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Derivation of Human Embryonic Stem Cells from Day-8 Blastocysts Recovered after Three-Step In Vitro Culture

^a Institute of Human Genetics, University of Newcastle, Newcastle upon Tyne, United Kingdom;
^b School of Biological and Biomedical Sciences, University of Durham, Durham, United Kingdom;
^c Newcastle Fertility Centre at Life, International Centre for Life, Newcastle upon Tyne, United Kingdom

Key Words. Human embryonic stem cells • In vitro culture • Pluripotency • Differentiation

Correspondence: Miodrag Stojkovic, M.D., Institute of Human Genetics, University of Newcastle, Central Parkway, Newcastle upon Tyne, NE1 3BZ, U.K. Telephone: 44-191-219-4746; Fax: 44-191-219-4747; e-mail: miodrag.stojkovic{at}ncl.ac.uk

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human embryonic stem cells (hESCs) have been derived from the inner cell mass (ICM) of day 5–7 blastocysts and hold great promise for research into human developmental biology and the development of cell therapies for the treatment of human diseases. We report here that our novel three-step culture conditions successfully support the development of day-8 human blastocysts, which possess significantly (p <.01) more ICM cells than day-6 blastocysts. Plating of ICMs isolated from day-8 blastocysts resulted in the formation of a colony with hESC morphology from which a new hESC line (hES-NCL1) was derived. Our stem cell line is characterized by the expression of specific cell surface and gene markers: GTCM-2, TG343, TRA1-60, SSEA-4, alkaline phosphatase, OCT-4, NANOG, and REX-1. Cytogenetic analysis of the hESCs revealed that hES-NCL1 line has a normal female (46, XX) karyotype. The pluripotency of the cell line was confirmed by the formation of teratomas after injection into severely combined immunodeficient mice and spontaneous differentiation under in vitro conditions.

	INTRODUCTION

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Human embryonic stem cells (hESCs) have the ability to generate cells from all three embryonic germ layers and represent a great potential source of cells for therapeutic uses [1–4]. Pluripotent hESC lines have now been derived from the inner cell mass (ICM) of day-5 to day-7 surplus and donated human blastocysts after routinely using two-step in vitro culture [5–11]. Previously we reported that more complex media for long-term in vitro culture of bovine embryos is necessary to improve the quality (i.e., cell number, as well as blastocyst and hatching rates) of in vitro recovered embryos [12, 13]. In humans, in vitro culture conditions are also suboptimal and result in low blastocyst rate or blastocysts with low cell number and lacking a distinct ICM [9, 11].

Therefore, in this study we evaluated the effects of more complex three-step culture conditions on in vitro development of late human blastocysts (day 8), on the number of ICM cells, and whether these blastocysts could be used for derivation of hESCs.

	MATERIALS AND METHODS

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Culture of Embryos
Fresh day-2 human embryos, produced by in vitro fertilization (IVF) for clinical purposes, were donated by individuals (average age 34.5 ± 4.1 years) after informed consent and after Human Fertilisation and Embryology Authority (HFEA, U.K.) approval. Embryos were cultured in G1 medium (step one) until day 3 (IVF = day 0) and transferred to G2.3 medium (step two) until day 6 (both G1 and G2.3 from Vitrolife, Kungsbacka, Sweden, http://www.vitrolife.com). On day 6 recovered blastocysts were cultured for an additional 2 days in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Paisley, Scotland, http://www.invitrogen.com) supplemented with 15% (v/v) Glasgow medium conditioned (GLCM) by Buffalo rat liver (BRL) cells (step three). Embryos that reached different blastocyst stages (blastocyst [bl], expanded blastocyst [ebl], hatching or hatched blastocyst [hbl]) on day 6 or day 8 were scored as grade A (ICM very well formed and with tightly grouped cells) or grade B (ICM with only several and loosely grouped cells) blastocysts and used for further experiments.

