Human embryonic stem cells (HESc) isolated from early embryos can be grown in vitro while maintaining pluripotency and therefore can be different- iated into many of the cell types that constitute the body including neural precursors,1,2 hematopoietic cells,3 cardiomyocytes4,5 and insulin-secreting cells.6,7 These cells can potentially be used in regenerative medicine and drug discovery. The biggest obstacle for their use in regenerative medicine is transplant rejection. Additionally, there are well documented and marked differences in differentiation potential and drug susceptibility between HESc lines due to various genetic backgrounds.8 It is necessary to obtain more HESc lines representing the diverse Chinese population with different differentiation potentials. Since HESc lines were successfully derived for the first time by Thomson et al9 in 1998, many other HESc lines with different human leukocyte antigen (HLA) types have been isolated. However, the majority of reported HESc lines are from North America and Europe.10 Several Chinese HESc lines have been reported, but this number is not sufficient for the large population of China. For better management and potential use of HESc cell lines, it is necessary to fully describe the characteristics of the cell lines when reported, especially regarding their directed differentiation ability. The directed differentiation ability of the Chinese HESc cell lines are yet to be described.11,12 Currently in China, the most frequently used HESc lines for directed differentiation into pancreatic and hepatic cells are foreign HESc lines, such as the H1 line. It would be advantageous for Chinese researchers if there was a domestic cell line that could be efficiently differentiated into pancreatic and hepatic cells not only for development research but also for future applications in regenerative medicine.
We reported the establishment of a Chinese human embryonic stem cell line, designated CH1, in our in vitro fertilization-embryo transfer (IVF-ET) center. This cell line was established from donated day 6 blastocysts with informed consent. Similar to all other reported Chinese HESc lines, CH1 showed normal morphology, karyotype and pluripotency characteristics as described previously.13 Moreover, we found that CH1 cells differentiated into pancreatic and hepatic cells in vitro with the same efficiency as H1 cells. This newly established cell line will be a valuable resource for studying early embryo development and regenerative medicine, especially within the Chinese population.
Embryos collection, thawing and culture
All embryos used in this research were donated by couples that gave their signed and informed consent for this work. This study was approved by the Ethics Committee of Peking University Medicine School. Embryos were thawed according to a method developed by the IVF-ET center, where frozen blastocysts were first cultured in 0.3 mol/L sucrose for 2 minutes, then in 0.2 mol/L sucrose for 3 minutes, followed by Quinn's sperm washing medium (Sage, USA) for 5 minutes and finally cultured in blastocyst culture medium (Sage) for approximately 2 hours. The quality of three thawed embryos was evaluated according to the scoring system for human blastocysts.14
Derivation of the Chinese human embryonic stem cells line (CH1)
The medium used for HESc derivation was designated HESc derivation medium (HDM) and was composed of 80% DMEM/F12 (Gibco, USA), 15% knockout serum replacement (KSR; Invitrogen, USA), 5% embryonic stem (ES) cell screened fetal bovine serum (ES-FBS, Hyclone, USA), 2 mmol/L Glutamax-I, 0.055 mmol/L β-mercaptoethanol, 1% nonessential amino acids (all from Invitrogen), 10 ng/ml basic fibroblast growth factor (bFGF; Invitrogen) and 10 µg/ml recombinant human leukemia inhibitory factor (hLIF; Chemicon International, USA). The medium used for HESc in early passages was HDM with 5% KSR replacing the 5% ES-FBS, and called HESc growth medium (HGM). The medium used for established HESc cultures was HESc culture medium (HCM), and was HGM without hLIF.
Throughout the establishment of CH1, a batch of three cryopreserved blastocysts at day 6 were obtained. After thawing and removal of the zona pellucida of the embryos, with acidic Tyrode's solution, the three embryos were individually plated on mitomycin C-treated mouse embryonic fibroblast (MEF) feeders in 4-well plates containing HDM within 48 hours. The medium was changed, first to fresh HDM, followed by replenishment with HGM every day. At 7–12 days post-plating, outgrowths were observed and only those which included compact ES cell-like clumps were passaged, with HDM switched to HGM. Three more passages were performed following the emergence of primary HESc colonies. HESc colonies were then passaged normally with HCM using the isolation procedure outlined in Figure 1A.
