Defining Reprogramming Checkpoints from Single-Cell Analyses of Induced Pluripotency

Abstract

Elucidating the mechanism of reprogramming is confounded by heterogeneity due to the low efficiency and differential kinetics of obtaining induced pluripotent stem cells (iPSCs) from somatic cells. Therefore, we increased the efficiency with a combination of epigenomic modifiers and signaling molecules and profiled the transcriptomes of individual reprogramming cells. Contrary to the established temporal order, somatic gene inactivation and upregulation of cell cycle, epithelial, and early pluripotency genes can be triggered independently such that any combination of these events can occur in single cells. Sustained co-expression of Epcam, Nanog, and Sox2 with other genes is required to progress toward iPSCs. Ehf, Phlda2, and translation initiation factor Eif4a1 play functional roles in robust iPSC generation. Using regulatory network analysis, we identify a critical role for signaling inhibition by 2i in repressing somatic expression and synergy between the epigenomic modifiers ascorbic acid and a Dot1L inhibitor for pluripotency gene activation.

Figure 1.. Combining Epigenomic and Signaling Modifiers Leads to High-Efficiency Generation of Bona Fide iPSCs

(A) Top: schematic of FBS reprogramming experiment. Cells were harvested and immunofluorescence performed on the days indicated by the arrows. Bottom: number of NANOG+ colonies counted at each indicated time point (on date) or after 4 additional days after doxycycline (dox) was removed (withdrawal). Bars represent SD between two replicate samples. Right panel – immunofluorescence images of NANOG. Scale bar, 250 μm. (B) Top: schematic of A2S reprogramming experiment. Cells were harvested and immunofluorescence performed on the days indicated by the arrows. Bottom: number of NANOG+ colonies counted at each indicated time point (On Date) or after 4 additional days after dox was removed (withdrawal). Bars represent SD between two replicate samples. (C) Top: schematic of single-cell reprogramming experiment. MEFs infected with tdTomato virus were sorted and plated in a 96-well plate. Dox-independent colonies were stained with alkaline phosphatase (AP). Bottom: number of AP+ wells observed in each condition. Percentages indicate how many of the wells were AP+ out of the total number of wells with tdTomato+ cells. Data from two independent experiments are presented. (D) Monocle clustering plot showing ESCs or iPSCs cultured in A2S or FBS media.

Figure 2.. A2S Accelerates FBS Reprogramming

(A) Monocle t-SNE plots showing clustering of reprogramming cells from FBS and A2S, MEFs, and FBS-cultured ESCs. Samples were grouped into 14 clusters. Cells colored by sample (i) and cluster (ii). (B) Graph showing the composition of each cluster from Figure 2A by sample. (C) Heatmap representing the percentage of cells expressing the top 10% differentially expressed genes that define the 14 t-SNE clusters in Figure 2A. Each row represents a single gene. Genes were grouped by k-means into 15 groups labeled A to O, and the number of genes within each group are in parentheses. The 14 t-SNE clusters labeled 1–14 are presented in columns approximating their similarity to ESCs. Significant gene ontology terms associated with a specific group are labeled on the right. n.s., not significant. Arrows indicate pattern of expression change between MEFs and ESCs.

Figure 3.. Reprogramming-Specific Gene Expression Patterns Are Important for Conversion to iPSCs

(A) t-SNE plots based on Figure 2A highlighting the expression of MEF-associated mesenchymal genes that are downregulated as cells transition from MEFs to pluripotency. Top schematic indicates the pattern of expression. (B) Percentage of Cdh1+ cells that also co-express the indicated MEF genes on the x axis. The percentage of MEF gene-expressing cells that express Cdh1 is presented in brackets on the x axis. Note that because of the limit of detection of single-cell transcriptional analysis, co-expression may be underestimated. (C) (i) t-SNE plots based on Figure 2A illustrating co-expression of Cdh1 with Twist1. Note that because of the limit of detection of single-cell transcriptional analysis, co-expression may be underestimated. (ii) Immunofluorescent staining for CDH1 and TWIST1. Percentage of CDH1+/TWIST1+ colonies on A2S day 4 shown below image. Scale bar, 10 μm. (D) t-SNE plots based on Figure 2A highlighting the expression of DNA replication and cell-cycle-associated genes. Top schematic indicates the pattern of expression. (E) Left: percentage of cells that are Ki67+ at each indicated reprogramming time point in FBS or A2S systems. Right: immunofluorescent staining of Ki67 during FBS and A2S reprogramming (day 9 and day 4, respectively). Scale bar, 50 μm. (F) t-SNE plot based on Figure 2A for the anti-proliferation gene Cdkn1c. Top schematic indicates the pattern of expression. (G) Percentage of Cdh1+ cells that co-express cell cycle or anti-proliferative genes. Note that because of the limit of detection of single-cell transcriptional analysis co-expression may be underestimated. (H) Number of NANOG+ colonies on day 4 of A2S reprogramming after small interfering RNA (siRNA)-mediated knock down of Ehf. Error bars represent SD of two replicates.

