Isolation and Characterisation of Putative Ovarian Germ Line Stem Cells

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2 Isolation and Characterisation of Putative Ovarian Germ Line Stem Cells Prof. Evelyn E Telfer Institute of Cell Biology and Centre for Integrative Physiology University of Edinburgh

3 Disclosures Evelyn E Telfer is a receipt of a MRC research grant and a research grant from OvaScience

4 Store of resting primordial follicles utilized throughout reproductive life Primordial Preantral Decreasing numbers with development Late Preantral Increasing levels of apoptosis Antral Does the ovary have any capacity for regeneration?

5 Fast developing field of adult stem cells Skeletal Muscle Skeletal Muscle Smooth muscle Bone Cartilage Fat Heart Brain Brain Nerves Blood cells Muscle All tissues Bone Marrow Marrow Bone Cartilage Tendon Muscle Fat Liver Brain/Nerve Blood cells Heart All Tissues Cornea Retina Skin Heart Lung Pancreas Liver Spermatogonia Peripheral Blood Bone Marrow Blood cells Nerves Fat Cells Bone Cartilage Muscle Gastrointestinal Esophagus Stomach Small Intestine Large Intestine Placenta CORD BLOOD Bone Various Tissue Cartilage Muscle Nerve Bone Marrow Tendon Blood vessel It is likely that the ovary has stem cells for all ovarian cell components

6 Putative Human Oogonial Stem Cells (OSCs) isolated from adult woman ovarian tissue Fluorescent Activated Cell Sorting (FACS) approach DDX4:BrdU DDX4 DDX4:BrdU double staining human cultured OSCs: DDX4=germ cell marker BrdU=incorporates into proliferating cells

7 OSCs are unipotent progenitor germ cells Characteristic Stem Cell Progenitor Cell OSCs In vivo self-renewal Unlimited; lifetime Limited; transient Limited; transient In vitro self-renewal Unlimited Limited Limited Potentiality Pluripotent Unipotent Unipotent Adapted from Seaburg 2003

8 Putative human OSCs form oocyte like structures Isolation of OSCs based on DDX4 protein being extracellular Telfer & Albertini, 2012

9 Testing the potential of putative OSCs isolated from human ovaries Can human ovarian tissue support the formation of follicles from putative OSCs in vitro?

10 In Vitro Growth of Human Oocytes / Follicles: Three Step Process Step 1: Micro-cortex culture Step 2: Culture isolated follicles Follicles prior to isolation Antral development from in vitro grown human primordial follicles within 10 days Step 3: Cumulus complex culture (oocyte maturation) Telfer et al., 2008 Remove oocyte and surrounding cells

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12 GFP-positive cells in different stages of development Type I Type II GFP+ GFP+ GFP+ Type III Type IV GFP+ GFP+ Telfer et al., unpublished Scale bar 50 µm

13 Only oocyte like structures are GFP positive GFP-ve GFP +ve Telfer et al, unpublished Positive and negative GFP Oocyte like structures in same sections is an indication that the injected cells are forming structures and recruiting somatic cells

14 Human putative OSCs form follicles within 6 days and are multilaminar structures within days C Cells that are capable of recruiting somatic cells and forming oocyte like follicles have been isolated from human ovaries in the Tilly lab Initial culture stages A 6 days and 12 days of culture C B D Need to independently isolate cells: How reproducible is this? Telfer et al, unpublished

15 Isolation of putative OSCs from human ovarian tissue using FACs based sorting in our lab Marie McLaughlin, Yvonne Clarkson, Paul Skehel, Martin Waterfall, Cheryl Dunlop, Hamish Wallace, Richard Anderson and Evelyn Telfer

16 Fluorescence Activated Cell Sorting Disaggregated Ovarian Cortex Sample OSC Fluorescent Tag Secondary Antibody Primary Antibody DDX4 Nozzle Cells pass through in single file Forward Scatter (FS) = cell size Side Scatter (SS) = Granularity Sort cells on basis of DDX-4 on cell surface and cell size Adapted from Woods & Tilly, 2013

17 PCR analysis of human FACS sorted cells GERM LINE MARKERS a) Positive control b) DDX4 positive cells P2 c) DDX4 positive cells P3 d) DDX4 positive cells P1 e) DDX4 negative cells f) Negative control STEM CELL MARKERS FOLLICLE/OOCYTE MARKERS P1 P3 Dunlop, Clarkson, McLaughlin, Waterfall, Skehel, Anderson & Telfer (Unpublished) HOUSEKEEPING DDX4 P2

18 McLaughlin, Clarkson, Waterfall, Dunlop, Skehel, Anderson, Telfer (unpublished) DDX4 protein is present in FACS sorted positive cells but not in negative cells

19 DDX4 cells can be observed in human ovaries DDX4 positive cells in the ovary Negative controls Telfer et al, unpublished Scale bar 25 µm

20 Controversy on DDX4 localisation RNA helicase FACS sorted DDX4 +ve cells with external epitope Meikar et al., 2011 Hernandez et al., 2015

21 C-terminus of DDX4 detected on cell surface Myc + Cy2 Myc + Cy2 Myc-Vap- B DDX4 Non-permeabilised Permeabilised Scale bar 20 µm Clarkson, Liao, McLaughlin, Skehel, Anderson, Telfer (unpublished)

22 Testing the potential of putative OSCs isolated in our lab (bovine) Chimeric ovary experiments Combine putative OSCs (bovine) with murine somatic cells Transplant under kidney capsule of SCID mouse PhD student Kelsey Grieve in collaboration with Williams lab Oxford 22

23 Chimeric ovary studies: Mouse somatic cells only Isolated mouse somatic cells transplanted under kidney capsule of SCID mouse (no follicles observed)

24 Chimeric ovary studies: Bovine putative OSCs combined with mouse somatic cells Follicle Follicle Follicle Follicle Bovine putative OSCs combined with mouse somatic cells, and transplanted to SCID mouse oocyte / follicle structures formed

25 Functional oocyte development In vivo Oocyte Growth 19-30µm 40-80µm 80-90µm µm µm primordial primary preantral Early antral Mid antral Pre-ovulatory Growth / Meiotic Arrest Acquisition of Meiotic Competence Acquisition of Developmental Competence Transcription / Transcriptional Repression Genomic imprinting

26 What does this mean? The menopause happens! But cells with germ and stem cell markers can be isolated from ovaries of women and prepubertal girls Useful model of human germ cell development / maturation Potential Clinical Applications: Fertility preservation / restoration. In vivo and in vitro

27 The future The field of germ line stem cells is new Concentration of effort has been on doubting their existence Putative OSCs have been identified in a range of species (bats, cows, pigs, rats, rhesus monkey, Lemur) Time to concentrate on defining the populations of cells that reside within the mature ovary Applying cell based strategies to infertility treatment and fertility preservation

28 Acknowledgements Marie McLaughlin Yvonne Clarkson Cheryl Dunlop Kelsey Grieve Paul Skehel Martin Waterfall John Binnie Joan Creiger Richard Anderson Hamish Wallace David Albertini (IVG work) Jon Tilly (Boston) Suzanne Williams (Oxford) All the patients who kindly donate their ovarian tissue and the clinical teams that support this