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Keywords:

  • human embryonic stem (hES) cells;
  • pronucleus (PN);
  • aneuploid zygote;
  • pluripotency

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Human embryonic stem (hES) cell lines have been derived from normally or abnormally fertilized zygotes. However, the similar and different properties of these two types of hES cell lines are not well-known. To address this question, we generated nine hES cell lines from zygotes containing normal (2PN) and abnormal (0PN, 1PN, 3PN) pronuclei. A side-by-side comparison showed that all cell lines exhibited distinct identity and karyotypical stability. They expressed similar “stemness” markers and alkaline phosphatase activity and differentiated into three embryonic germ lineages in embryoid bodies and teratomas. Under neural differentiation-promoting conditions, they were directed into neural progenitors and neurons. However, a variation in cell cycle and the relative abundance of gene expression of undifferentiated and differentiated markers were observed. These variations were also seen among individually derived normal hES cell lines. Thus, normal hES cell lines can be developed from fertilized zygotes with abnormal pronuclei usually excluded from clinical use. Developmental Dynamics 239:425–438, 2010. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Human embryonic stem (hES) cell lines can be derived from the inner cell mass (ICM) of preimplantation blastocysts (Thomson et al.,1998) and are capable of self-renewal and differentiating into multiple cell lineages representing three embryonic germ layers. Their differentiation potential has raised hope that these cells could provide a renewable source for cell transplantation for severe degenerative diseases.

The blastocysts used for the establishment of hES cell lines have been donated by individuals undergoing infertility treatment. Pronuclear formation takes place 6–20 hr after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) showing various appearances. Routinely, the embryos selected for transfer are developed from zygotes having two pronuclei, one of female and one of male origin and showing a second polar body (Nagy et al.,1998). Although zygotes with abnormal pronuclei are not usually used for transfer, reports have shown that transfer of single-embryos with excessive polypronuclear zygote formation (Matt et al.,2004) or embryos developed from single-nucleated zygotes has resulted in healthy births (Munne et al.,1993; Feenan and Herbert,2006). Therefore, a normal hES cell line may also be established by blastocysts developed from abnormal fertilized zygotes.

It is estimated that 3–5% of fertilized human zygotes containing abnormal pronuclei arise in IVF. These zygotes cannot develop into human beings and are usually disposed of by IVF centers. Previous studies have shown that it was difficult to derive hES cells from poor-quality IVF embryos maintained in culture (Mitalipova et al.,2003; Lerou et al.,2008). Recently, a diploid human embryonic stem cell line was derived from a mononuclear zygote (Suss-Toby et al.,2004). This new strategy may facilitate hES cell line derivation; however, a comparative analysis of the unique properties and behavior of the hES cell lines derived from normally and abnormally fertilized zygotes is critical to identify self-renewal, genetically stable and pluripotent cell lines for research and therapeutic use. In addition, diploid human embryonic stem cell lines derived from trinuclear zygotes have not been reported. It remains uncertain whether all types of abnormally fertilized zygotes can be used for generation of normal hES cell lines. In the present study, we established nine hES cell lines derived from fertilized zygotes with normal (2PN) and abnormal (0PN, 1PN, 3PN) pronuclei. An extensive comparison of these cell lines demonstrated that normal hES cell lines can be developed from fertilized zygotes with abnormal (0PN, 1PN, 3PN) pronuclei which are usually excluded from clinical use.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Derivation of Nine hES Cell Lines From Fertilized Zygotes With Normal and Abnormal Pronuclei

We established nine hES cell lines (mSDU-hES 1–9 cell lines) from 46 fresh blastocysts that were clinically discarded. Of the 46, 9 were derived from 2PN-zygotes, 13 from 0PN-zygotes, 9 from 1PN-zygotes, and 5 from 3PN-zygotes (Fig. 1). ICMs were isolated mechanically and cultured on mitotically inactivated mouse embryonic fibroblast (MEF) (Fig. 2A). ICMs were attached to MEFs feeder layers and continuously proliferated in the initial passage. After 7–8 days, each of the small ICM-derived colonies was mechanically dissociated and replated on a fresh feeder layer. Then these colonies were split onto newly prepared feeder layers every 4–6 days. Each cell line of different PN zygotes showed similar round compact colony morphology with a defined border toward the feeder layer (Fig. 2B–E). Under high magnification (×400), they were found to have a high ratio of nucleus to cytoplasm, and at least two prominent nucleoli (Fig. 2F). Each of the cell lines was successfully cryopreserved and thawed. The mSDU-hES 1 had a survival rate as high as 70% after 18 months of cryopreservation. A period of replicative crisis was not observed for any of the cell lines. A summary of main characteristics of the nine established hES cell lines is shown by Table 1.

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Figure 1. Zygotes with different pronuclei generated 20–22 hr after in vitro fertilization (IVF) in our IVF laboratory. A: 0PN zygote. B: 1PN zygote. C: 2PN zygote. D: 3PN zygote.

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Figure 2. There are no significant differences found in colony morphology of human embryonic stem (hES) cells derived from zygotes with normal and abnormal pronuclei. A: Outgrowth of inner cell masses (ICMs) at 3 days post plating. The ICMs were isolated from blastocysts developed from fresh PN stage embryos mechanically. Scale bar = 100 μm. B–E: Similar morphology of colonies derived from the mSDU-hES 2 (0PN) (B), 4 (1PN) (C), 5 (2PN) (D), 8 (3PN) (E). Scale bars = 100 μm. (F). Higher magnification of central regions of the mSDU-hES 2 colony. Scale bar = 10 μm.

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Table 1. Summary of Characteristics of the Nine Established hES Cell Lines
hES cell linemSDU- hES1mSDU- hES2mSDU- hES3mSDU- hES4mSDU- hES5SDU- hES6mSDU- hES7mSDU- hES8mSDU- hES9
  1. hES, human embryonic stem; ICM, inner cell mass; MEF, mouse embryonic fibroblast; AP, alkaline phosphatase; EB, embryoid bodies.

