The generation of induced pluripotent stem (iPS) cells presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. Although several intermediate populations have been described1, 2, 3, 4, 5, 6, 7, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPS cells. The rapid expansion of minor reprogrammed cells in the heterogeneous population can also obscure investigation of relevant transition processes. Understanding the biological mechanisms essential for successful iPS cell generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that in mouse embryonic fibroblasts, reprogramming follows an orderly sequence of stage transitions, marked by changes in the cell-surface markers CD44 and ICAM1, and a Nanog–enhanced green fluorescent protein (Nanog–eGFP) reporter. RNA-sequencing analysis of these populations demonstrates two waves of pluripotency gene upregulation, and unexpectedly, transient upregulation of several epidermis-related genes, demonstrating that reprogramming is not simply the reversal of the normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and the improved understanding of the reprogramming process will lead to new reprogramming strategies.
At a glance
Several reports have suggested that reprogramming progresses in an ordered manner3, 5, 6, 8, 9, 10. To identify markers whose expression changed concurrent with pluripotency gene expression, we performed time course microarray analysis using a piggyBac transposon-based secondary reprogramming system3, 11 (Supplementary Fig. 2a). Of a number of candidate cell-surface markers, Cd44 and Icam1 (also known as CD54) demonstrated the most dynamic expression changes throughout secondary mouse embryonic fibroblast (MEF) reprogramming (Supplementary Fig. 2b). For further investigation, we generated an efficient secondary reprogramming system in which doxycycline-mediated induction of the reprogramming factors could be monitored by an mOrange reporter placed after the 2A-peptide-linked reprogramming cassette c-Myc-Klf4-Oct4-Sox2 (MKOS)12, and endogenous Nanog promoter activation could be followed by expression of enhanced green fluorescent protein (eGFP)13 (Supplementary Fig 3). Reprogramming cultures were supplemented with vitamin C and an Alk inhibitor, both of which enhance reprogramming efficiency10, 14, 15. In this secondary reprogramming system, Nanog–eGFP+ cells appeared as early as day 6, and >60% of mOrange+ transgene-expressing cells were found to be Nanog–eGFP+ by day 12 (Supplementary Figs 4 and 5a). Most mOrange+ transgene-expressing cells lost expression of Thy1 (also known as CD90) and gained E-cadherin (also known as Cdh1) expression by day 4 (Supplementary Fig. 5b, c). Expression of stage-specific embryonic antigen 1 (SSEA-1, also known as Fut4) barely changed after day 8, with a gradual gain of Nanog–eGFP+ cells in both SSEA-1+ and SSEA-1− cell populations (Supplementary Fig. 5d). Consistent with heterogeneous expression of SSEA1 in iPS and embryonic stem (ES) cells, it was not possible to delineate the reprogramming process accurately using SSEA-1 (Supplementary Fig. 6). By contrast, the appearance of CD44− and ICAM1+ cells at later time points closely correlated with Nanog–eGFP expression (Supplementary Fig. 5e, f). Double staining for CD44 and ICAM1 revealed that a distinct series of population changes occur during reprogramming (Fig. 1). Initially, MEFs displayed high CD44 and broad ICAM1 expression, with most becoming ICAM1− by day 6, along with the appearance of a minor CD44− ICAM1− cell population. By day 8, CD44− populations appeared enriched, and at day 12 almost all cells displayed an iPS/ES-cell-like CD44− ICAM1+ profile, of which more than 60% expressed Nanog–eGFP. Consistent with the observation that Nanog expression is not necessarily a sign of completed reprogramming16, Nanog–eGFP+ cells were observed even before cells obtained this iPS/ES-cell-like phenotype (CD44− ICAM1+). Both ICAM1+- and ICAM1−-sorted MEFs demonstrated similar fluorescence-activated cell sorting (FACS) profile changes during reprogramming (Supplementary Fig. 7). Immunofluorescence for CD44 and ICAM1 revealed that reprogramming is not synchronized even within individual colonies (Supplementary Fig. 8). Secondary reprogramming of the non-polycistronic iPS cell line 6c (refs 3, 11) and primary reprogramming using MKOS and Oct4-P2A-Sox2-T2A-Klf4-E2A-cMyc (OSKM)17 piggyBac transposons resulted in similar ICAM1 and CD44 profile changes, indicating their suitability for use in other systems and contexts (Supplementary Fig. 9). These findings demonstrated the asynchronous but stepwise manner of reprogramming, and highlighted the potential usefulness of CD44 and ICAM1 to isolate intermediate reprogramming subpopulations.
