Self-renewal and differentiation capabilities are variable between human embryonic stem cell lines I3, I6 and BG01V
© Tavakoli et al; licensee BioMed Central Ltd. 2009
Received: 19 August 2008
Accepted: 05 June 2009
Published: 05 June 2009
A unique and essential property of embryonic stem cells is the ability to self-renew and differentiate into multiple cell lineages. However, the possible differences in proliferation and differentiation capabilities among independently-derived human embryonic stem cells (hESCs) are not well known because of insufficient characterization. To address this question, a side-by-side comparison of 1) the ability to maintain an undifferentiated state and to self-renew under standard conditions; 2) the ability to spontaneously differentiate into three primary embryonic germ lineages in differentiating embryoid bodies; and 3) the responses to directed neural differentiation was made between three NIH registered hES cell lines I3 (TE03), I6 (TE06) and BG01V. Lines I3 and I6 possess normal XX and a normal XY karyotype while BG01V is a variant cell line with an abnormal karyotype derived from the karyotypically normal cell line BG01.
Using immunocytochemistry, flow cytometry, qRT-PCR and MPSS, we found that all three cell lines actively proliferated and expressed similar "stemness" markers including transcription factors POU5F1/Oct3/4 and NANOG, glycolipids SSEA4 and TRA-1-81, and alkaline phosphatase activity. All cell lines differentiated into three embryonic germ lineages in embryoid bodies and into neural cell lineages when cultured in neural differentiation medium. However, a profound variation in colony morphology, growth rate, BrdU incorporation, and relative abundance of gene expression in undifferentiated and differentiated states of the cell lines was observed. Undifferentiated I3 cells grew significantly slower but their differentiation potential was greater than I6 and BG01V. Under the same neural differentiation-promoting conditions, the ability of each cell line to differentiate into neural progenitors varied.
Our comparative analysis provides further evidence for similarities and differences between three hESC lines in self-renewal, and spontaneous and directed differentiation. These differences may be associated with inherited variation in the sex, stage, quality and genetic background of embryos used for hESC line derivation, and/or changes acquired during passaging in culture.
Human embryonic stem cells (hESCs) possess the ability to self-renew in an undifferentiated state in culture while retaining the ability to differentiate into all of the cell types in the human body. These unique capabilities make hESCs a renewable source of a wide range of cell types for potential use in research and cell-based drug screening and therapies for many diseases. These cells have been in high demand for use in basic and applied biomedical research. As of January 1, 2006, at least 414 human ES cell lines have been derived worldwide . Large numbers of cell lines with genetic diversity are necessary to cover the vast spectrum of HLA isotypes to avoid transplant rejection [2, 3]. However, many of these cell lines are not fully characterized and differences among these cell lines are uncertain , although recent studies have revealed similarities and differences among individually developed human embryonic stem cell lines [3–12].
The comparison of the unique properties and behavior of each individually derived cell line is critical in identifying the safe and efficacious lines for research and therapeutic use [3, 13]. It is also essential to understand how the inherited variation in the sex, stage, quality and genetic background of embryos, as well as environmental influences such as derivation methods and passage procedures can affect the ability of hES cell lines to self-renew and differentiate. Directly comparing hES cell lines is challenging since all the genetic, environmental and methodological variables complicate the assessments. Previous studies have attempted setting up a core set of standard assays to characterize the status of "stemness" and pluripotency  and to define a reasonable set of markers that would serve as reliable indicators for self-renewal and differentiation of hESCs [10, 12]. In the present study, a side-by-side comparison of the ability to maintain an undifferentiated state and to self-renew under standard conditions, the ability to spontaneously differentiate into cell types of three germ layers in embryonic bodies, and directed differentiation under neural differentiation-promoting conditions was made between three NIH registered hESC lines I3, I6 and BG01V. I3 (NIH Registry Name TE03) and I6 (NIH Registry Name TE06) which were derived using rabbit anti-human whole antiserum with a normal XX and a normal XY karyotype respectively ; BG01V contains known chromosomal aberrations (XXY, +12 and +17) possesses characteristics similar to its normal parental line BG01 [16, 17]. The hESC lines I3, I6 and BG01V have been extensively characterized and tested in our laboratory for potential reference standard cell lines, because these three lines represent consensus standard human ES cell lines and a karyotypically abnormal human ES cell variant respectively.
