A Method for the Selection of Human Embryonic Stem Cell Sublines with High Repla - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-4 23:58 |只看该作者 |倒序浏览 |打印

作者：Kouichi Hasegawaa,b, Tsuyoshi Fujiokaa,c, Yukio Nakamurac, Norio Nakatsujib, Hirofumi Suemoria作者单位：aLaboratory of Embryonic Stem Cell Research, Stem Cell Research Center, andbDepartment of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan;cCell Engineering Division, BioResource Center, RIKEN, Tsukuba, Ibaraki, Japan ! b2 ^9 S/ P+ i3 [

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      【摘要】
   Human embryonic stem cells (hESCs) exhibit pluripotency and indefinite proliferation and are a potential source of cells for transplantation therapies and drug discovery. These applications will require large amounts of hESCs. However, hESCs are difficult to culture and maintain at larger scales, in part because of their low resistance to dissociation during passaging. To circumvent this, we developed a simple and easy method for establishing hESC sublines tolerant of complete dissociation. These cells exhibit high replating efficiency and also high cloning efficiency, and they maintain their ability to differentiate into the three germ layers. Several sublines have no detectable abnormalities in their karyotypes, and they retained their characteristics under feeder-free culture conditions and after freeze-thawing. Thus, these hESC sublines would be valuable for hESC applications.
      【关键词】 Human embryonic stem cells Replating efficiency Cloning efficiency% Y8 \( m4 M" z+ W' f- R0 L
   INTRODUCTION  p4 {4 t5 S. T& z3 I0 a- B3 Q; c
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Human embryonic stem cells (hESCs) are derived from preimplantation embryos and can be maintained in an undifferentiated state for long periods while retaining a normal karyotype. In addition, they have the ability to differentiate into all three germ layers . hESCs could potentially provide an unlimited supply of any cell type for transplantation therapy, drug screening, or toxicology studies, as well as functional genomic and proteomic experiments. These applications require large amounts of differentiated cells derived from hESCs. However, given that differentiated cells generally proliferate slowly or not at all, large-scale culture of undifferentiated hESCs will be necessary./ x! p4 Q! Z7 p8 j1 x# m7 k
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Unlike the large-scale culture of mouse ESCs (mESCs), that of hESCs is difficult and requires prolonged incubation times because hESCs grow slowly compared with mESCs. The population doubling time for hESCs is more than twice that of mESCs  (supplemental Fig. 1B). Therefore, additional factors likely contribute to the difficulty associated with culturing hESCs.

We propose that the sensitivity of hESCs to physical damage is one of these factors. Generally, hESCs rarely survive after dissociation into single cells during passaging . Therefore, increased tolerance of complete dissociation may permit easier expansion of hESC culture.) Z+ o, M$ a! n4 \+ i

Here, we report the development of a novel method for isolating hESC sublines compatible with larger culture scales. The hESC sublines showed higher tolerance to complete dissociation and much higher replating efficiency and cloning efficiency compared with typical hESC lines. In addition, a part of these subline cells did not possess detectable karyotype abnormalities. We examined several molecules that influence the replating efficiency of hESCs and discovered that Wnt signaling is one factor mediating replating efficiency.
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MATERIALS AND METHODS* d! \; r! Y: ^2 h
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Culture of hESCs2 d+ b$ u, M- C/ t+ L- X1 d
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The hESC lines KhES-1, -2, and -3 were established in our laboratory . They were then collected as a larger cell mass by centrifugation for 3 minutes at 1,000 rpm and seeded onto a MEF feeder layer. The use of hESC lines were performed in conformity with The Guidelines for Derivation and Utilization of Human Embryonic Stem Cells (2001) of MEXT (the Ministry of Education, Culture, Sports, Science and Technology), Japan.' E+ j( X4 ?4 M, D0 Z" S
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Selection of hESC Sublines Tolerant ofComplete Dissociation
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KhES-1 (passage numbers 15, 15, 19, 19, 21, 21, 81, 81), KhES-2 (passage numbers 34, 34, 96, 96), and KhES-3 (passage numbers 41, 41, 42, 42) cells were completely dissociated using 0.05% trypsin-EDTA (Invitrogen). Briefly, the cells were washed once with PBS, treated with the 0.05% trypsin-EDTA for 2¨C5 minutes at 37¡ãC, and gently dissociated to single cells by pipetting in the culture media (supplemental Fig. 2A). The cells and feeder cells in the single-cell suspension were counted using a hematocytometer. Then, the hESCs were seeded at 50,000 cells per 60-mm feeder-coated dish and cultured for 7¨C10 days. To select hESCs resistant to complete dissociation, we repeated the complete dissociation during passaging. The replating efficiency was calculated as the colony number per seeded cell number. When an elevation in replating efficiency was observed, each cell line was seeded at 1,000 cells every passage. Then, we examined the karyotype, expression of hESC marker molecules, and the expansion after cryopreservation for each cell line .
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Karyotype Analysis

