1

Running title: In Vitro Derived Human Sperm

Title: Derivation of Human Sperm from Embryonic Stem Cells

JAE HO LEE1, 2, 7, MAJLINDA LAKO1, 2, 7, LYLE ARMSTRONG1, 2, MARY HERBERT1, 2, 3, MANYU LI4, WOLFGANG ENGEL4, 8, Xin Zhang1, 2, DAVID

ELLIOTT2, MIODRAG STOJKOVIC5, JOHN PARRINGTON6, ALISON MURDOCH1, 3, TOM STRACHAN2, KARIM NAYERNIA1, 2, 9

1 North East England Stem Cell Institute (NESCI), Newcastle University, Newcastle upon Tyne, UK

2 Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, UK

3 Department of Reproductive Medicine, Newcastle University, Newcastle upon Tyne, UK

4 Institute of Human Genetics, University of Gttingen, Germany

5 Centro de Investigacin Prncipe Felipe (CIPF), Valencia, Spain

6 Department of Pharmacology, University of Oxford, Oxford, UK

7 These authors contributed equally to this work.

8 Wolfgang Engel did not work with human embryonic stem cells

9 Corresponding Author:

Prof. Dr. Karim Nayernia Chair of Stem Cell Biology NESCI/IHG International Centre for Life Newcastle University, Newcastle upon Tyne Central Parkway Newcastle upon Tyne NE1 3BZ UK Telephone: 0044-191-2418643 Fax: 0044-191-2418810 Email: karim.nayernia@ncl.ac.uk

Acknowledgement: This study was supported by Newcastle University, Newcastle upon Tyne and ONE NorthEast.

Key words: Human embryonic stem cells, Differentiation, Germ cells, Sperm

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ABSTRACT

Differentiation of human embryonic stem cells (hESCs) to specific cell types can be

achieved using methods that mimic in vivo embryonic developmental programs.

Human egg and sperm cells (gametes) are derived from a founder population of

germline stem cells (GSCs), primordial germ cells (PGCs), that are set aside early in

embryogenesis which give raise to gonocytes in males. After birth, gonocytes

differentiate to adult male germline stem cells, spermatogonial stem cells (SSCs),

which can self-renew and generate sperm cells, are unique stem cells in that they are

solely dedicated to transmit genetic information from generation to generation.

Understanding the mechanisms of germ cell specification, development and its

differentiation to sperm is important for elucidating the causes of male infertility.

Here, we developed an in vitro strategy for establishing of male GSCs from human

embryonic stem cells. These in vitro derived GSCs express markers which are

specific for PGCs, SSCs, meiotic and postmeiotic germ cells indicating maturation of

the primordial germ cells to haploid male gametes. In vitro derived germ cells are able

to enter meiosis and generate haploid motile sperm-like cells in vitro. While full

potential of the human ES derived germ cells and sperm remains to be demonstrated,

this in vitro modeling of human gametogenesis provides a new approach for studying

biology of human germ cells and establishment of therapeutic approaches in

reproductive medicine.

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INTRODUCTION

Germ cells are a pre-requisite for fertility [1, 2]. Derivation of both male and female

gametes in vitro raises the possibility of using these gametes to gain a better

understanding of basic reproductive biology and, in particular, to extend the potential

for therapeutic cloning, transgenic technologies and the treatment of infertility. Our

understanding of the molecular regulation of normal germ cell development in

mammals has progressed significantly due to the utility of the mouse as a model

system [3-5]. However, the molecular and cellular regulation of human germ cell

development is almost completely unknown due to the lack of an in vitro model. The

creation of such a model will provide powerful tools for undertaking new types of

reproductive studies, and might permit the development of new technologies and

novel approaches in assisted reproductive medicine. Human embryonic stem cells

(hESCs) could potentially fill this need, as embryonic stem cells (ESCs) in general

have a remarkable capacity for pluripotency. Recent studies have reported that

successful differentiation of mouse ESCs into primordial germ cells (PGCs) as well as

into mature male [6-8] and female [9] gametes can be achieved in vitro and in vivo.

