generation and characterization of mouse parthenogenetic embryonic stem cells containing genomes...

Cell Biology International 31 (2007) 1336e1344www.elsevier.com/locate/cellbi

Generation and characterization of mouse parthenogenetic embryonicstem cells containing genomes from non-growing

and fully grown oocytes

Hua Shao a,1, Zhuying Wei a,1, Lingling Wang a,1, Lihua Wen b,c, Biao Duan a,Lie Mang a, Shorgan Bou a,*

a Key Laboratory for Mammal Reproduction Biology and Biotechnology of Education Ministry, Inner Mongolia University, Hohhot 010021, Chinab Department of Cellular and Molecular Medicine, University of Ottawa, Canada

c Ottawa Health Research Institute, Civic Campus, 725 Parkdale Avenue, Ottawa, ON K1Y 4E9, Canada

Received 25 March 2007; revised 27 April 2007; accepted 12 May 2007

Abstract

It is known that oocytes can be activated without male contribution in vitro and develop to blastocysts which are used to isolate partheno-genetic embryonic stem cells. Unfortunately, differentiation capacity of the parthenogenetic embryonic stem cells was rather lower than fertil-ized embryos derived ES cells, which might be the result of the absence of male genome. It had been found that some maternally expressedgenes were repressed and some paternally expressed genes were expressed in the non-growing oocytes. Therefore, maternal genome fromnon-growing oocytes can partially act as ‘‘sperm genome’’. In the present study, parthenogenetic blastocysts containing genome from non-growing and fully grown oocytes (named as NF-pBlastocysts) were produced by germinal vesicle transfer, and three newly established parthe-nogenetic embryonic stem (named as NF-pES) cell lines were derived from the resulting parthenogenetic blastocysts. All three NF-pES cell lineswere positive for ES cell markers, such as alkaline phosphatase (AKP), stage-specific embryonic antigen 1 (SSEA-1) and octamer-bindingtranscription factor (Oct-4). They have a normal chromosome karyotype (40) and can be maintained in an undifferentiated state for extendedperiods of time. When NF-pES cells were injected into severe combined immunodeficient mice, teratomas with all three embryonic germ layerswere obtained. The in vitro differentiation potential of NF-pES cells was analyzed by embryonic bodies (EB) formation. The expression of germlayer markers, such as nestin (ectoderm), desmin (mesoderm), and a-fetoprotein (endoderm) demonstrated that the NF-pES cells can differen-tiate into all three germ layers.� 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Parthenogenesis; ES; Non-growing oocytes; Germinal vesicle transfer; Differentiation; Chimeras; Teratomas

1. Introduction

Embryonic stem (ES) cells are pluripotent cells derived fromthe inner cell mass of blastocysts and capable of proliferatingindefinitely and differentiating into a wide variety of cells types

* Corresponding author. Research Center for Laboratory Animals, Inner

Mongolia University, 235 Daxuexilu, Hohhot 010021, China. Tel./fax: þ86

471 4995071.

E-mail address: [email protected] (S. Bou).1 These authors contributed equally to this work.

1065-6995/$ - see front matter � 2007 International Federation for Cell Biology

doi:10.1016/j.cellbi.2007.05.008

both in vivo and in vitro (Evans and Kaufman, 1981; Thomsonet al., 1998; Brivanlou et al., 2003). Human ES cells-based ther-apies, in which virtually any tissue or cell type could be pro-duced, have raised the hope that degenerative disease, such asParkinson’s disease and diabetes, could be treated (Smith,1998; Rippon and Bishop, 2004; Sun et al., 2006). However,generation of human ES cell lines need to destroy the compe-tent embryo with the potential to form an individual, which isviewed as ethically problematic (Cibelli et al., 2002). One of so-lutions to avoid such ethical issues is to generate pluripotentcells derived from parthenogenetic embryos, which, lacking

. Published by Elsevier Ltd. All rights reserved.

