MAKING GAMETES FROM STEM CELLS

Anna Veiga1,2, Cristina Eguizabal1 1.- Centre de Medicina Regenerativa de

Barcelona

2.- Institut Universitari Dexeus. Barcelona

Pluripotent Stem Cells (PSC)

• Gamete differentiation from PSC(ES/iPS) - Derivation of Primordial Germ Cells (PGCs)- Oocyte derivation (mouse/human)- Sperm derivation (mouse/human)

• Oogenesis in the adult female

Pluripotent Stem Cells can be obtained from cells located in the inner cell mass of blastocysts (hESC) and from nuclear reprogramming (SCNT and iPS)

Hochedlinger, Development 2009 (adapted from Waddington, 1957)

Cell. 2007 IInduction of pluripotent stem cells from adult

human fibroblasts by defined factors.Takahashi K, Tanabe K, Ohnuki M, Narita M,

Ichisaka T, Tomoda K, Yamanaka S.

Science, 2007

Science,1998

Derivation of PGCs from PSCs

2008

• Generation a novel protocol based on the expression of CXCR4 receptor during the in vitro differentiation of PGCs from hES.

• This differentiation process is accompanied by the development of putative Sertoli-like cells.

• Generation a novel protocol (EBs + BMP4) for differentiating in vitro PGCs from hES.• These PGCs expressed specific germ cell markers and showed a removal of the

parental imprints and chromating modification changes.

2008

2009

• The generation and characterization of hESC lines stably carrying a VASA-pEGFP-1 reporter construct that expresses GFP in a population of differentiating hESC that show expression of characteristic markers of primordial germ cells.

2009

• Pluripotent stem cells (both hESC and iPS) give rise to in vitro PGC following 7 days of differentiation on hFGC

• They correspond to immature PGCs (developmental stage in vivo between specification and less than 9 week of gestation)

• Initiation of imprinting erasure is dependent on the epigenetic status of the

pluripotent stem cell from which iPGCs are generated.

Oocyte derivation

• After 2 weeks in culture, structures similar to advanced follicle-like structures appear. Most of them degenerate but around 20% give rise to

“oocytes”> 40 µm.

• The functional activity of the follicles is evidenced by estradiol production

Formation of follicular structures

• At D26 of culture, oocyte-like cells ( 50-70 µm) are released from surrounding somatic cells and float freely in the supernatant.

• All ZP markers are + except ZP1 (accounting for fragile

ZP structure)

• Addition of gonadotrophins to isolated follicles results in extrusion of “oocytes” and formation of a polar body-like structure

Oocyte-like structures derived from ES cells

• At about day 43, structures similar (morphology and markers) to preimplantation embryos are found.

These blastocyst-like structures represent parthenotes (probably) induced by culture conditions

Blastocyst-like structures derived from ES cells

Differentiation, 2007

• Granulosa cells are effective in inducing the differentiation of ES derived PGCs into oocyte-like cells through direct cell-to-cell contacts

• Novel in vitro system to study oogenesis

• Stem cells isolated from the skin of porcine fetuses have the intrinsic ability to differentiate into oocyte-like cells.

• When differentiation was induced, a subpopulation of these cells expressed markers (Oct4, GDF9b,DAZL and Vasa), consistent with germ-cell formation.

• On further differentiation, these cells formed follicle-like aggregates that secreted oestradiol and progesterone and responded to gonadotropin stimulation.

Nature Cell Biology 2006

• Some of these aggregates extruded large oocyte-like cells that expressed oocyte markers, such as ZP, and meiosis markers,(SCP3).

• Some of these oocyte-like cells spontaneously developed into parthenogenetic embryo-like structures.

2006

• Differentiation protocol using testicular cell conditioned media for derivating in vitro putative ·oocytes” from hES.

• These oocytes expressed some oocyte-specific markers.

Sperm derivation

• Culture conditions: Embryoid body differentiation

• Marker expression• pluripotency (Oct4).• GC development markers (Stella ,Fragilis)• Genes exclusively expressed in GC line

(Dazl, Piwil2, Rnf17, Rnh2, Tdrd1 y Tex14)

During embryoid body formation pluripotency markers expression decreased with some expression of GC specific genes

NATURE, 2004

• Up-regulation of genes associated with male germ cell development (Sry, Acrosin, Haprin, markers of Sertoli cells)

• No expression of Zp proteins.

• Within the context of EB differentiation, the default programm of female gametogenesis is suppressed.

PGC differentiation to functional gametes

Meiosis

EB microenvironment is permissive for male germ cell development and meiotic maturation, even though highly inefficient

• EB derived cells have a similar morphology to testis derived haploid cells. Round spermatids?