Zygotes derived from2PN0PN0PN1PN2PN2PN2PN3PN2PN
IRB approvalYesYesYesYesYesYesYesYesYes
OriginICMICMICMICMICMICMICMICMICM
FeederMEFMEFMEFMEFMEFMEFMEFMEFMEF
AP+++++++++
SSEA-1
SSEA-3+++++++++
SSEA-4+++++++++
TRA-1-60+++++++++
TRA-1-81+++++++++
OCT4+++++++++
Karyotypep32p85p80p60p78p96p66p79p75
 46, XY46, XX46, XY46, XY46, XX46, XX46, XY46, XY46, XX,
FingerprintingYesYesYesYesYesYesYesYesYes
Teratoma+++++++++
EB+++++++++
Freezing and thawing+++++++++
Tubes of frozen (106 hES cells/tube)40381239071761115970
Passage(2008-03-02)P38P90P97P88P89P114P87P90P88

Expression of Alkaline Phosphatase Activity and “Stemness” Markers

Like previously reported hES cells, our hES cells possessed high levels of alkaline phosphatase (AP) activity (Fig. 3A1–A9). Scoring of at least 300 randomly chosen cells of each hES cell line revealed greater than 90% of the colonies remained undifferentiated and expressing AP. The expression of several undifferentiated (stemness) markers was confirmed by immunocytochemistry. Cells of each hES cell line were positive to transcription factors and glycolipids markers: OCT-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81(Fig. 3B–F), but were negative for SSEA-1 (data not shown). Feeder cells were negative for all surface markers used in this experiment.

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Figure 3. Expression of undifferentiated (or “stemness”) markers and alkaline phosphatase activity in the mSDU-hES four cells. A,B: (A1-A9) alkaline phosphatase, (B1-B9) transcription factor OCT4. C–F: (C-F) glycolipids markers SSEA-3 (C1-C9), SSEA-4 (D1-D9), TRA-1-60 (E1-E9), and TRA-1-81 (F1-F9). Scale bar = 100 μm.

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Short Tandem Repeat Analysis Shows Distinct Identity of Each hES Cell Lines

All hES cell lines could be distinguishable because they showed different short tandem repeat (STR) profiles in 16 STR loci (Fig. 4). Each cell line was diploid shown by single peak or double peak at every locus. A single peak indicates a homozygote and a double peak, a heterozygote at this locus. The first red locus represents the sex of the embryo that the hES cell line was derived from. If the hES cell line was derived from a female embryo, only one red peak appears at the first locus, while a male embryo-derived-hES cell line shows two different red peaks. Results of sex detection by this method were done by karyotype analysis. Each of the nine hES cell lines showed distinct 16 loci according to the distances from the loci to the markers, indicating that each of the nine hES cell lines has its own genetic label.

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Figure 4. Distinct short tandem repeat (STR) profiles of nine human embryonic stem (hES) cell lines defined by DNA fingerprinting. The isogenic analysis of four blue peaks (from left to right): loci D8S1179 (8), D21S11 (21q11.2-q21), D7S820(7q11.21-22), CSF1PO (5q33.3-34); five green peaks: D3S1358 (3p), TH01 (11p15.5), D13S317 (13q22-31), D16S539 (16q24-qter), D2S1338 (2q35-37.1); four black peaks: D19S433 (19q12-13.1), vWA (12p12-pter), TPOX (2p23-2per), D18S51 (18q21.3); three red peaks: Amelogenin (Xp22.1-22.3 Yp11.2), D5S818 (5q21-23), FGA (4q28). These peaks represent locations of polymorphisms for each STR marker. Each single peak or double peaks of one locus shows how the STR is expressed at this locus. Orange peaks are markers with lengths of 50, 75, 100, 139, 150, 160, 200, 0 (250), 300, 340, 350, 400, 450, 490, 500 bp from left to right, respectively. According to the different distances from the same locus to the markers, we can distinguish one cell line from another easily by its own genetic labels.

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All Cell Lines Exhibit Normal Karyotype

Karyotyping was performed every 6 month and revealed a normal diploid karyotype in each hES cell line: The mSDU-hES 1, 3, 4, 7, and 8 cell lines with 46, XY; and the mSDU-hES 2, 5, 6, and 9 cell lines with 46, XX. No chromosome aberration was detected. Karyotype analysis of all the nine cell lines are shown in Figure 5.

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Figure 5. Karyotypes of the nine cell lines show that they all have a normal complement of 46 chromasomes. Karyotypes of mSDU-hES 1, 3, 4, 7, and 8 cell lines are all 46, XY and karyotypes of mSDU-hES 2, 5, 6, and 9 cell lines are all 46, XX.

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Formation of Teratomas

Severe combined immunodeficiency (SCID) -beige mice were killed 10 to 12 weeks after being injected with undifferentiated hES cells to confirm the formation of teratomas. Teratomas were formed in the mouse gastrocnemius injected with mSDU-hES 5 cells and appeared as well-differentiated tumor-like structures, including a kidney-like structure, primitive neural tube, skin, gastrointestinal epithelium, tooth-like structure, and cartilage (Fig. 6). Mice injected with hES cells from the other eight cell lines also formed teratomas containing three embryonic germ lineages.

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Figure 6. Differentiation of the mSDU-hES 5 cells into three embryonic germ lineages in teratomas. Histological examination of teratomas formed in the gastrocnemius of severe combined immunodeficiency (SCID) mice following innoculation of mSDU-hES 5 cells. A: Primitive neural epithelium-like cells. B: Gastrointestinal tract epithelium. C: Glandular structure. D: Cartilage. Scale bars = 100 μm.