The hESCs were processed for G-banding and fluorescent in situ hybridization (FISH) at various passage numbers. For G-banding, hESCs were treated with 100 ng/ml colcemid (KaryoMax; Invitrogen), dispersed, and fixed. Fifty cells at metaphase were analyzed for G-banding at 300¨C500 band levels by trypsinize and Giemza's staining. FISH was performed according to the manufacturer's protocol (STAR FISH; Cambio Ltd, Cambridge, U.K., http//www.cambio.co.uk) using the following probes: fluorescein isothiocyanate-labeled chromosome 12-specific probe and biotin-labeled chromosome 17-specific probe. Hybridized ESCs were analyzed by fluorescence microscopy.
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Copy Number Analysis and Single-Nucleotide Polymorphism Genotyping
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The genomic DNA of the hESCs was used for hybridization to oligonucleotide arrays containing 58,946 single-nucleotide polymorphisms (SNPs) (GeneChip Human Mapping 50K Array Xba; Affymetrix, Santa Clara, CA, http://www.affymetrix.com) in accordance with the manufacturer's protocol. The SNP call rate was more than 97% for all samples. The gene copy number of the samples was analyzed using the Affymetrix Chromosome Copy Number Analysis Tool. To reduce noise, a five-SNP moving window was scored as copy number change. SNP genotyping was performed using and the Affymetrix GeneChip Genotyping Analysis Software." \( `8 ~0 a- l. M2 B" C

Characterization of Undifferentiated hESCs by Molecular Markers& F9 U* l, C1 I: I: s3 H* H

The hESC colonies were fixed with 4% paraformaldehyde, and alkaline phosphatase (ALP) activity was determined with a Vector Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Immunostaining using anti-tumor rejection antigen (TRA)-1-60 antibody (Chemicon International, Temecula, CA, http://www.chemicon.com), anti-stage specific embryonic antigen (SSEA)-3 and -4 antibody, anti-Oct-3 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com), and anti-Nanog antibodies (a gift from Dr. Takashi Tada, Kyoto University, Japan) was carried out as previously described .
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For reverse transcription-polymerase chain reaction (RT-PCR) analysis, total RNA was isolated from growing hESCs using Sepazol I (Nacalai Tesque, Inc., Kyoto, Japan, http://www.nacalai.co.jp). Quantitative RT-PCR was then performed using SYBR Green PCR Master Mix and an ABI PRISM 7700 Sequence Detection system (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) according to the manufacturer's protocol. The following primers were used for the PCR of OCT-3, NANOG, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5'-GGTATTCAGCCAAACGACCA-3' and 5'-CACGAGGGTTTCTGCTTTGC-3' for OCT-3, 5'-CTGCTGAGATGCCTCACACG-3' and 5'-TGCCTTTGGGACTGGTGGA-3' for NANOG, and 5'-GAAGGTGAAGGTCGGAGTC-3' and 5'-GAAGATGGTGATGGGATTTC-3' for GAPDH. The PCR results were determined by the ratio of OCT-3 or NANOG versus GAPDH using nine PCRs from three dishes for each cell line.  r6 x7 `4 @% q6 a7 }
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Embryoid Body Formation6 u# _2 F# W$ R; N1 O% M; U

Confluent hESC colonies were detached with CTK solution, collected by sedimentation, and seeded onto low-attachment dishes (Costar Ultra Low Attachment; Corning Life Sciences, Acton, MA, http://www.corning.com/lifesciences). Embryoid bodies were cultured for 3¨C6 weeks in the same culture medium as the hESCs and then plated onto gelatin-coated dishes.