Hubner et al. reported the first study of in vitro gamete production [9]. These authors

cultured mouse ESCs carrying a Pou5f1-reporter gene without feeder cells or added

growth factors. The formation of ovarian follicle-like structures was observed, which

included oocyte-like cells expressing Pou5f1 and Ddx4. These cells also expressed

meiotic marker genes (e.g., Dmc1, Sycp3), and were released from follicles after 34

weeks of culture. Although the germ cell potential of oocyte-like cells was not tested

by fertilization, structures that closely resemble blastocysts emerged in 6 weeks,

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suggesting spontaneous parthenogeneic activation. Thus, this study suggested that

ESC-derived cells have a potential to undergo gametogenesis in vitro.

Toyooka et al. next reported the derivation of male germ cells from mouse ESCs [6].

These authors took a different approach from Hubners, and used embryoid bodies

(EBs) as starting material. Using ESCs carrying a Ddx4-reporter construct, the authors

induced EBs and found that Ddx4+ cells spontaneously appeared, suggesting the

presence of cells with characteristics of postmigratory PGCs in EBs. Ddx4+ cells

were purified and aggregated with male genital ridge cells of wild-type embryos.

Following implantation into adult mouse testes, the cell aggregates formed

seminiferous tubules that supported complete spermatogenesis derived from purified

Ddx4+ cells. Although the ability of spermatozoa to activate eggs was not examined,

this study suggested that the germ line specification and the emergence of

postmigratory PGCs can occur spontaneously or be induced in EBs.

Using EBs, Geijsen et al. also suggested a spontaneous emergence of male PGCs

from mouse ESCs in vitro [7]. Furthermore, the authors detected and isolated haploid

cells from EBs, and showed that the injection of the cells into eggs resulted in the

formation of blastocyst-like structures. Although the isolated cells did not resemble

spermatozoa and analyses of further embryonic development were not completed, this

study suggested that male PGCs arise from ESCs and spontaneously become

postmeiotic cells that are capable of activating eggs.

Recently, Nayernia et al. reported not only the induction of male gametes from ESCs

but also successful production of offspring [8]. These authors cultured mouse ESCs,

which were transfected with a Stra8 (stimulated by retinoic acid gene 8)-reporter

construct, and later, also with a Prm1 (protamine 1)-reporter construct, in monolayer

culture. Following repeated RA treatments and selection of Stra8+ cells, they isolated

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Prm1+ cells and injected them into eggs. When the resulting 65 embryos were

transferred to surrogate mothers, seven live pups carrying the Prm1-reporter gene

were derived, which apparently had growth abnormalities and died within 5 months

after birth.

The ability of human ESCs to enter the germ line was examined by Clark et al. [10].

These authors generated EBs from female and male human ESCs and found using

PCR or immunohistochemistry that some cells in EBs expressed marker genes

specific to different stages of germ line development (e.g., DDX4, DAZL).

Furthermore, cells expressing a meiotic marker gene, SYCP3, were identified in

human EBs. Thus, this study suggested the possibility that human ESCs of both sexes

may spontaneously enter the germ line and undergo meiosis.

In present study, we have developed a strategy for the establishment of germline stem

cells from human embryonic stem cells which are able to enter and complete meiosis

and produce sperm-like cells.

MATERIALS AND METHODS

Culture of Human Embryonic Stem Cells

Human ESC were grown on mitotically inactivated mouse embryonic fibroblasts and

passaged essentially as described [11, 12]. The human ESC medium contains

Knockout-DMEM (Invitrogen, Paisley, UK), 100 M -mercaptoethanol

(Sigma,Dorset UK), 1 mM L-glutamine (Invitrogen), 100 mM non-essential amino

acids (Invitrogen), 20% serum replacement (SR, Invitrogen), 1% penicillin-

streptomycin (Sigma) and 4 ng/ml FGF2 (Invitrogen).Medium was changed daily.

Human ESC were passaged by incubation in 1 mg/ml collagenase IV (Invitrogen) for

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5-8 minutes at 37 C or mechanically dissociated and then removed to freshly

prepared feeders.

In Vitro Derivation of Male Germ Cells

Construction of the Stra8-EGFP construct was previously described [13]. Human

ESCs were maintained under antibiotic free conditions for at least 3 passages before

transfection. On the day of transfection, human ESC were incubated with collagenase

IV (1mg/ml; Sigma) for five minutes to generate small clumps which were

subsequently transfected using the Nucleofector kit from Amaxa (mouse embryonic

stem cell transfection kit) using manufacturers instructions. Two days after the

transfection, stable clones were selected using G418 selection (150 g/ml) for 10 days.