Immunocytochemistry, flow cytometry, quantitative RT-PCR and MPSS were used to assess the self-renewal and differentiation capabilities. We found that all three cell lines actively proliferated and expressed similar "stemness" and pluripotency markers and alkaline phosphatase activity. All the cell lines differentiated into phenotypes representing ectoderm, endoderm, and mesoderm and were directed into neural cell lineages in vitro. However, the significant differences were observed in growth rate, BrdU incorporation, relative abundance of pluripotency marker expression and the ability to differentiate. These differences between the cell lines may depend on a combination of genetic, environmental and methodological factors , implicating the importance of establishing standard protocols for hESC derivation and culture.
Human embryonic stem cell lines I3, I6, and BG01V used in this study were cultured on mitomycin C-treated mouse embryonic fibroblasts CF-1 (ATCC, SCRC-1040.2; http://www.atcc.org). Cells were cultured at 37°C, in a 5% CO2 atmosphere, in the ES medium of Dulbecco's modified Eagle's medium (D-MEM)/F12 (ATCC 30-2006)80%, supplemented with 2.0 mM L-alanyl-L-glutamine (ATCC 30-2115), 0.1 mM non-essential amino acids (ATCC 30-2116), 0.1 mM 2-mercaptoethanol (Sigma Catalog No. M-7522) and 4 ng/ml basic fibroblast growth factor (bFGF; R & D Systems Catalog No. 233-FB), 5%; Knockout serum replacement (Invitrogen Catalog No. 10828), 15%; fetal bovine serum (ATCC SCRR-30-2020), penicillin (100 I.U./mL) and streptomycin (100 μg/mL) (ATCC 30-2300). An additional 4 ng/ml of bFGF was added in the first 24 hours after thawing the cells. Daily medium changes began after the first 48 hours in culture. The BG01V colony formation was visible within 2–3 days and the other two cell line's colony formation was observed in 3–4 days. Cells were passaged every 4–5 days using collagenase IV (200 Units/mL) (Invitrogen Corporation) for BG01V, I6 was passaged every 6–7 days and I3 was passaged every 7–8 days.
Embryoid body formation
hESCs in culture were removed from feeder cells using collagenase IV (200 Units/mL) (Invitrogen Corporation; http://www.invitrogen.com). hESC clusters were transferred to 10 × 10-cm ultra-low-attachment dishes (corning; http://www.corning.com) and cultured in medium D-MEM/F12 (80%) (ATCC 30-2006) supplemented with ES-Qualified FBS (15%) (ATCC SCRC-30-2020), knockout serum replacement (KSR) (5%) (Invitrogen Corporation), L-alanyl-L-glutamine (2.0 mM) (ATCC 30-2115), non-essential amino acids (1×) (ATCC 30-2116), β-mercaptoethanol (0.1 mM) (Invitrogen Corporation), penicillin (100 I.U./mL)/streptomycin (100 μg/mL) (ATCC 30-2300). The medium was changed every second day. To evaluate the growth rates of EBs, phase-contrast photographs of EBs were taken and the total areas of EBs were measured using Scion Image. The percent increase in total areas of the cell spheroids was compared between different cell lines. Data were calculated as mean ± S.E.M. of at least 3 separate cultures. The statistical significance was determined using the Student's t-test with p < 0.05 considered significant.