Next, we aimed to confirm that the observed CD44/ICAM1 profile changes reflected the transition of individual cells from one stage to the next, and not merely the loss of one major population and expansion of another minor population. CD44+ ICAM1− (gate 1), CD44− ICAM1− (gate 2) and CD44− ICAM1+ (gate 3) cell populations, either Nanog–eGFP+ (that is, 1NG+, 2 NG+ and 3NG+) or Nanog–eGFP− (1NG−, 2NG− and 3NG−), were isolated by cell-sorting at day 10 of reprogramming and re-plated in reprogramming conditions (Fig. 2a). After 3 days, both NG+ and NG− cells progressed in the order of gates 1 to 2 to 3 (Fig. 2b). This progression correlated well with increased Nanog–eGFP+ colony-forming potential (c.f.p.), with 3NG+ cells displaying similar clonogenicity to fully reprogrammed iPS cells (Fig. 2c). Of cells with the same CD44/ICAM1 profile, Nanog–eGFP expression correlated with a higher c.f.p. (for example, 1NG− versus 1NG+).
To examine the progression of the reprogramming process more accurately, cells from each gate were sorted, and their expression of CD44/ICAM1/Nanog–eGFP was re-analysed after 24 h (Fig. 2d). On the basis of total cell numbers in each gate after 24 h (Supplementary Fig. 10), we generated a reprogramming route map representing differences in the efficiency of these stage transitions and in Nanog–eGFP+ c.f.p. (Fig. 2e). Similar results were obtained when each subpopulation was sorted at day 8 (Supplementary Fig. 11). This analysis revealed that reaching a Nanog–eGFP+ state is a rate-limiting step—as few cells overcame this barrier in the 24 h assay—and those that do so reprogram more efficiently than their Nanog–eGFP− counterparts, consistent with the role of Nanog as an accelerator of reprogramming and the gateway to pluripotency18, 19.
To determine global gene expression changes during these stage transitions, we carried out RNA-sequencing analysis using a highly multiplexed sample bar-coding system20, 21, 22, 23, 24, 25, 26 (see Methods and Supplementary Table 1). Hierarchical clustering using the complete list of differentially expressed genes (DEGs) revealed four major branches: (1) MEFs; (2) 1NG−/+ and 2NG−; (3) 2NG−/+ and 3NG−/+; and (4) 3NG+ sorted at day 15 (3NG+D15), iPS and ES cells (Fig. 3a). There was a prominent gene expression difference between 3NG+ and 3NG+D15 cells, with the latter being more similar to iPS and ES cells (Fig. 3a and Supplementary Fig. 12), possibly reflecting the observed difference in the c.f.p. in the absence of doxycycline (Supplementary Fig. 13). The DEGs between these two populations may be involved in the establishment of an exogenous-factor-independent self-renewal state. Principal component analysis clearly distinguished 2NG+ from 3NG− cells, consistent with the higher probability of the former to reach the 3NG+ state within 24 h (Supplementary Figs 10 and 12b). DEGs could be classified into five distinct expression pattern groups (A–E) (Fig. 3a and Supplementary Tables 2 and 3). Group A contained readily downregulated fibroblast-related genes. Group D comprised factors gradually upregulated towards iPS cells, in which ES cell genes were highly enriched (P ≤ 0.000367) (Fig. 3c). However group C, which contained genes upregulated at early stages and maintained throughout reprogramming, also included some pluripotency-related factors. To extend this finding, we examined the expression pattern of 22 pluripotency-related genes in our data set27, 28. Interestingly, 8 pluripotency genes, including endogenous Oct4 (also known as Pou5f1), were already upregulated at the 1NG+/2NG− stages to the level found in 3NG+ cells (Fig. 3b, left), whereas 14 pluripotency genes were more gradually upregulated in the later stage reprogramming populations (Fig. 3b, right, and Supplementary Table 4). This early and late pluripotency gene upregulation was confirmed at the single cell level5 (Fig. 3e), highlighting the high resolution of the CD44/ICAM1 sorting system.