The efficiency of transfection was 7 colonies for Ncl-3 and 10 colonies for Ncl-4 per

1,000,000 cells. The resulting colonies were pooled together to generate the stable

cells lines Ncl3-Stra8 and Ncl4-Stra8.

Thereafter, cells were cultured on a feeder layer of mitomycin C-inactivated mouse

embryonic fibroblasts with human ESCs medium. To induce differentiation, hESCs

were spontaneously differentiated for 3 weeks with differentiation ESCs medium,

which was replaced 20% serum replacement (SR, Invitrogen) with 20% FBS

(Hyclone) and excluded 4 ng/ml FGF2 (Invitrogen). After this, these ESCs were

cultured with conditioned medium containing retinoic acid (RA) (Sigma) at a final

concentration of 106 M for another 3 weeks. Differentiated GFP-positive cells were

sorted by FACS. Sorted cells were cultured in medium containing Knockout-DMEM

(Invitrogen, Paisley, UK), 1 mM L-glutamine (Invitrogen), 100 mM non-essential

amino acids (Invitrogen), 20% serum replacement (SR, Invitrogen), 1% penicillin

streptomycin (Sigma), 1,000 U/ml human recombinant leukemia inhibitory factor

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(LIF; Sigma) and 4 ng/ml human recombinant basic fibroblast growth factor (hrbFGF;

GIBCO). Furthermore, 10 mM forskolin (Sigma) is a mediator that enhances

intracellular cAMP levels by stimulating adenylate cyclase (protein kinase A) and it

has also been reported to significantly promote PGC proliferation [14, 15]. In

particular, to help aggregation, sorted cells were cultured in 96 well plate and

centrifuged at 1400 rpm for 4 min and for ROCK inhibitor treatment, Y-27632

(Calbiochem; water soluble) was added to culture medium at 10 mM 1 h before

detaching the cells from the feeder layer and also upon seeding the cells onto a

newMEF layer. A single halfday treatment of Y-27632 was sufficient for enhanced

survival of dissociated hES cells in low-density adhesion culture on MEF cells [16].

Multi-cellular colonies emerged in approximately 20-25 days.

SemiQuantitative RTPCR Analysis

Total RNA was isolated by using the RNeasy Mini Kit (Qiagen) according to

manufacturers instructions. Single strand cDNAs were prepared from 5 g RNA

using a reverse transcriptase reaction (Invitrogen, Karlsruhe, Germany) and analysis of

gene expression was done RT-PCR with Platinum Taq (Invitrogen). The primer pairs

are shown in Table 1.

Real-Time RT-PCR Analysis

Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to

manufacturers instructions and For mRNA, reverse transcription was performed

using random primers and the Superscript III kit (Invitrogen). Real-time quantitative

PCR for mRNA was conducted with 1 l of cDNA, 10 l of QuantiTect SYBR Green

PCR Master Mix, 1 l each of specific 10 M forward and reverse primers (MWG

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Biotech, Ebersberg, Germany), and 7 l of sterile water (all reagents from Qiagen,

Hilden, Germany) using primer sets shown in Table 1.

The data were analyzed by using the RelQuant software (Roche Applied Science).

Genomic PCR

PCR analysis was performed on genomic DNA extracted from human ESCs with

NEO, EGFP and Stra8-EGFP-specific primers: GFP (5-GAC CCT GAA GTT CAT

CTG CA-3, 5-ACT CCA GCA GGA CCA TGT GAT-3), NEO (5-AGG CTA

TTC GGC TAT GAC TG-3, 5-TCA TCC TGA TCG ACA AGA CC-3), Stra8-

EGFP (5-CGT AGT GTG CCA AAC TGA TGT GG-3, 5-CAG GGT CAG CTT

GCC GTA GGT GG-3), respectively. The cycling conditions were as follows: 5 min

at 94C, five cycles of 30 s at 94C, 30 s at 5761C, 1 min at 72C, 30 cycles of 30 s

at 94C, 45 s at 56C, 1 min at 72C and 72C for 10 min.