Directed neural differentiation of hESCs
The directed neural differentiation method was described previously . Briefly, colonies of the three hESC lines I3, I6, BG01V were removed from MEF feeders and dissociated into small clumps by incubating with collagenase IV (200 Units/ml) (Invitrogen Carlsbad, CA; http://www.invitrogen.com) at 37°C for 35 minutes. The hESC clumps were pelleted and cultured in suspension in low attachment dishes with hESC medium without bFGF for 5 days (the end of this stage is considered as 5 days of differentiation). hESC grew into floating aggregates or embryoid bodies (EBs). The neuroectodermal induction began with EBs transferred into the neural differentiation medium (NDM) that consisted of two parts of a modified Eagle's medium (ATCC 30-2002; http://www.atcc.org), one part F12k- (ATCC 30-2004), 1× N-2 supplement (Gibco Catalog No. 317740; http://www.invitrogen.com), 0.1 mM non-essential amino acids (ATCC 30-2116), penicillin (100 IU/ml)/streptomycin (100 μg/ml) (ATCC 30-2300) and 5 ng/ml bFGF (R& D Systems Catalog No. 233-FB) for 10 days. At days 15–17 of differentiation, EBs were plated on PDL/laminin substrate-coated 35 mm dishes (corning; http://www.corning.com). Although some neural rosettes were formed in floating embryoid bodies (EBs), increased rosettes were visualized after plating of the EBs on substrates. Neuroectodermal cells in rosettes were further differentiated into neural progenitors and their progeny on PDL/laminin substrates.
To compare the growth rate between I3, I6 and BG01V, all three hES cells were plated into 6 well plates containing a feeder layer of mitomycin C-treated fibroblast (MEF). The cells were cultured at 37°C in a 5% CO2 atomosphere. Basic fibroblast growth factor (4 ng/ml) was added to each cell culture after the first 24 hours. The medium was changed daily after 48 hours. The cells from three separate wells were harvested using a 0.25% (w/v) trypsin/0.53 mM EDTA solution (ATCC cat # 30-2101) each day. The cell counts were performed using Cedex Analysis System, Innovatis. Data were calculated as mean ± S.E.M. of at least 3 separate cultures. The statistical significance was determined using the Student's t-test with p < 0.05 considered significant.
Bromodeoxyuridine (BrdU) incorporation and counterstaining with propidium iodide (PI)
To monitor cell proliferation within colonies of hES cells, bromodeoxyuridine (BrdU) incorporation with 5-bromo-2-deoxy-uridine Labeling and Detection Kit I (Roche, Indianapolis, IN; http://www.roche.com) was used as described previously . Briefly, cultures were exposed to 20 μM BrdU for 4 hours and then fixed with 70% alcohol containing 50 mM glycine at PH 2.0. After rinsing with the kit wash buffer, cells were incubated overnight with mouse anti-BrdU (1:1000) followed by incubation with FITC-conjugated donkey anti-mouse IgG (1:50) (Jackson Immunological Research, West Grove, PA) for 45 min. Some cultures that were not exposed to BrdU were used as negative controls which showed no immunoreactivity, demonstrating the specificity of BrdU antibody. In order to quantify the cell proliferation rate, cell nuclei were counterstained by the addition of 5 μg/ml propidium iodide (PI) for 10 min. PI+ and BrdU+ cells were examined and photographed with Nikon eclipse TE 300 microscope. The proliferation index was defined as the percentage of BrdU+ nuclei in the total number of PI+ cells. At least 5 labeled colonies were counted from each dish and three dishes were evaluated. Data were calculated as mean ± S.E.M. which were statistical significance determined by using the Student's t-test with p < 0.05 considered significant.
For the immunostaining of undifferentiated hES colonies for "stemness" markers, undifferentiated hESCs were cultured on mitomycin C-treated feeder cells in 35 mm tissue culture dishes (Corning, Corning, NY, http://www.corning.com). Colonies were rinsed twice before fixation with 4% paraformaldehyde (EMS, Hatfield, PA, http://www.emsdiasum.com) in 1× PBS for 15 min at room temperature. Cells were permeabilized with 0.5% saponin (Sigma, St. Louis, MO, http://www.sigma-aldrich.com) in PBS for 10 min. Primary antibodies against Oct3 (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com, 1:250), NANOG (1:100), and SSEA4 (1:100), and TRA-1-81 (1:50) (all from Millipore, Billerica, MA http://www.millipore.com) 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, http://www.invitrogen.com; 1:50), or FITC-conjugated donkey anti-Mouse IgM (Jackson ImmunoResearch, West Grove, PA, http://www.jacksonimmuno.com, 1:50), or Alexa flour 488-conjugated rabbit anti-goat IgG1 (Invitrogen/Molecular Probes, Eugene, OR, http://www.invitrogen.com). Colonies were incubated with secondary antibodies for 45 min at room temperature.
