Stem Cell Rev and Rep DOI 10.1007/s12015-011-9233-y
Surface Marker Epithelial Cell Adhesion Molecule and E-cadherin Facilitate the Identification and Selection of Induced Pluripotent Stem Cells Hsin-Fu Chen & Ching-Yu Chuang & Wen-Chih Lee & Hsiang-Po Huang & Han-Chung Wu & Hong-Nerng Ho & Yu-Ju Chen & Hung-Chih Kuo
# Springer Science+Business Media, LLC 2011
Abstract The derivation of induced pluripotent stem cells (iPSCs) requires not only efficient reprogramming methods, but also reliable markers for identification and purification of iPSCs. Here, we demonstrate that surface markers, epithelial cells adhesion molecule (EpCAM) and epithelial cadherin (E-cadherin) can be used for efficient identification and/or isolation of reprogrammed mouse iPSCs. By viral transduction of Oct4, Sox2, Klf4 and n- or c-Myc into mouse embryonic fibroblasts, we observed that the conventional mouse embryonic stem cell (mESC) markers, alkaline phosphatase (AP) and stage-specific embryonic antigen 1
(SSEA1), were expressed in incompletely reprogrammed cells that did not express all the exogenous reprogramming factors or failed to acquire pluripotent status even though exogenous reprogramming factors were expressed. EpCAM and E-cadherin, however, remained inactivated in these cells. Expression of EpCAM and E-cadherin correlated with the activation of Nanog and endogenous Oct4, and was only seen in the successfully reprogrammed iPSCs. Furthermore, purification of EpCAM-expressing cells at late reprogramming stage by FACS enriched the Nanog-expressing cell population suggesting the feasibility of selecting successful
Electronic supplementary material The online version of this article (doi:10.1007/s12015-011-9233-y) contains supplementary material, which is available to authorized users. H.-F. Chen : H.-N. Ho Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, Taipei, Taiwan H.-F. Chen : H.-N. Ho Graduate Institute of Clinical Genomics, College of Medicine, National Taiwan University, Taipei, Taiwan W.-C. Lee : H.-C. Wu : H.-C. Kuo Institute of Cellular and Organismic Biology, Academia Sinica, No. 128, Sec. 2, Academia Road, Nankang, Taipei, Taiwan C.-Y. Chuang Genomics Research Center, Academia Sinica, Taipei, Taiwan H.-P. Huang Division of Medical Research, National Taiwan University Hospital, Taipei, Taiwan
Y.-J. Chen Institute of Chemistry, Academia Sinica, Taipei, Taiwan
H.-C. Kuo Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan
W.-C. Lee : H.-C. Kuo Institute of Biotechnology, National Taiwan Ocean University, Keelung, Taiwan
H.-C. Kuo (*) Stem Cell Program, Genomics Research Center, Academia Sinica, No. 128, Sec. 2, Academia Road, Nankang, Taipei, Taiwan e-mail:
[email protected]
Stem Cell Rev and Rep
reprogrammed mouse iPSCs by EpCAM expression. We have thus identified new surface markers that can efficiently identify successfully reprogrammed iPSCs and provide an effective means for iPSC isolation. Keywords Induced pluripotent stem cells . EpCAM . E-cadherin . Reprogramming . Embryonic stem cells
Introduction Reprogramming somatic cell types into embryonic stem cell (ESC)-like induced pluripotent stem cells (iPSCs) provides a means to generate patient-specific pluripotent stem cells without the use of embryos; therefore, iPSCs are a very attractive option for future stem cell-based therapies [1–4]. However, some technical issues such as low derivation rate, genome integration, and difficulties in selecting fully reprogrammed iPS clones, remain to be resolved before iPSC production can be efficient, safe, and fulfill the requirements for clinical application [5, 6]. Since their first derivation by lenti- or retro- viral vectors [5], major improvements have been made in transduction systems for iPSC derivation and iPSCs can now be reprogrammed from somatic cells using genome integration-free transduction systems [7, 8]. However, reliable marker/reporter systems that can efficiently distinguish successfully reprogrammed iPSCs from their unreprogrammed or partially reprogrammed counterparts are also essential for efficient isolation of iPSCs. Takahashi et al. used Fbx15, a pluripotency-related gene, to identify iPSC clones generated by over-expression of various transcription factors in mouse fibroblasts [4]. The iPSC clones selected by transgenic Fbx15 reporter were indistinguishable from their embryonic counterparts in morphology, growth requirements, proliferation, markers and gene expression, epigenetic, and