Flow Cytometry

Single cell suspensions were generated using acutase (Chemicon). Single cells were

suspended in PBS at concentration of 106 cells/ml. Cells were washed again three

times and resuspended in PBS before being analysed with FACS Calibur instrument

(BD) equipped with CellQuest software. For haploidization, cells were prepared as

described above and stained with using a Kit supplied by Partec (CyStain DNA). Cell

count was ensured to be below 5x106/ml. After that, extraction buffer (200ul to 100ul

of cells) was added for 30 mins at room temperature. For counter staining, 800ul of

DAPI added and incubated in the dark for 30 mins. The sample was analysed at 10PSI

using a 60mw UV laser (355) at cell count of 200 cells / second.

Immunohistochemistry

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Cytospin preparations of single cell suspension were made by using approximately

10,000 cells per slide. Cells were fixed in 4% paraformaldehyde solution for 10 min at

room temperature, washed three times in PBS and then incubated with an anti-Piwil2

[24], TSPY [25], RBM [26], hnRNPG-T [26], PLCzeta [27], antibody in a

humidifying chamber at 4C overnight. After washing, culture slides were incubated

with a 1:300 FITC-conjugated in goat anti-sheep IgG, 1:300 FITC-conjugated in goat

anti-rabbit IgG and/or 1:300 Cy3-conjugated in goat anti-rabbit IgG (Sigma-Aldrich)

secondary antibody. Slides were washed three times in PBS and mounted in DAPI

mounting solution (Vector Laboratories, Burlingame, CA, USA).

Fluorescence In Situ Hybridization Analysis

Moving cells were washed with PBS and then resuspended in 30% glycerol-PBS.

Briefly, cells were fixed with 0.5% formaldehyde-PBS for 2 hrs and decondensed in

10 mM DTT, 0 to 0.5 mg/ml heparin in PBS for 30 minutes at room temperature.

Treatment with DTT-heparin induces uniform nuclear swelling, while preserving

nuclear shape; the higher the heparin concentration, the higher the resulting nuclear

decondensation. Such swelling is a prerequisite to perform FISH in moving cells.

Decondensed cells were loaded onto microscope slides, air-dried, washed in 2x SSC at

37 C for 2 mins, water, dehydrated in a 70% to 100% ethanol series for each 1 min

and air-dried again. For denaturation of the FISH probe, a wet towel was placed in a

sealed box/container and leave in a 37 C incubator. A hot plate was set at 75 C and

allowed to reach temperature before moving onto the next step. After that, probe (7 l

buffer + 2 l dH20 + 1 l probe) 10 l was aliquoted into the tube and spun for a few

seconds in a micro-centrifuge. 10ul of probe was distributed onto slide and place a

22x22mm coverslip (0-thickness) and the edges of the cover-slip were sealed with

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vulcanizing rubber glue solution and then left to dry at 37 C for approximately 5

minutes. Finally, the cover-slip placed face down onto the slide and left for 1 min

for the probe to denature. The slide was put in humid box and left in a 37 C incubator

overnight. For post hybridization wash, approximately 50mls of post hybridization

buffer was washed into a coplin jar and placed it into a cool water bath before setting

the temperature to 72 C. 50mls of 2x SSC was aliquoted into a coplin jar, carefully

removed vulcanizing rubber glue solution and cover-slip from slide, washed slide in

2xSSC for 2mins using tweezers and mounted slide with preferably Vectashield anti-

fade mountant with DAPI.

Poseidon fluorescent labeled specific DNA probes SE X (DXZ1)/SE Y (DYZ3) and

SE 18 (D18Z1)-specific probes from KREATECH Biotechnology. SE X/SE Y

KB20030 is a ready made mix of the two probes with the X centromere being labelled

in green and the Y centromere being labelled in red. Additionally, SE 18 (D18Z1)

KB-20018 is labelled in red. DNA from appropriate, chromosome-specific probes are

first labeled with reporter molecules. The labeled DNA probe is then hybridized to the

metaphase chromosomes or interphase nuclei on a slide. After washing, the specimen

is screened for the reporter molecules by fluorescence microscopy.

RESULTS AND DISCUSSION

Embryonic stem (ES) cells are pluripotent cell lines established from undifferentiated

embryonic cells characterized by nearly unlimited self-renewal and differentiation

capacity. During differentiation in vitro, ES cells were found to be able to develop

into specialized somatic cells types and to recapitulate processes of early embryonic

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development. Despite the well-recognized pluripotency of hESC, it has proven rather

challenging to direct the differentiation of hESC into specific lineages. However, an

ESC-based strategy could permit the generation of an unlimited supply of desirable,

fully functional cell types from an abundant, renewable, and readily-accessible source.