Cell-Number Analysis
In this experiment we investigated whether our three-step embryo culture supported development of day-8 blastocysts and whether these blastocysts possess more ICM cells than day-6 blastocysts that were cultured in two-step conditions. The cell numbers of 11 ICMs isolated from day-6 blastocysts (4 blA, 1 blB, 4 eblA, and 2 eblB) were compared with the cell numbers of 13 ICMs isolated from day-8 blastocysts (5 eblA, 2 eblB, 5 hblA, and 1 hblB) using 1.5 µg/ml 4'-6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO, http://www.sigmaaldrich.com) labeling as previously described [14].

Derivation of hESCs
For this experiment, 11 donated day-2 embryos were cultured in G1 and then in G2.3 steps as described above. On day 6, recovered blastocysts were cultured in BRL-conditioned medium until day 8 (step three). Seven ICMs were isolated from blastocysts recovered on day 8 using the procedure described by Reubinoff et al. [6]. Initially, isolated ICMs were cultured on a -irradiated mouse embryonic fibroblasts (MEF) monolayer (75,000 cells/cm²) in DMEM supplemented with 10% (v/v) Hyclone defined fetal calf serum (FCS; Hyclone, Logan, UT, http://www.hyclone.com) for 10 days. After 17 days, the primary hESC colony was mechanically dispersed into several small clumps, which were cultured on a fresh MEF layer with embryonic stem cell (ES) medium containing Knockout-DMEM (Invitrogen), 100 µM ß-mercaptoethanol (Sigma), 1 mM L-glutamine (Invitrogen), 100 mM nonessential amino acids, 10% serum replacement (SR; Invitrogen), 1% penicillin-streptomycin (Invitrogen), and 4 ng/ml basic fibroblast growth factor (bFGF; Invitrogen). ES medium was changed daily. hESCs were passaged by incubation in 1 mg/ml collagenase IV (Invitrogen) for 5–8 minutes at 37°C or mechanically dissociated and then removed to freshly prepared feeders or plates precoated with Matrigel (BD Biosciences, Bedford, MA, http://www.bdbiosciences.com) for feeder-free growth, as previously described [15].

Characterization of hESCs
Live staining was performed by adding primary antibodies (GCTM-2 and TG343, a kind gift from Dr. M. Pera, and TRA1-60, a kind gift from Dr. P. Andrews; SSEA-4 from Developmental Studies Hybridoma Bank [Iowa City, IA, http://www.uiowa.edu/~dshbwww/list)] to hESCs for 20 minutes at 37°C. The primary antibodies were used at the following dilutions: GCTM-2, 1:2; TG343, 1:2; TRA1-60, 1:10; SSEA-4, 1:5. The samples were gently washed three times with ES medium before being incubated with the secondary antibodies (antimouse immunoglobulin G and anti-mouse immunoglobulin M, both 1:100 dilution [Sigma]) conjugated to fluorescein isothiocyanate (FITC) at 37°C for 20 minutes. The samples were again washed three times with ES medium and subjected to fluorescence microscopy. For OCT-4 immunostaining, hESCs were fixed in 3.7% formaldehyde (BDH, Coventry, U.K.) for 20 minutes at room temperature, followed by incubation in 3% hydrogen peroxide for 10 minutes. The hESCs were permeabilized with 0.2% Triton x100 (Sigma) diluted in 4% sheep serum (Sigma) for 30 minutes at 37°C. The hES colonies were incubated with the primary antibodies (OCT-4 from Santa Cruz Biotechnologies, Heidelberg, Germany, http://www.scbt.com) to a final concentration of 10 µg/ml for 30 minutes at room temperature. The hES colonies were washed twice with PBS for 5 minutes and then incubated with the secondary antibody (biotinylated rat antimouse immunoglobulin [DAKO, Cambridgeshire, U.K.] used at 1:100 dilution) for 30 minutes at room temperature. After that, hESCs were washed again with PBS, incubated with avidinbiotin complex/horseradish peroxidase (ABC/HRP) solution for 25 minutes at room temperature, and washed again with PBS. The detection was carried out by incubation with diaminobenzidine (DAB) solution (Sigma) at room temperature for 1 minute. Final washes were done with distilled water. The primary antibody was omitted for the negative control. The alkaline phosphatase (AP) staining was carried out using the Alkaline Phosphatase Detection Kit following manufacturer’s instructions (Chemicon International, Temecula, CA, http://www.chemicon.com). Briefly, cells were fixed in 90% methanol and 10% formamide for 2 minutes and then washed with rinse buffer (20 mM Tris-HCl at pH 7.4, 0.05% Tween-20) once. Staining solution (Naphthol/Fast Red Violet) was added to the wells, and plates were incubated in the dark for 15 minutes. The bright field images were obtained using a Zeiss microscope and AxioVision software (Carl Zeiss, Jena, Germany).

Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis
The reverse transcription was carried out using the cells to cDNA II kit (Ambion, Huntingdon, U.K.) according to manufacturer’s instructions. In brief, hESCs were submerged in 100 µl of ice-cold cell lysis buffer and lysed by incubation at 75°C for 10 minutes. Genomic DNA was degraded by incubation with DNAse I for 15 minutes at 37°C. RNA was reverse transcribed using Moloney murine leukemia virus (M-MLV) reverse transcriptase and random hexamers following manufacturer’s instructions. PCR reactions were carried out using the following primers:

OCT-4F: 5'-GAAGCTGGAGAAGGAGAAGCTG-3'

OCT-4R: 5'-CAAGGGCCGCAGCTTACACATGTTC-3'

REX-1F: 5'-GCGTACGCAAATTAAAGTCCAGA-3'

REX-1R: 5'-CAGCATCCTAAACAGCTCGCAGAAT-3'

NANOGF: 5'-GATCGGGCCCGCCACCATGAGTGTGGATCCAGCTTG-3'

NANOGR: 5'-GATCGAGCTCCATCTTCACACGTCTTCAGGTTG-3'

TERTF: 5'-CGGAAGAGTGTCTGGAGCAAGT-3'

TERTR: 5'-GAACAGTGCCTTCACCCTCGA-3'

GAPDHF: 5'-GTCAGTGGTGGACCTGACCT-3'

GAPDHR: 5'-CACCACCCTGTTGCTGTAGC-3'

PCR products were run on 2% agarose gels and stained with ethidium bromide. Results were assessed on the presence or absence of the appropriate size PCR products. Reverse transcriptase negative controls were included to monitor genomic contamination.

Karyotype Analysis of hESCs
The karyotypes of hES-NCL1 cells grown on MEF cells or in feeder-free conditions were determined by standard G-banding procedure.

Tumor Formation in Severe Combined Immunodeficient (SCID) Mice
All procedures involving mice were carried out in accordance with institution guidelines and institution permission. Approximately 3,000 hESCs were injected beneath the capsule of the kidney or the testis of adult male SCID mice. After 21–90 days, mice were sacrificed and tissues were dissected, fixed in Bouins overnight, processed, and sectioned according to standard procedures and counterstained with either hematoxylin and eosin or Weigert’s stain. Sections were examined using bright field light microscopy and photographed as appropriate.

In Vitro Differentiation of hESCs
Colonies of hES-NCL1 passage 21 were grown in feeder-free conditions in ES medium. After 5–14 days, spontaneous differentiation was observed, and differentiated cells were passaged and cultured under the same conditions. Cells were fixed in 4% paraformaldehyde in PBS (Sigma) for 30 minutes and then permeabilized for an additional 10 minutes with 0.1% Triton X (Sigma). The blocking step was 30 minutes with 2% FCS in PBS. Cells were incubated with antibody against nestin (1:200 dilution; Chemicon) or human alpha smooth muscle actin (1:50 dilution; Abcam, Cambridge, U.K., http://www.abcam.com) for 2 hours. Each antibody was detected using corresponding secondary antibodies conjugated to FITC.