pluripotent features [4]. However, iPSCs selected by Fbx15 expression were not germline permissive indicating that Fbx15 is not suitable for detecting fully reprogrammed iPSCs, although it is able to report the pluripotency status of the reprogrammed cells [4]. Later, Nanog and Oct4 were used as marker genes for iPSC selection, and iPSCs with germline competency were successfully isolated [9–13]. Although transgenic reporter systems involving pluripotency-related genes can effectively isolate iPSCs with fully reprogrammed status, the nature of the genetic modification could potentially cause unwanted genetic defects rendering these systems unsuitable for future clinical application. IPSCs have also been successful isolated by morphological traits, though with conceivably lower accuracy [3, 4]. Because of the drawbacks of these approaches, identification of iPSCs by surface markers is an attractive alternative. At present, stage-specific embryonic
antigen 1 (SSEA1) and alkaline phosphatase (AP) are the most commonly used surface markers for characterization of mouse ESCs. However, the correlation between their expression and the pluripotent status of the reprogrammed somatic cells is not specific. Therefore, identification of other surface markers that have high specificity to pluripotency is important for both basic research and future clinical applications. Epithelial cells adhesion molecule (EpCAM) is a type I transmembrane glycoprotein which is expressed in many progenitor cells [14], most epithelial cells [15], and human cancer cells [16]. EpCAM has also been used to identify hepatic stem cells, germs cells, and cancer-initiating cells [14, 15, 17–19]. Recently, it has been reported that EpCAM is expressed in human and mouse ESCs [20–24]. Moreover, the expression of EpCAM was shown to be involved in the maintenance of the undifferentiated status of human ESCs, through directly regulating the expression of reprogramming genes, such as Oct4, Sox2, Klf4, c-Myc and Nanog [24]. As these reprogramming factors (Oct4, Sox2, Klf4, c-Myc) are also used for iPSC production, it is interesting to examine the role and the expression pattern of EpCAM during the course of iPSC reprogramming. Cadherins are calcium-dependent transmembrane glycoproteins that mediate cell-cell adhesion. It has long been recognized that cadherins are the most important cell-cell receptors for the formation of intercellular interaction and maintenance of normal tissue morphology [25]. Differentiation of ESCs has been associated with down-regulation of cell surface epithelial cadherin (E-cadherin) [26] and expression of E-cadherin was found markedly higher in undifferentiated ESCs [27]. In addition, the small GTPase, Rap1, affects the endocytic recycling pathway involved in the formation and maintenance of E-cadherin-mediated cell-cell cohesion, which is essential for colony formation and self-renewal of human ESCs [28]. This study was designed to examine the roles of EpCAM and E-cadherin in improving iPSC isolation, focusing particularly on their usefulness in identifying truly pluripotent iPSCs. Our results suggest that EpCAM and E-cadherin are more suitable than SSEA1 and AP as surface markers to screen for mouse iPSC generation.
Materials and Methods Cell Culture Mouse embryonic fibroblasts (MEFs), 293T cells and Platinum-E (Plat-E) packaging cells were cultured in MEF medium: DMEM medium supplemented with 15% fetal bovine serum (FBS), 1× non-essential amino acids (NEAA, Invitrogen 11140-050), 1× L-glutamine (Invitrogen 25030081) and 1× penicillin/streptomycin (Invitrogen 15140-122).
Stem Cell Rev and Rep
Plat-E packaging cells were maintained in 10 mg/ml blasticidin and 1 mg/ml puromycin. The D3 mESCs (ATCC) and iPSCs were cultured in ESC medium: DMEM supplemented with 20% FBS, 1× NEAA, 1× L-glutamine, 0.0007% β-mercaptoethonal and 103 leukemia inhibitory factor (LIF, Millipore) on a monolayer of mitomycin C-treated MEF feeder cells. MEFs used for iPSC generation underwent less than seven passages.
used in IF staining included rabbit anti-mouse Nanog (Reprocell, RCAB002P-F), mouse anti-mouse Oct3/4 (Santa Cruz, sc-5279), rabbit anti-mouse Oct3/4 (Santa Cruz, sc-9081), mouse anti-mouse Sox2 (R&D, MAB2018), rabbit anti-mouse KLF 4(Santa Cruz, sc-20691), mouse anti-mouse n-Myc (Santa Cruz, sc-56729), mouse anti-mouse SSEA1 (Chemicon, MAB4301), rat anti-mouse EpCAM (eBioscience, 14-1579; BD, 552370) and rat anti-mouse ECadherin (Sigma, U3254).