The differentiation of hESC into specific lineages could provide a tool for studying

the molecular mechanisms underlying lineage specification, early embryonic

developmental pathways, and the pathological basis of genetic disorders, as well as

provide a source of transplantable cells and tissue. In all these studies, strategies for

manipulating stem cell differentiation (selection of differentiated cell types) are based

on knowledge of the mechanisms by which lineage decisions are made during early

embryogenesis. A combination of approaches, including analysis of molecular

markers specifically expressed in relevant cell types and morphological and/or

histochemical approaches should be used to define differentiated cell types. Therefore,

in order to identify germ cells in different stages of formation and differentiation

during human ES differentiation, we compiled a list of markers that are germ cell-

specific (not expressed in somatic lineages), and germ cell-enriched (highly expressed

in germ cells with limited expression in somatic cells) and are expressed in defined

stages of germ cell development in vivo (from primordial germ cells to sperm). From

this list, a profile of the expected sequential expression of markers were generated that

would define development of the male germ cell lineage during ES cell differentiation

in vitro (Fig. 1)

Mimicking the cellular interactions of the embryo by providing appropriate signalling

molecules in culture has enabled the differentiation of ES cells to be directed

predominately toward particular lineages. Multistep strategies incorporating the

provision of soluble factors known to influence lineage choices in the embryo,

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coculture with other cells or tissues, genetic modification, and selection for desirable

cell types by reporter gene constructs have allowed the production of ES cell derived

specific cell types. We used a promoter based selection strategy that can visualize the

initial step of male germ cell formation. We used promoter region of Stra8 gene for

generation of reporter construct (Fig. 2A). The Stra8 gene encodes a cytoplasmic

protein and is expressed specific to the developing male gonad during mouse

embryogenesis. In adult mouse, its expression is restricted to the premeiotic germ

cells.

Two XY (hES-NCL3, hES-NCL4) [12] and one XX (H9) [28] human ES cell lines

were transfected with the Stra8-EGFP fusion gene and stable cell lines were

established (Fig. 2B, C). After culture of cells in conditioned medium containing

retinoic acid (RA), positive cells were sorted by fluorescence-activated cell sorting

(FACS), and after culturing, expression of green fluorescence protein (GFP) under the

control of the germline stem cell specific Stra8 promoter region [13] was observed

(Fig. 2D-O). The expression of Stra8 gene which is required for spermatogenesis is

directly related to the availability of retinoic acid.

RT-PCR analyses were employed to evaluate the expression in the GFP+ sorted cells

of different markers for premeiotic, meiotic, and postmeiotic male germ cells. Positive

expression was found for markers for the premeiotic (OCT4 [29], GDF3 (Growth and

Differentiation Factor 3) [30], C-KIT [29], STELLA [30], VASA [31, 32], , PLZF [20],

PIWIL2 [14, 24], DAZL (Deleted in Azoospermia Like) [31]), meiotic ( synaptonemal

complex protein (SCP3) [33], DMC1 [34], acrosin (ACR) [22], BOULE [35], PGK2

[36]) and haploid (TP2 [23], protamine 1 (PRM1) [23]) male germ cells (Fig. 3A).

These results could be confirmed by quantitative real-time RT-PCR analysis (Fig. 3B).

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Therefore, Stra8-EGFP positive cells appear to be heterogeneous and contain

premeiotic, meiotic and postmeiotic cells.

Expression of germ cell-specific genes in the sorted cells revealed that these cells are

able to differentiate to different stages of postnatal male germ cell development. On

protein level, male germ cell differentiation could be confirmed by

immunohistochemical analysis with antibodies against VASA [31, 32], RNA Binding

Motif (RBM) [37], PIWIL2 [14, 24], Testis Specific Protein Y-encoded (TSPY) [25,

38], HNRNPG-T [26] , Synaptonemal Complex Protein 1 (SCP1) [39], PLCzeta [40]

and PRM1 [23] proteins which are expressed in premeiotic, meiotic and postmeiotic

stages (Fig. 4).

To investigate whether ES-derived germ cells undergo meiosis in vitro, we

determined the DNA content of the cells by flow cytometric analysis. As shown in

Figure 5A and 5B, about 3.1 % of cells were haploid indicating completion of meiotic

process (Fig. 5). In contrast, in germline stem cells derived from XX embryonic stem

cells (H9), only male specific premeiotic and early meiotic markers could be observed,

indicating that XX derived germ cells are not able to enter meiosis in vitro (Fig. 5).