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1337H. Shao et al. / Cell Biology International 31 (2007) 1336e1344

the paternal genetic materials, are not capable of developing toterm (McGrath and Solter, 1984). Therefore, parthenogeneticembryonic stem (pES) cells provide a potential source with his-tocompatible tissues (Kim et al., 2007) for autologous cell ther-apy in females, without destroying a competent embryo (Vranaet al., 2003). However, it had been demonstrated that develop-mental ability of pES cells was restricted, particularly in form-ing mesoderm and endoderm (Fundele et al., 1989; Fundeleet al., 1991; Allen et al., 1994).

In chimeric mouse, a considerable contribution of partheno-genetic stem cells was found in all the tissues of chimericembryos before day 13 of embryonic development. After day13, parthenogenetic stem cells were given severe selective pres-sure, which led to significant decrease in all chimeric tissues(Jagerbauer et al., 1992). In common, these pES cells were de-rived from parthenogenetic embryo containing the same twosets of genomes from activated fully grown oocytes. Obviously,there were absence of paternally derived genes in pES cells,which seem to be participated in the regulation of cell growthand differentiation, because a very good contribution of fertil-ized blastocyst-derived ES cells was seen in all the tissues ofchimeras obtained by injecting the ES cells into blastocysts(Bradley, 1987; Clarke et al., 1988). Maternal genome fromnon-growing oocytes can partially act as ‘‘sperm genome’’, be-cause disruption of maternal imprinting in non-growing oocytescan result in some other regulatory mechanism for gene expres-sion, which is induced by epigenetic modification during sper-matogenesis (Obata et al., 1998; Bao et al., 2000). Previousstudies have verified that developmental potential of partheno-genetic embryos can be markedly improved following nucleartransfer using a combination of non-growing oocytes and fullygrown oocytes. However, little is known about developmentalpotential of NF-pES cells, which were derived from the parthe-nogenetic blastocysts with genomes from non-growing andfully grown oocytes.

In the present study, we established firstly the NF-pES cellline derived from reconstructed parthenogenetic blastocystcontaining genomes from non-growing and fully grownoocytes. It gives insights into the study of differentiation andgenomic imprinting of parthenogenetic embryonic stem cells.

2. Materials and methods

2.1. Animals

KunMing (KM) white mice (Laboratory Animal Center, Inner Mongolia

University), C57BL/6J EGFP-transgenic mice (Model Animal Research Cen-

ter, Nanjing University) and severe combined immunodeficient (SCID) mice

(Shanghai Center for Laboratory Animal, Chinese Academy of Sciences)

were purchased and housed in a temperature- and light-controlled room

(14 h light; 10 h dark) and were feed freely. All animal usage protocols

were made according to the Guide for Care and Usage of Laboratory Animals

by the Research Center for Laboratory Animal, Inner Mongolia University.

2.2. Production of reconstituted diploid parthenogeneticembryos

In this study, parthenogenetic embryos were constructed by combining two

genetically different mouse oocytes. Germinal vesicle (GV) stage oocytes

were collected from KM white mouse ovarian follicles, 48 h after PMSG

treatment (Shao et al., 2006). The cumulus cells were then removed by hyal-

uronidase (300 U/ml, Sigma) digestion within M2 medium supplied with 3-

isobutyl-1-methylxanthine (IBMX, Sigma). ZP of GV oocytes were digested

by 0.03% (W/V) pronase (Sigma) (Bao et al., 2000), and then the GV was

removed by micromanipulation. Non-growing oocytes were obtained from

ovaries of 1-day-old C57BL/6J EGFP-transgenic mice, and then were com-

bined with GV-free oocytes in the presence of 10% (W/V) PHA-P (Sigma).

The resulted complex was induced to fuse with one pulse of direct current

at 1.8 kV/cm for 20 ms by using electro cell manipulator (ECM, Australia).

One hour after fusion, the result was confirmed by microscopic examination.