Immunostaining of hESC-derived neural cells for Nestin and TuJ1 were performed as described previously . Briefly, neural differentiation medium-treated EB were plated on PDL/laminin coated 35 mm Tissue Culture plates (Corning, Corning, NY, http://www.corning.com). Differentiated cells were fixed with 4% paraformaldehyde and permeabilized in 0.5% saponin as described above. Primary antibodies used were rabbit anti-nestin, 1:200, mouse anti-tubulin clone TUJ-1, 1:300, chicken anti-SOX1, 1:200, (all from Millipore, Billerica, MA http://www.millipore.com). Secondary antibodies used were either rhodamine-conjugated donkey anti- rabbit IgG-(H+L) (Jackson Immunoresearch, West Grove, PA; http://www.jacksonimmuno.com), Alexa Fluor 488 conjugated goat anti-mouse- IgG (H+L) (Molecular Probes, Eugene, Oregon; http://www.invitrogen.com) or FITC-conjugated donkey anti-chicken IgG (Millipore, Billerica, MA http://www.millipore.com, 1:50). Cells were counterstained with the 4'-6-diamidino-2-phenylindole (DAPI) with dilution of 1:1000 (Sigma; http://www.sigmaaldrich.com). Immunofluorescence signals were observed and photographed with a Nikon TE 300 epifluorescence microscope (Nikon, Inc. Melville, NY) equipped with a Qicam FAST1394 digital camera (Surrey, BC, Canada) and Openlab vs. 4.0.4 software http://www.improvision.com.
To quantify the percentage of hESC-differentiated neural progenitors, cell counting was performed on cultures immunostained for nestin, together with nuclear DAPI counterstaining, in 35 mm culture dishes coated with different laminin substrates from at least three independent experiments. All data were expressed as mean ± SEM, and Student's t test was used for statistical evaluation. In all instances p < 0.05 was considered statistically significant.
Alkaline Phosphatase staining
Endogenous alkaline phosphatase activity in BG01V, I3 and I6 cells was detected using the ELF® 97 Endogenous Alkaline Phosphatase Detection Kit (ATCC catalog # SCRR-3010) according to the manufacturer's instructions. Cells cultured in 6 well plates (Corning Life Sciences; http://www.corning.com) were treated with 4% paraformaldehyde for 15 minutes at room temperature. The cells were washed with 1× PBS, treated with 0.2% Tween-20 for 10 minutes at room temperature and rinsed with 1× PBS. Fixed cells were then incubated with a filtered 1:20 dilution of the phosphatase substrate in situ, and the reaction was monitored using an epifluorescence microscope. The reaction was terminated using a stop solution consisting of PBS, 25 mM EDTA, 5 mM levamisole, pH 8.0. Cells were rinsed with PBS before mounting on glass microscope slides.
Total RNA was isolated from three hESC lines I3, I6, and BG01V using an RNAeasy Plus Mini kit (Qiagen catalog NO 74134; http://www1.qiagen.com/). The isolated RNAs were quantified using a RNA 6000 Nano Kit (Catalog NO 5067-1511). The integrity of RNA was checked on Agilent 2100 Bioanalyzer (Agilent Technologies; http://www.chem.agilent.com part No G2940CA). Equal amounts of RNA (1 μg) was taken for all samples and reverse transcription was done using RT2 First Strand kit from Superarray Biosciences (SuperArray, catalog No C-03; http://www.superarray.com. The total volume of the reaction was 20 μL and was diluted to 100 μL. PCR reactions were performed using a ABI Fast 7900 using RT2 Real-Time™ SYBR Green PCR master mix PA-012 and qRT-PCR primers from SuperArrray Biosciences. The total volume of the PCR reaction was 10 μL. The qRT-PCR Primers sets catalog numbers are 18srRNA PPH00073A, B-actin PPH05666A, POU5F1, Nanog, UTF1 (undifferentiated) PPH02394A, PPH02391A and PPH17032A, keratin C and NEFL (ectoderm) PPH-21369A and PPH02430A, alpha-globin and Beta-globin (Mesoderm) PPH09054A and PPH12971A, alpha-1AT PPH02413A, nestin and Musashi 1, SOX1 (Neural Progenitor) PPH02388A, PPH13090A and PPH02390A, MAP2, GAD-65 (Neural) PPH02419A, PPHo5950A, S100B and GFAP (astrocytes) PPH02408A and PPH02472A; http://www.superarray.com. The thermocycler parameters were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Each gene for each sample was run in quadruplicate. Relative changes in gene expression were calculated using the ΔΔCt (threshold cycle) method. This method first subtracts the ct (Threshold cycle number) of the gene-avg ct of the two house keeping genes (18srRNA and ACTB) to normalize to the RNA amounts. Finally, the delta delta ct is calculated by subtracting the normalized average ct of the treated cells from the normalized average ct of the undifferentiated cells. Then this delta ct is raised to the negative power of 2 in order to calculate the fold change .