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Comparison of Proliferative Potential Between hES Cell Lines Derived From Zygotes With Normal and Abnormal Pronuclei

Previous study (Klaus et al.,2006) has shown that there are fundamental differences in cell cycle parameters of hES cells and committed somatic cells. The hES cell cycle maintains the four canonical cell cycle stages G1, S, G2, and M, but the duration of G1 is dramatically shortened while 65% of asynchronously growing hES cells are in S phase. To study a possible difference in cell cycle between hES cell Lines derived from zygotes with normal and abnormal pronuclei, we examined the cell cycle phase distributions of the mSDU-hES 2 (0PN), 4 (1PN), 5 (2PN), and 8 (3PN) cell lines at 72 hr after passage using flow cytometry. The cell cycle distributions of these hES cell lines showed a unique pattern of four cell cycle stages G1, S, G2, and M characterized by a large proportion of cells in S phase and a small proportion of cells in G0/G1 and G2/M phase (Fig. 7). Fluorescence-activated cell sorting analysis for DNA content showed 62.7 ± 1.0% of cells reside in S phase, 35.5 ± 0.6% in G0/G1 and 1.5 ± 0.1% in G2/M phase fin mSDU-hES 2 (0PN) cell line. The mSDU-hES 4 (1PN) cell line exhibited 56.7 ± 0.6% of cells reside in S phase, 22.4 ± 0.7% in G0/G1 and 21.5 ± 0.7% in G2/M phase; the mSDU-hES 5 (2PN) cell line showed 54.4 ± 0.6% of cells reside in S phase, 27.4 ± 0.6% in G0/G1, and 18.2 ± 0.2% in G2/M phase; and for mSDU-hES 8 (3PN) cell line, 50.4 ± 0.4% of cells reside in S phase, 18.8 ± 0.4% in G0/G1 and 30.8 ± 0.1% in G2/M phase. Compared with mSDU-hES 5 (2PN) cell line, mSDU-hES 2 (0PN) cell line showed higher proportion of cells in S phase and slightly higher composition of proliferating cells while 1PN and 3PN-zygote-derived hES cell lines showed similar composition of proliferating cells with mSDU-hES 5 (2PN) cell line (Fig. 7).

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Figure 7. Flow cytometric analysis of human embryonic stem (hES) cell cycles showing the similar proliferative potential between 2PN-zygote-derived and 1PN- and 3PN-zygote-derived hES cells. But 0PN-zygote-derived hES cell lines show a slightly higher composition of proliferating cells than 2PN-zygote-derived hES cell lines (P < 0.05). Undifferentiated mSDU-hES 1-9 cells were fixed and stained with propidium iodide. DNA content is measured by fluorescence-activated cell sorting analysis. Proportions in different phases of four cell lines are shown. The fractions of G0/G1, S, and G2/M phase cells of mSDU-hES 2 are 35.5 ± 0.6%, 62.7 ± 1.0%, and 1.5 ± 0.1%, respectively. The fractions of G0/G1, S, and G2/M phase cells of mSDU-hES 4 are 22.4 ± 0.7%, 56.7 ± 0.6%, and 21.5 ± 0.7%, respectively. The fractions of G0/G1, S, and G2/M phase cells of mSDU-hES 5 are 27.4 ± 0.6%, 54.4 ± 0.6%, and 18.2 ± 0.2%, respectively. The fractions of G0/G1, S, and G2/M phase cells of mSDU-hES 8 are 18.8 ± 0.4%, 50.4 ± 0.4%, and 30.8 ± 0.1%, respectively. Each test is repeated three times independently.

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To investigate differences in the proliferation rate of cells between different cell lines, 5-bromo-2′-deoxyuridine (BrdU) incorporation assay was performed. The mSDU-hES 2 (0PN), 4 (1PN), 5 (2PN), and 8 (3PN) cell lines derived from different pronucleus zygotes showed the following average percentages of BrdU incorporation: 98.5%, 91.5%, 94.0%, and 97.9%, respectively (Fig. 8). Multiple comparisons between every two groups by statistic analysis software SPSS 13.0 indicated that the percentage of BrdU incorporation of mSDU-hES-2 (0PN) was significantly higher than that of mSDU-hES-5 (2PN) (P < 0.05), and mSDU-hES-4 (1PN). The mSDU-hES-8 (3PN) showed no significant difference in average percentage of BrdU incorporation with that of mSDU-hES -5 (P > 0.05).

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Figure 8. Flow cytometric analysis of 5-bromo-2′-deoxyuridine (BrdU) incorporation showing the similar proliferative rate between 2PN-zygote-derived and 1PN- and 3PN-zygote-derived human embryonic stem (hES) cells (P > 0.05), but 0PN-zygote-derived hES cell lines show a slightly higher proliferating rate than 2PN-zygote-derived hES cell lines (P < 0.05). The percentages of BrdU incorporation of mSDU-hES-2, 4, 5, 8 are 98.5%, 91.5%, 94.0%, 97.9%, respectively. Each test is repeated four times independently. Statistic analysis was performed by software SPSS 13.0. In these instances, P < 0.05 was considered statistically significant.

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In summary, at 72 hr after passage, a large population of cells in mSDU-hES 2 (0PN) cell line were found in S phase (Fig. 7), and this result was consistent with a higher percentage of cells exhibiting BrdU incorporation than other hES cell lines tested (Fig. 8). The proportion of cells in G2/M phase of mSDU-hES 2 (0PN) cell line is relatively smaller than that of mSDU-hES 4 (1PN), 5 (2PN), and 8 (3PN) cell lines (Fig. 7). Our finding indicates that mSDU-hES 2 (0PN) cell line may have a shorter cell cycle duration than other cell lines tested due to the higher proliferation rate.