All fused complexes were washed three times in maturation medium (TYH

medium), and were incubated under 37 �C for 19 h. After incubation, polar

bodies (PB) extrusion was checked under microscope. The resulting MII oo-

cytes were enucleated and the MII spindles were transferred underneath ZP

of fully grown MII oocytes of KM white mouse, whose PBs were removed be-

forehand (Bao et al., 2000; Obata and Kono, 2002; Sotomaru et al., 2002;

Kono et al., 2004). The reconstituted units were induced to fuse with the

same condition as above. All fused units were further activated with 10 mg/

ml SrCl2 (Sigma) in CZB medium (Ca2þ- and CCB-free) for 6 h (Wakayama

et al., 1998). PBs were examined at the same time. The reconstituted units with

two PBs and two pronuclear were further developed to blastocysts in KSOM

medium (Nagy, 2003).

2.3. NF-pES cells isolation and maintenance

To isolate NF-pES cells, ZP of PG blastocyst was digested with 0.03% (W/

V) pronase. After digestion, zona-free blastocysts were cultured on mitotically

inactive mouse embryonic fibroblasts. Culture medium was DMEM (Gibco)

supplemented with 15% fetal bovine serum (Hyclone), 1 mM Glutamine

(Gibco), 0.1 mM b-mercaptoethanol (Sigma), 1% nonessential amino acids

(Gibco), and 1000 U of 1 ml leukemia inhibitory factor (LIF, Gibco) (Fundele

et al., 1990). After 4e6 days culture, ICM-derived outgrowths were separated

into clumps by the combination of both enzyme and mechanical dissociation

with a micropipette, and plated on mitomycin C-treated mouse embryonic

fibroblasts feeder layer. After another 4e6 days culture, clones resembling

ES cells in morphology were then picked and dissociated into single-cell

solution. Clones obtained from the second culture were termed as passage 1,

they were then passaged or frozen until the usage.

2.4. Immunohistochemistry

Alkaline phosphatase staining was performed according to the manufactur-

er’s protocol (Sigma). Expression of stem cells markers were examined by

immunofluorescence staining using ES cells characterization kit (Chemicon)

as recommended by the manufacturer. The primary antibodies used were

stage-specific embryonic antigens-1 (SSEA-1), and Oct-4.

2.5. Karyotype analysis

Karyotype analysis was carried out on the cells from passage 5. The

mouse feeder layer were eliminated from undifferentiated NF-pES cells by

plating cells on the feeder layer-free tissue culture dishes for three times.

The resulting cells were incubated with 0.05 mg/ml demecolcine (Sigma)

for 2 h. Cells were then trypsinized and pipetted to produce a single-cell sus-

pension. Centrifugation was performed to pellet the cells. The pellet was pi-

petted and resuspended in 0.075 M KCl for 30 min at 37 �C. The cells were

fixed in methanol:acetic acid (3:1) for 10 min at room temperature, and drop-

ped on to precleaned slides. Chromosome spreads were stained with Giemsa

and photographed. Approximately 20 metaphase nuclei were examined for

each cell line.

2.6. Generation and analysis of teratomas

About 5� 105 undifferentiated NF-pES cells of passage 10 were in-

jected into the hind limb muscle of SCID mice. Three weeks after

1338 H. Shao et al. / Cell Biology International 31 (2007) 1336e1344

injection, the resulting teratomas were embedded in OCT and sectioned.

The sections were fixed in the acetone and stained with antibodies against

a-fetaprotein (AFP) (DAKO, 1:100), nestin (DAKO, 1:100), desmin

(DAKO, 1:100), and GFP (Clontech, 1:100). Counterstain was performed

using Hoechst 33342.

2.7. Production of chimeric mice

Blastocysts injections were performed as described (Wakayama et al.,

2001). Briefly, KM white females were mated with KM white male, and blas-

tocysts were collected at day 4 post coitum (dpc). Then, 10e15 of the NF-pES

cells were injected into each blastocyst, and the injected embryos were trans-

ferred into 2.5 dpc pseudopregnant KM white females. All pregnant females

were delivered naturally and the chimeric offspring were examined for coat

color contribution.