Motility is a characteristics function of the male gamete, which allows sperm to

actively reach and penetrate the female gamete. Motile cells were released to the hES

cell culture medium with human recombinant leukemia inhibitory factor (LIF), human

recombinant basic fibroblast growth factor (hrbFGF), forskolin and ROCK inhibitor

treatment, Y-27632 after culturing the cells for two months in the hES cell culture

medium with retinoic acid (RA) indicating formation of the specialized structure of

the sperm flagellum (Supplemental Video online). The formation of tail-like

structures could be observed in some motile cells (Fig. 6A). Meiosis of these cells was

confirmed by fluorescence in situ hybridization (FISH) analysis on chromosomes 18,

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X and Y (Fig. 6B). Using the X-Y-18 assay, chromosome X is green, chromosome Y

is red and 18 is red. A normal human sperm carrying 23 single chromosome is labeled

in chromosome X, Y and 18 respectively and ES-IVD is also shown in each labeled

chromosome X, Y and 18 like normal human sperm.

These results clearly demonstrate that human embryonic stem cells are able to

differentiate to adult male germline stem cells, spermatogonial stem cells, which can

undergo meiosis and produce motile haploid sperm-like cells. These models are very

useful tools for understanding of molecular and cellular mechanisms of male

infertility, establishment of therapeutic approaches for male infertility and provide

testing systems to examine toxicological effects of drugs on human germ cells.

Approximately 1 in 7 couples have fertility problems. In 20% of couples, low sperm

count/quality is the only cause of infertility, and it is a contributory factor in a further

25% of couples. In 30-50% of men, no cause is identified for the poor sperm

characteristics. Although assisted reproductive technologies such as IVF and ICSI

have dramatically improved the prospects for infertile couples, there are some types

of male factor infertility that remain untreatable. These include arrested

spermatogenesis which may be due to defective germ cells, an abnormal testicular

environment or aberrations in endocrine pathways regulating testis function. One of

the essential bases for treatment of male infertility is the understanding of the human

spermatogenesis by modelling human germ cell development. There has previously

been no robust cell-based model for examining the genetic and epigenetic

mechanisms of human germ cell formation.

Human spermatogenesis consists of three biologically significant processes: stem cell

self-renewal and differentiation, meiosis, and haploid cell morphogenesis.

Understanding the molecular mechanisms behind these processes might provide clues

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to the puzzle of species preservation and evolution. From the biological point of view,

in vitro derived germ cells provide a very useful experimental tool for undertanding of

basic mechanisms of male germ cell specification, development and differntiation

including meiosis, germ cell morphogenesis and epigenetics. We demonstarted clearly

that germ cells dereived from XY hES cells are able to enter meiosis and complete

this process. In contrast, germ cells derived from XX hES cells express spermatogonia

specific markers, but are not able to complete meiotic process in vitro. There are

several lines of evidence demonstrating that genes on Y chromosomes are essential

for meiosis. DAZ (Deleted in Azoopsermia) and BOULE proteins paly imortant role

during meiosis. The defects in spermatogenesis that are associated with an absence of

BOULE or DAZ function have substantial similarities. Spermatocytes are formed in

boule homozygotes, but fail to udergo meiotic devisions. In individuals deletd for

DAZ containing region of Yq, the postmeiotic stages of spermatogenesis are similary

rarer of absent [41, 42].

Our results clearly demonstrated that germ cells derived from XY human embryonic

stem cells undergo meiotic division and produce haploid sperm-like cells. However,

these proof-of-principle experiments should be followed by detailed molecular,

cellular, morphological and functional analysis in further experiments.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interests

ACKNOWLEDGEMENTS

This research was supported by The University of Newcastle upon Tyne, One

NorthEast and BBSRC. We thank I. Dimmick, Ch. Mueller and J. Barel for excellent

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technical assistance. We thank J. Schmidtke, D. Elliott and H. Cooke for providing

antibodies against Tspy and hnRNPG-T, Dazl and RBM antibodies.

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Online Supplemental Video: Release of motile sperm-like cells in medium

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LEGEND TO FIGURES

FIG. 1. Schematic representation of the different stages of germ cell differentiation in

prenatal and postnatal development. Expected expression patterns of genes used to

predict each stage of germ cell development are shown by the name of the gene

together with the black bars extending to the right of each gene name.