Reverse Transcription-Polymerase Chain Reaction Analysis of Differentiated Gene Expression in Embryoid Bodies Derived From Zygotes With Normal and Abnormal Pronuclei

Spontaneously differentiated embryoid bodies (EBs) in two differentiating stages, 5-day (data not shown) and 14-day, and undifferentiated hES cells (Fig. 9) were assessed for expression of seven genes (NF-68, KERATIN, CMP, KALLIKREINF, ENOLASE, α-FETOPROTEIN, and α1-AT) representing three embryonic germ lineages. The hES cells cultured in ES growth medium without basic fibroblast growth factor (bFGF) formed either simple (Fig. 9A) or cystic (Fig. 9B) EBs. Some cells in cystic EBs resembled a vascular structure, which is showed by an arrow in Figure 9B. The initial hES cells of the nine cell lines could express OCT4 (Niwa et al.,2000) of high level (Fig. 9C) but these undifferentiated cells did not express the seven differentiating markers (data not shown). The EBs derived from mSDU-hES 1–8 cell lines expressed OCT4 and the tissue-specific genes representing ectoderm, mesoderm and endoderm. But the mSDU-hES 9 (2PN)-derived EBs expressed genes representing ectoderm and mesoderm, but not endoderm (Fig. 9C).

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Figure 9. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of differentiated gene expression in undifferentiated human embryonic stem (hES) cells and 14-day-old embryoid bodies (EBs) derived from zygotes with normal and abnormal pronuclei. A: The 5-day-old EBs (simple EBs). B: The 14-day-old EBs (cystic EBs). The arrowhead points to the vascular structure. C: Expression of OCT4 in undifferentiated hES cells and of three embryonic germ layer-specific genes in spontaneously differentiated, 14-day-old EBs derived from nine mSDU-hES cell lines. Markers that the arrowheads point to mean 500 bp. The EBs derived from all cell lines express the tissue-specific markers representing ectoderm, mesoderm, and endoderm except for those derived from mSDU-hES 9 (2PN), which only express the tissue-specific markers representing ectoderm and mesoderm but not endoderm. Each test is repeated three times independently.

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Assessing Undifferentiated and Differentiated Gene Expression in hES Cell Lines Derived From Zygotes With Normal and Abnormal Pronuclei by Real-Time Quantitative Polymerase Chain Reaction

Real-time quantitative polymerase chain reaction (qPCR) was used to quantify the levels of mRNA expression of 11 selected genes in the hES cells. The selected genes included undifferentiated markers OCT4, NANOG, UTF1, DPPA5, LIN41, and SOX2and differentiated markers SOX1, H19, GATA4, IGF2, and HAND1. We quantitated and compared mRNA levels expressed in nine hES cell lines derived from zygotes with normal and abnormal pronuclei (Fig. 10).

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Figure 10. Quantitative polymerase chain reaction (qPCR) analysis of 11 genes expression in nine hES cell lines derived from zygotes with normal and abnormal pronuclei, of which six are undifferentiated markers and five are differentiating markers. The expression level of each gene in the mSDU-hES 1 (2PN) is considered as the control, and the expression level of each gene in other cell lines are indicated as a ratio to it. A fivefold change in expression level in an hES cell line compared to the mSDU-hES 1 is considered significant. The expression levels of OCT4, NANOG, LIN41, and SOX2 do not show significant difference among the nine cell lines, while UTF1 and DPPA5 expression in some cell lines was significantly higher than others. A variation in relative abundance of gene expression of differentiated markers in nine mSDUhES cell lines is observed. For example, SOX1 is expressed much higher in mSDU-hES 6 (2PN) and mSDU-hES 3 (0PN) than other cell lines.

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We found that the hES cell lines derived from normal or abnormal pronuclei zygotes expressed all 11 undifferentiated and differentiated markers, but their gene expression levels were variable. For the most undifferentiated genes OCT4, NANOG, LIN41, and SOX2, there was no significant difference among the hES cell lines. However, compared with other hES cell lines, the expression of DPPA5 was significantly lower in 2PN-derived mSDU-hES 5, while the expression of UTF1 was significantly higher in 3PN-derived mSDU-hES 8. A profound variation in relative abundance of gene expression in differentiated markers of the cell lines was observed. The ectoderm marker SOX1 was expressed much higher in mSDU-hES 6 (2PN) and mSDU-hES 3 (0PN); endoderm markers H19 and GATA4 were expressed much higher in mSDU-hES 1 (2PN) but lower in mSDU-hES 9 (2PN); mesoderm markers IGF2 and HAND1 were expressed much higher in mSDU-hES 1 (2PN) and mSDU-hES 3 (0PN) but lower in mSDU-hES 2 (0PN) and mSDU-hES 5 (2PN). Such differences in the relative abundance of gene expression in undifferentiated and differentiated markers were also found in normal hES cell lines (Tavakoli et al.,2009).

Similar Neural Differentiating Potential Among 2PN-zygote-derived and 0PN-, 1PN-, and 3PN-zygote-derived hES Cell Lines

Possible differences in pluripotency between hES cell lines derived from zygotes with normal and abnormal pronuclei were also examined during the directed neural differentiation. To test the possible differences, we used a reliable step-wise differentiation protocol (Ma et al.,2008). After 3 weeks of neural differentiation of hES cells in culture, both NESTIN+ neural progenitors and TUJ1+ neurons were generated from normally and abnormally fertilized zygote-derived cell lines (Fig. 11). NESTIN+ and TUJ1+ cells were manually counted and the neural differentiation potential was expressed as the percentage of NESTIN+ and TUJ1+ cells vs the total differentiated cells stained by DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride). The percentages of NESTIN+ cells differentiating from the nine hES cell lines were 70% ± 2.9, 75% ± 3.7, 74% ± 3.9, 70% ± 4.3, 72% ± 5.5, 74% ± 3.3, 75% ± 3.6, 69% ± 3.3, and 70% ± 3.9, respectively. The percentages of TUJ1+ neuron derived from the nine hES cell lines were 22% ± 3.1, 21% ± 2.9, 19% ± 2.5, 20% ± 3.0, 22% ± 3.2, 23% ± 2.6, 19% ± 2.9, 23% ± 2.8, and 21% ± 2.4, respectively. No significant differences were found in the percentage of NESTIN+ or TUJ1+ cells among the nine hES cell lines (P > 0.05).

