Isolation and Characterisation of Putative Ovarian Germ Line Stem Cells

Prof. Evelyn E TelferInstitute of Cell Biology

and Centre for Integrative PhysiologyUniversity of Edinburgh

Disclosures

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

Preantral

Antral

Decreasing numbers with development

Primordial

Increasing levels of apoptosisLate

Preantral

Store of “resting” primordial follicles utilized throughout reproductive life

Does the ovary have any capacity for regeneration?

Fat CellsBone

CartilageMuscle

Skeletal MuscleSkeletal MuscleSmooth muscle

Bone Cartilage

FatHeart

BrainBrain

NervesBlood cells

MuscleAll tissues

GastrointestinalEsophagusStomach

Small IntestineLarge Intestine

PlacentaBone

CartilageMuscleNerve

Bone MarrowTendon

Blood vessel

CORD BLOODVarious Tissue

Peripheral BloodBone MarrowBlood cells

Nerves

Bone MarrowMarrowBone

CartilageTendonMuscle

FatLiver

Brain/NerveBlood cells

HeartAll Tissues

Skin

CorneaRetina

Spermatogonia

HeartLung

PancreasLiver

Fast developing field of adult stem cells

It is likely that the ovary has stem cells for allovarian cell components

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

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

Putative human OSCs form oocyte like structures

Isolation of OSCs based on DDX4 protein being extracellular

Telfer & Albertini, 2012

Testing the potential of putative OSCs isolated from

human ovaries

Can human ovarian tissue supportthe formation of follicles from putative

OSCs in vitro?

In Vitro Growth of Human Oocytes / Follicles: Three Step Process

Follicles prior to

isolation

Step 2: Culture isolated folliclesStep 1: Micro-cortex culture

Step 3: Cumulus complex culture (oocyte maturation)

Remove oocyte and surrounding cells

Telfer et al., 2008

Antral development from in vitro grown human primordial follicles within 10 days

Type III Type IV

Type I Type II

GFP-positive cells in different stages of development

GFP+

GFP+

GFP+

GFP+

GFP+

Telfer et al., unpublishedScale bar 50 µm

Only oocyte like structures are GFP positive

GFP +ve

GFP-ve

Positive and negative GFP “Oocyte like structures” in same sections is an indication that the injected cells are forming

structures and recruiting somatic cellsTelfer et al, unpublished

C

Initial culture stagesA

B

6 days and 12 days of cultureC

D

Human putative OSCs form follicles within 6 days and are multilaminar structures within 10-14 days

Cells that are capable of recruiting somatic cells and forming oocyte like follicles have been isolated from human ovaries in the Tilly lab

Need to independently isolate cells: How reproducible is this?Telfer et al, unpublished

Isolation of putative OSCs fromhuman 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

Forward Scatter (FS) = cell sizeSide Scatter (SS) = Granularity

Fluorescence Activated Cell Sorting

Disaggregated Ovarian Cortex

OSCDDX4Primary Antibody

Secondary Antibody

Fluorescent Tag

Adapted from Woods & Tilly, 2013

Sample

Nozzle

Cells pass through in single file

Sort cells on basis of DDX-4 on cell surface and cell size

PCR analysis of human FACS sorted cells

a) Positive controlb) DDX4 positive cells

P2c) DDX4 positive cells

P3d) DDX4 positive cells

P1e) DDX4 negative

cellsf) Negative control

GERM LINE MARKERS

STEM CELL MARKERS

FOLLICLE/OOCYTE MARKERS

HOUSEKEEPING

P1

P2

P3

DDX4Dunlop, Clarkson, McLaughlin, Waterfall, Skehel, Anderson & Telfer (Unpublished)

DDX4 protein is present in FACS sorted positive cells but not in negative cells

McLaughlin, Clarkson, Waterfall, Dunlop, Skehel, Anderson, Telfer (unpublished)

DDX4 cells can be observed in human ovaries

Telfer et al, unpublished Scale bar 25 µm

Negative controls

DDX4 positive cells in the ovary

Controversy on DDX4 localisation

RNA helicase FACS sorted DDX4 +ve cells with external epitope

Meikar et al., 2011 Hernandez et al., 2015

C-terminus of DDX4 detected on cell surface

Scale bar 20 µm

Permeabilised

Non-perm

eabilised

Myc + Cy2 Myc + Cy2

Myc-Vap-B

DDX4

Clarkson, Liao, McLaughlin, Skehel, Anderson, Telfer (unpublished)

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

Chimeric ovary studies: Mouse somatic cells only

Isolated mouse somatic cells transplanted under kidney capsule of SCID mouse (no follicles observed)

Bovine putative OSCs combined with mouse somatic cells, and transplanted to SCID mouse – oocyte / follicle structures formed

Chimeric ovary studies: Bovine putative OSCs combined with mouse somatic cells

FollicleFollicle

FollicleFollicle

Oocyte Growth

In vivo

19-30µm

40-80µm 80-90µm

90-100µm 100-110µm

Pre-ovulatoryprimordial primary preantral Early antral Mid antral

Functional oocyte development

• Growth / Meiotic Arrest

• Acquisition of Meiotic Competence

• Acquisition of Developmental Competence

• Transcription / Transcriptional Repression

• Genomic imprinting

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

• 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

The future

Acknowledgements

All the patients who kindly donate their ovarian tissue and the clinical teams that support this

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)