Virus Production and Generation of iPSCs Flow Cytometric Analysis The procedures for virus production and iPSC generation were described previously [29]. Lentiviral vectors encoding mouse Oct4, Sox2, Klf4 and n-Myc were obtained from Addgene (pLOVE-Oct4, Addgene plasmid 15952; pLOVESox2, Addgene plasmid 15953; pLOVE-Klf4, Addgene plasmid 15950; pLOVE-Nmyc, Addgene plasmid 15951). Lentiviral vectors accompanied with pcDNA3.1-VSVG and pCMVDR8.9 were transiently transfected into 293T cells using FuGENE® 6 Transfection Reagent according to the manufacturer’s instructions (Roche, 11815-091001). Retroviral vectors encoding mouse Oct4, Sox2, Klf4 and c-Myc were also obtained from Addgene (pMXs-mOct4, Addgene plasmid 13366; pMXs-mSox2, Addgene plasmid 13367; pMXs-mKLF4, Addgene plasmid 13370; pMXs-c-MYC, Addgene plasmid 13375) and transiently transfected into Plat-E packaging cells. Viral supernatant fractions were harvested at 60 h and 84 h after transfection, followed by filtration through 0.45 mm filters (Millipore, SLHV033RB). Two rounds of viral transduction of MEFs were performed 24 h apart and cells were incubated with viruses for a subsequent 24 h before the medium was changed to MEF medium. After 4 days, transduced MEFs were cultured on fresh feeder cells and switched to ESC medium after 24 h. Finally ESC-like colonies were manually selected 20 days after viral transduction and scaled up for further analyses.
Cells were trypsinized for SSEA1 and EpCAM detection. For E-Cadherin staining, cells were suspended for 30 min in 5 mM EDTA to disperse the cells. Suspended single cells were fixed with 4% PFA for 20 min and washed with 1× PBS three times. Fixed cells were first blocked with 3% FBS in 1× PBS for 30 min, followed by antibody incubation, and finally subjected to flow cytometric analysis according to the manufacturer’s instructions (BD FACSCalibur™ platform). The antibodies used to label cells included mouse anti-mouse SSEA1 (Chemicon MAB4301), rat anti-mouse EpCAM (eBioscience 14-5791; BD 552370) and E-cadherin (Sigma U3254). Teratoma Formation and Histological Assay The teratoma formation assay was performed as described previously [30, 31]. Briefly, the mESC-like clones were suspended in ESC culture medium at a concentration of 1× 107 cells/ml, and 100 μl of cell suspension was intramuscular injected into rear legs of NOD-SCID mice. Four weeks after the injection, tumors were surgically dissected from the mice, freshly fixed in 4% PFA, and embedded in paraffin. Sections were stained with hematoxylin and eosin (HE) and examined by a pathologist.
RT-PCR Results RNA was isolated with TRIZOL, and first strand cDNA synthesis was performed using SuperScript® III Reverse Transcriptase (Invitrogen, 18080-044). The primer sets in the present study are listed in Supplementary Table 1. Alkaline Phosphatase and Immunofluorescence Staining Alkaline phosphatase (AP) staining was performed using the Leukocyte Alkaline Phosphatase kit (Sigma). To perform immunofluorescence (IF) staining, cells were first fixed with 4% paraformaldehyde (PFA) and washed with 1× PBS three times. For probing intracellular proteins, fixed cells were permeated with 1× PBS containing 0.2% TritonX-100 and 0.1% Tween 20. The primary antibodies
SSEA1 and AP are Expressed in Incomplete/Partially Reprogrammed Cells To generate iPS cells, we transduced MEFs with lentiviral vectors encoding Oct4, Sox2, Klf4 and n-Myc (Fig. 1a) [32–34]. Two days after transduction, small aggregated cells started to appear in culture and expanded rapidly in number. After enzyme dissociation and replating onto MEF feeder cells, mESC-like colonies began to appear in culture 9 days after viral infection. We selected 17 cell clones with mESC-like morphology and growth patterns for further characterization during the course of colony expansion (day 15–25). First, we examined the expression patterns of