FIG. 2. Isolation of germ cells derived human embryonic stem cells. (A) Schematic

depiction of the Stra8-EGFP reporter gene harbouring a 1.4 kb promoter region of

Stra8. (B, C) Genomic PCR using specific primers for GFP, neomycin (NEO) and

Stra8-EGFP regions on DNA isolated from transfected human embryonic stem cell

lines H9, hES-NCL3 (NCL3), hES-NCL4 (NCL4) and untransfected human

embryonic stem cell (-). Phase contrast (left), fluorescence (middle) and merged

images (right) of Stra8-EGFP+ sorted cells growing on a embryonic fibroblast layer

show expression of green fluorescence protein under germline stem cell specific

promoter region, (D-F) Stimulation of differentiation of germ cells treatment with

retinoic acid (RA) on spontaneous differentiation of hES-NCL4 (NCL4) and (J-L)

H9 cell lines. (G-I) After FACS, Stra8-EGFP (Green) expression in colonized hES-

NCL4 (NCL4) and (M-O) H9 cell lines. Bar, 50 m.

FIG. 3. (A) Transcription of germ cell specific genes in hES cell derived germ cells.

RT-PCR analysis of parental XY (1) and XX (2) embryonic stem cells under

nondifferentiating conditions compared to the sorted cells in differentiating conditions

(4:XY, 7:XX). Expression of premeiotic (OCT4, GDF3 (growth/differentiation factor

3), C-KIT, VASA, STELLA, PLZF (promyelocytic leukemia zinc finger), PIWIL2

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(piwi-like 2), DAZL, (deleted-in-azoospermia-like)); meiotic (SCP3 (synaptonemal

complex protein 3), DMC1 (disrupted meiotic cDNA), ACR (acrosin), BOULE, and

PGK2 (phosphoglycerate kinase 2)) and haploid (TP2 (transition nuclear protein 2)

and PRM1 (protamine 1)) genes could be detected in XY hES derived germ cells (4).

In XX hES cell derived germ cells, only expression of premeiotic germ cells and

SCP3 could be observed (7). RT-PCR analysis of mouse fibroblast (3), human testis

(5) and human heart (6) served as controls. (B) Quantitative expression analysis of

germ cell specific genes in undifferentiated XY and XX ES cells, germ cells derived

from XY and XX ES cells, mouse fibroblast, human testis and human heart served as

controls. A clear increase of expression of germ cell specific genes ( Stra8, c-Kit,

Vasa, plzf, Piwil2, SCP3, PGK2 and Prm1) has been detected.

FIG. 4. Expression of germ cell specific markers in differentiated hES cells. (A-I)

Immunohistochemical analysis showed expression of premeiotic (VASA, RBM,

PIWIL2, TSPY), meiotic (hnRNPG-T (GT), SYCP1 (synaptonemal complex protein

1) and sperm (PLCzeta, PRM1 (protamine 1)) specific proteins. (A-F and H, I) Bar,

20 m. (G) Bar, 50 m.

FIG. 5. Haploidization of hES cell derived germ cells. (A) Identification of haploid

(1N), diploid (2N) and tetraploid (4N) cell populations in ES-derived germ cells. XY

hES derived germ cells (XY-IVD) show about 3.1% haploid cells, whereas XX hES

(XX-IVD) derived germ cells show no haploid cells, DNA content of human sperm

and human Leucocytes serve as controls. (B) Balk diagram of FACS analysis.

Page 23 of 30St

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FIG. 6. Formation of haploid sperm-like cells. (A) Magnification of cells from

supernatant showing formation of tail and head in sperm-like cells. (B) Fluorescence

in situ hybridization analysis of ES-derived sperm-like cells (lower part) and control

sperm cells (upper part) have been shown by fluorescence in situ hybridization using

probes specific for chromosome 18 (red), X (green) and Y (red). Here, probes are

used for the mixture of chromosome X (DXZ1) / chromosome Y (DYZ3) together

and chromosome 18 (D18Z1). Bar scales: in (A) 5 m, in (B) 1 m.

Page 24 of 30St

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FIG. 1.

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FIG. 2.

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FIG. 3.

A

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FIG. 4.

FIG. 5.

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FIG. 6.

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