2.8. In vitro differentiation

To induce the NF-pES cell differentiation in vitro, undifferentiated NF-pES

cells were detached from the mouse feeder layers and cultured in suspension in

DMEM medium without LIF. To assess differentiated ability into three embry-

onic germ layers, the EBs cultured for 9 or10 days were embedded in OCT and

sectioned. The sections were fixed in the acetone and stained with antibodies

against a-fetaprotein (AFP) (DAKO, 1:100), nestin (DAKO, 1:100), and

desmin (DAKO, 1:100).

Restituted oocyte with two 2nd pb and twopronuclears after fusion and activation

GV oocyte from kunming whiteadult mice

Digest zona with pronase

Zona-free enucleated GV oocyte Non-growing oocyte from GFP-transgenicmice derived from C57

Zona-free enucleated GV oocyteadhered to non-growing oocyte

IVM after fusion, GVBD,and 1st pb extrusion

Transfer MII chromosome into perivitelline space offreshly ovulated kunming white MII oocyte

Fig. 1. Schematic diagram showing the production of NF-pEmbryo. NF-pEmbryos were produced by serial nuclear transfer. ZP-free technology was applied in first

nuclear transfer. The haploid chromosome complex (white) obtained by the first nuclear transfer was transferred to MII oocyte in the second nuclear transfer. The

reconstituted complex was fused and activated to form diploid NF-pEmbryo with two pronuclei (white: GFP-transgenic mice; black: KM white mice).


3. Results

3.1. Construction of NF-pBlastocyst

Parthenogenetic blastocyst containing genomes from non-growing and fully grown oocytes were produced by modifiedmicromanipulation, which was different from the proceduredescribed by Kono et al. (1996). The ZP-free technologywas applied in this study to improve GV transferring effi-ciency (Figs. 1 and 2). Totally, 383 GV stage oocytes were di-gested by pronase to remove ZP, and GVs in 246 of ZP-freeoocytes were successfully eliminated. After electronic shock,

125 complexes including non-growing oocyte and enucleatedGV oocyte were fused. Fusion rate was 54.82% (125/228)(Table 1). After maturation culture, 64 constructed complexesreached to MII stage, which has 1st PB. The resulted MII spin-dle, which was from C57BL/6J, was transferred into 1st PBfreed KM MII oocytes. The second fusion rate was 91.07%(51/56) (Table 2). After artificial activation by SrCl2,43.14% (22/51) of fused units formed, which include two2nd PBs and two female pronuclei (Fig. 2). The constructedembryos were cultured for 4 days, and 13 embryos developedinto blastocysts stage. The NF-pBlastocyst formation rate was25.49% (13/51) (Table 2).

Fig. 2. Construction of a diploid NF-pEmbryo. (A) GV stage oocytes from KM white mice (�200). (B) ZP-free GV stage oocytes after pronase digestion (�200).

(C) Oocyte with GV enucleated (�200). (D) Ovaries from 1-day old C57BL/6J mice (�100). (E) Non-growing oocytes from 1-day old C57BL/6J mice ovaries

under UV light (�200). (F) Enucleated GVoocytes adhered to non-growing oocytes with PHA-P (�200). (G) Oocyte extruding first PB after fusion and maturation

(�400). (H) Reconstituted NF-pEmbryos with two pronuclei and two PBs (�200). (I) The NF-pEmbryos under UV light (�200).