Stem Cell Rev and Rep Fig. 1 Generation and characterization of partially reprogrammed cells from MEFs by transduction with lentiviruses encoding Oct4, Sox2, Klf4 and n-Myc. a Scheme for reprogramming MEFs into iPSCs with lentiviruses encoding Oct4, Sox2, Klf4, and n-Myc. b Morphology and expression of pluripotency-associated markers AP, SSEA1 and Nanog of colonies isolated based on their mESC-like morphology 14 days after the induction of reprogramming. (Four representative clones, #12, #14, #11 and #17 are shown.) Only Clone #17 expresses AP homogenously while Clones #4 and #11 exhibit AP unevenly. SSEA1 (red) can be found in Clones #11 and #17 in a mosaic pattern. None of examined clones expresses endogenous Nanog (red). Scale bar=60 μm (phase contrast), 200 μm (AP) and 50 μm (SSEA1 and Nanog)
SSEA1 and AP in the mESC-like clones and their association to Nanog expression, a commonly accepted criterion by which to determine the status of successful reprogramming. IF analysis revealed that the expression patterns of SSEA1 and AP varied among these clones. We found that 3 out of the 17 clones did not express AP (Fig. 1b and Table 1) and the others were either homogenously (3 clones) or heterogeneously (11 clones) expressed AP (Fig. 1b and Table 1). Eleven out of the 17 clones expressed SSEA1, but SSEA1 expression was not homogenous in all 11 clones including two representative clones (Clones #17 and #37), which showed homogeneous AP expression (Fig. 1b and Table 1). Next, we examined the Nanog expression in the 17 selected cell clones, and we found that none of them expressed Nanog (Figs. 1b, 2a, and Table 1). Taken together, these data suggest that SSEA1, AP and morphological traits are not sufficient to identify the successfully reprogrammed mouse iPSCs that have acquired pluripotent characteristics. To explore the relationship between the exogenous transgenes, Oct4, Sox2, Klf4 and n-Myc and surface markers, SSEA1 and AP, five representative mESC-like clones (Clones #11, #17, #35, #37 and #38), which expressed AP and SSEA1 were selected for further studies. RT-PCR analysis, showed that endogenous Klf4 and n-Myc were expressed in all 5 clones as well as MEFs. Endogenous Nanog, Oct4 and Sox2, however, were not expressed in any of the 5 clones (Fig. 2a). Interestingly, the expression of surface proteins EpCAM and E-Cadherin were not detected in any of the 5 clones. In addition, only Clone #17 expressed all the four transgenes;
only two transgenes were expressed in the other 4 clones. IF analysis showed that the expression of four reprogramming– associated proteins was either absent or generally unevenly distributed in the colonies of the 5 clones, in striking contrast to homogeneous expression in most cells of the mESCs colony (Fig. 2b). In Clone #17 colonies, Sox2 and Klf4 were evenly distributed in most cells, whereas Oct4 and n-Myc distributions showed mosaic expression patterns (Fig. 2b). In colonies of the other 4 clones (Clone #11, #35, #37, and #38), the protein expression, if present, showed an uneven expression pattern (Fig. 2b). The expression of Sox2 transgene appeared to be correlated with AP expression; Clones #17 and #37 expressed exogenous Sox2 gene (Fig. 2a) which and concomitantly more intense AP staining than the other clones (Fig. 1b). RT-PCR and IF, thus indicated that none of the 5 clones were reprogrammed successfully. Absence of EpCAM and E-cadherin Expression in Incompletely Reprogrammed Cells To further determine the expression patterns of EpCAM and E-cadherin in the unsuccessfully reprogrammed somatic cells, we then examined the expression of these two surface markers in the 17 incompletely reprogrammed cell clones described above by IF. Normally EpCAM and SSEA1 are expressed on the cell membranes of mESCs, and E-Cadherin is located intercellularly. Consistent with the RT-PCR result above (Fig. 2a), double immunostaining SSEA1 with EpCAM or E-cadherin showed that EpCAM and E-cadherin were not