Sperm morphology

• ICSI with haploid cells into recipient oocytes

• 50% of the embryos cleaved to 2 cell stage

• 20% reach the blastocyst stage.• FISH: normal for sex chromosomes

Biological functionality

• Germ cell in vivo production from mESC.• Transplantation of ESC aggregates to testicular

capsule.• ESC derived cells can participate in spermatogenesis

and produce functional germ cells in vitro

PNAS, 2003

• Establishment of two cell lines (Spermatogonial stem cells- SSCs) with patterns of differentiation towards male germ cells .

• Derived from mESC (directed gene expression for premeiotic and haploid male germ cells)

• Motility of cells• Formation of tail-like structures

Nayernia et al, Developmental Cell 2006

• Positive for PGCs, premeiotic (Oct 4, Fragilis, Stella,..),, meiotic (Scp3, Acr) and postmeiotic male germ cells markers

• Formation of sperm structures.

• Acrosome like structures

• Condensation of the nucleus

• ICSI of in vitro-generated cells (haploid

cells) into oocytes of wildtype females.• Polar body extrusion, PN formation and

normal features of embryo development.• Transfer into the oviducts of

pseudopregnant females

• 65 embryos transferred • 12 animals born.• Larger or smaller offspring • Premature death (5 days to 5

months after birth). • Abnormal methylation patterns

and phenotypic abnormalities

interference with the essential reprograming events in gametogenesis

• BM stem cells are able to transdifferentiate into male germ cells that express molecular markers of PGC (fragilis, stella,Mvh..)

• Expression of spermatogonial stem cells and spermatogonia markers (CKIt, DAZL..)

Hum Reprod, 2009

• hESC can form PGC and post meiotic spermatids in vitro.

• Gene expression profiles characteristics of oocyte development and follicle like structures were detected. No oocyte with ZP was recognised.

• hESC create a favourable niche for spermatogenesis?

Nature, 2009

• Generation of in vitro PGC from male and female hESC

• Silencing and overexpression of genes encoding germ-cell specific cytoplasmic RNA binding proteins

• The human gene DAZL functions in PGC formation

• DAZ and BOULE promote germ-cell progression to meiosis and formation of haploid germ cells that ressemble round spermatids

2011

• iPS cells can form in vitro meiotic and post-meiotic haploid cells over-expressing DAZ, DAZL and BOULE.

2011

• Generation of a novel differentiating 2 step-protocol without overexpressing any genes.

• iPS can form in vitro meiotic and haploid like-cells.

• The post-meiotic-like cells generated have features of human spermatids expressing Acrosin.

2011

• Generation of in vitro PGC-like cells from mES and miPS through epiblast-like cells having a normal global transcription profiles, epigenetic reprogramming,and cellular dynamics.

• These PGCLCs express Integrin-b3 and SSEA1 markers of spermatogonias.

• Healthy offspring with normal methylation patterns of imprinted genes.

Oogenesis in the adult female?

Nature 2004

• Juvenile and adult mouse ovaries possess mitotically active germ cells that, based on rates of oocyte degenetration are needed to replenish the oocyte pool.

• Presence of cells expressing meiotic markers in juvenile and adult mouse ovaries

• Wild type ovaries grafted into transgenic mice (GFP) become infiltrated with GFP positive cells that form follicles

• Rapid generation of 100s of oocytes• Extragonadal source of germ cells?• Germline markers in BM • BM transplantation restores oocyte

production in wild type mice sterilized by chemotherapy

• Donor derived oocytes observed in females following peripheral blood transplantation

Cell, 2005

BM is a potential source of GC that sustain oocyte production in adulthood

• Bone marrow cells do not give rise to oocytes and do not enhance ovulation of endogenous oocytes in transplanted mice

Nature,2006

• Circulating cells do not give rise to mature ocytes in long term parabionts

• Circulating cells do not give rise to mature oocytes in injured parabionts

Transplantation and parabiotic mouse model.

Capacity of circulating BM to generate ovulated oocytes?

• PGCs have been obtained both from hESC and iPS cells• Oocyte-like cells can be obtained from embryonic and fetal

stem cells. • Male germ cells and functional sperm have been obtained

through ESC• Offspring generated from ESC derived sperm are highly

abnormal • The use of such gametes in ART remains a “distant

prospect"• Stem cell derived gametes can become a valuable

resource for research: germ cell development , epigenetic reprogramming and germline gene modification

• Oogenesis does not take place in the adult human female

Conclusions

THANK YOU FOR YOUR ATTENTION!

aveiga@cmrb.eu

anavei@dexeus.com