We also identified two additional gene expression patterns displaying transient upregulation (group B) or downregulation (group E) exclusively in the intermediate stages of reprogramming. This finding indicates that reprogramming from MEFs to iPS cells is not simply the loss of MEF genes and gain of ES cell genes. Gene Ontology analysis revealed that genes related to ectoderm/epidermis development and keratinocyte differentiation were highly enriched in group B (P ≤ 0.000274) (Fig. 3c, d and Supplementary Tables 3–5). Although SFN and KRT17 were barely detectable by immunofluorescence in MEFs and iPS cells, transient upregulation was observed in the intermediate stages of reprogramming (Supplementary Fig. 14). Single-cell PCR confirmed the co-expression of epidermis genes (Ehf and Ovol1) with early pluripotency genes in the 1NG−/+ stage (Fig. 3e). Consistent with our data, analysis of three published microarray data sets incorporating partially reprogrammed iPS cells1, a time course experiment3 and a subpopulation analysis with Thy1, SSEA-1 and Oct4–eGFP (ref. 6) confirmed transient epidermal gene expression during reprogramming (Supplementary Figs 15–17 and Supplementary Tables 6–8). Partially reprogrammed cells from B cells also displayed similar epidermis gene expression4, whereas two factor-reprogramming (Oct4 and Sox2) of MEFs did not29. Therefore, this intermediate state could be a consequence of the use of Klf4 that is important for efficient reprogramming, and demonstrates that the reprogramming process is not simply a reversion of normal differentiation (summarized in Supplementary Fig. 1). It would be intriguing to investigate whether similar transient gene expression changes can be seen in reprogramming of ectoderm or endoderm lineages. Downregulation of these epidermis genes coincided with upregulation of ‘late’ pluripotency genes. Future examination of this rapid switch in gene expression may provide a new insight into the molecular mechanism of reprogramming.
The integrative data analysis described above demonstrated that this CD44/ICAM1/Nanog–eGFP marker system could uniquely provide high-resolution information during late pluripotency gene upregulation, enabling the discrimination of ‘reprogramming’ from ‘expansion of reprogrammed cells’ (Fig. 3b and Supplementary Figs 16b and 17f). This system also refines investigation of the kinetics of reprogramming. It has recently been shown that vitamin C increases reprogramming efficiency by facilitating histone 3 Lys 9 (H3K9) demethylation7, and that reprogramming factors fail to bind trimethylated H3K9-rich regions in the initial stages of reprogramming30. We carried out reprogramming in the absence of vitamin C and observed not only a decrease in the iPS cell colony number, but also a marked delay in the transition from one stage of reprogramming to the next (Supplementary Fig. 18). Similar analyses can be performed using our marker system to investigate the mechanism of action of other factors that alter reprogramming efficiency. Isolation and analysis of subpopulations affected by these factors could reveal the downstream genes specifically involved in, and required for, successful reprogramming. Further studies using this high-resolution analysis system have the potential to make a considerable contribution towards revealing the molecular mechanisms of reprogramming.
The piggyBac transposon PB-TAP containing the tetO2 promoter, an attR1R2 Gateway cloning cassette (Invitrogen) and rabbit β-globin poly A signal, was provided by A. Nagy. To minimize silencing of the reprogramming vector, a chicken β-globin insulator31 was inserted into the PacI site between the piggyBac 3′-terminal repeat (3′-TR) and the tetO2 promoter, and a human lamin B2 (LMB2) replicator32 plus another chicken β-globin insulator were inserted into the EcoRV site between the rabbit β-globin poly A signal and the piggyBac 5′-TR, to generate PB-TAP IRI. The BamHI fragment containing loxP-flanked MKOS reprogramming cassette followed by ires-mOrange (2LMKOSimO) from pCAG2LMKOSimO (ref. 12) was inserted into a Gateway entry vector pENTR 2B (Life Technologies), to generate attP2LMKOSimO pENTR. Finally the attP2LMKOSimO cassette was Gateway-cloned into the PB-TAP IRI to yield reprogramming piggyBac transposon PB-TAP IRI attP2LMKOSimO. Similarly, reprogramming piggyBac transposon PB-TAP IRI 2LOSKMimO was generated after transferring the OSKM reprogramming cassette17 into attP2LMKOSimO pENTR replacing the MKOS cassette. Plasmid sequences are available on request.
Generation of a primary iPS cell line D6s4B5
Embryos at 12.5 days post coitum (d.p.c.) were obtained from RosartTA/rtTA, NanogeGFP/+, Col1a1+/+ mice, which were derived by crossing TNG mice13 and B6;129-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm2(tetO-Pou5f1)Jae/J (Jackson Laboratory). The embryos were decapitated, eviscerated, dissociated with 0.25% trypsin and 0.1% EDTA, and plated in MEF medium (GMEM, 10% FBS, penicillin–streptomycin, 1× non-essential amino acids (Invitrogen), 1 mM sodium pyruvate and 0.05 mM 2-mercaptoethanol). The PB-TAP IRI attP2LMKOSimO (500 ng) and pCyL43 piggyBac transposase expression vector33 (2 μg) were introduced into the MEFs by nucleofection (Amaxa) as before12, and cells were cultured in ES cell medium (MEF medium supplemented with 1,000 U ml−1 leukaemia inhibiting factor (LIF)) in the presence of 1.0 μg ml−1 doxycycline (Sigma) for an initial 8 days, and thereafter 0.5 μg ml−1 doxycycline. Pluripotency of a clonal iPS cell line D6 was confirmed by teratoma formation, and a subclone D6s4B5 was used for secondary reprogramming. To compare CD44 and ICAM1 profiles of primary reprogramming with PB-TAP IRI attP2LMKOSimO and PB-TAP IRI 2LOSKMimO vectors, MEFs were nucleofected as above and cultured in the presence of 1.0 μg ml−1 doxycycline, 10 μg ml−1 vitamin C (Sigma) and 500 nM Alk inhibitor A 83-01 (TOCRIS Bioscience).