Stem Cell Rev and Rep Table 1 Summary of expression of pluripotency genes and surface markers of mouse mESC-like clones derived from MEFs. The expression of EpCAM and E-Cadherin correlates with the expression of endogenous Oct4 and Nanog (shown in gray)
Oct4
Sox2
Klf4
Myc
Surface markers Nanog
Tg
Endo
Tg
End
Tg
End
Tg
End
AP
SSEA1
MEFs
-
-
-
-
-
+
-
-
mESCs
-
+
-
+
-
+
-
11
+
-
-
-
-
+
17
+
-
+
-
+
35
-
-
-
-
37
-
-
+
38
+
-
18
-
24
-
-
-
-
-
+
+
+
+
+
+
+
-
-
+/-
+/-
-
-
+
+
-
-
+
+/-
-
-
+
+
-
-
-
+/-
+/-
-
-
-
+
+
-
-
-
+
+/-
-
-
-
-
+
+
-
-
-
+/-
+/-
-
-
-
-
-
-
+
-
-
-
+/-
+/-
-
-
-
-
-
-
-
+
-
-
-
+/-
+/-
-
-
29
-
-
+
-
+
+
+
-
-
+/-
+/-
-
-
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
-
+/-
+/-
-
-
5
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
-
+/-
+/-
-
-
10
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
-
+/-
+/-
-
-
4
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
-
+/-
-
-
-
6
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
-
+/-
-
-
-
ESC-
8
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
-
+/-
-
-
-
like
12
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
-
-
-
-
-
clones
15
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
-
-
-
-
-
21
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
-
-
-
-
-
7
+
+
+
+
+
+
+
+
+
+
+
+
+
8
+
+
+
+
+
+
+
+
+
+
+
+
+
34
+
+
+
+
-
+
+
+
+
+
+
+
+
69
+
+
+
+
-
+
+
+
+
+
+
+
+
72
+
+
+
+
-
+
+
+
+
+
+
+
+
75
-
+
-
+
-
+
+
+
+
+
+
+
+
77
+
+
+
+
+
+
+
+
+
+
+
+
+
81
+
+
+
+
+
+
+
+
+
+
+
+
+
42
-
+
+
+
+
+
-
+
+
+
+
+
+
45
+
+
+
+
+
+
+
+
+
+
+
+
+
6
+
+
+
+
-
+
+
+
+
+
+
+
+
14
+
+
+
+
-
+
+
+
+
+
+
+
+
22
+
+
+
+
+
+
-
+
+
+
+
+
+
29
-
+
+
+
-
+
+
+
+
+
+
+
+
30
-
+
+
+
+
+
+
+
+
+
+
+
+
Lentiviral
transduction 3 (N=17)
EpCAM E-Cad
Retroviral transduction (N=15)
Tg, transgene; End, endogenous gene; “+” indicates expression was detected; and “−” indicates that expression was not detected. For easy comparison, the clones showing mosaic patterns of specific gene expression are designated as “+/−”
expressed in all 17 partially reprogrammed clones (Fig. 3a & Table 1). This result contrasted strikingly with the mESCs. To further confirm the IF results, flow cytometric analysis was performed to examine the percentages of SSEA1+,
EpCAM+, and E-cad+ cells in 5 representative partially reprogrammed clones. More than 95% of mESCs expressed the three markers (Fig. 3b and Supplementary Fig. 1). In agreement with the IF data, SSEA1 was partially expressed
Stem Cell Rev and Rep Fig. 2 Expression of pluripotency genes in representative partially reprogrammed Clones #11, #17, #35, #37 and #38. a RT-PCR analysis shows that none of the cell clones expresses endogenous pluripotency genes Oct4, Sox2, Nanog or surface proteins EpCAM and E-Cadherin. Endogenous Klf4 and n-Myc are detected in all cell clones including parental MEF. Of the representative lines, only Clone #17 expresses all four transgenic pluripotency genes. b Pluripotency factors Oct4, Sox2, Klf4 and n-Myc (green) are either absent or expressed unevenly in colonies. The cell clones are stained with antibodies as indicated and localization of the nucleus is shown by DAPI stain (blue). Endo, endogenous genes; Tg, transgenes. Scale bar=50 μm
in the incompletely reprogrammed clones (28.7% in Clone #37, 18.5% in Clone #35 and lower percentages in other clones). Notably, the expression levels of EpCAM and Ecadherin were negligible (Fig. 3b). Expression of EpCAM and E-cadherin are Correlated with Reprogramming Status Since the above data showed that except for Clone #17, the other 4 selected clones expressed only two transgenes (Fig. 2), we speculated that the failure in reprogramming and subsequent abnormal AP, SSEA1, EpCAM and Ecadherin expression might be due to lack of reprogramming factors or proper epigenetic conditions for reprogramming. To test this notion, we performed a two-step rescue