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Figure 11. Neural progenitors and neurons differentiated from all of nine human embryonic stem cell lines. There are no significant differences in the neural differentiating efficiency among them. A: (A1–A9) Phase contrast images showing neural progenitor-like and neuron-like cells migrating out from embryoid bodies. B: (B1–B9) All nuclei were stained by DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride). C: (C1–C9) Immunofluorescent staining for TUJ1 (neuronal marker). D: (D1–D9) Immunofluorescent staining for NESTIN (neural progenitor marker). To estimate the percentage of differentiated cells expressing NESTIN or TUJ1, the number of labeled cells was counted from a double-immunolabeled culture and normalized with the total number of cells determined by counting DAPI nuclear counterstained cells. Scale bars = 100 μm. Statistic analysis was performed by software SPSS 13.0. In these instances, P < 0.05 was considered statistically significant.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In the present study, we have collected 46 blastocysts donated by patients undergoing treatment of infertility in our IVF center and nine hES cell lines were established, including cell lines derived from zygotes with 0PN, 1PN, 2PN, and 3PN. These cell lines have been maintained in culture for 2 years. Using immunocytochemistry, BrdU incorporation, flow cytometry, STR profiling, and qRT-PCR, these cell lines were characterized for self-renewal, identity, “stemness,” stability, and pluripotency. All nine cell lines showed a similar expression pattern of “stemness” markers, including transcription factors and glycolipids markers: OCT-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81; the ability to proliferate and differentiate into cell lineages in ectoderm, endoderm, and mesoderm in differentiating embryoid bodies and teratomas. Under directed neural differentiation, all the cell lines differentiated into neural progenitors and neurons. Interestingly, all nine hES cell lines derived from zygotes with normal and abnormal pronuclei shared the majority of these characteristics.

Previous studies showed that normal human embryos can be developed from zygotes that manifest a single pronucleus after IVF. In fact, uninucleate human zygotes are relatively common following that procedure (2–5%; Munne et al.,1993). Embryos derived from single-nucleated human zygotes may contain two types. The first type is parthenogenetic, and the second type of single-pronucleated zygote is produced by a fusion of the paternal and maternal genomes during the course of syngamy. Asynchronous pronuclear inflation may be the mechanism responsible for the development of diploid embryos from unipronucleate zygotes (Munne et al.,1993; Staessen et al.,1993). Normal hES cell lines we derived from zygotes with 0PN may be really from 2PN-zygotes because of missing the phase of presence of both pronuclei. However, as the limitation of technology and expenditure, most IVF labs have not taken a genetic test to identify the number of pronuclei of the zygotes whether they are to be implanted or discarded.

Several different events may lead to hyperploidy of the human zygote: 1. Multiple sperm penetrations, of which dispermy is the most common; 2. Fertilization by a diploid sperm or oocyte; and 3. Inhibition or delay of second polar body extrusion and formation of two female pronuclei (or possibly one diploid) together with a single sperm pronucleus (monospermic digyny) (Palermo et al.,1995). Polyspermy may be correlated with other factors, including oocyte immaturity and postmaturity (Wentz et al.,1983), sperm concentration (Wolf et al.,1984), inherent defects or fractures of the zona pellucida occurring during oocyte collection (Wentz et al.,1983), progesterone levels in the follicular fluid (Ben-Rafael et al.,1987), and more recently, high serum estradiol levels (Andrea et al.,2000). In natural conception, it has been reported that triploid embryos may proceed to term, and result in a liveborn child (Uchida and Freeman,1985).

Using human eggs in development of hES cell lines is controversial. The comparative analysis of characteristics of hES cell lines generated in our IVF Center from abnormal zygotes further confirms the possibility of recycling clinical nonvaluable zygotes to derive normal hES cell lines. Our results suggest that hES cell lines established by zygotes with morphologically abnormal pronucleus, 0PN, 1PN, 3PN, may also show normal phenotypes and characteristics of hES cells. Thus, poor quality cleavage stage human embryos, whether fresh or frozen, will not be discarded and have the value of being used for scientific research. The sources of establishment of hES cell lines will be expanded widely.

Studies using karyotypes have revealed that 1PN zygotes can represent fertilized oocytes. The delivery of normal offspring has been reported after the replacement of such embryos, provided that a second pronucleus was observed at a later assessment (Staessen et al.,1993). The results of karyotyping of mSDU-hES 2, 3, 4, 8 were in accordance with the report above. Karyotypes of hES cell lines derived from zygotes with abnormal pronucleus (0PN, 1PN, 3PN) were all normal diploid. Furthermore, the male karyotype, 46, XY, demonstrates that hES cell lines derived from zygotes with 0PN, 1PN, 3PN are not parthenogenetical or multiploid generated. DNA fingerprinting analysis showed these cell lines had double peaks at each STR locus, which is also an emphatic proof for their normal karyotypes and nonparthenogenic derivation.

As far as the ability of differentiation concerned, hES cell lines derived from zygotes with normal and abnormal pronuclei showed no significant difference with most marker expression. Like most cell lines derived from 2PN-zygote, cell lines derived from 0PN, 1PN, 3PN-zygote could also differentiate into cells of all three embryonic germ layers, ectoderm, mesoderm and endoderm in vitro as well as in vivo. While one cell line derived from 2PN-zygote could only differentiate into cells of two embryonic germ layers, ectoderm and mesoderm in vitro. Under neural differentiation-promoting conditions, normally and abnormally fertilized zygote-derived cell lines were directed into neural progenitors and neurons. No prominent differences of differentiating rates were observed among them. These results demonstrate that the ability of differentiation of hES cells derived from zygotes with abnormal pronuclei is similar to the hES cells derived from zygotes with normal pronuclei. The pronuclei cannot predict the potential of differentiation of hES cells. Maybe this potential is more related to the quality of the embryo which is assessed after in vitro fertilization in the IVF laboratory.