Culture, cryopreservation and thawing of CH1
To maintain the CH1 line, the medium was changed daily, and colonies were passaged every 4–7 days. For bulk culture of cells, cultures were passaged enzymatically using dipase II (Chemicon).
The cultures were cryopreserved by slow freezing as previously described.15 Briefly, colonies were detached mechanically from the dishes after incubation in HCM supplemented with 1 µm Y27632 (Chemicon) for 1 hour. Colonies were collected and HCM was added, followed by an equal volume of 2× freezing medium consisting of 60% FBS, 20% dimethyl sulfoxide (DMSO) and 20% HCM. Cells were dispensed in 1 ml aliquots into pre-labeled cryogenic vials, which were then placed in cell freezing boxes and stored in a –80°C freezer overnight. The next day, the frozen cell stocks were transferred into liquid nitrogen for long-term storage.
For thawing, a vial of HESc were placed in a 37°C water bath with gentle agitation until thawed, then centrifuged and transferred into cell solution with fresh feeders. Cultures were incubated at 37°C/5% CO2. Media was changed two days post-thawing and daily thereafter, with the cultures split before they became too confluent.
Alkaline phosphatase (AP) staining and immunocytochemical analysis
AP staining was performed with an alkaline phosphatase kit (Promega, USA) according to the manufacturer's instructions. Immunostaining was performed as previously described.16 Briefly, cells were fixed in 4% paraformaldehyde and then blocked with 10% goat or donkey serum plus 0.2% Triton X-100 for 60 minutes at room temperature, then incubated at 4°C overnight with primary antibodies. The primary antibodies and the respective dilutions they were used at were: rabbit anti-Oct4 antibody (1:100; Abcam, UK), rabbit anti-Sox2 antibody (1:200; Abcam), goat anti-Nanog antibody (1:20; R&D Systems, USA), mouse anti-SSEA4 antibody (1:100; Santa Cruz Biotechnology, USA), mouse anti-TRA-1-60 antibody (1:100; Santa Cruz Biotechnology), mouse anti-TRA-1-81 antibody (1:100; Santa Cruz Biotechnology), rabbit anti-βIII-Tubulin (Tuj1) antibody (1:400; Covance Research Products, USA), goat anti-SOX17 antibody (1:200; R&D Systems), goat anti-FOXA2 antibody (1:200; R&D Systems), rabbit anti-PDX1 antibody (1:1000; Abcam), goat anti-AFP antibody (1:200; Invitrogen), goat anti-ALB antibody (1:500; DAKO, USA) and rabbit anti-BRACHYURY antibody (1:200; Santa Cruz Biotechnology). The appropriate secondary antibodies were FITC/TRITC- conjugated IgG (Santa Cruz Biotechnology) and incubated for 45 minutes at room temperature. Cells with only secondary antibody staining served as negative controls. Cell nuclei were identified with DAPI (Roche, USA). Images were captured with an Olympus phase contrast microscope (IX-71; Olympus, Tokyo, Japan).
In vitro differentiation and teratoma formation
In vitro differentiation of HEScs was performed by the embryoid body (EB) formation method. HEScs were dissociated into small clumps and plated in suspension to form EBs in a differentiation medium of HCM without bFGF. After six days in suspension, EBs were replated onto 0.1% gelatin-coated dishes in differentiation medium. Immunostaining was performed to detect the expression of markers for the three germ layers after seven days.
For teratoma formation, serially passaged HEScs were dissociated into small clumps mechanically, then 100 μl cells (1×107 cells/ml) were resuspended in PBS with 30% Matrigel (BD) and injected under the skin of Scid mice. After 4–8 weeks, the teratomas were collected and prepared for paraffin sections, then stained with hematoxylin and eosin.
HES cells were prepared for karyotype analysis using standard protocols.17 The karyotype was finally determined by microscopic examination after conventional Giemsa staining and G-banding analysis.