Figure 4.. Co-expression Clusters of Core Pluripotency Factors with Specific Subsets

(A) Percentage of cells expressing each representative pluripotency-associated gene within the t-SNE clusters from Figure 2A, namely, C10, C6, C9, and in all clusters other than C1, C10, C6, and C9. (B) (i) Co-expression measured by Jaccard index clustering of genes in group N from Figure 2C for genes within Box 1 from Figure S4B in clusters C10, C6, C9, and C1. Note that because of the limit of detection of single-cell transcriptional analysis, co-expression may be underestimated. (ii) Violin plots depicting the level of expression of Sall4 and Tdgf1 in Nanog+ cells in clusters C10, C6, C9, and C1. (C) Same as (B) for genes within Box 2 of Figure S4B. (D) Same as (B) for genes within Box 3 of Figure S4B. (E) Reprogramming results upon knockdown of Phlda2 during A2S reprogramming. (i) Number of NANOG+ and DPPA4+ colonies on day 6 of A2S reprogramming after siRNA-mediated knock down of Phlda2. Error bars represent SD of two replicates. (ii) Knock down efficiency of the Phlda2 siRNAs compared to a nontargeting control. Bars represent SD between two replicate samples. (iii) Immunofluorescence images for representative NANOG+/DPPA4+ and NANOG+/ DPPA4− colonies. Scale bar, 50 μm.

Figure 5.. Roadblocks to High-Efficiency Reprogramming

(A) Pseudotime trajectory generated by Monocle for the A2S reprogramming system. Left - trajectory colored by pseudotime. Middle- trajectory colored by sample. Asterisk indicates that MEFs cannot ontogenically convert to ESCs, but pseudotime reflects transition to a pluripotent state. Right-trajectory colored by individual sample. (B) Heatmaps for clustering of genes that define the branchpoints (q-value, <1E-40)from BEAM analysis for early branch (left panel) and late branch (right panel). Center of the gray bar above heatmap is the start of the branchpoint. Red represents cells at the end of the branchpoint. Blue represents cells at the end of the continuing branch. (C) Pseudotime plots that display how the expression of the representative genes differs as cells either exit at the late branchpoint (solid line) or continue along the path toward successful reprogramming (dashed line) colored by sample. (D) Violin plots depicting the level of expression of Twist1 in Nanog+ cells (top left) and the expression of Nanog, Sall4, and Tdgf1 in Epcam+ cells in both the late branch and in the continuing segment of the trajectory. (E) Left: schematic of EPCAM sort experiment. MEFs were reprogrammed in A2S conditions for 3 days and sorted based on EPCAM expression (high or medium). These two populations underwent 3 more days of reprogramming and were sorted again based on high, medium, or no expression of EPCAM. Right: graphs depicting the percentage of the day 6 population that have high, medium, or no EPCAM expression from cells that were EPCAM-high on day 3 (top) or medium on day 3 (bottom). (F) Left: number of NANOG+ colonies on day 4 of A2S reprogramming after siRNA-mediated knock down of Eif4a1. Error bars represent SD of two replicates. Right: cell counts on each day of Eif4a1 knock down reprogramming experiment.

Figure 6.. A2S Concurrently Enhances Downregulation of MEF Genes and Upregulation of ESC Genes

(A) NANOG+ colonies on specified day or after 4 days of dox withdrawal in each dual combination (A2, AS, and S2). Dashed line: NANOG+ colonies on day 6 of A2S. Bars represent standard deviation between two replicate samples. (B) Heatmap generated from the MERLIN module analysis indicating the level of expression for the differentially expressed genes from the FBS+A2S analysis. Each row is a separate gene. Values are normalized to zero mean from the FBS and A2S reprogramming. Each column is a separate cell grouped based on the clusters in Figure 2A (left) or duration of chemical combination exposure (right). MERLIN modules are labeled as M1 through M11. (C) Violin plots of representative genes from expression patterns in Figure 6B. (D) Network wiring of regulatory connections inferred using MERLIN, colored by each reprogramming condition for the genes of a transiently expressed module. The edge color corresponds to the regression coefficient between the regulator and target connected by the edge (ranging from −0.5 (blue) to 0 (white) to 0.5 (red)) estimated using the data from the specific treatment. Edge width corresponds to edge confidence (from 80% [1] to 100% [5]). Node color corresponds to percentage of cells in a condition in which that gene was expressed (from 0% [white] to 100% [green]). Node border indicates gene membership in a module: pink if the gene is in the given module and gray if it is not. The node size is proportional to the out-degree of the node. Network corresponds to M8. (E) Same as (D) for genes in an upregulated, pluripotency-associated gene module (M10).

Figure 7.. Model Depicting Regulation of Key Genes during MEF Reprogramming

Four general gene expression patterns are observed during MEF reprogramming: down-regulation, transient downregulation, transient up-regulation, and gene upregulation. Mesenchymal genes are downregulated independently of each other and their expression is compatible with epithelial (Cdh1) or early pluripotency (Nanog) gene expression.Transiently regulated genes include cell cycle and anti-proliferative genes. Completion of reprogramming is enhanced by co-expression of markers, such as EpCAM with pluripotency genes (represented by colored circles), and the complete activation of the pluripotency network (represented by red and white networks). The addition of acceleration factors can impact specific gene expression patterns, whereas only the combination of A2S can lead to complete rewiring of the pluripotency network.