Teratoma Formation
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hESCs were dissociated using 0.05% trypsin-EDTA, and 200 µl of suspension containing approximately 107 cells was subcutaneously injected into severe combined immunodeficiency (SCID) mice (CLEA Japan, Inc., Tokyo, http://www.clea-japan.com). After 2¨C3 months, the resulting teratomas were dissected and fixed with Bouin's fixative solution. Samples were embedded in paraffin, sectioned at 5-µm thickness, and subsequently stained with hematoxylin and eosin.4 }. m& [3 c7 s7 g/ R

Determination of Population Doubling Time, d4 [) R  s4 M5 U- V+ n8 @

hESCs were seeded at 105 cells per well onto 24-well plates containing feeder cells. Twenty-four, 48, 72, and 96 hours after seeding, three wells of hESCs plus feeder cells were trypsinized into a single cell suspension. The cells from each well were distinguished as either hESCs or feeder cells by their morphology, and each type was counted with a homocytometer. Cell numbers at each time point were plotted and analyzed using Microsoft Excel (Microsoft, Redmond, WA, http://www.microsoft.com).5 Y& ]) D  I. k5 o5 `

Clonal Proliferation Assay
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hESCs were dissociated completely with 0.05% trypsin-EDTA (Invitrogen), seeded at 104 cells per well in feeder-coated six-well culture dishes, and cultured in hESC medium supplemented with 5 ng/ml bFGF. For feeder-free culturing, dishes were coated with growth factor-reduced Matrigel diluted 20-fold (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), 10 µg/ml Fibronectin (Sigma-Aldrich), 20 µg/ml laminin (BD Biosciences), 10 µg/ml collagen type I (BD Biosciences), 10 µg/ml collagen type IV (BD Biosciences), or 0.1% gelatin (Sigma-Aldrich) instead of using feeder cells. MEF-conditioned media (MEF-CM) were used instead of hESC culture media. To examine the effect of various signaling pathways on replating efficiency, general Caspase inhibitor (Z-VAD-FMK; R&D Systems, Inc., Minneapolis, http://www.rndsystems.com), nuclear factor (NF)-B inhibitor (ammonium pyrrolidine dithiocarbamate ; Dojindo Laboratories, Kumamoto, Japan, http://www.dojindo.co.jp), Amifostine (Biomol International, PA, http://www.biomol.com), phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), interleukin (IL)-3 (ProSpec-Tany TechnoGene, Rehovot, Israel, http://www.prospec.co.il), tumor necrosis factor- (TNF-) (ImmunoKontact, Abingdon, U.K., http://www.immunok.com), granulocyte macrophage-colony-stimulating factor (GM-CSF) (ReliaTech GmbH, Braunschweig, Germany, http://www.reliatech.de), Noggin (PeproTech EC Ltd, London, http://www.peprotechec.com), Activin A (PeproTech EC Ltd), transforming growth factor (TGF)-ß1 (R&D Systems), Wnt-3a (R&D Systems, Inc.), or Wnt-1 (Cell Sciences, Inc., Canton, MA, http://cellsciences.com) was added to the culture medium at the onset of seeding, then continuously until the end of culturing. For all assays, the replating efficiency was examined by counting the number of colonies after 7 days of culturing.
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Limiting Dilution Assays
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hESCs were diluted and seeded at 0.5, one, two, or three cells per well onto Matrigel-coated 96-well plates and cultured in 200 µl of MEF-CM. After 5 days of culturing, the cells were stained for ALP activity and scored as ALP-positive and -negative wells at each dilution. The frequency of colony forming and the percentage of cloning efficiency were calculated with L-Calc limiting dilution analysis software (StemSoft Software, Inc., Vancouver, BC, Canada, http://www.stemsoft.com) and Microsoft Excel.6 l2 i' d9 e4 c8 ]; k1 M1 u