It is clear that no single marker is sufficient to define the state of hES cells; therefore, we selected six different markers to test their undifferentiated state. Our qPCR results showed that to some extent each of the nine cell lines had the ability to maintain the undifferentiated state when cultured in proper condition. For most of the six genes, the expression of hES cell lines derived from zygotes with normal and abnormal pronuclei showed slight difference. Normal pronuclei derived hES may have lower mRNA level of one or two genes, while abnormal pronuclei derived hES may even have higher mRNA level of some gene. That is to say, this phenomenon may be determined by the special biological property of different cell lines, but have no direct connection with the normal or abnormal pronuclei-derivation.

For the differentiating ability, the result of qPCR testing gene expression of hES cell was corresponding to RT-PCR testing the gene expression of EBs. Abnormal pronuclei derived hES may not have lower differentiating abilities to three germ layers than normal pronuclei derived hES cells. Some normal pronuclei derived hES cells may have deficiency in one germ layer oriented differentiation. Not all of the normal pronuclei derived hES cells have the same biological activity of differentiation. We can take use of their special characters to establish different models of diseases or to study the different gene expression during biological development.

We also compared cell cycles and capacity of proliferation of cell lines derived from zygotes of different pronuclei. Cell lines derived from diploid zygotes had proliferating cells of S phase ranging from 48.3% to 53.6%. Cell lines derived from zero-pronuleus-zygotes had more proliferating cells of S phase than normal pronuleus-zygotes derived cell lines. Cell lines derived from triploid and haploid zygotes showed no difference from normal zygotes derived cell lines. The results of BrdU incorporation assay were corresponding to that of flow cytometric analysis of hES cell cycles. The capacity of proliferation of cell lines derived from triploid and haploid zygotes showed no difference with normal zygotes derived cell lines. And the interesting phenomenon is that the proliferating capacity of cell lines derived from zero-pronuleus-zygotes was even higher than that of normal zygotes derived cell lines.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Establishment and Maintenance of hES Cell Lines

Fresh blastocysts developed from different pronucleated zygotes donated by couples undergoing IVF-embryo transfer (ET) or ICSI-ET treatment in the reproductive medical center after informed consent and institutional review board approval was obtained. ICMs were isolated from blastocysts mechanically by glass needle under microscope, then they were transferred onto an MEF-feeder layer. ICM outgrowths were passaged to plates with new MEFs by mechanical dissection approximately 7–10 days. For maintaining established stable hES cells, hES cells were digested into small clumps by collagenase IV propagated on a new feeder layer every 4–6 days. The hES cells were cultured in knockout-DMEM supplemented with 20% knockout serum replacement (SR; all from GIBCO), 0.1 mM β-mercaptoethanol (Roche), 0.1 mM nonessential amino acids (Invitrogen), 1 mM glutamine (Sigma), and 4 ng/ml human recombinant basic FGF (hrbFGF;R&D). For cryopreservation, hES cells were isolated entirely from the feeder cells after cultured on the feeder cells for 3–4 days and put into CRYO.P, then freezing-protection liquid composed of 90% Knockout SR, 10% dimethyl sulfoxide containing sucrose of final concentration 7% were added into it slowly, and cryopreservation were carried out in PLANER (Sunbury on Thames England).

Feeder Preparation

MEFs were cultured from fetuses of day-13.5 postcoitum BALB-C mice. Feeder cells were cultured in high glucose DMEM (Sigma) supplemented with 10% fetal bovine serum (Hyclone). They were treated with 10 μg/ml mitomycin C (ALEXES Biochemicals) for 1.5 hr to arrest mitosis, then thoroughly washed in phosphate buffered saline (PBS), and replated at a concentration of 60,000 cells/cm2 in gelatin-coated cell culture dishes.

AP Staining

AP activity of hES was detected by the Alkaline Phosphatase Detection Kit (Chemicon). The hES cells were fixed by a fixative (4% paraformaldehyde in PBS) for 1–2 min. Then after washed with Rinse Buffer, colonies were covered by stain solution thoroughly according to the kit. The resulting color reaction was stopped with PBS.

Immunocytochemistry

The hES cell colonies were rinsed twice before fixation with 4% paraformaldehyde in 1× PBS for 15 min at room temperature. Cells were permeabilized with 0.1% Triton X-100 for 10 min (Henderson et al.,2002). Primary antibodies against OCT4 (BD Biosciences, San Jose, CA, 1:250), SSEA-1, SSEA-3, and SSEA-4 (1:100), TRA-1-60, and TRA-1-81 (1:50) (Millipore, Billerica, MA) were incubated with colonies overnight at 4°C. The secondary antibodies used were either, Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L), (Invitrogen/Molecular Probes, Eugene, OR, 1:50), or fluorescein isothiocyante (FITC) -conjugated donkey anti-Mouse IgM (Jackson ImmunoResearch, West Grove, PA, 1:50), or Alexa Flour 488-conjugated rabbit anti-goat IgG1 (Invitrogen/Molecular Probes). Colonies were incubated with secondary antibodies for 45 min at room temperature.

