Slovenia

    Martin Stimpfel, Branko Cvjeticanin, Eda Vrtacnik-Bokal & Irma Virant-Klun

  2. Institute for Anatomy and Cell Biology, Medical Faculty, University of Heidelberg, Im Neuenheimer Feld 307, 69120, Heidelberg, Germany

    Thomas Skutella

  3. Institute of Anatomy, Medical Faculty, University of Ljubljana, Korytkova 2, 1000, Ljubljana, Slovenia

    Marija Meznaric

  4. Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230, Domzale, Slovenia

    Peter Dovc

  5. Department of Molecular Diagnostics, Institute of Oncology Ljubljana, Zaloska 2, 1000, Ljubljana, Slovenia

    Srdjan Novakovic & Petra Cerkovnik

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  1. Martin Stimpfel
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  8. Eda Vrtacnik-Bokal
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  9. Irma Virant-Klun
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Corresponding author

Correspondence to Irma Virant-Klun.

Additional information

Martin Stimpfel and Irma Virant-Klun contributed equally to this work.

The authors state that they have no competing financial interests.

This study was supported by the Slovenian Research Agency (grant J3-0415 to I.V.-K.) and by the German Federal Ministry of Education and Research (grant 01GN1001 to T.S.).

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Supplementary Fig. S1

Ovarian stem cell cultures positively stained on mesenchymal stem cell markers. a M-CAM. b Thy-1. c STRO-1. d Negative control (CD 14). e Negative control (CD 19). Bar 100 μm (JPEG 21 kb)

High resolution image (TIFF 896 kb)

Supplementary Fig. S2

Positive staining of mesenchymal-like stem cells for the expression of markers of pluripotency, namely OCT-4, SSEA-4, SOX-2 and NANOG, as revealed by immunofluorescence. a OCT-4-positive cells. b Nuclei stained with DAPI. c Merged image of a, b. d SSEA-4-positive cells. e Nuclei stained with DAPI. f Merged image of d, e. g SOX-2-positive cells. h Nuclei stained with DAPI. i Merged image of g, h. j NANOG-positive cells. k Nuclei stained with DAPI. l Merged image of j, k. Bar 100 μm (JPEG 32 kb)

High resolution image (TIFF 1734 kb)

Supplementary Fig. S3

Amplification plot showing expression of telomerase (TERT telomerase reverse transcriptase) in samples of ovarian stem cells (T1 ovarian stem cells, T2 putative ovarian stem cells in 2-week-old culture, T3 human embryonic stem cells [H1 line], T4 putative ovarian stem cells in 7-month-old culture, gapdh glyceraldehyde 3-phoshate dehydrogenase) (JPEG 53 kb)

High resolution image (TIFF 1399 kb)

Supplementary Fig. S4

Western blot analysis in ovarian cell cultures developed without primary antibody on hESCs. Membrane was probed with secondary antibody only. Immunoreactive protein corresponded to upper band at around 75 kDa and was non-specific (JPEG 7 kb)

High resolution image (TIFF 1930 kb)

Supplementary Fig. S5

Teratoma formation in SCID (severe combined immunodeficiency) mice after transplantation of ovarian cancer and melanoma cells (positive controls) (JPEG 27 kb)

High resolution image (TIFF 1286 kb)

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Stimpfel, M., Skutella, T., Cvjeticanin, B. et al. Isolation, characterization and differentiation of cells expressing pluripotent/multipotent markers from adult human ovaries. Cell Tissue Res 354, 593–607 (2013). https://doi.org/10.1007/s00441-013-1677-8

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