RESULTS; u# n6 e6 f# v# J' \" {6 i

Isolation of hESC Sublines Showing HighReplating Efficiency
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After dissociation into the single cells, hESCs exhibit decreased survival and self-renewal as well as a low replating efficiency . To investigate whether hESC sublines with high replating efficiency could be isolated, we examined the consecutive passaging of cell lines after complete dissociation, termed clonal passaging (supplemental Fig. 2A). All three hESC lines, KhES-1 (46, XX), -2 (46, XX), and -3 (46, XY), showed low replating efficiencies (0.5%¨C4%) after the first clonal passage and maintained this rate throughout the first few passages (Fig. 1A). However, these three hESC lines displayed different and distinct properties at later passages. For KhES-1, the replating efficiency increased to 25%¨C30% within the first 10 clonal passages, and this efficiency was maintained for more than 15 clonal passages (eight trials). When the KhES-1 sublines were continuously cultured using clonal passaging, the replating efficiency was elevated to 50%¨C60% after 26¨C30 passages (six trials). The increased replating efficiencies were maintained after freeze-thawing for the KhES-1 sublines (Fig. 1A). Similar to KhES-1 sublines, the KhES-3 sublines showed high replating efficiencies after 10 clonal passages (four trials). In contrast, the KhES-2 sublines retained their replating efficiencies for the first 20 clonal passages (four trials).

Figure 1. The establishment and karyotyping of human embryonic stem cell sublines with high replating efficiencies. (A): Clonal passage numbers and replating efficiencies of two typical KhES-1, KhES-2, and KhES-3 sublines. The discontinuous line for the KhES-1 sublines indicates freeze-thaw points. G-band karyotype of KhES-1 subline 1 cells at passage 16; 46, XX (B), KhES-1 subline 1 cells at passage 38; 47, XX,  12 (C), and KhES-3 subline 1 cells at passage 12; 46, XY, add  (p11) (D). The arrowhead indicates the additional band. Fluorescent in situ hybridization analysis of KhES-1 subline 1 cells at passages 15 (E) and 35 (F). Chromosome 12 was detected as green signals, chromosome 17 as red signals, and the other chromosomes were detected as blue signals. Copy number analysis of chromosome 7 for KhES-1 subline 2 at passage 41 (G). Arrowheads indicate the hemizygous deletion.

Given that consecutive enzymatic bulk passaging was reported to induce karyotype abnormalities in hESCs , we examined the karyotypes of the hESC sublines with high replating efficiency by G-banding analysis. As shown in Figure 1B and 1E, all KhES-1 sublines showing 25%¨C30% replating efficiency had no detectable karyotype abnormalities. However, five of six KhES-1 sublines cells showing 50%¨C60% replating efficiency had karyotypic abnormalities. KhES-1 subline one had a trisomic chromosome 12 (Fig. 1C), which was confirmed by FISH analysis (Fig. 1F). KhES-1 subline two did not have detectable abnormalities (data not shown). The other sublines had undetermined trisomic chromosomes or additional copies of chromosome 1 or 4 (data not shown). Unlike the KhES-1 sublines, all KhES-3 sublines showing 25%¨C30% replating efficiency had additional copies of chromosomes (Fig. 1D and data not shown). The KhES-2 sublines that maintained their replating efficiencies had normal karyotypes.

To investigate in more detail whether the hESC sublines undergo karyotypic alterations, we performed gene copy number analysis of KhES-1 sublines showing normal karyotype and high replating efficiency (four sublines with 25%¨C30% replating efficiency, and one subline with approximately 50% efficiency) using microarray. Consistent with G-banding analysis, parental KhES-1 and all KhES-1 sublines showing 25%¨C30% replating efficiency had no alteration in their gene copy number (data not shown). Although in the later passaging of KhES-1 subline 2, showing approximately 50% replating efficiency, had a normal G-banding pattern, two deletions in chromosome 7 were detected by copy number analysis (Fig. 1G). Although in the later passaging of KhES-1 sublines and KhES-3 sublines resulted in high replating efficiencies and abnormal karyotypes, we could not determine or generalize the relationships between increased replating efficiency and karyotypic changes.2 ^8 g' ?$ P$ K3 v3 o
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All of the hESC sublines with high replating efficiency proliferated and formed normal morphological colonies. To assess whether these subline cells maintained hESC characteristics, we examined the expression of cellular markers of the undifferentiated state and their ability to differentiate. All sublines expressed hESC surface markers, such as TRA-1-60, SSEA-3, SSEA-4, and ALP, and the transcription factors OCT-3 and NANOG (Fig. 2). These results indicate that the subline cells were maintained in an undifferentiated state. We next examined the differentiation capacity of the subline cells in vitro and in vivo (Fig. 3). For the differentiation assays in vitro, the subline cells differentiated into elongated neurons, beating cardiomyocytes and mesenchymal cells, and in teratoma assays in vivo, they formed various tissues consisting of the three germ layers. From these results, we conclude that the hESC sublines with high replating efficiency maintained their hESC characteristics.

Figure 2. The expression of human embryonic stem cell (hESC) markers in the hESC sublines with high replating efficiency. Alkaline phosphatase activity was detected as blue signals in KhES-1 subline colonies at passage 18 (A). Antibodies raised against stage specific embryonic antigen (SSEA)-3 (B), SSEA-4 (C), TRA (tumor rejection antigen)-1-60 (D), OCT-3 (E), and NANOG (F) were used for the immunostaining of KhES subline colonies at passage 23. Scale bar = 100 µm.
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Figure 3. Differentiation of the human embryonic stem cell sublines with high replating efficiency in vitro and in vivo. Three-week-old embryoid body (EB) from KhES-1 sublines at passage 11 displayed cystic morphology (A). Attached EB showed pigmented epithelium and mesenchymal cells (B) and neural cells (C). Hematoxylin and eosin-stained sections of an 8-week-old teratoma formed from KhES-1 sublines at passage 22 (D). The teratoma showed endodermal epithelium with mucosa (E), gut-like epithelium (F), muscular tissue (G), cartilage (H), neuroepithelium (I), and pigmented epithelium (J). Scale bar = 50 µm.0 \6 S5 f, n& i
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Characterization of hESC Sublines with High Replating Efficiency
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The hESC sublines with high replating efficiency retained normal morphology, expression of undifferentiated hESC-specific markers, and the ability to differentiate. However, the difference in replating efficiency between normal hESCs and the hESC sublines may have been due to alterations in the expression levels of self-renewal factors, proliferation, survival, and/or attachment to the feeder layer. To compare these characteristics among the hESC sublines, we categorized them according to their characteristics during clonal passaging (Table 1). Cells in the first phase had low (original) replating efficiencies and normal karyotypes. Those in the second phase had high replating efficiencies and no detectable abnormalities in their karyotypes, whereas those in the third phase had high replating efficiencies and karyotypical abnormalities.+ {* r0 @# H. f

Table 1. Classification of the three phases of the hESC sublines3 K5 ]+ m: v: V" \; z0 Y+ R: @5 h

To assess what alterations in cellular physiology may contribute to these different characteristics, we first examined the expression levels of the self-renewal factors NANOG  in parental and two sublines for each phase. Quantitative RT-PCR analysis revealed that OCT-3 and NANOG were not dramatically different between the three phases (Fig. 4A, 4B). Thus, the expression levels of self-renewal factors may not be directly related to the replating efficiency. We next examined the growth of cells in each phase by counting the cell numbers every 24 hours after clonal passaging. As shown in Figure 4C, 24 hours after seeding, the attached and surviving cells were very low in number (approximately 15%), and many floating cells and debris were present in culture for hESCs with the lowest replating efficiencies (phase 1). This was similar to that for the normal hESCs. However, for sublines with greater replating efficiencies (phases 2 and 3), most cells were attached and alive (>85%), and the number of floating cells and the amount of debris were low. Between 24 and 48 hours after seeding, the cell numbers were not different or had decreased for cells with normal karyotypes (phases 1 and 2). However, the cells lines with aberrant karyotypes (phase 3) displayed log-phase growth. After 48 hours, the cells showed log-phase growth for all three phases. During log-phase growth, the cells of each phase had a similar doubling time (Fig. 4D). Thus, the expression levels of the self-renewal factors and proliferation rate were not altered for all phases during growth, but the initial hESC attachment to the feeder layer and/or the survival rate after dissociation was increased for cells in phases 2 and 3. In addition, the initial ability for self-renewal after attachment and survival was increased in the cells in phase 3.