Table 1

MII oocytes derived from non-growing oocytes by the GV transfer

No. of

ovaries

No. of

oocytes

No. of GV

enucleated

oocytes

No. of oocytes

adhered to

non-growing oocytes

No. of oocytes

fused with

non-growing oocytes

No. of MII

oocytes

Exp. 1 6 68 34 34 20 12

Exp. 2 6 68 44 40 23 14

Exp. 3 6 47 30 28 17 6

Exp. 4 6 90 51 50 18 12

Exp. 5 8 110 87 76 47 20

Total 32 383 246 228 125 64

Table 2

Diploid parthenogenetic blastocysts contained genomes from non-growing and fully grown oocytes

No. of the

resulting MII

oocytes

No. of

reconstituted

oocytes

No. of fused

oocytes

No. of activated

oocytes with one

PD

No. of activated

oocytes with two

PD

No. of blastocysts

from two-PD

activated oocytes

Exp. 1 12 10 10 2 6 3

Exp. 2 12 11 8 3 2 1

Exp. 3 6 3 3 0 2 2

Exp. 4 12 12 12 3 6 4

Exp. 5 20 20 18 4 6 3

Total 62 56 51 12 22 13

Fig. 3. Morphology and karyotype of NF-pES cell colonies. (A) NF-pES cell colonies after 2 days culture (�100). (B) NF-pES cell colonies under UV light

(�100). (C) Representative karyotype of NF-pES cell (�1000).

Fig. 4. Character of NF-pES cells derived from reconstituted NF-pEmbryo. (A) The NF-pES cell colony stained positive for alkaline phosphatase (�400). (B) The

NF-pES cells stained positive for SSEA-1 (�400). (C) The NF-pES cells stained positive for Oct4 (�400).


Fig. 5. Immunohistological staining of NF-pES cells-derived teratomas. (A) Nestin-positive tissue with neural tube-like structure. (B, F, J, N, R) Expression of GFP

under UV light. (C, G, K, O, S) Nuclei stained by Hoechst 33342. (D) Merged image of A, B and C. (E) Desmin-positive tissue with striated muscle-like structure

(slit section). (H) Merged image of E, F and G. (I) Desmin-positive tissue with striated muscle-like structure (transverse section). (L) Merged image of I, J and K.

(M) Cytokeratin seven-positive tissue with digestion tube-like structure (transverse section). (P) Merged image of M, N and O. (Q) Cytokeratin seven-positive

tissue with hepatic cord-like structure (slit section). (T) Merged image of Q, R and S. Magnification: �400.


Fig. 6. Morphology and immunohistological analysis of EB, derived spontaneously from differentiating pES cells. (A) Phase contrast imaging of EB from NF-pES

cells (�40). (B) The same EB under UV light (�40). (C) EB section was positive with ectoderm marker: nestin (red) (�40). (D) EB section was positive with

mesoderm marker: desmin (red) (�40). (E). EB section was positive with ectoderm marker: a-fetaprotein (red) (40). Counterstain was with Hoechst (blue).

3.2. Establishment of NF-pES cells

After digested by pronase, all 13 ZP-free NF-pBlastocystswere seeded onto mitomycin C-treated MEF feeder layers inthe ES cell culture medium. Eight blastocysts were found inhealthy morphology. Three NF-pES cell lines, which had nor-mal ES cell morphology, were obtained from the outgrowths(Fig. 3A). All the NF-pES cell lines were GFP (Fig. 3B) andstem cell markers positive. Immunostaining showed that NF-

pES cell colony was positive for SSEA-1 and Oct-4 (Fig. 4Band C). In addition, high level of alkaline phosphatase activityin their cytoplasm was detected in NF-pES cell colony(Fig. 4A). Karyotype by Giemsa staining showed that theNF-pES cells contained normal diploid (40) chromosomes(Fig. 3C). To confirm the NF-pES cells containing genomesfrom KM white and C57BL/6J mice, these two lines micewere cross-fertilized to analyze phenotype of F1 mice coatcolor. The results showed that F1 hybrid mice had two types

Fig. 7. Coat color of hybrid and chimaeric mice. (A) Coat color of F1 hybrid mice from KM white and C57BL/6J mice. (B) Coat color of chimeric offspring

obtained by NF-pES cells injected into blastocyst cavity of KM white mice.


of coat color: black or yellow brown (Fig. 7A). There also ex-isted black or yellow brown chimeric coat color in chimerasobtained by the NF-pES injection into KM white blastocysts(Fig. 7B). Therefore, the data suggested that NF-pES cellscontained two sets of chromosomes derived from non-growingand fully grown oocytes.