procedure in an attempt to convert the incompletely reprogrammed cells into reprogrammed iPSCs with pluripotent characteristics, by reintroduction of the missing reprogramming factors into each of the incompletely reprogrammed cell clones and subsequent treatment with two chromatin-modifying agents (histone deacetylase inhibitor, Trichostain A, TSA; and DNA methyltransferase inhibitor, 5-Aza-2′-deoxycytidine, (AzaC)). This approach was based on a report that both chromatin-modifying agents could re-activate the endogenous pluripotency-associated gene Nanog in neurosphere cells [35]. The expression of the endogenous Oct4, Sox2, n-Myc, EpCAM, E-cadherin and Nanog remained undetectable on day 15 after viral reinfection of the missing reprogramming factors into the incompletely reprogrammed cell clones, although all four
Stem Cell Rev and Rep Fig. 3 Absence of expression of surface proteins EpCAM and E-Cadherin in partially reprogrammed clones. a Immunostaining analysis demonstrates that the representative partially reprogrammed cell lines express SSEA1 (red) in a mosaic pattern. Neither EpCAM nor E-Cadherin (green) is detected in any partially reprogrammed clones. Scale bar=50 μm. b Percentage of cells with surface marker expression. The population of EpCAM+ and E-Cadherin+ cells of the representative cell clones are as low as that of the MEFs. Detached cells were probed with antibodies against SSEA1, EpCAM and E-Cadherin followed by flow cytometric analysis
transgenes were expressed in the incompletely reprogrammed cell clones by day 15 (Fig. 4b). Next, we selected, respectively, 2 and 10 subclones from Clone #17 and #37 and subjected them to AzaC/TSA treatment. Among the subclones, Clones #17-5, #17-7 and #37-1 expressed all four transgenes (Fig. 4c). As demonstrated by RT-PCR analysis, the expression of endogenous Nanog, Oct4, Sox2, EpCAM and E-cadherin were still absent from these clones after AzaC/TSA treatment (Fig. 4d). Collectively, these results suggest that reintroduction of the missing exogenous reprogramming factors and chromatin-modifying agents did not result in the conversion of the incompletely reprogrammed cells into iPSCs and the activation of EpCAM and E-cadherin expression. Expression of EpCAM and E-cadherin in Reprogrammed iPSCs Since we were not successful in reprogramming MEFs into iPSCs by transduction of lentiviral vectors encoding Oct4, Sox2, Klf4 and n-Myc, we then transduced MEFs with retroviral vectors encoding Oct4, Sox2, Klf4 and c-Myc (Fig. 5a) to try to generate iPSCs. On day 15 after viral transduction, we selected 85 cell colonies with morphology resembling mouse ESC colonies for further culture. After 15 days, 21 clones retained the morphological traits of
mouse ESCs and 15 clones were randomly selected for further characterization. RT-PCR analysis with primer sets specific for the transgenic and endogenous Oct4, Sox2, Klf4, and c-Myc revealed that all 15 clones expressed endogenous Oct4, Sox2, Klf4, c-Myc and Nanog, suggesting successful reactivation of the pluripotency-related genes in MEFs (Fig. 5b & Table 1). Furthermore, IF assay demonstrated that all these clones homogeneously expressed AP, SSEA1 EpCAM, and E-cadherin (Fig. 5d & Table 1). We also demonstrated that representative cell clones #34 and #75 were able to form teratomas in SCID mice (Fig. 5e), suggesting that these two putative iPSC clones were to be true iPSCs. A comparison of the expression patterns of the endogenous genes, Oct4, Sox2, Klf4, c-Myc, Nanog, and surface markers in all incompletely reprogrammed cell clones and reprogrammed iPSC clones generated by lentiviral or retroviral infection showed that the expression of EpCAM and E-cadherin appeared to be closely associated with the onset of endogenous Oct4, and Sox2 expression (Figs. 2b, 5b–e, and Table 1). In addition, the activation of Nanog was also directly related to EpCAM and E-cadherin expression (Fig. 5d and Table 1). Taken together, our results suggest that the expressions of EpCAM and E-cadherin are effective indicators for the successful reprogramming of somatic cells into iPSCs.