Figure 4. Characterization of human embryonic stem cell sublines with high replating efficiency. The expression levels of NANOG (A) or OCT-3 (B) in growing KhES-1 subline cells in each phase were compared with normal KhES-1 by quantitative polymerase chain reaction. The growth curves (C) and doubling times (D) of KhES-1 sublines at each phase. The replating efficiency of KhES-1 sublines in phase 2 under feeder-free conditions (E) or at various densities on seeding (F). The bars indicate standard deviations, and the asterisk (*) indicates the presence of morphologically differentiated colonies. Abbreviation: MEF-CM, mouse embryonic fibroblast-conditioned medium.7 z8 F2 o0 u8 Y3 E4 _% z

To further analyze the alterations in the hESC sublines with high replating efficiency, we examined whether initial attachment was dependent on direct attachment to feeder cells or on extracellular matrix (ECM) components of the feeder layer. Given that the combination of ECM (Matrigel) and MEF-CM is sufficient to maintain the undifferentiated state of hESCs , clonal proliferation assays, as described in Materials and Methods, were performed on feeder cells or Matrigel for hESCs in phase 2. When cells were seeded on Matrigel, the replating efficiency was not different from cells seeded on feeder cells and remained high for at least 10 passages (Fig. 4E and data not shown). Most hESCs were attached to the Matrigel 24 hours after seeding. However, the surviving and proliferating cells were reduced to approximately half that of the initially attached cells after 48 hours. These results suggest that the replating efficiency is affected by initial attachment to the ECM component and survival after attachment, rather than by direct adhesion to feeder cells.
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Next, we examined the density effect on the replating efficiency for hESCs in phase 2. The replating efficiency was slightly increased in a density-dependent manner (Fig. 4F). Although the opportunity for attachment between hESCs is higher for high-density cultures, fixed-point observations revealed that the hESCs proliferated and formed colonies from single cells (data not shown). These results suggest that the elevation of the replating efficiency according to the density of hESCs was not due to cell-cell interaction between hESCs.4 V# ^% z# y1 i- E5 ?5 @1 X( z

To determine in more detail whether the subline cells proliferate clonally from single cells without cell-cell adhesion, the cloning efficiency of the subline cells was evaluated by limiting dilution assays in feeder-free culture. As shown in Table 2, the cloning efficiency was very low in phase 1 subline cells but was increased in phase 2 and 3 cells. In addition, the cloning efficiency measured by limiting dilution assays did not display a significant difference from the replating efficiency (Tables 1 and 2). These results suggested that the subline cells were able to proliferate clonally from single cells, and the replating efficiency is thought to represent the cloning efficiency in clonal propagation assays.7 H$ ]" Q4 a- x; v" |
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Table 2. Cloning efficiency of the human ESC sublines' X" T. W3 j$ ^7 }8 U  s

Replating Efficiency Elevating Factors
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The increased replating efficiency in hESC sublines may arise from increased attachment and survival after dissociation, as well as from the inherent ability of self-renewal. Activating the signaling pathways that regulate these characteristics may increase the replating efficiency of nonselected wild-type hESCs. Therefore, we examined the effects of candidate molecules that might influence attachment, survival, or self-renewal in our clonal proliferation assays using the three original hESC lines.