Reverse transcription-polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted from HEScs with an RNeasy micro kit (Qiagen, USA) and cDNA was subsequently generated with SuperScript III (Invitrogen). The PCR was performed with TAP DNA polymerase (CLP, San Diego, CA, USA) and 0.2 mmol/L deoxynucleoside triphosphates. Cycling conditions were as follows: 97°C for 5 minutes followed by 28–32 cycles of amplification involving 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, and 72°C elongation for 30 seconds, with a final extension at 72°C for 7 minutes. Primers specific for the markers of undifferentiated HEScs are listed in Table.
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Table. Primers used in RT-PCR analysis of stemness genes for human embryonic stem cells
Directed differentiation into pancreatic and hepatic cells
CH1 cells were differentiated into pancreatic and hepatic cells as previously described.18,19 In brief, for differentiating into pancreatic cells, CH1 cells were dissociated into small clumps with 200 U/ml collagenase IV (4 minutes, 37°C) and the undifferentiated colonies were collected by sedimentation. The dissociated colonies were plated onto Matrigel (1:50; BD Biosciences, USA)-coated dishes (Falcon, USA) for attachment with a coverage of 60% and then incubated with DMEM/F12 supplemented with 0.2% bovine serum albumin (BSA) (Sigma), 0.5× N2 and 0.5× B27, 100 ng/ml activin A and 1 µmol/L wortmannin for 4 days. Differentiated cells were then cultured in F12/IMDM (1:1) supplemented with 0.5% BSA, 0.5% insulin-transferrin-selenium (ITS), 0.5× B27, 2 µmol/L retinoic acid (RA; (Sigma, USA), 20 ng/ml fibroblasts growth factor (FGF)7 and 50 ng/ml NOGGIN for 4 days. Cells were then cultured in DMEM (high glucose) supplemented with 0.5% BSA, 1% ITS, 1× N2 and 50 ng/ml epidermal growth factor (EGF) (Sigma, USA) for five days, with cells exhibiting obvious expansion and reaching nearly 100% confluence.
For hepatic differentiation, CH1 cells were incubated for 24 hours with RPMI 1640 medium (Invitrogen/Gibco, Rockville, MD, USA), supplemented with 0.5 mg/ml albumin fraction V (Sigma-Aldrich, USA) and 100 ng/ml activin A. On the following 2 days, 0.1 and 1% ITS was added to this medium, respectively. The differentiated HEScs were cultured in Hepatocyte Culture Medium (Cambrex, Baltimore, MD, USA) containing 30 ng/ml FGF4 and 20 ng/ml bone morphogenetic protein (BMP2) (both from Peprotech, USA) for 4 days. Differentiated cells were then incubated in Hepatocyte Culture Medium containing 20 ng/ml hematopoietic growth factor (HGF) and 20 ng/ml keratinocyte growth factor (KGF) (both from Peprotech) for 6 days.
Derivation of the CH1 cell line
We obtained embryos and established HESc lines according to the ethical standards of international stem cell banks. The donated embryos were used to establish HESc lines by researchers who did not participate in the process of collecting the embryos. A total of three blastocysts were obtained and processed to establish HESc lines according to our procedures. After thawing, the first embryo was designated top quality: 6AA with a tightly defined inner cell mass (Figure 1B), the cyst of the second one did not expand but the embryo hatched completely, and the third one was termed poor: 5CC, and had a poorly defined inner cell mass. The zona pellucida of 5CC was removed by treatment with acidic Tyrode's solution. Whole embryos were plated on fresh feeders, with all three embryos attached after 48 hours incubation. Two of the embryos flattened out and were eventually overtaken by trophectoderm cells. No ES-like outgrowth emerged until day nine and only one developed an ES-like outgrowth at day eight in the 6AA well (Figure 1C). The clump was mechanically dissociated from the flattened trophectoderm cells at day 9, and was seeded on fresh feeder cells (Figure 1D) with HDM and then switched to HGM. The compact clump grew and primary HESc colonies (Figure 1E) emerged. Three further passages were performed mechanically with medium switch from HDM to HGM. Following this, HESc colonies (Figure 1F) were passaged normally with HCM, successfully propagated and developed into a new cell line we designated CH1.