Statistical Analysis
Cell numbers of ICMs isolated from day-6 or day-8 blastocysts were compared using the Wilcoxon rank-sum test. The data are presented as mean ± standard deviation.

	RESULTS

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Traditionally, one- or two-step conditions for in vitro culture of human embryos are used to obtain day 5 or day 6 (IVF = day 0) early blastocysts. Then, these blastocysts have been used for the derivation of new hESC lines. We developed a three-step culture system that successfully supports the development of late (day-8) blastocysts. Analysis of cell numbers of ICMs revealed that day-8 blastocysts possess significantly (p<.01) more ICM cells than day-6 blastocysts (51.3 ± 9.6 versus 36.8 ± 11.9, respectively). In view of this result, we used day-8 blastocysts to derive hESCs. Of the 11 day-2 donated embryos, 7 (63.6%) blastocysts developed to day 6. All 7 of these blastocysts expanded or hatched on day-8 after transfer to the third step (GLCM). The representative pictures of day-6 and day-8 blastocysts are presented in Figures 1A and 1B, respectively. After isolation of ICMs by immunosurgery, three primary hESC colonies showed visible outgrowth (Fig. 1C), and one stable hESC line (hES-NCL1) with typical hESC morphology was successfully derived (Fig. 1D and 1E).

View larger version (44K): [in this window] [in a new window]   Figure 1. Morphology of human blastocysts and hES-NCL1 cells. (A): Day-6 blastocysts and (B) hatched day-8 blastocysts. Note the presence of very well-organized inner cell mass (*) in the day-8 blastocyst recovered after three-step in vitro culture. (C): Inner cell mass cells (arrow) grown on irradiated MEFs 10 days after immunosurgery. (D): Primary hESC colony grown on inactivated MEF cells. (E): Same colony at high magnification. Note the typical morphology of hESCs: They are small cells with large nucleoli. Scale bars: 100 µm (A, B, D); 200 µm (C); 50 µm (E). Abbreviations: hESC, human embryonic stem cell; MEF, mouse embryonic fibroblast.

View larger version (65K): [in this window] [in a new window]   Figure 2. (A, C, E, G): Phase contrast and (B, D, F, H) fluorescence microscopy images of hES-NCL1 cells. The human embryonic stem cells were stained with antibody recognizing the GTCM-2 (B), TG343 (D), TRA1-60 (F), SSEA-4 (H), OCT-4 (I), and alkaline phosphatase (K) epitopes. (J) Negative OCT-4 control. Scale bars: 200 µm (A, B, K); 100 µm (C, D, G–J); 50 µm (E, F).

View larger version (42K): [in this window] [in a new window]   Figure 3. (A): Reverse transcription PCR and (B) karyotype analysis of undifferentiated hES-NCL1 cells grown on mouse embryonic fibroblasts. The PCR products obtained used primers specific for OCT-4, REX-1, NANOG, and TERT. The housekeeping gene, GAPDH, was used to normalize the samples for the cDNA content. A plus sign (+) indicates the presence of reverse transcriptase, and a minus sign (–) indicates the lack of the enzyme. Normal female karyotype (46, XX) of hES-NCL1 cell line (B). Abbreviation: PCR, polymerase chain reaction.

View larger version (27K): [in this window] [in a new window]   Figure 4. (A): Morphology and (B) TRA1-60 staining of undifferentiated hES-NCL1 cells grown in feeder-free conditions (Matrigel and mouse embryonic fibroblast–conditioned medium) maintaining (C) normal karyotype (46, XX) after 11 passages.