Stem Cell Rev and Rep
Fig. 4 Endogenous pluripotency genes remain inactive after second induction of reprogramming and drug treatment in partially reprogrammed clones. a Schema of re-induction of reprogramming of partially reprogrammed clones. All five Clones #11, #17, #35, #37 and #38 are infected with lentiviruses encoding Oct4, Sox2, Klf4 and n-Myc. Subclones of infected cells are isolated for AzaC/TSA treatment. b RT-PCR assay shows that all four transgenic pluripotency genes Oct4, Sox2, Klf4 and n-Myc are detected in the representative partially reprogrammed clones after re-infection. None of the
EpCAM Facilitates the Isolation of the Nanog-expressing iPSC Population The observation that the expression of EpCAM was strongly correlated with expression of Nanog in the putative iPSCs clones implies the possibility that EpCAM can be used to enrich Nanog-expressing iPSCs by FACS cell sorting. To test this notion, we infected MEFs with retroviral vectors encoding Oct4, Sox2, Klf4 and c-Myc. On day 12 after viral infection, cells were collected and hybridized with antibodies against SSEA1 and/or EpCAM and subjected to FACS cell sorting. The level of Nanog
re-infected clones expresses endogenous pluripotency-associated genes Nanog, Oct4 and Sox2. Neither endogenous EpCAM nor ECadherin is detected in these clones. c Expressions of transgenic pluripotency factors of subclones derived from Clone #17 and #37 after second infection are examined by RT-PCR. All four transgenes are detected in Subclones #17-5, #17-7 and #37-1. d Endogenous Nanog, Oct4, Sox2, EpCAM and E-Cadherin remain inactive in subclones of #17 and #37 after AzaC and TSA treatment. Cells are subjected to RT-PCR assay 7 days after AzaC/TSA treatment Fig. 5 IPSC clones derived from MEFs by transduction with retroviruses encoding Oct4, Sox2, Klf4 and c-Myc expressed EpCAM and ECadherin. a Schema for generation of miPSCs with retroviruses. b RTPCR analysis showed that endogenous EpCAM, E-Cadherin, Oct4, Sox2, Klf4, c-Myc and Nanog were all detected in 6 representative iPSC clones (Clones #7, #8, #34, #69, #72 and #75). Transgenes are partially repressed in Clones #34, #69, #72 (Klf4) and #75 (Oct4, Sox2 and Klf4). Messenger RNA of iPSC clones were harvested 30 days after isolation. c All four transgenes were integrated in the selected clones. d Homogenous expressions of endogenous AP (blue), SSEA1 (red), EpCAM, ECadherin (green) and Nanog (red) are detected in Clone #34 and #75 by IF analysis. DAPI stain (blue) indicates the localization of nucleus. Scale bar=60 μm (phase contrast), 200 μm (AP) and 50 μm (SSEA1, EpCAM, E-Cadherin and Nanog). e All three germ layers are recognized in teratomas generated by Clone #34 and #75. Scale bar=300 μm
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expression in the SSEA1 and/or EpCAM expressing cell populations was then compared by quantitative RT-PCR. As shown in Fig. 6a, 5.34% and 29.6% of cells were SSEA1-positive and EpCAM-positive, respectively, while 3.37% of cells were SSEA1/EpCAM-positive (6a). The level of Nanog expression was significantly increased in the isolated cell population based on EpCAM expression (6b). Moreover, IF analysis showed that number of Nanog+
colonies is significantly increased after selection of EpCAMexpressing cells by FACS sorting (Fig. 6c). These results demonstrated that EpCAM is an effective surface marker for distinguishing Nanog-positive iPSCs from Nanog-negative incompletely reprogrammed cells. In contrast, cells sorted based on SSEA1 expression were not able to effectively enrich the Nanog-expressing population. Interestingly, Nanog-positive cells could be isolated most effectively by
Fig. 6 Expression of Nanog is enriched in subpopulation of reprogramming MEFs sorted with EpCAM antibody. a After transduction with retroviral pluripotency genes Oct4, Sox2, Klf4 and c-Myc, MEFs were cultured for 12 days and then trypsinized. Trypsinized cells were then subjected to cell sorting with antibodies against SSEA1, EpCAM or both as indicated. The percentages of
SSEA1-positive, EpCAM-positive and SSEA1/EpCAM-positive cells in sorted cells were 5.34%, 29.6% and 3.37%, respectively. Red: MEFs; blue: reprogrammed MEFs. b mRNA of sorted cells was extracted and the expression of endogenous Nanog was analyzed by Q-PCR. * indicates p