To explore attachment, we performed a clonal proliferation assay using various ECM components (Fig. 5A) and examined several ECM molecules, including Matrigel, fibronectin, laminin, collagen type I, and collagen type IV. All of these substrates generated a lower number of adhered cells than by feeder culturing for KhES-1 cells. When KhES-2 and KhES-3 cells were seeded onto Matrigel, the replating efficiencies were indistinguishable from those of cells seeded onto feeder cells. However, the other ECM molecules reduced the replating efficiency for KhES-2 and KhES-3 cells. We did not find an ECM molecule that promoted attachment better than a feeder layer.+ Y# a7 G1 U, I4 ~: I# x) J/ s
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Figure 5. The effect of various molecules on the replating efficiency of human embryonic stem cells (hESCs). The replating efficiencies of KhES-1 (black bars), KhES-2 (gray bars), and KhES-3 (white bars) cell lines are shown for various culturing conditions such as cells grown in the presence of various ECM substrates in feeder-free culturing (A), different concentrations of general Caspase inhibitor Z-VAD-FMK (B), various NF-B activators and an inhibitor (C), several concentrations of bFGF (D), or the combination of bFGF and molecules involved in hESC self-renewal (E). The bFGF concentration was 5 ng/ml in (A), (B), (C), and (E). The bars indicate the standard deviation, the asterisk (*) indicates the presence of morphologically differentiated colonies, and the dagger () indicates that the morphology of the feeder cells was different from the control experiment. Abbreviations: APDC, ammonium pyrrolidine dithiocarbamate; bFGF, basic fibroblast growth factor; DMSO, dimethyl sulfoxide; GM-CSF, granulocyte macrophage¨Ccolony-stimulating factor; IL-3, interleukin-3; MEF-CM, mouse embryonic fibroblast-conditioned medium; NF-B, nuclear factor-B; PMA, phorbol 12-myristate 13-acetate; TGF, transforming growth factor; TNF-, tumor necrosis factor-.
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To examine the survival rate, we performed a clonal proliferation assay in the presence of a general caspase inhibitor to inhibit apoptosis . We did not observe any increase in replating efficiency with the caspase inhibitor over a broad concentration range, including doses (10 mM) several orders of magnitude greater than those typically used (100 nM). Although the NF-B inhibitor APDC decreased the replating efficiency, typical and 10-fold doses of various activators of NF-B signaling, IL-3, GM-CSF, TNF-, Amifostine, and PMA did not increase the replating efficiency. Thus, we could not find any factors that promoted hESC survival under our conditions (Fig. 5A, 5B).

To increase the self-renewal of hESCs after dissociation, we examined various concentrations of bFGF, which maintains the undifferentiated state of hESCs . The addition of Noggin, Activin A, and TGF-ß1 did not increase the replating efficiency (Fig. 5D). However, the addition of Wnt3a and Wnt1 increased the replating efficiency of KhES-2 and KhES-3 cells but not of KhES-1, and they altered the morphology of the hESCs (Figs. 5D and 6). To further ascertain the effect of activating canonical Wnt signaling, we examined the replating efficiency, morphology, and expression of hESC markers for various concentrations of Wnt3a. In KhES-1, Wnt3a did not affect the replating efficiency, although the colonies differentiated in a dose-dependent manner (Fig. 6A). Unlike in KhES-1 cells, Wnt3a increased the replating efficiency in a dose-dependent manner in KhES-2 and KhES-3 cells. However, the colonies displayed differentiated morphologies at these concentrations, which achieved the highest replating efficiency (Fig. 6A, 6B). These data indicate that the canonical Wnt pathway affects the replating efficiency and also the differentiation of KhES-2 and KhES-3 cells.
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Figure 6. The effect of various concentrations of Wnt on human embryonic stem cells. (A): The replating efficiencies of KhES-1 (black bars), KhES-2 (gray bars), and KhES-3 (white bars) cell lines are shown for various concentrations of Wnt3a. The bars indicate the SD, the asterisk (*) indicates containing morphologically differentiated colonies, and the double asterisk (**) indicates that all colonies were differentiated. (B): The morphology, ALP activity (blue signals), and immunochemical staining of OCT-3 (brown signals) of KhES-2 colonies cultured for clonal proliferation assay at various concentrations of Wnt3a are shown. Scale bar = 500 µm. Abbreviation: ALP, alkaline phosphatase.+ q) y- t, Z/ p3 e) \$ W1 r8 _$ C