Immunostaining of hES cell-derived neural cells for NESTIN and TUJ1 were performed as described previously (Ma et al.,2008). Briefly, differentiated cells were fixed with 4% paraformaldehyde and permeabilized in 0.1%Triton X-100. Primary antibodies used were rabbit anti-NESTIN, 1:200, mouse anti-TUJ1, 1:300 (Millipore, Billerica, MA). Secondary antibodies used were either rhodamine-conjugated donkey anti-rabbit IgG-(H+L) (Jackson Immunoresearch, West Grove, PA), Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Molecular Probes, Eugene, Oregon) or FITC-conjugated donkey anti-chicken IgG (Millipore; 1:50). Cells were counterstained with the 4′-6-diamidino-2-phenylindole (DAPI) with a dilution of 1:1,000 (Sigma).

Karyotypic Analysis

Samples of cells were treated with colcemid KaryoMAX (0.07 μg/ml, Invitrogen) for 3–4 hr and harvested. After washing, the cells were incubated in 0.4% trypsin solution (GIBCO) for 2–3 min. hES cells were incubated in 0.075 mol/L KCl solution for 40 min at 37°C, and fixed in methanol: acetic acid (3:1, v/v). After Giemsa staining, a cytogenetics specialist examined the karyotypes of the hES cells at a resolution of 300 bands. At least 50 cells from each sample were examined.

DNA Fingerprinting

DNA fingerprinting analysis was performed with genomic DNA from each hES cell line. The 16 short tandem repeat (STR) loci were amplified by PCR to identify established hES cell lines. The STR loci used were D8S1179 (8), D21S11 (21q11.2-q21), D7S820 (7q11.21-22), CSF1PO (5q33.3-34), D3S1358 (3p), TH01 (11p15.5), D13S317 (13q22-31), D16S539 (16q24-qter), D2S1338 (2q35-37.1), D19S433 (19q12-13.1), vWA (12p12-pter), TPOX (2p23-2per), D18S51 (18q21.3), Amelogenin (Xp22.1-22.3 Yp11.2), D5S818 (5q21-23), FGA (4q28). Specific human STR markers were analyzed using an AmpFlSTR Identifiler kit (ABI) on an automated ABI 310 GeneScan software.

Flow Cytometry

The distribution of cells at specific cell cycle stages was evaluated by flow cytometry. hES colonies at day 3 after passaged were harvested mechanically, dissociated into single cells with 0.1% trypsine (Sigma) (37°C for 3 min), and washed with phosphate-buffered saline. For DNA content analysis, cells were fixed in 75% ethanol at 4°C overnight, rehydrated in PBS, treated for 30 min with 20 μg/ml RNase A and for 30 min with 40 μg/ml propidium iodide (Sigma) on ice. The samples were analyzed for cell cycle distribution by flow cytometry (Beckman Coulter Epics XL-4) using multicycle software (Beckman Coulter).

Cell proliferation was assessed by detecting incorporation of 5-bromo-2′-deoxyuridine (BrdU) into DNA in four independent experiments. For the BrdU incorporation assay, cells at day 3 after passaged were allowed to incorporate BrdU(10 μmol/L, Bioscience) for 24 hr at 37°C. Then cells grown on feeder cells were collected mechanically, trypsinized to single-cell suspensions, and then fixed with 70% ethanol overnight. After rehydrated by PBS twice, the cells were treated by 2 N HCl for 20 min and then rehydrated by PBT (PBS+0.5%Tween20+0.5%BSA [bovine serum albumin]) twice. Then the cells were incubated by BrdU-FITC–labeled monoclonal antibody for 30 min at room temperature and again rehydrated by PBT twice. Finally, BrdU incorporation assay was detected by flow cytometry (Beckman Coulter Epics XL-4) using system II software (Beckman Coulter).

Teratoma Formation

At the time of routine passaging, clumps consisting of 300–400 undifferentiated hES colonies were harvested and injected with a sterile 26G needle into gastrocnemius of 4- to 8-week-old severe combined immunodeficiency (SCID) mice (Slac laboratory, Shanghai). At 10 to 12 weeks later, the resulting tumors were fixed in 10% neutral buffered formalin, embedded in paraffin, and examined histologically by staining with hematoxylin and eosin (Gardner,2002).

RT-PCR Analysis

To induce the formation of EBs, hES cell colonies at good state were dissociated into small parts mechanically, and grown in suspension in the same hES culture medium but lacking bFGF. Total RNA isolation from 7, 14, and 21 days-EBs was performed using TRIzol reagent according to the manufacturer's protocol (TaKaRa Biotechnology Co., Ltd. [Dalian]). First-strand cDNAs were generated using 2 μg of RNA, Superscript reverse transcriptase (Promega Corporation), and random hexamer primers (Promega Corporation) and oligo d(T) (Invitrogen). For the PCR, first-strand cDNA (the equivalent of 40 ng of reverse-transcribed RNA) was amplified in a final volume of 20 μl with 1 U of Taq DNA polymerase (TaKaRa Biotechnology Co., Ltd. [Dalian]) and 10 pmol of each primer. Gene-specific primers were designed with Primer3 software (Whitehead Institute/MIT Center for Genome Research) as Table 2. Each gene transcript was amplified in a Biometra thermal cycler by 5 min of initial denaturation at 94°C, followed by 30 cycles of 1 min denaturation at 94°C, 1 min of annealing at primer-specific temperature 58°C, and 1 min of primer extension at 72°C. Final extension was carried out for 10 min at 72°C. To ensure quantitative results, the number of PCR cycles for each set of primers was checked to be in the linear range of amplification. In addition, all cDNA samples were adjusted to yield equal amplification of β-ACTIN as an internal standard. PCR products were visualized by ethidium bromide staining following 1.2% agarose gel electrophoresis.

Table 2. Primers of Genes From Three Germinal Layers Detected by RT-PCR
ClassificationGenesPrimers
  1. RT-PCR, reverse transcriptase-polymerase chain reaction.