Each chimaeric embryo was collected at 12.5 d.p.c., dissociated and cultured in MEF medium. One-twentieth of the dissociated cells were exposed to doxycycline (300 ng ml−1) for 2 days, and the proportion of transgenic MEFs was measured by FACS analysis of mOrange expression. For FACS time course and colony counting experiments, secondary transgenic MEFs were diluted to 5% and 30% by addition of 129 wild-type MEFs and plated in a gelatinized 6-well-plate at 1 × 105 cells per well (5,000 and 30,000 transgenic MEFs per well, respectively). For sorting experiments, MEFs were plated at 2 × 105 cells per gelatinized 100 mm plate (1 × 104 transgenic MEFs per plate). Cells were cultured in reprogramming medium, which is ES cell medium supplemented with 300 ng ml−1 doxycycline, 10 μg ml−1 vitamin C and 500 nM Alk inhibitor. Medium was changed every 2 days.
Flow cytometry and cell sorting
Cell-surface marker analysis was performed with the following eBioscience antibodies: ICAM-1-biotin (13-0541; 1/100), CD44-biotin (17-0441; 1/100), CD44- allophycocyanin (APC) (17-0441; 1/300), streptavidin-phycoerythrin (PE)-Cy7 (25-4317-82; 1/1500), SSEA-1-647 (51-8813; 1/50), E-cadherin-biotin (13-3249; 1/100), Thy1-APC (17-0902, 1/300) and CD2-biotin (13-0029; 1/100). For sorting experiments, dead cells were excluded using 4′,6-diamidino-2-phenylindole (DAPI) nucleic acid stain (Invitrogen) (0.5 ng ml−1). Cells were incubated in 0.25% trypsin and 1 mM EDTA (Life Technologies) for 1–2 min at 37 °C, collected in GMEM media containing 10% FCS and counted. Staining was carried out in FACS buffer (2% FCS in PBS) at ~1 × 106 cells ml−1 for 15–30 min at 4 °C, and followed by washing with FACS buffer, sorting and/or analysis with FACSAriaII and LSRFortessa (both BD Biosciences), respectively. Excitation laser lines and filters used for each fluorophore are summarized in Supplementary Table 9. Data were analysed using FlowJo software (Tree Star). Intact cells were identified based on forward and side light scatter, and subsequently analysed for fluorescence intensity. Additional gating was carried out as outlined in Supplementary Fig. 2. For colony formation assays, sorted cells were plated on γ-irradiated MEFs in 12-well plates at 3.5 × 103 cells per well. Nanog–eGFP+ colonies were quantified 10 days after sorting. For 24 h or time-course analysis, sorted cells were plated in gelatinized 48-well plate at 1 × 104 cells per well. In both cases, cells were cultured in reprogramming medium after sorting.
Immunofluorescenceand confocal microscopy imaging
Images of cells stained with ICAM-1-biotin (1/100), CD44-APC (1/300) and streptavidin-PE-Cy7 (1/1,500) antibodies described above were captured with a confocal microscope (Leica TSC SP2) and Leica confocal software. Cells stained with anti-Krt17 (LifeSpan BioSciences) and anti-Sfn (Sigma) antibodies and anti-Rabbit IgG CF633 secondary antibody (Sigma) were imaged with a fluorescence microscopy (Olympus).
Multiplexed RNA sequencing and data analysis
RNA was isolated with TRI reagent (Sigma) following the manufacturer’s instructions. RNA quality and concentration was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies). Using 10 ng RNA, reverse transcription with bar-coded primers, complementary DNA amplification, and sequencing with Illumina HiSeq 2000 were performed as previously described20, 21. Quality control of the obtained reads and alignment to the mouse reference genome (NCBI37/mm9) were performed using the GeneProf web-based analysis suite with default parameters22. Gene expression read counts were exported and analysed in R to identify DEGs, using the edgeR and DESeq Bioconductor libraries23, 24, 25. For both methods, low expression transcripts (less than 13 reads in all samples) were filtered out, and P values were adjusted using a threshold for false discovery rate (FDR) ≤ 0.05. Genes listed as DEGs by both methods in any two subpopulation comparison indicated in Supplementary Table 1 and Supplementary Fig. 12a (total 3,171) were used for further analysis. Hierarchical clustering and K-means clustering (K = 5) was performed using Cluster 3.0, and Java Treeview was used for visualization34, 35. This multiplexed RNA-sequencing technology reads only the 5′ end of transcript, thus detecting only endogenous Oct4 and Sox2. Nanog expression was detectable in Nanog–eGFP− populations owing to the reporter system. Principal components analysis was performed in R and plotted with the scatterplot3d library36. Gene Ontology enrichment was calculated using the DAVID functional annotation bioinformatics tool26. Gene Ontology term enrichment analysis was carried out with a modified Fisher exact P value. The three additional published studies1, 3, 6 (GEO accession numbers GSE21757, GSE14012 and GSE42379) were analysed in a similar way. For the time course data, the analysis was performed as following: data were robust multi-array average (RMA)37 normalized using the expression console from Affymetrix and, because no replicates were provided, fold changes between two samples were calculated in Excel. Genes with more than 1.5-fold changes were classified as DEGs. For the Plath and Polo data set, data were RMA-normalized using the ‘affy’ package38 in R, and DEGs were identified using the ‘limma’ package38 in R with fold change ≥ 1.5 and FDR ≤ 0.05, or fold change ≥ 1.5 where no replicates were available. Subsequently, K-means clustering of the identified DEGs was performed for all studies. Selected gene expression data are shown as the relative expression against the highest signal among the samples using an averaged signal value (reads per million) of duplicates/triplicates.
Single-cell gene expression analysis
Single-cell qPCR was performed as described previously5 with slight modifications. In brief, 22 sets of TaqMan gene expression assays (Applied Biosystems; Supplementary Table 9) were pooled at a final concentration of 180 nM per primer set and 50 μM per probe. Individual cells were sorted directly into 10 μl RT-PreAmp Master Mix (5 μl of CellsDirect reaction mix (Invitrogen), 2.5 μl of pooled assays, 0.2 μl of SuperScript III (Invitrogen), 1.3 μl of water) using FACSAria II. Cell lysis and sequence-specific reverse transcription were performed at 50 °C for 15 min. Reverse transcriptase was inactivated by heating to 95 °C for 2 min. Subsequently, in the same tube, cDNA went through sequence-specific amplification by denaturing at 95 °C for 15 s, and annealing and amplification at 60 °C for 4 min for 22 cycles. Preamplified products were diluted fivefold with water and analysed in 48.48 dynamic arrays on a biomark system (Fluidigm) following the Fluidigm protocol. Ct values were calculated and visualized using BioMark real-time PCR analysis software (Fluidigm). Each assay was performed in replicate.
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We thank A. Nagy and K. Woltjen for providing the 6c iPS cell line, I. Chambers for providing TNG mice, S. Monard and O. Rodrigues for assistance with flow cytometry, and T. Kunath, T. Burdon, S. Lowell and N. Festuccia for discussions and comments on the manuscript. We also thank L. Robertson for technical assistance, and Biomed unit staff for mouse husbandry. This work was supported by ERC grants ROADTOIPS (no. 261075) and BRAINCELL (no. 261063), and the Anne Rowling Regenerative Neurology Clinic. J.O.’M. and T.R. are funded by an MRC PhD Studentship and a Darwin Trust of Edinburgh Scholarship, respectively.
- Supplementary Figures (3.6 MB)
This file contains Supplementary Figures 1-18.
- Supplementary Data (3 MB)
This zipped file contains Supplementary Tables 1-9. Supplementary Table 1 shows differentially expressed genes (DEGs) between samples, Supplementary Table 2 shows genes in A-E category from O'Malley subpopulation data, Supplementary Table 3 displays gene ontology from O'Malley subpopulation data, Supplementary Table 4 contains signal values of pluripotency and epidermis genes, Supplementary Table 5 shows epidermis genes EST profile, Supplementary Table 6 shows genes in pA-pD’ category from piPSC data, Supplementary Table 7 contains genes in tA’-tD category from time course data, Supplementary Table 8 shows genes in TSO A1-TSO E category from Thy1/SSEA1/Oct4-GFP subpopulation data and Supplementary Table 9 shows flow cytometry conditions and TaqMan Gene Expression Assay ID.