View larger version (86K): [in this window] [in a new window]   Figure 5. Histological analysis of teratomas formed from grafted colonies of hES-NCL1 cells in severe combined immunodeficient mice. (A): Neural epithelium (ne). (B): Structures of the skin including epidermis (ed), dermis (dm), and cornified layer (c). The stratum granulosum (arrow) is characterized by intracellular granules that contribute to the process of keratinization. (C–E):A wall of respiratory passage showing epithelium (ep), submucosa (sm), submucosal glands (sg), smooth muscle (mus), neural ganglia (ng), and supporting cartilage (cart). (F): High-magnification image of respiratory pseudostratified columnar epithelium containing occasional cells expressing cilia (arrow) and goblet cells secreting mucin (m). Histological staining: hematoxylin and eosin (A, D, E) and Weigert’s (B, C, F). Scale bars: 100 µm (A, D); 25 µm (B, C, E); 12.5 µm (F).

View larger version (32K): [in this window] [in a new window]   Figure 6. Spontaneous differentiation of hES-NCL1 cells into (A) smooth muscle and (B) neuronal cells, demonstrating differentiation into cells of mesoderm and ectoderm, respectively. Human embryonic stem cells were stained with smooth muscle actin antibody (A) and nestin antibody (B). Scale bars: 100 µm.

	DISCUSSION

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Published data suggest that the success rate in deriving hESC lines is highly dependent on the isolation conditions [9], experience of the group and quality of recovered blastocysts [22], and, most important, on the numbers and quality of early embryos donated for research purposes. For instance, Thomson et al. [5] used 14 blastocysts to derive five hESC lines, Reubinoff et al. [6] derived two hESC lines from four blastocysts, Park et al. [10] derived three hESC lines from 13 isolated ICMs, Pickering et al. [11] derived three hESC lines from 58 embryos, Mitalipova et al. [9] derived four hESC lines from 19 embryos, and previously Cowan et al. [16] described the derivation of 17 hESC lines from 344 embryos. An exact comparison of the success rate among different groups is difficult to make at this point, because some of the groups report the numbers of blastocysts used rather than the total number of embryos. In addition, some of the groups have used a small number of embryos from which it is difficult to extract statistically significant data. We have succeeded in deriving one hESC line from seven blastocysts developed from 11 embryos, a rate that is similar to other derivation studies described above. We feel, however, that our three-step culture has resulted in a high rate of embryos (63%) reaching the blastocyst stage, even after prolonged in vitro culture.

Our hESC line shows typical expression of ES cell and surface markers: GTCM-2, TG343, TRA1-60, SSEA-4, OCT-4, NANOG, and REX-1. Undifferentiated hESCs displayed a high level of telomerase reverse transcriptase expression, had a normal female karyotype, and maintained the potential to form derivates of all three embryonic germ layers after injection in SCID mice. Spontaneously differentiated hES-NCL1 cells expressed markers for smooth muscle actin and neuronal cells, which demonstrated that under in vitro conditions the hES-NCL1 line can form tissues that are derivative of mesoderm and ectoderm.

In conclusion, we have demonstrated that more complex three-step conditions successfully support in vitro development of human blastocysts until day 8 and that these blastocysts possess more ICM cells than their day-6 counterparts. We also demonstrated that day-8 blastocysts can be successfully used for derivation of karyotypically stable and pluripotent hESCs and have established a new, fully characterized hESC line. We are now in the process of improving in vitro culture conditions and quality of embryos using animal-free ingredients for culture of embryos, isolated ICM cells, and hESCs in an attempt to establish new clinical-grade hESC lines.

	ACKNOWLEDGMENTS

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We thank M. Choudhary, J. Fenwick, S. Harbottle, V. Lamb, C. Leary, S. Cassley, S. Parker, K. Yallop, and A. Elliott for their support and Drs. P. Andrews and M. Pera for their donation of the specific antibodies. This work was supported by Newcastle University Hospitals Special Trusties, Newcastle Health Charity, and Life Knowledge Park. Miodrag Stojkovic and Majlinda Lako contributed equally to this work.

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