Massively Parallel Signature Sequencing
Massively parallel signature sequencing (MPSS) was performed using 1–2 μg purified total RNA from each of the three human ESC lines (BG01V, I3 and I6) from undifferentiated cells (day 0) and cells at different stages of differentiation (days 7, 14 and 21); the presence and absence of ESC markers and markers of differentiation were evaluated. The quality of total RNAs was evaluated using an Agilent Bioanalyzer. mRNA isolation was processed according to the MPSS protocol as described previously  with some modification. In brief, the mRNA was reverse-transcribed, the cDNA synthesized and digested with Dpn II, then GEX adaptors ligated with Dpn II and amplified by PCR, the cDNA library was ready to sequence. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparison between samples. Only reliable and annotatable signatures against updated human signature database were considered for further analysis.
To generate a complete, annotated human signature database, all the possible signatures from the human genome sequence, the human UniGene sequences, and human mitochondrion were extracted. Each virtual signature was ranked based on its position and orientation in the original sequence. The annotation database is established based on the virtual signatures, their classes and their corresponding genes so that each signature only has one corresponding annotation. The database is then used to annotate the data from the experiment.
Different hESC lines exhibit different colony morphologies
Differences in growth curves between undifferentiated hESC lines
To examine potential differences in the ability to self-renew between the three hESC lines, the percent increase in cell numbers relative to cell numbers at day 1 were calculated up to 6 days (Figure 1D). The relative growth was determined, followed by the plating of approxmately 105 cells on a MEF feeder, and trypsinized cells were counted using a Cedex Analysis System. The differences in the cell number between each cell lines should represent the differences in relative growth of hESCs since the fibroblast feeder cells were mitomycin C-treated. The growth curves in Figure 1D show significant differences in percent increase in the number of cells at day 6 in culture between the three cell lines. I6 and BG01V cell colonies reached an average size of 300–400 cells for splitting at 5–6 days after passaging. In contrast, I3 cells grew slower than the other two cell lines and were not ready to passage until culture day 8. It appeared more difficult to maintain the I3 cell line in an undifferentiated state since it had more tendency to differentiate.
MPSS expression analysis of three human ESC lines
All undifferentiated hESC lines express pluripotency markers, but their gene expression levels are variable
The Y-axis plots the fold change of the undifferentiated I6 and BGO1V cell lines in comparison to the undifferentiated I3 cell line. The expression level of the undifferentiated genes implicates that the I3 hES cells express much lower levels of "stemness" (undifferentiated) genes compared to the I6 and BG01V cell lines (Figure 3B and 3C).
Differences in BrdU incorporation between undifferentiated I3, I6 and BG01V cells
Embryoid body formation and growth rate vary among the three hESC lines
All three hESC lines are able to differentiate into cells expressing markers of all three germ layers and neural cells
Differences in expression of neural phenotypes and genes in directed neural differentiation between three hESC lines
To assess differences in the gene expression during neural differentiation between the three cell lines, total RNA was harvested from the EBs at 2 days post plating on poly-D-lysine/laminin substrates. The qRT-PCR analysis of the expression levels for the neural progenitor markers SOX1 and MSI1, the mature neuron marker MAP2, and the astrocyte marker GFAP showed that the all these genes were strongly expressed in the I3 cells (Figure 9G). The Y-axis plots the fold changes of gene expression for each cell line when compared to its own undifferentiated levels at Day 0. The high levels of expression for the neural progenitor genes SOX1 and MSI1 in the I3 and I6 cells were consistent with their high expression of nestin immunoreactivity (Figure 9A, B).