Because we could not find molecules that increase the replating efficiency of parental hESC lines to those of hESC sublines, we next analyzed the genetic alteration of the sublines to identify the genes affecting the replating efficiency. For close examination of genetic changes in the sublines, SNPs in parental KhES-1 and subline genomes were compared by SNP array, because the gene copy number analysis did not exhibit a difference among parental and the sublines. As shown in Table 3, 32 SNPs were changed in the sublines from parental line. Among the genes with the SNP changes, proteins involved in cell adhesion (NRXN1, ROBO1, and COL19A1) and in signal transduction (VAV3, OR10J5, NR5A2, NRXN1, ROBO1, LPHN3, LRRC4C, and FGF-13) were found.( v. ~/ Y7 a4 T+ Z5 A1 d) Y

Table 3. Changed SNPs in the hESC sublines# d3 p8 H! b' J8 I; T+ k# R
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DISCUSSION
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Culturing hESCs is not easy, and refined skills regarding passaging are required to handle, maintain, and expand hESC lines. One of the factors associated with this difficulty is the low tolerance of these cells to dissociation. To circumvent this, we developed a method for the isolation of hESC sublines with high replating efficiency using clonal passaging. The sublines survived at a higher rate after dissociation, had a normal morphology, maintained an undifferentiated state, and had the ability to differentiate into the three germ layers. In addition, those established from one of three parental hESC lines had not only high replating efficiencies but also a normal karyotype through 20 passages. Such hESC sublines, the phase 2 KhES-1 sublines, permit not only easy scaling-up by 50¨C100 times per week, but also easier handling, similar to mESCs. Furthermore, the increased replating efficiency is retained under feeder-free conditions and after freeze-thawing. These hESC sublines could become a valuable tool for investigating self-renewal mechanisms by comparing them with parental hESC lines, for developing differentiation methods for transplantation therapies, and for drug discovery and screening.

There have been several reports on accidental and naturally selected high-replating efficiency hESC sublines displaying abnormal karyotypes but maintaining an undifferentiated state and ability to differentiate  among hESC lines. Therefore, it is not surprising that establishing hESC sublines with high replating efficiency using clonal passaging is not always effective., D) p6 ~, _2 e$ J# {5 y
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A recent report revealed that hESCs undergo subtle genomic alterations, which are undetectable in some cases by standard karyotype analysis, during culturing . Our results suggest that a part of the downstream Wnt pathway may affect the replating efficiency of hESCs even if it is not sufficient to increase the replating efficiency.; E8 e, z8 C/ _: s, A' [: S. V2 ]4 e
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We also found that the replating efficiency increased with increasing hESC density when culturing hESC sublines. Unlike supplementing the culture medium with Wnts, the density effect on the replating efficiency did not affect the undifferentiated state of the hESCs. This suggests that hESC sublines may produce soluble molecules, which enhance the replating efficiency without affecting differentiation. One of the altered SNPs found in our SNP analysis was associated with a gene encoding a soluble ligand, FGF-13; however, there is no report that the relationship between the SNPs affects the expression of FGF-13 or the function of FGF-13 in hESCs. Further analysis will be necessary to elucidate the molecular mechanism responsible for increasing the replating efficiency of hESCs by comparing conventional hESCs and the hESC sublines by, for example, gene expression profiling, proteome analysis, or further examination of genetic changes and epigenetic changes. These studies may facilitate increases in replating efficiency without the selection of stable hESCs against dissociation.; f$ t$ l! W7 Z# G! S8 Y

hESC lines with high-replating efficiencies may become an important tool for various studies and applications. They could provide large amounts of undifferentiated cells easily, quickly, and reliably. In addition, they could facilitate the production of large amounts of uniform human differentiated cells. Such production of uniform human cells will be necessary for regenerative medicine, drug screening, toxicological, functional genomics, and proteomic studies. Therefore, studies on improvement of the replating efficiency of hESCs will be important for future applications of hESCs.

DISCLOSURES

The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS

We thank Dr. Takashi Tada for providing the anti-NANOG antibodies and the members of our laboratory for their helpful discussions. This study was supported by the National Bioresource Project, Science Technology Innovative Cluster Creation Project, and MEXT, Japan. K.H. and T.F. contributed equally to this work.
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