3.3. Differentiation of NF-pES cells in vivo

The differentiation ability of NF-pES cells was analyzed byteratomas formation. Three weeks later, NF-pES developedinto teratomas. Immunohistochemistry staining showed thatNF-pES cells-derived teratomas were GFP positive (Fig. 5B,F, J, N, and R). In addition, the teratomas were positive fornestin (Fig. 5A), desmin (Fig. 5E and I) and cytokeratin-7(Fig. 5M and Q), which were ectoderm, mesoderm and endo-derm markers, respectively.

3.4. In vitro differentiation of NF-pES cells

The in vitro differentiation potential of NF-pES cells wasalso examined. After 9e10 days of culture in absence ofMEF feeder layers and LIF, the NF-pES cells could formEBs spontaneously (Fig. 6A). All the EB are GFP positive(Fig. 6B). Representative markers for the three germ layerswere detected, including nestin (ectoderm), desmin (meso-derm), and AFP (endoderm) (Fig. 6CeE).

4. Discussion

This research establishes new parthenogenetic embryonicstem (pES) cell lines, designated NF-pES cell lines fulfillsthe standards for mouse ES cells, including (a) derivationfrom the NF-blastocyst, (b) karyotypically normal, (c) prolif-eration for long periods of time in the undifferentiated state(more than 10 months), (d) recovery after freezing and thaw-ing and (e) differentiation into a variety of cell types in vitroand in vivo. Importantly, NF-pES cell lines were derivedfrom reconstituted parthenogenetic blastocysts containinggenomes from non-growing and fully grown oocytes. In previ-ous reports, pES cells were derived from activated fully grownoocytes (MII), containing the same two sets of maternal chro-mosome. The participation of these pES cells in chimeras hasbeen analyzed extensively as well (Clarke et al., 1988;Thomson and Solter, 1988; Fundele et al., 1989; Paldi et al.,1989; Fundele et al., 1990; Jagerbauer et al., 1992). Differen-tiation capacity of pES cells is limited and the severe selectionagainst the contribution of pES cells in certain tissues has beenfound (Fundele et al., 1991). Parthenogenetic embryos withgenomes from non-growing and fully grown oocytes, whichcan further develop to 13.5 days of gestation (Kono et al.,1996; Obata et al., 1998), 3 days longer than parthenogeneticembryos from the activated MII oocytes (Surani and Barton,1983; Barton et al., 1984; McGrath and Solter, 1984; Suraniet al., 1986). Parthenogenetic mice were born following nu-clear transfer using a combination of H19 gene knockoutmice oocytes and non-growing oocytes (Kono et al., 2004).

It was reported that some maternally expressed genes were re-pressed and some paternally expressed genes were expressedin the non-growing oocytes (Obata et al., 1998).

These results suggest that paternal genome plays a signi-ficant role in improvement of embryo developmental abilityand in the maintenance of specific differentiated cell types(Fundele et al., 1990). As NF-parthenogenetic embryos con-taining a number of paternally expressed genes, could devel-opmental capacity of NF-ES cells be improved? So wegenerated NF-pES cells containing genomes from non-grow-ing and fully grown oocytes. Although there was epigenetic in-stability in ES cells during in culture (Humpherys et al., 2001),no research had been performed on NF-pES cells. The NF-pES cell line we established, although its mechanism hadnot been clarified, might be a useful tool for assessing theeffects of genome imprinting on cell differentiation. Further-more, the establishment of the NF-pES cell line might givea novel insight of isolating human pES cells from humanparthenogenetic embryos.

Acknowledgements

We thank Dr Zhihong Niu, Dr Dongshan Yang and Dr ZhenYan for helpful discussion and critical reading of the manu-script. We are grateful to Mr Zhankuan Li and Mrs Xiao Shifor the housing of mice. This work was supported by the Na-tional High Technology Research and Development Project ofChina (2002AA242061 to SB) and the Inner MongoliaUniversity.

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