In the present study, we demonstrated that although all the three hESC lines I3, I6 and BG01V can maintain their ability to proliferate and give rise to the progeny of the three embryonic germ layers, their self-renewal and differentiation capabilities are variable. The overall gene expression profiles of the three lines were similar; however, in most cases, the relative abundance of expression of the same "stemness" and differentiation genes were highly variable between the cell lines. We also found that under the same neural induction conditions, the ability of each of the three lines to differentiate into neural progenitors was also distinct. Previous studies that compared hESC lines focused on the expression of pluripotency and the differentiation marker genes [9, 10, 12, 26]. In the present study, in addition to the variable gene expression in undifferentiated and EB differentiated states, we detected a profound variation in the cell growth rate, BrdU incorporation and the directed neural differentiation.
The work presented here is part of continuing efforts to develop a database of the properties and behaviors of different hESC lines and to understand the similarities and differences between individual hES cell lines by side-by-side comparison. This comparative analysis of individually-derived hESC lines is critical, because the properties and behavior of each line are uniquely shaped by their histories. It has become clear that different derivations produce hESC lines that are similar with regards to "stemness", but with inherent differences in gene expression, methylation status, X chromosome inactivation, rate of self-renewal and the ability to differentiate [6, 26, 27]. More importantly, the behavior of cells and their overall state changes as culture conditions and the stress they are subjected to is altered, and permanent genomic changes frequently occur as passage numbers increase [28–30]. Variability in genetic, environmental and methodological factors has led to a great difficulty in comparing results of studies of the hESC lines among laboratories.
In this study, our side-by-side comparison between the three hESC lines was made under the same culture conditions in an effort to minimize the influences of environmental and methodological factors. The differences we found between the three cell lines may be due to the genetic variation and epigenetically inherited alterations from previous culture history. Lines I3 and I6 were derived in the same laboratory but differ in sex . BG01V is a variant cell line with an abnormal karyotype . Another factor that may contribute to the cell line differences is the variation in passage number between the three cell lines. It is more challenging to directly compare differences in directed differentiation between different human ES cell lines because the differentiation protocols most likely are cell line-specific. Previous study has shown that the reliable dopaminergic differentiation was induced by co-culture with the mouse stromal cell line PA6 . However, in the present study, under culture conditions that favored neural differentiation of the I3 and I6 cells, the BG01V barely produced neural progenitors and expressed much fewer neural-specific genes compared to I3 and I6 cells. The inconsistency in BG01V cell neural differentiation data between Zeng's and our studies points out that each hES cell line needs an optimized protocol for a specific phenotype differentiation.
hESC lines have a great potential to provide new research tools that support clinical applications. The frequency of non-obvious changes in the hESC behavior and potency is of great concern for the future of cell replacement therapies. Physicians who transplant hESC-derived cells into patients must be in confident as to the safety and stability of the cells they use. Thus it is necessary not only to establish a set of characterization tests which are sensitive enough to detect small but harmful changes, but these tests must also be simple and inexpensive enough to be used routinely. The comparison made in this study also shows that the individually derived hESC lines from different laboratories are variable to various extents. Therefore reference standards, such as cell lines that provide consistent, predictable results and are not difficult to culture are needed. We believe that the database of hESC characterization data and standard reference materials will permit the research community to readily monitor and compare hESC lines.
Our side-by-side comparison confirms the general finding that hESC lines share the properties of self-renewal, expression of "stemness" and pluripotency markers and the ability to differentiate, but many differences remain between cell lines. These differences include the ability to maintain an undifferentiated state, to self-renew, and to differentiate. In addition to inherited variation in the sex, stage, quality and genetic background of embryos used for hESC line derivation, these differences may be associated with derivation methods and changes acquired during passaging in culture. To this end, it is important to set up standards shared by multiple laboratories for routine analysis of undifferentiated state ("stemness"), identity, stability, pluripotency and sterility of hESC lines.
This work was supported by contract grant N01AG40002 from NIH/National Institute on Aging, and by the intramural research program of the National Institute on Aging. We thank Dr. Dezhong Yin for assistance with the qRT-PCR analysis and Tina Zadeh for performing immunocytochemistry.
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