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Figure 1. Establishment of CH1 cell line. A: The scheme for derivation of the ES cells. The thawed day 6 blastocyst of highest quality (6AA) (B, scale bar = 200 μm) was seeded onto MEF feeders in HDM for 3 days, then the medium was changed to HGM. After 4–9 days, the outgrowth included compact ES-like clumps (C, scale bar = 80 μm) that emerged, grew bigger, and were passaged mechanically without cutting and finally seeded onto fresh MEF feeders also in HDM, followed by HGM. This was recorded as passage 0 (P0) (D, scale bar = 80 μm). After another 5–7 days, the ES-like clones (E, scale bar = 80 μm) emerged, were passaged mechanically, dissected into small clumps and seeded on fresh MEF feeders. These were recorded as P1. Then, ES clones (F, scale bar = 80 μm) emerged and were further passaged.
Up to this point, CH1 cells were passaged mechanically up to passage 30 (P30) without differentiation. When passaged in bulk in 60 mm dishes at P16, cells were cryopreserved. To improve the survival rate after thawing, the colonies were treated with Y27632 for 1 hour before freezing. The HESc colonies could be first observed at day three post-thawing and the recovery rate was up to 60%. As previously described, Y27632, a ROCK inhibitor, was beneficial to the survival of HEScs.20 In this study, treatment with this small molecule helped us to achieve a high recovery rate.
Characterization of the CH1 line
The CH1 cells had the typical morphology of HEScs as described previously:9,13 flat and compact colonies with small cells having a high nucleus/cytoplasm ratio and prominent nucleoli (Figure 1F). The cells expressed a number of molecular markers for undifferentiated pluripotent human stem cells including octamer binding protein 4 (OCT4), SOX2, NANOG, SSEA-4, TRA-1-60, TRA-1-81 and alkaline phosphatase (Figure 2E–2G). RT-PCR analysis also confirmed the expression of stemness marker genes such as OCT4, SOX2, C-MYC, NANOG and KLF4 (Figure 2H). After about 20 passages, they exhibited normal karyotypes: 46 XX (Figure 3E). CH1 cells efficiently formed EBs (Figure 3A) through the suspension method in differentiation medium. After replating, the EBs also yielded cells representing three germ layers: SOX17 (endoderm), BRACHYURY (mesoderm) and β-III tubulin (ectoderm) (Figure 3B–3D). Additionally, teratomas were harvested 6 weeks after injection of the cells under the skin of Scid mice. Histological examination showed that the teratoma contained various tissues of the three germ layers (Figure 3F–3H).
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Figure 2. Characterization of CH1 cells (A–F). Staining of HESc markers such as OCT4 (A, scale bar = 80 μm), NANOG (B, scale bar = 80 μm), SOX2 (C, scale bar = 80 μm), TRA-1-60 (D, scale bar = 80 μm), TRA-1-81 (E, scale bar = 80 μm), SSEA-4 (F, scale bar = 80 μm). Alkaline phosphatase (AP) staining of CH1 (G, scale bar = 80 μm). RT-PCR analysis of the expression of HESc specific genes (H, from left to right: SOX2, OCT4, C-MYc, Klf4, NANOG and marker).
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Figure 3. Spontaneous differentiation of CH1 cells in vitro and in vivo. EBs (A, Phase contrast micrographs, scale bar = 200 μm) were formed through a suspension method, and after replating they were differentiated into cells of the three germ layers and were examined by immunostaining: brachyury (mesoderm; B, scale bar = 80 μm), βIII-Tubulin (ectoderm; C, scale bar = 80 μm) and Sox17 (endoderm; D, scale bar = 80 μm). Normal karyotypes of CH1 cells were shown by G-banding: (E) female karyotype, 46 XX. Teratomas were generated from CH1 cells. Histological examination showed the teratomas contained various tissues, including smooth muscle (mesoderm; F, scale bar = 80 μm), gut-like stratified squamous epithelium (endoderm; G, scale bar = 80 μm) and cartilage (ectoderm; H, scale bar = 80 μm).
CH1 cells were differentiated into pancreatic and hepatic cells
To test their differentiation potential, CH1 cells were induced to differentiate into pancreatic and hepatic cells. H1, a well-established HESc line, was differentiated at the same time with the same protocols as the control. For pancreatic cell differentiation, CH1 cells were induced with a step-wise protocol established in our lab.21 CH1 cells were first induced to differentiate into definitive endoderm with activin A and wortmannin. By day 4, the definitive endoderm marker genes Foxa2 and SOX17 were detected. These derived definitive endoderm cells were treated with RA, NOGGIN and FGF7 to induce pancreatic cells for 5 days. At the end of this stage, the pancreatic cells marked with PDX1 were detected (Figure 4E–4H). Finally, when these pdx1-positive cells were treated with EGF for 5 days, they rapidly proliferated. The results of this differentiation process were similar to those achieved with H1 cells (data not shown).
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Figure 4. Directed differentiation of CH1 cells into pancreatic and hepatic cells. With a step-wise protocol, CH1 cells were differentiated into hepatic cells marked by Alb (A) and Afp (B) (DAPI, C; merged, D). Also, pancreatic cells were marked by Pdx1 (F) and detected. βIII-Tubulin (E) was co-immunostained with Pdx1 on these cells (DAPI, G; merged, H). The scale bars for all images were 80 μm.
As for differentiation into hepatic cells, a step-wise protocol composed of definitive endoderm induction, hepatic specialization and expansion was used. After 12 days of induction, the hepatic marker genes AFP and Alb were detected and found to be expressed in differentiated CH1 cells. Most of AFP-expressing cells also expressed ALB (Figure 4A–4D). Moreover, these differentiated cells could also secrete Alb, which was detected by ELASA (data not shown), showing that they were functional hepatic cells.
We established a cell line from three frozen embryos, with the derivation rate equal to that reported by Chen et al.22 According to previous reports, several factors influenced the rate of derivation, these were: treatment methods before plating, timing of plating, the percentage of FBS in the derivation medium, the genetic background, and the quality of the embryos. The final two factors were critical in deriving a new cell line. In this study, although we plated whole blastocysts without isolation of the inner cell mass, we achieved a high success rate which might be due to the high quality of the embryo. Recently, other researchers also derived two HESc lines and deposited them into the UK Stem Cell Bank,23 their derivation rate was about 9.5%. This low derivation rate was likely due to the poor quality of the embryos initially.
CH1 had normal morphology and karyotype, expressed stemness genes and can spontaneously differentiated into cells representing three germ layers both in vivo and in vitro. All these indicated CH1 was a new pluripotent stem cell line. To test the ability of directed differentiation of the CH1 cells, we chose to induce the cells to directly differentiate into pancreatic and hepatic cells. The results demonstrated that CH1 cells had high potential to differentiate into pancreatic and hepatic cells. However, our research remains limited regarding the targeted differentiation ability of CH1 cells. The ability of the specialized pancreatic and hepatic cells to mature and differentiated into specific cells of the other two germ layers must be further investigated.
We reported a new Chinese HESc line, CH1, of which the establishment process complied with the standards of the international stem cell banks. The cell line was found to be fully pluripotent and had high potential to differentiate into pancreatic and hepatic cells. The line has now been submitted to the UK Stem Cell Bank. Once it is accepted, it will provide not only a new tool to study the mechanism of human pancreatic and hepatic development in vitro, but will also be a resource for diabetes and hepatic disease therapies.
Acknowledgments: We are grateful to Prof. DENG Hong-kui of College of Life Sciences of Peking University for his critical advice during the experiments, and revision of this paper. We thank LIU Hai-song for his advice in the writing of this paper, CHI Xiao-chun for teratomas analysis, Rebecca Amstrong for her revision of this paper and all the workers of IVF-ET Center of Peking University People's Hospital for collecting embryos.
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