EctodermNF-68Forward 5′-ACGCTGAGGAATGGTTCAAG-3′
  Reverse 5′-TAGACGCCTCAATGGTTTCC-3′
 KERATINForward 5′-AGGCCCAATACGAGGAGATT-3′
  Reverse 5′-ATAGCCACTGGAGATGGTGG-3′
MesodermCMPForward 5′-AAAAAGGGCAATGACACCAG-3′
  Reverse 5′-TGTGCAGTCTCTGAGGTGG-3′
 KALLIKREINFForward 5′-GCTTTCTCAGCCAGGACATC-3′
  Reverse 5′-TATTCTTTGCCTCCCAGGTG-3′
 ENOLASEForward 5′-GTTCAATGTCATCAATGGCG-3′
  Reverse 5′-GTGAACTTCTGCCAAGCTCC-3′
Endodermα-FETOPROTEIN(α-FP)Forward 5′-TGAAAACCCTCTTGAATGCC-3′
  Reverse 5′-TCTTGCTTCATCGTTTGCAG-3′
 α1-ATForward 5′-ACTGTCAACTTCGGGGACAC-3′
  Reverse 5′-CCCCATTGCTGAAGACCTTA-3′
Stemness markerOCT4Forward 5′-GAGTCCCAGGACATCAAAGC-3′
  Reverse 5′-CTTCCTCCACCCACTTCTGC-3′
Confidential reference itemsβ-ACTINForward 5′-GTGGGGCGCCCCAGGCACCA-3′
  Reverse 5′-CTCCTTAATGTCACGCACGATTTC-3′

Real-Time qPCR Analysis

Eleven genes (OCT4, NANOG, SOX2, UTF1, DPPA5, LIN41, SOX1, H19, IGF2, GATA4, and HAND1) were selected to assess self-renewal and differentiation in human embryonic stem cell (Cai et al.,2006). OCT4, NANOG, UTF1, DPPA5, LIN41, SOX2 represent the undifferentiated hES cells' state. Early markers of differentiation are as follows: (a) ectoderm markers-SOX1; (b) endoderm markers, including H19, GATA4; (c) mesoderm markers-IGF2, HAND1. Real-time qPCR was used to quantify the levels of mRNA expression of 11 selected genes in hES cells. Total RNA was extracted from undifferentiated hES cells and retro-transcription was performed as just mentioned. Gene-specific primers were designed with Primer3 software (Whitehead Institute/MIT Center for Genome Research) as Table 3. PCR reactions were carried out by a 7500 sequence detection system (Applied Biosystems) using a SYBR Green qPCR kit (Applied Biosystems) according to the manufacturer's instructions. The content of selected genes was normalized to the content of GAPDH, and nontemplate control were performed for every genes as blank control. Standard curves were generated using 10 ng cDNA per 20-μl reaction volume. All PCR products were checked by melting curve analysis to exclude the possibility of multiple products or incorrect product size. PCR analyses were conducted in triplicate for each sample.

Table 3. Primers of Genes Detected by Real-Time qPCR
ClassificationGenesPrimers
  1. qPCR, quantitative polymerase chain reaction.

Undifferentiated genesOCT4Forward 5′-GCAGCTTAGCTTCAAGAACATGTG-3′
  Reverse 5′-GCTTTGCATATCTCCTGAAGATTTT-3′
 NANOGForward 5′-ACAACTGGCCGAAGAATAGCA-3′
  Reverse 5′-GGTTCCCAGTCGGGTTCAC-3′
 UTF1Forward 5′-CGACATCGCGAACATCCT-3′
  Reverse 5′-CCAGGGACACTGTCTGGTC-3′
 DPPA5Forward 5′-GCGAGGGATGCTCAAACTTG-3′
  Reverse 5′-CTTCATTGCATTGGCTGGAA-3′
 HAND1Forward 5′-CTGAGAGCATTAACAGCGCATT-3′
  Reverse 5′-GTGGCTAGGCGCAGAGTCTT-3′
 SOX2Forward 5′-GCAAGATGGCCCAGGAGAA-3′
  Reverse 5′-CGCTTAGCCTCGTCGATGA-3′
Differentiated genes EctodermSOX1Forward 5′-GAAGCCCAGATGGAAATACGTT-3′
  Reverse 5′-GGACAGTCCATACTCCAGGACAA-3′
EndodermH19Forward 5′-GAAGCGGGTCTGTTTCTTTACTTC-3′
  Reverse 5′-CTGGGTAGCACCATTTCTTTCAT-3′
 GATA4Forward 5′-TGTCCCAGACGTTCTCAGTCAGT-3′
  Reverse 5′-CCTGCTTGGAGCTGGTCTGT-3′
MesodermIGF2Forward 5′-TTCCAGACACCAATGGGAATC-3′
  Reverse 5′-GCCGCACAGGGTCTCACT-3′
 LIN41Forward 5′-GCAGGGACGACAGGCAAT-3′
  Reverse 5′-CGTTCCTCCAGGGCTTTCTT-3′
Confidential reference itemsGAPDHForward 5′-GAAGGTGAAGGTCGGAGTC-3′
  Reverse 5′-GAAGATGGTGATGGGATTTC-3′

Directed Neural Differentiation of hES Cells

The hES cell colonies were removed from MEF feeders and dissociated into small clumps by incubating with collagenase IV at 37°C for 15 min. The hES cell clumps were pelleted and cultured in suspension in low attachment dishes with hES cell medium without bFGF for 5 days. The neuroectodermal induction began with EBs transferred into the neural differentiation medium that consisted of DMEM:F12 medium (HyClone), N-2 supplement (Gibco), 0.1 mM nonessential amino acids (Invitrogen), penicillin (100 U/ml)/ streptomycin (100 μg/mL) (Gibco), and 5 ng/ml bFGF (R&D) for 10 days. At days 15–17 of differentiation, EBs were plated on substrate-coated dishes (Ma et al.,2008).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The authors are grateful for the technical support provided by all members in the IVF laboratory (Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, Jinan) and all members in the Central Laboratory, Shandong Provincial Hospital.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES