HK40089154B - Generation of induced pluripotent stem cells with polycistronic sox2, klf4, and optionally c-myc - Google Patents

Description

Induced pluripotent stem cells were generated using polycistronic SOX2, KLF4, and optional C-MYC.

This application claims priority to U.S. Provisional Application Serial No. 62/916,830, filed on October 18, 2019, the contents of which are expressly incorporated herein by reference in their entirety.

By referencing the sequence list provided in the Text file and incorporating

The sequence list is provided here as a text file “373038WOSEQ LIST.txt”, created on October 16, 2020, and is 53,248 bytes in size. The contents of the text file are incorporated herein by reference in their entirety.

Background Technology

This study first demonstrated that differentiated somatic cells could be reprogrammed into induced pluripotent stem cells (iPSCs) utilizing the ectopic expression of four factors: Oct4 (O), Sox2 (S), Klf4 (K), and c-Myc (M) (Takahashi and Yamanaka, 2006). Oct4 has long been considered indispensable in the reprogramming process because it is the only one of the four capable of inducing pluripotency independently, and its family members cannot substitute for its function (Kim et al., 2009a; Kim et al., 2009b; Nakagawa et al., 2008). Mechanistic studies have shown that reprogramming is initiated by the overall collaborative engagement of three precursor factors, Oct4, Sox2, and Klf4, followed by genome-wide epigenetic remodeling and two transcriptional waves (Chen et al., 2016; Chronis et al., 2017; Polo et al., 2012; Smith et al., 2016; Soufi et al., 2012; Sridharan et al., 2009). These studies highlight the synergistic effect of Oct4, Sox2, and Klf4 (Chronis et al., 2017; Sridharan et al., 2009), but do not explain why Oct4 is unique, and the functions of Sox2 and Klf4 in this process remain poorly understood.

Invention Overview

This article describes methods and compositions for precisely controlling factor stoichiometry during cell reprogramming using polycistronic boxes. Surprisingly, the data described herein indicate that, in the absence of ectopic Oct4, polycistronic Sox2, Klf4, and c-Myc (e.g., the S _2A K _2A M polycistronic construct) are sufficient to establish pluripotency in several types of differentiated somatic cells. In some cases, c-Myc is optional and the use of polycistronic Sox2 and Klf4 (e.g., S _2A K) is sufficient. The stoichiometry of Sox2 and Klf4 is more important for this reprogramming (e.g., compared to the stoichiometry of c-Myc) because disruption of the Sox2 and Klf4 factor balance leads to a significant reduction or failure of iPSC production. Genome-wide studies have revealed the synergistic binding of Sox2 and Klf4, resulting in the gradual activation and establishment of pluripotency networks. Furthermore, parallel transcriptomic analyses using secondary _{S2A K2A} M embryonic fibroblasts (2°MEF) and neural progenitor cells (2°NPC) revealed convergent reprogramming trajectories and similar efficiencies. The results presented in this paper illustrate the stoichiometric adequacy of Sox2 and Klf4 in pluripotency induction in the absence of ectopic Oct4. The data presented in this paper demonstrate the core functions of Sox2 and Klf4 in pluripotency induction.

Brief description of the attached diagram

Figures 1A to 1P illustrate the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) using a polycistronic _{S2A K2A} M expression cassette (expressing Sox2, Klf4, and Myc-C, as well as 2A cleavable linkers between Sox2 and Klf4 and between Klf4 and Myc-C). Figure 1A shows a schematic diagram depicting the _{S2A K2A} M polycistronic expression system and the reprogramming procedure. Figure 1B shows an image of colonies obtained from _{S2A K2A} M reprogramming, illustrating EGFP expression in colonies on day 7 of reprogramming (scale bar, 100 μm). pH, phase contrast. MEFs express Oct4-GFP (OG2 cells) as a marker of pluripotency, where the Oct4 promoter is operatively linked to a segment encoding Enhanced Green Fluorescent Protein (EGFP). Figure 1C shows an image of _{S2A K2A} M colonies, illustrating EGFP signaling in situ and at passages 1 and 20 (scale bar, 100 μm). Figure 1D shows complete DNA demethylation at the Oct4 promoter in _{S2A K2A} M induced pluripotent stem cells (iPSCs). Figure 1E shows the detection of Nanog, Sox2, and SSEA1 proteins in _{S2A K2A} M iPSCs (scale bar, 100 μm). Figure 1F schematically illustrates the correlation between overall gene expression in _{S2A K2A} M iPSCs and R1 embryonic stem cells (ESCs). Figure 1G shows an image of chimeric mice generated by injecting _{S2A K2A} MiPSCs into blastocysts implanted in pseudopregnant females, serving as confirmation of the pluripotency of _{S2A K2A} M iPSCs. Figure 1H shows mouse embryos constructed via a tetraploid complementation assay, which involves electrofusion of cellular stage CD1 (ICR) embryos to produce tetraploid embryos and injection of _{S2A K2A} M iPSCs into the embryos to form reconstructed tetraploid blastocysts, which are then implanted into pseudopregnant CD1 (ICR) female mice. Figure 1I shows how _{S2A K2A} M iPSCs facilitate the implantation of germ cells in the blastocyst. Figure 1J shows a schematic diagram of additional polycistronic cassettes for _{O2A S2A K2A} M, _{O2A S2A} M, _{O2A K2A} M, and _{S2A K2A} M, where O stands for Oct4, S for Sox2, K for Klf4, and M for c-Myc. Figure 1K shows a Western blot illustrating protein expression in MEFs from the _{O2A S2A} M, _{O2A K2A} M, and _{S2A K2A} M expression cassettes after 48 hours of induction. Figure 1L shows a Western blot demonstrating efficient cleavage of the polycistronic peptide at the 2A site in the transduced MEFs after prolonged exposure. Figures 1M-1 through 1M-4 schematically illustrate the Oct4-EGFP colony counts during 14 days of induction of _{O2A S2A K2A} M, _{O2A S2A} M, _{O2A K2A} M, and _{S2A K2A} M in 100,000 initial OG2 MEFs. Figure 1M-1 schematically illustrates the Oct4-EGFP colony counts after _{O2A S2A K2A} M induction. Figure 1M-2 schematically illustrates the Oct4-EGFP colony counts after _{O2A S2A} M induction. Figure 1M-3 schematically illustrates the number of Oct4-EGFP colonies after _{O2A K2A} M induction. Figure 1M-4 schematically illustrates the number of Oct4-EGFP colonies after _{S2A K2A} M induction. Figure 1N schematically illustrates the expression of pluripotency gene markers in _{S2A K2A} M iPSCs compared to the expression of the same marker in embryonic stem cells (ESCs). Figure 1O shows EGFP-positive colonies generated from reprogrammed neural progenitor cells (NPCs) using _{S2A K2A} M (scale bar, 100 μm). Figure 1P schematically illustrates the expression of pluripotency gene markers in _{S2A K2A} M iPSCs reprogrammed from neural progenitor cells (NPCs).

Figures 2A to 2S illustrate how secondary _{S2A K2A} M MEFs (2°MEFs) can be efficiently reprogrammed to pluripotency. Figure 2A shows a schematic diagram of _{S2A K2A} M 2°MEFs and NPCs derived from embryos obtained from tetraploid complementation assays. Figure 2B is a Western blot showing Sox2 and Klf4 protein expression at specified time points following doxycycline-induced polyprotein expression in _{S2A K2A} M secondary (2°) MEFs. Figure 2C shows cells exhibiting Sox2 and Klf4 activation in 2°MEFs and NPCs (scale bar, 50 μm). Figures 2D-1 to 2D-4 show morphological changes in MEFs during day 0 and the first 3 days of reprogramming (scale bar, 100 μm). Figure 2D-1 shows an image of a MEF on day 0. Figure 2D-2 shows an image of a MEF on day 1. Figure 2D-3 shows an image of a MEF on day 2. Figure 2D-4 shows an image of a MEF on day 3. Figures 2E-1 to 2E-4 schematically illustrate the activation of multiple mesenchymal epithelial transition (MET) genes during the first 4 days of reprogramming. Figure 2E-1 schematically illustrates Cdh1 activation during the first 4 days of reprogramming. Figure 2E-2 schematically illustrates EpCAM activation during the first 4 days of reprogramming. Figure 2E-3 schematically illustrates Krt8 activation during the first 4 days of reprogramming. Figure 2E-4 schematically illustrates Ocln activation during the first 4 days of reprogramming. Figure 2F shows the activation of Oct4-EGFP in 2° MEF when cultured under normal ESC (DMSO) and AF conditions (AF: medium containing A83-01+ lorantin) (scale bar, 100 μm). Figure 2G shows the activation of Oct4-EGFP as examined by flow cytometry. Figures 2H-1 and 2H-2 schematically illustrate the activation of Oct4 and Nanog during MEF reprogramming. Figure 2H-1 schematically illustrates Oct4 activation during MEF reprogramming. Figure 2H-2 schematically illustrates Nanog activation during MEF reprogramming. Figure 2I schematically illustrates EGFP-positive colony formation efficiency with and without small molecules (A: A83-01; F: trichomoniasis). The three conditions (A, F, and AF) were compared with the control sample (DMSO). Figure 2J schematically illustrates EGFP-positive colony formation efficiency at different cell densities. Figure 2K schematically illustrates EGFP-positive colony formation efficiency as measured by initial nuclear count. Figure 2L schematically illustrates EGFP-positive colony formation efficiency as measured by single-cell seeding. Figure 2M shows cells immunofluorescence staining for Oct4 and Nanog proteins at the end of reprogramming. Figure 2N schematically illustrates the time to EGFP-positive colony formation (i.e., iPSC generation) induced by S _2A K _2A M in 2° MEF. Data in Figures 2E, 2I, and 2J represent mean + SD (n ≥ 3). p-values were determined by one-way ANOVA and Bonferroni post-hoc test. *p <0.05; **p <0.01; ns, not significant. Figure 2O shows in situ and P1 iPSC colonies obtained by reprogramming ₂ °MEF (scale bar, 100 μm). Figure 2P schematically shows the expression of pluripotency gene markers in _S2AK2AM 2°iPSCs compared to embryonic stem cells (ESCs). Figure 2Q schematically shows the number of colonies generated by 2°NPCs (AF: A83-01, saliva) with or without AF. Figure 2R schematically shows the efficiency of EGFP-positive colony formation by 2°NPCs as measured by counting cell nuclei before and after doxycycline addition. Figure 2S schematically shows the time to generate iPSCs from 2° _NPCs expressing _S2AK2AM . The induction of polyprotein expression by doxycycline was removed within the days shown.

Figures 3A through 3O illustrate the importance of Sox2 and Klf4 stoichiometry for _{S2AK2AM reprogramming} . Figure 3A shows a schematic diagram illustrating the three factor combinations S+ _K2AM , K+ _S2AM , M+ _S2AK , and S+K+M, where a plus sign indicates expression of a single (“monocistronic”) factor under a polycistronic factor or under other single (“monocistronic”) factors. Figures 3B-1 and 3B-2 show Sox2 and _Klf4 immunofluorescence-stained cells transduced with polycistronic _S2AK2AM and monocistronic S+K+M expression vectors (scale bar, 100 μm). The three single cells shown in the left figure are magnified and highlighted on the right. Figure 3B-1 shows Sox2 and Klf4 immunofluorescence-stained cells transduced with a single _{polycistronic S2AK2AM} expression vector (scale bar, 100 μm). Figure 3B-2 shows immunofluorescence staining of Sox2 and Klf4 cells transduced with the monocistronic S+K+M expression vector (scale bar, 100 μm). Figures 3C-1 and 3C-2 show scatter plots of Sox2 and Klf4 fluorescence intensity in single cells. The y and x axes represent the intensity of _Sox2 and Klf4, respectively, and each point represents one cell. RFU: relative fluorescence unit. Figure 3C-1 shows a scatter plot of Sox2 and Klf4 fluorescence intensity in single cells under the polycistronic _S2AK2AM expression vector. Figure _3C -2 shows a scatter plot of Sox2 and Klf4 fluorescence intensity in single cells under the monocistronic _S2AK2AM expression vector. Figure 3D schematically shows the number of EGFP-positive colonies in cell types transduced with _S2AK2AM , S+ _K2AM , K+ _S2AM , M+ _S2AK , and S+ _K +M. Figure 3E is a schematic diagram of the expression cassette used to add Sox2 (+Sox2) or Klf4 (+Klf4) expression within the S _2A K _2A M 2°MEF in Figure 3F. Figures 3F-1 to 3F-3 show scatter plots of Sox2 and Klf4 signal intensities in single cells of the control, +Sox2, and +Klf4 cell types shown in Figure 3E. The y and x axes represent the intensities of Sox2 and Klf4, respectively. Figure 3F-1 shows a scatter plot of Sox2 and Klf4 signal intensities in a single control cell. Figure 3F-2 shows a scatter plot of Sox2 and Klf4 signal in a single cell with added +Sox2. Figure 3F-3 shows a scatter plot of Sox2 and Klf4 signal in a single cell with added +Klf4. Equations shown in Figure 3F-1 are provided to indicate the diagonal distribution of cells. This equation is used to measure cells that tend to have high Sox2 or Klf4 under the +Sox2 and +Klf4 conditions shown in Figure 3E. The percentage of cells with high Sox2 and Klf4 is shown in Figures 3F-1 to 3F-3. RFU: Relative fluorescence unit. Figures 3G-1 and 3G-2 schematically show the Sox2 and Klf4 expression levels in cell lines expressing added Sox2 (+Sox2) or added Klf4 (+Klf4) on day 2. Figure 3G-1 schematically shows the Sox2 expression level in a cell line expressing added Sox2 (+Sox2) on day 2. Figure 3G-2 schematically shows the Klf4 expression level in a cell line expressing added Klf4 (+Klf4) on day 2. Figure 3H schematically shows endogenous Oct4 activation in +Sox2 and +Klf4 cells on day 4 when using the expression system shown in Figure 3E. Figure 3I schematically shows the number of EGFP-positive colonies per 8000 cells for +Sox2 and +Klf4 cell cultures on day 12 when using the expression system shown in Figure 3E. Efficiency for each cell type is shown. Figure 3J shows a schematic depiction of the following three polycistronic expression cassettes: _K2AM , _S2AM , _S2AK , and monocistronic expression cassette S+K. Figure 3K schematically shows the number of EGFP-positive colonies per 100,000 cells for the _K2AM , _S2AM , _S2AK , and S+K cell types as a measure of efficiency in iPSC production. Figure 3L schematically shows the expression of the pluripotency gene marker in _S2AK iPSCs. R1 mouse ESCs were used as controls. Figure 3M shows Oct4-EGFP colonies generated by _S2AK expression in situ and in passage 1 (scale bar, 100 μm). Data in Figures 3D, 3G, 3H, and 3I represent mean ± SD (n ≥ 3). p-values were determined by one-way ANOVA and Bonferroni post-hoc test. **p < 0.01. Figures 3N-1 to 3N-3 show the Sox2 and Klf4 signal intensities in single cells under the expression systems shown. Figure 3N-1 shows the Sox2 and Klf4 signal intensities in a single S+K _2A M cell type. Figure 3N-2 shows the Sox2 and Klf4 signal intensities in a single K+S _2A M cell type. Figure 3N-3 shows the Sox2 and Klf4 signal intensities in a single M+S _2A K cell type. The y-axis and x-axis represent the intensities of Sox2 and Klf4, respectively, 48 hours after doxycycline induction, and the dashed lines represent the thresholds for positive Sox2 and Klf4 staining signals. The numerical percentage of cells co-expressing Sox2 and Klf4 is also provided. RFU: Relative fluorescence unit. Figure 3O schematically shows the percentage of cells expressing both Sox2 and Klf4 in S _2A K _2A M, S+K _2A M, K+S _2A M, M+S _2A K, and S+K+M cultures (co-expression efficiency).

Figures 4A through 4I illustrate the identification of transcriptional conversions in MEF reprogramming and the converging trajectories in MEF and NPC reprogramming. Figure 4A shows a schematic diagram of RNA samples collected for RNA sequencing at different time points. Figure 4B shows a principal component analysis (PCA) of MEF reprogramming, illustrating the reprogramming process from MEF to iPSC. Data for samples from days 0 (heart), 2 (star), 4 (triangle), 8 (pentagon), 12 (rhombus), and iPSC/ESC (circle) are shown. Each sample, except for iPSC and ESC, has two replicates. Figure 4C shows a hierarchical clustering analysis of MEF reprogramming intermediates. Figure 4D shows a correlation analysis of MEF reprogramming intermediates. Two replicates were used for each time point. Figure 4E schematically shows the number of differentially expressed genes (DEGs) found between successive intermediates during MEF reprogramming. Figure 4F schematically shows a comparison of MEF and NPC reprogramming trajectories. Cells were projected onto the first two (dashed lines) or three principal components of principal component analysis (PCA). Circles and squares represent MEF and NPC reprogramming intermediates, respectively. Sample data at days 0 (heart), 2 (star), 8 (pentagon), 12 (diamond), and iPSC/ESC (circle) are shown. Each sample had two replicates except for iPSC and ESC. Figure 4G schematically illustrates the number of differentially expressed genes (DEGs) among intermediates from the same time points of MEF and NPC reprogramming. Figure 4H shows a schematic model of the convergence trajectories of MEF and NPC reprogramming over time. Figure 4I schematically illustrates the number of EGFP-positive colonies from EGFP-positive and EGFP-negative populations during MEF and NPC reprogramming. EGFP-positive and negative populations were sorted and re-plate-inoculated on day 6 for continued reprogramming.

Figures 5A through 5G illustrate the removal of MEF identity and activation of pluripotency networks during MEF reprogramming. Figure 5A shows the expression profiles of genes altered during the day 0/day 2 transcriptional transition. Upregulated and downregulated genes are further subgrouped based on their further expression changes. Gene numbers are shown in parentheses. Figures 5B-1 through 5B-3 show the downregulation of Thy1, Col6a2, and S100s4 on day 2 during MEF reprogramming. Figure 5B-1 shows the downregulation of Thy1 on day 2 during MEF reprogramming. Figure 5B-2 shows the downregulation of Col6a2 on day 2 during MEF reprogramming. Figure 5B-3 shows the downregulation of S100s4 on day 2 during MEF reprogramming. Figure 5C shows the expression profiles of genes upregulated during MEF reprogramming. The genes are further grouped according to the time of their first activation doubling. Activated pluripotency genes are listed on the right according to their activation time shown on the left. Figure 5D shows a heatmap illustrating the activation kinetics of pluripotent genes during MEF reprogramming. The highest level during reprogramming was set to 1 (100%) for normalization. Figure 5E schematically illustrates the activation of Oct4, Zfp296, and Lin28a/b as validated by qPCR at different reprogramming days. Figure 5F shows a correlation analysis of MEF and NPC reprogramming intermediates using 112 pluripotency-related genes. Cell populations from the same time points are highlighted with boxes. Figure 5G shows a schematic model of the convergent trajectories of MEF and NPC reprogramming. The original cell identity was removed during transcriptional conversion at day 0/day 2, and a pluripotency network was gradually established thereafter.

Figures 6A through 6M illustrate the collaboration between Sox2 and Klf4 to activate the pluripotency network in _{S2AK2AM reprogramming} . Figure 6A shows the de novo discovery of peak motifs bound by Sox2 and Klf4 in chromosome immunoprecipitation experiments. Figure 6B shows the distance analysis of Sox2 and Klf4 motifs in the Sox2 peak. Figure 6C shows the direct interaction between Sox2 and Klf4 as verified by co-immunoprecipitation in reprogramming MEF on day 2. Figure 6D shows a Venn diagram showing the overlap of Sox2 and Klf4 peak sites. Figure 6E shows a heatmap of the Sox2, Klf4, and H2K27 acetylated ChIP-seq signals for a specified peak group, sorted by the intensity of Sox2 in Sox_Klf and Sox_solo and by the intensity of Klf4 in Klf_solo. Figure 6F shows the quantification of the signal intensity of Sox2, Klf4, and H3K27 acetylated signals from the data in Figure 6E. Figures 6G-1 to 6G-3 show box plots illustrating gene expression associated with the Sox_Klf, Sox_solo, and Klf_solo peaks. Figure 6G-1 shows a box plot illustrating gene expression associated with the Sox_Klf peak. Figure 6G-2 shows a box plot illustrating gene expression associated with the Sox_solo peak. Figure 6G-3 shows a box plot illustrating gene expression associated with the Klf_solo peak. Figure 6H shows a Venn diagram illustrating the binding overlap of Sox2 under S _2A K _2A M and Sox2_tetO conditions. Figure 6I shows the de novo discovery of motifs with Sox2 binding peaks under Sox2_tetO conditions. Figure 6J shows the quantification of signal intensity of Sox2 and H3K27 acetylation in the Sox2 binding peaks of three different groups. Sox2_co represents the shared peak under S _2A K _2A M and Sox2_tetO conditions, Sox_SKM represents the peak specific to S _2A K _2A M reprogramming, and Sox_tetO represents the peak specific to the Sox2_tetO condition. In the upper right corner, for the top three plots, SKM (solid line) represents S _2A K _2A M reprogramming, and Sox2 (dashed line) represents the Sox2_tetO condition. In the lower right corner, for the bottom three plots, solid lines represent day 0 of reprogramming, and dashed lines represent day 2 of reprogramming. Figure 6K shows the Sox2 and Klf4 binding at the Oct4 enhancer along the Oct4 regulatory region of chromosome 17, as well as the H3K27 acetylation site. The positions of the superenhancer and ChIP-qPCR amplicon (a to i) are also shown. Figure 6L shows the Sox2 and Klf4 binding at the Oct4 enhancer as examined by ChIP-qPCR on day 2 of reprogramming, where a to i are shown in Figure 6K. Figure 6M shows the binding of Sox2 and Klf4 at the Oct4 enhancer as examined by ChIP-qPCR on day 5 of reprogramming, where a to i are shown in Figure 6K.

Invention Details

As described herein, in the absence of ectopic Oct4 expression, polycistronic Sox2, Klf4, and c-Myc are sufficient to establish pluripotency in several types of differentiated somatic cells. In some cases, c-Myc is not required. The stoichiometry of Sox2 and Klf4 is important for this reprogramming because disruption of factor balance leads to a significant reduction or failure of iPSC production. To optimize the stoichiometry of Sox2 and Klf4, a polycistronic expression cassette is described herein, comprising a promoter operatively linked to a nucleic acid segment encoding Sox2, Klf4, and optionally c-Myc. The nucleic acid segment may also contain one or more peptide linkers between the coding regions of Sox2, Klf4, and optionally c-Myc. For example, a 2A “self-cleaving” peptide can be used as a peptide linker between the coding regions of Sox2, Klf4, and optionally c-Myc. Such a linker provides cleavage between the Sox2, Klf4, and optionally c-Myc peptides. For example, an example of a polycistronic expression cassette may contain an open reading frame containing the coding regions of Sox2, Klf4, and c-Myc, wherein there are cleavable 2A peptide linkers between and within the Sox2 and Klf4 coding regions, and wherein there are 2A peptide linkers (referred to as S _2A K _2A M) between and within the Klf4 and c-Myc coding regions. Examples of cleavable linker sequences are provided herein.

"Klf polypeptide" refers to any of the following: a naturally occurring member of the Krüppel-like factor (Klf) family (i.e., a zinc finger protein containing an amino acid sequence similar to that of the Drosophila embryonic model regulator Krüppel), or a variant of a naturally occurring member that maintains similar transcription factor activity (at least 50%, 80%, or 90% of the activity) compared to a most recently associated naturally occurring family member, or a polypeptide that at least contains the DNA-binding domain of a naturally occurring family member and may also contain a transcriptional activation domain. See Dang, D.T., Pevsner, J. & Yang, V.W. Cell Biol. 32, 1103-1121 (2000). Exemplary Klf family members include Klf1, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, Klf16, and Klf17. Klf2 and Klf-4 have been found to be factors capable of generating iPS cells in mice, as have the associated genes Klf1 and Klf5, although with reduced efficiency. See Nakagawa, et al., Nature Biotechnology 26:101-106 (2007). In some embodiments, the variants have at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% amino acid sequence identity throughout their entire sequence compared to naturally occurring Klf polypeptide family members (e.g., compared to those listed above or, for example, those listed in GenBank). Klf peptides (such as Klf1, Klf4, and Klf5) can be derived from humans, mice, rats, cattle, pigs, or other animals. Typically, proteins from the same species will be used with the cell species being manipulated.

The Klf4 peptide can be used as a pluripotency factor encoded in a polycistronic expression cassette. For example, the Klf4 peptide used can have NCBI accession number CAX16088 (mouse Klf4), NP_004226.3 (GI: 194248077) (human Klf4), or NP_001300981.1 (GI: 930697457) (human Klf4). The sequence of human Klf4 accession number NP_004226.3 (GI: 194248077) is shown below as SEQ ID NO: 1.

SEQ ID NO: 1 The Klf4 polypeptide is encoded by cDNA, for example, NCBI accession number Klf4 NM_004235.6.

The sequence of human Klf4 accession number NP_001300981.1 (GI: 930697457) is shown below as SEQ ID NO: 2.

SEQ ID NO: 2 The Klf4 polypeptide is encoded by cDNA, for example, NCBI accession number Klf4 NM_001314052.2.

"Sox polypeptide" refers to any of the following: a naturally occurring member of an SRY-associated HMG-box (Sox) transcription factor characterized by the presence of a high-mobility group (HMG) domain, or a variant thereof that maintains similar transcription factor activity (at least within 50%, 80%, or 90% of the activity) compared to the most recently associated naturally occurring family member, or a polypeptide that at least contains the DNA-binding domain of a naturally occurring family member and may also contain a transcriptional activation domain. See, for example, Dang, D.T., et al., Int. J. Biochem Cell Biol. 32: 1103-1121 (2000). Exemplary Sox peptides include, for example, Sox1, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox-21, and Sox30. Sox1 has been shown to generate iPS cells with similar efficiency to Sox2, and the genes Sox3, Sox15, and Sox18 have also been shown to generate iPS cells, although with slightly lower efficiency than Sox2. See Nakagawa, et al., Nature Biotechnology 26:101-106 (2007). In some embodiments, the variants have at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% amino acid sequence identity throughout their entire sequence compared to naturally occurring members of the Sox polypeptide family (e.g., those listed above or, for example, those listed in Genbank). Sox polypeptides (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18) can be derived from humans, mice, rats, cattle, pigs, or other animals. Typically, proteins from the same species will be used with the cell species being manipulated. The Sox2 polypeptide can be used as a pluripotency factor encoded in a polycistronic expression cassette.

For example, the Sox2 polypeptide encoded in a polycistronic expression cassette can have accession number CAA83435 (human Sox2) and has the following sequence (SEQ ID NO: 3).

The Sox2 polypeptide is encoded by cDNA, for example, under NCBI accession number NM_003106.4.

"Myc polypeptide" refers to any of the following: a naturally occurring member of the Myc family (see, for example, Adhikary, S. & Eilers, M. Nat. Rev. Mol. Cell Biol. 6: 635-645 (2005)), or a variant thereof that maintains similar transcription factor activity (within at least 50%, 80%, or 90% of the activity) compared to a most recently related naturally occurring family member, or a polypeptide that at least contains the DNA-binding domain of a naturally occurring family member and may also contain a transcription activation domain. Exemplary Myc polypeptides include, for example, c-Myc, N-Myc, and L-Myc. In some embodiments, the variant has at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% amino acid sequence identity throughout its entire sequence compared to naturally occurring Myc polypeptide family members (e.g., compared to those listed above or, for example, those listed in Genbank). Myc peptides (e.g., c-Myc) can be derived from humans, mice, rats, cattle, pigs, or other animals. Typically, proteins from the same species will be used with the cell species being manipulated. Myc peptides can be pluripotency factors. For example, in some cases, a Myc peptide can be the human Myc peptide (human Myc) with accession number CAA25015, which has the following sequence (SEQ ID NO: 4).

The Myc polypeptide under SEQ ID NO: 4 is encoded, for example, by the nucleic acid portion under NCBI accession number X00196.1.

“Oct polypeptide” means any of the following: a naturally occurring member of the Octamer family of transcription factors, or a variant thereof that maintains similar transcription factor activity (at least within 50%, 80%, or 90% of the activity) compared to a most recently related naturally occurring family member, or a polypeptide that at least contains the DNA-binding domain of a naturally occurring family member and may also contain a transcription activation domain. Exemplary Oct polypeptides include Oct-1, Oct-2, Oct-3/4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11. For example, Oct3/4 (referred to herein as “Oct4”) contains a POU domain, which is a 150-amino acid sequence conserved in Pit-1, Oct-1, Oct-2, and uric-86. See Ryan, A.K. & Rosenfeld, M.G. Genes Dev. 11, 1207-1225 (1997). In some embodiments, variants of the Oct polypeptide family (e.g., those listed above or, for example, those listed in Genbank accession numbers NP002692.2 (human Oct4) or NP038661.1 (mouse Oct4)) have at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% amino acid sequence identity throughout their entire sequence. Oct polypeptides (e.g., Oct3/4) can be derived from humans, mice, rats, cattle, pigs, or other animals. Typically, proteins from the same species will be used with the cell species being manipulated. Oct polypeptides can be pluripotency factors.

An example of the Oct4 polypeptide sequence is available in the NCBI database under accession number NP002692.2 (human Oct4), shown below as SEQ ID NO: 5.

The cDNA nucleotide sequence of the human Oct4 polypeptide having SEQ ID NO: 5 is available in the NCBI database with accession number NM_002701.4 (GI: 116235483), which is shown below as SEQ ID NO: 6.

Nucleic acid segments encoding Sox2, Klf4, and optional c-Myc are linked together to form a larger polycistronic nucleic acid segment. As shown in this paper, the positions of the Sox2, Klf4, and optional c-Myc coding regions within the polycistronic nucleic acid can vary. In some cases, the Klf4 coding region is located at the 5' of the Sox2 and optional c-Myc coding regions. In other cases, the Sox2 coding region is located at the 5' of the Klf4 and optional c-Myc coding regions. In some cases, the cMyc coding region is not included in the polycistronic nucleic acid. Typically, the polycistronic nucleic acid is constructed so that the Sox2 and Klf4 peptides are expressed at approximately equal levels.

The cleavage site can be contained within a box between the regions encoding Sox2, Klf4, and optionally c-Myc. The cleavable peptide linker used between the Klf4, Sox2, and/or c-Myc coding regions can include, for example, 2A or LP4 sequences (deFelipe et al., Trends Biotechnol 24(2):68-75(2006); Sun et al. Processing and targeting of proteins derived from polyprotein with 2A and LP4/2A as peptide linkers in a maize expression system, PLOS(2017)).

Cleavable linkers can have a variety of sequences. The 2A-mediated “self-cleavage” mechanism involves ribosome skipping at the C-terminus of 2A to form a glycyl-prolyl peptide bond. Therefore, cleavable linkers can have Gly-Pro at their C-terminal linker. The conserved sequence GDVEXNPGP (SEQ ID NO: 7) (where X is any amino acid) is shared by different 2A linkers at its C-terminus and is necessary for generating steric hindrance and ribosome skipping.

The first 2A variant discovered was F2A (foot-and-mouth disease virus), followed by E2A (equine rhinitis virus), P2A (swine chezinvirus-12A), and T2A (thosea asigna virus 2A). The LP4 linker peptide is derived from a naturally occurring polyprotein found in the seeds of *Impatiens balsamina* and can cleave between the first and second amino acids during post-translational processing. Examples of cleavable linkers that can be used to link Sox2 and Klf4, along with optional c-Myc proteins, include (where an N-terminal GSG may be present but is not required in some cases):

P2A connector: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 8)

T2A connector: GSGEGRGSLL TCGDVEENPGP (SEQ ID NO: 9)

E2A connector: GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 10)

F2A connector: GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 11)

LP4 connector: SNAADEVAT (SEQ ID NO: 12)

LP4/2A connector: SNAADEVATQLLNFDLLKLAGDVESNPGP (SEQ ID NO: 13)

2Am1 connector: APVKQLLNFDLLKLAGDVESNPGP (SEQ ID NO: 14)

2Am2 connector: SGSGQLLNFDLLKLAGDVESNPGP (SEQ ID NO: 15)

Some examples of the amino acid sequence of the S _2A K _2A M polypeptide are shown below as SEQ ID NO: 16.

An example of the amino acid sequence of the S _2A K polypeptide is shown below as SEQ ID NO: 17.

Cell transformation

Polycistronic nucleic acid segments encoding Sox2, Klf4, and optionally c-Myc can be introduced into cells to promote cell transformation into stem cells (e.g., pluripotent stem cells) or other cell types. The nucleic acid segments encoding Sox2, Klf4, and optionally c-Myc can be inserted into or used with any suitable expression system. The polycistronic Sox2, Klf4, and optionally c-Myc nucleic acids can be part of an expression cassette or expression vector containing a promoter segment operatively linked to the nucleic acid segments encoding Sox2, Klf4, and optionally c-Myc.

Recombinant expression can be efficiently accomplished using vectors. Vectors include, but are not limited to, plasmids, viral nucleic acids, viruses, bacteriophage nucleic acids, bacteriophages, kinases, and artificial chromosomes. Vectors may also contain other elements required for transcription (or translation if the vector contains marker genes or other protein-coding segments). Such expression cassettes and/or expression vectors can express sufficient amounts of Sox2, Klf4, and optionally c-Myc to improve the conversion of starting cells into stem cells or into cells of another phenotypic lineage.

Expression vectors and/or expression cassettes encoding polycistronic Sox2, Klf4, and optionally c-Myc may contain promoters for driving the expression (transcription) of polycistronic Sox2, Klf4, and optionally c-Myc. The vector may contain a promoter operatively linked to a polycistronic nucleic acid segment encoding Sox2, Klf4, and optionally c-Myc. Expression may include transcriptional activation, wherein transcription is increased by 10-fold or more, 100-fold or more, or, for example, 1000-fold or more, compared to basal levels in target initiation cells.

The term "vector" as used herein refers to any vector containing exogenous DNA. Therefore, a vector is an agent that transports exogenous nucleic acids into cells without degradation and includes a promoter that produces the expression of polycistronic Sox2, Klf4, and optionally c-Myc in the cells to which it is delivered. A variety of prokaryotic and eukaryotic expression vectors are suitable for carrying, encoding, and/or expressing polycistronic Sox2, Klf4, and optionally c-Myc mRNA. Such expression vectors include, for example, TetO-fuw, pET, pET3d, pCR2.1, pBAD, pUC, viral, and yeast vectors. Vectors can be used, for example, in various in vivo and in vitro settings. For example, some experimental work shown herein involves the use and modification of the TetO-FUW vector.

The expression cassette, expression vector, and sequence within the cassette or vector can be heterologous. The promoter and/or other regulatory segments can be heterologous to the polycistronic segments encoding Sox2, Klf4, and optionally c-Myc.

As used in this article, the term "heterologous" refers to an expression cassette, expression vector, regulatory sequence, promoter, or nucleic acid that has been manipulated in some way. For example, a heterologous promoter can be a promoter that is not naturally linked to a target nucleic acid segment, or a promoter that has been introduced into cells through a cell transformation procedure. Heterologous nucleic acids or promoters also include nucleic acids or promoters that are naturally present in an organism but have been altered in some way (e.g., placed in a different chromosomal location, mutated, added in multiple copies, linked to a non-natural promoter or enhancer sequence, etc.).

Heterologous coding regions can be distinguished from endogenous coding regions, for example, when a heterologous coding region is linked to a nucleotide sequence containing a regulatory element (e.g., a promoter to which no natural association has been found with the coding region), or when a heterologous coding region is linked to a chromosomal portion that does not exist in nature (e.g., a gene expressed at a locus whose protein is not normally expressed). Similarly, a heterologous promoter can be a promoter linked to a coding region to which it is not naturally associated.

Useful viral vectors include those associated with lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia virus, poliovirus, AIDS virus, neurotrophic viruses, Sindbis virus, and other viruses. Additionally, any family of viruses possessing these viral characteristics that make them suitable for use as vectors is also available. Useful retroviral vectors include those described in: Verma, I.M., Retroviral vectors for gene transfer. In MICROBIOLOGY-1985, AMERICAN SOCIETY FOR MICROBIOLOGY, pp. 229-232, Washington, (1985). For example, such retroviral vectors may include Murine Maloney Leukemia virus (MMLV) and other retroviruses expressing the desired characteristics. Typically, viral vectors contain non-structural early genes, structural late genes, RNA polymerase III transcripts, inverted terminal repeats necessary for replication and capsidation, and promoters that control viral genome transcription and replication. When modified into vectors, viruses usually remove one or more of the early genes, and a gene or gene/promoter cassette is inserted into the viral genome to replace the removed viral nucleic acid.

Expression cassettes and/or expression vectors may contain a variety of regulatory elements, including promoters, enhancers, translation initiation sequences, transcription termination sequences, and other elements.

A promoter is typically one or more sequences of DNA that function when located at a relatively fixed position relative to the transcription start site. For example, a promoter may be located upstream of the coding regions of Sox2, Klf4, and (optionally) c-Myc. A promoter contains the core elements required for the basic interactions of RNA polymerase and transcription factors, and may include upstream and responsive elements. An enhancer is typically a DNA sequence that functions at no fixed distance from the transcription start site, and may be located at the 5' or 3' of the transcription unit. Furthermore, enhancers can be within introns and within the coding sequence itself. They are typically 10 to 300 bases long and function in cis. Enhancers function to boost transcription from a nearby promoter. Like promoters, enhancers typically also contain responsive elements that mediate transcriptional regulation. Enhancers often define the regulation of expression.

Expression vectors used in eukaryotic host cells (e.g., animal, mammalian, or nucleated cells) may also contain sequences that affect mRNA expression and are essential for terminating transcription. For mRNA, these regions are transcribed into polyadenylated segments within the untranslated portion of the mRNA encoding tissue factor proteins. The 3' untranslated region also contains a transcription termination site. The identification and use of 3' untranslated regions containing polyadenylation signals in expression constructs has been well-established.

Expression of Sox2, Klf4, and optionally c-Myc from polycistronic expression cassettes or expression vectors can be controlled by any promoter capable of expression in prokaryotic or eukaryotic cells. Such promoters can include universally active promoters, inducible promoters, or developmentally regulatory promoters. Universally active promoters include, for example, the CMV-β-actin promoter. Inducible promoters can include promoters active in a specific cell population or promoters responsive to the presence of drugs such as tetracycline or doxycycline. Some examples of prokaryotic promoters that can be used include, but are not limited to, SP6, T7, T5, tac, bla, trp, gal, lac, or maltose promoters. Some examples of eukaryotic promoters that can be used include, but are not limited to, constitutive promoters, such as viral promoters like CMV, SV40, and RSV promoters, and regulatory promoters, such as inducible or repressive promoters like the tet promoter, hsp70 promoter, and CRE-regulated synthetic promoters. Vectors for bacterial expression include pGEX-5X-3, and vectors for eukaryotic expression include pCIneo-CMV.

Expression cassettes or vectors may contain a nucleic acid sequence encoding a marker product. This marker product is used to determine whether a gene has been delivered to a cell and is expressed once delivered. Preferred marker genes are fluorescent proteins, such as red fluorescent protein, green fluorescent protein, and yellow fluorescent protein. The *E. coli* lacZ gene can also be used as a marker. In some embodiments, the marker may be a selectable marker. When such a selectable marker is successfully transferred to a host cell, the transformed host cell can survive under selection pressure. There are two widely used distinct categories of selection protocols. The first category is based on cell metabolism and the use of mutant cell lines lacking the ability to grow independently of supplemental culture medium. The second category is dominant selection, which refers to selection protocols used for any cell type and do not require the use of mutant cell lines. These protocols typically use drugs to inhibit the growth of host cells. Those cells with the novel gene will express proteins that transfer drug resistance and will survive the selection. Some examples of such dominant selection use the drugs neomycin (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin (Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)).

Gene transfer can be achieved by direct transfer of genetic material, including but not limited to plasmids, viral vectors, viral nucleic acids, bacteriophage nucleic acids, bacteriophages, kinases, and artificial chromosomes, or by transferring genetic material into cells or vectors (e.g., cationic liposomes). Such methods are well known in the art and readily adaptable to the methods described herein. Transfer vectors can be any nucleotide construct (e.g., plasmids) used to deliver genes into cells, or as part of a general strategy for gene delivery, such as as part of a recombinant retrovirus or adenovirus (Ramet et al. Cancer Res. 53: 83-88, (1993)). Suitable means for transfection, including viral vectors, chemical transfectants, or physical-mechanical methods such as electroporation and direct diffusion of DNA, have been described, for example, by Wolff, J.A. et al., Science, 247, 1465-1468, (1990); and Wolff, J.A. Nature, 352, 815-818, (1991).

For example, polycistronic Sox2, Klf4, and optional c-Myc nucleic acid segments, expression cassettes, and/or vectors can be introduced into cells by any method, including but not limited to calcium-mediated transformation, electroporation, microinjection, lipid transfection, particle bombardment, etc. Cells can be expanded in cultures and subsequently administered to subjects, such as mammals, like humans. The amount or number of cells administered can vary, but can be used in quantities ranging from about ^10⁶ to about ^10⁹ cells. Cells are typically delivered in physiological solutions such as saline or buffered saline. Cells can also be delivered in groups of carriers such as liposomes, efflux bodies, or microcapsules.

Polycistronic expression cassettes and/or expression vectors encoding Sox2, Klf4, and optionally c-Myc can be introduced into starter cells or any cells subjected to the methods described herein. For example, cells can be contacted with viral particles containing the expression cassettes. Retroviruses and/or lentiviruses, for example, are suitable for the expression of Sox2, Klf4, and optionally c-Myc. Commonly used retroviral vectors are “deficient,” meaning they do not produce the viral proteins required for generating sexual infection. Instead, vector replication requires growth in packaging cell lines. To produce viral particles containing the target nucleic acid, retroviral nucleic acid containing the target nucleic acid is packaged into a viral capsid via a packaging cell line. Different packaging cell lines provide different envelope proteins to be incorporated into the capsid, which determine the cell specificity of the viral particles. Envelope proteins are of at least three types: tropophilic, amphiphilic, and heterophilic. Retroviruses packaged with sympathophilic envelope proteins, such as MMLV, are capable of infecting most mouse and rat cell types and are produced using sympathophilic packaging cell lines such as BOSC23 (Pear et al. (1993) Proc. Natl. Acad. Sci. 90: 8392-8396). Retroviruses carrying amphophilic envelope proteins (e.g., 4070A) (Danos et al., ibid.) can infect most mammalian cell types, including humans, dogs, and mice, and are produced using amphophilic packaging cell lines such as PA12 (Miller et al. (1985) Mol. Cell. Biol. 5: 431-437), PA317 (Miller et al. (1986) Mol. Cell. Biol. 6: 2895-2902), and GRIP (Danos et al. (1988) Proc. Natl. Acad. Sci. 85: 6460-6464). Retroviruses packaged with heterophilic envelope proteins (e.g., AKR env) can infect most mammalian cell types except mouse cells. Appropriate packaging cell lines can be used to ensure that target cells are targeted by the packaged viral particles. Suitable methods for introducing a retroviral vector containing an expression cassette into a packaging cell line and for collecting viral particles produced by the packaging cell line are well known in the art.

Polycistronic expression cassettes and/or expression vectors encoding Sox2, Klf4, and optionally c-Myc can be integrated into the cell's genome, or polycistronic expression vectors can be kept free for the period of time required to redirect cells into stem cell lineages. The free introduction and expression of pluripotency factors is desirable because the mammalian cell genome is not altered by the insertion of free vectors, and because free vectors are lost over time. Therefore, using free expression vectors allows for short-term expression of pluripotency factors required to convert non-pluripotent mammalian cells into pluripotent cells, while avoiding potential chromosomal mutations and subsequent pluripotency factor expression during the desired differentiation into another cell type.

Free plasmid vectors having polycistronic expression cassettes encoding Sox2, Klf4, and optionally c-Myc can be introduced into mammalian cells, as described, for example, in: Yu et al., Human induced pluripotent stem cells free of vector and transgene sequences, Science 324(5928):797-801(2009); U.S. Patent Application 20120076762, and Okita et al., A more efficient method toogenerate integration-free human iPS cells, NATURE METHODS 8:409-412(2011), the contents of which are expressly incorporated herein by reference in their entirety.

For example, a polycistronic expression cassette can be included, and Sox2, Klf4, and optional c-Myc can be expressed from a free vector having EBNA-1 (Epstein-Barr nuclear antigen-1) and oriP or Large T and SV40ori sequences, such that the vector can exist and replicate freely without being incorporated into the chromosome.

Polycistronic expression cassettes and/or vectors can be introduced into mammalian cells in the form of DNA, protein, or mature mRNA via techniques such as lipid transfection, binding to cell membrane-permeable peptides, liposome transfer/fusion, or microinjection. When in DNA form, vectors such as viruses, plasmids, or artificial chromosomes can be used. Some examples of viral vectors include retroviral vectors, lentiviral vectors (e.g., according to Takahashi, K. and Yamanaka, S., Cell, 126: 663-676 (2006); Takahashi, K. et al., Cell, 131: 861-872 (2007); Yu, J. et al., Science, 318: 1917-1920 (2007)), adenoviral vectors (e.g., Okita K, et al., Science 322: 949 (2008)), adeno-associated virus vectors, and Sendai virus vectors (Proc Jpn Acad Ser B Phys Biol Sci. 85: 348-62, 2009), the contents of which are incorporated herein by reference in their entirety. In addition, some examples of artificial chromosome vectors that can be used include human artificial chromosome (HAC), yeast artificial chromosome (YAC), and bacterial artificial chromosome (BAC and PAC) vectors. As plasmids, plasmids targeting mammalian cells can be used (e.g., Okita K, et al., Science 322:949 (2008)). Vectors can contain regulatory sequences, such as promoters, enhancers, ribosome-binding sequences, terminators, and polyadenylation sites, to enable the expression of pluripotency factors.

Initiating cells

The starting cells are targeted cells transformed by polycistronic Sox2, Klf4 and optional c-Myc expression cassettes or expression vectors.

The starting cell population can be derived from virtually any source and can be heterogeneous or homogeneous. The terms "selected cells" or "selected multiple cells" may also be used to refer to the starting cells. In some embodiments, the cells to be transformed, as described herein, are adult cells, including virtually any available adult cell type. For example, the cells can be autologous or allogeneic cells (relative to the subject to be treated or the subject of acceptable cells). In some cases, the starting cells are adult progenitor cells or adult somatic cells. In other embodiments, the starting cells include any type of cells derived from a newborn, including but not limited to newborn cord blood, progenitor cells, and tissue-derived cells (e.g., somatic cells). In some embodiments, the starting cell population does not include pluripotent stem cells. In other embodiments, the starting cell population may include pluripotent stem cells. Therefore, the starting cell population transformed by the polycistronic Sox2, Klf4, and optionally the c-Myc expression cassette or expression vector described herein can be virtually any live cell type, particularly somatic cell types.

As illustrated in this article, fibroblasts can be reprogrammed to cross lineage boundaries and directly transform into pluripotent stem cells. However, polycistronic expression cassettes and vectors can be used to transform initiating cells or initiate the transformation into another cell type. Multiple cell types from all three germ layers have been shown to be suitable for somatic cell reprogramming via genetic manipulation, including but not limited to liver and stomach cells (Aoi et al., Science 321(5889): 699-702(2008)); pancreatic β cells (Stadtfeld et al., Cell Stem Cell 2: 230-40(2008)); mature B lymphocytes (Hanna et al., Cell 133: 250-264(2008)); human dermal fibroblasts (Takahashi et al., Cell 131, 861-72(2007); Yu et al., Science 318(5854)(2007); Lowry et al., Proc Natl Acad Sci USA 105, 2883-2888 (2008); Aasen et al., Nat Biotechnol 26(11): 1276-84 (2008)); meningiocytes (Qin et al., J Biol Chem 283(48): 33730-5 (2008)); neural stem cells (DiSteffano et al., Stem Cells Devel. 18(5): (2009)); and neural progenitor cells (Eminli et al., Stem Cells 26(10): 2467-74 (2008)). Any starting cell can be transformed with the polycistronic Sox2, Klf4 and optional c-Myc expression cassettes or expression vectors described herein to initiate reprogramming into other cell types.

In some implementations, the starting cells can be transiently or continuously expressed by incubation under cell culture conditions, expressing Sox2, Klf4, and optionally c-Myc.

Reprogramming methods

The starting cells are treated under conditions and for a period of time sufficient to transform them across lineages and/or differentiation boundaries to form stem cells, particularly pluripotent stem cells or dedifferentiated stem cells that may be incompletely pluripotent. This process is called “reprogramming.” In some cases, the pluripotent stem cells or dedifferentiated cells thus formed can differentiate into other cell types (e.g., neural, cardiac, pancreatic, liver, and other cell types, or progenitor cells of such cells).

The time required to convert starting cells into induced pluripotent stem cells or dedifferentiated stem cells that can be incompletely pluripotent can vary. For example, starting cells can be incubated until stem cell markers are expressed. Such stem cell markers may include Nanog, SSEA1, Oct4, and combinations thereof. In another instance, starting cells can be incubated until markers for different cell types are expressed. In some cases, the incubation of starting cells may be sufficient to form teratomas containing all three germ layers, or may be sufficient to produce chimeric mice.

Therefore, the time required to convert the starting cells into induced pluripotent stem cells can vary. For example, the starting cells can be incubated under cell culture conditions for at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days, or at least about 11 days, or at least about 12 days, or at least about 13 days, or at least about 14 days, or at least about 15 days, or at least about 16 days, or at least about 17 days, or at least about 18 days, or at least about 19 days.

In some implementations, the stem cells thus formed can be expanded or further incubated under cell culture conditions for about 5 to about 35 days, or about 7 to about 33 days, or about 10 to about 30 days, or about 12 to about 27 days, or about 15 to about 25 days, or about 18 to about 23 days.

The embodiments illustrate some experiments conducted and results obtained during the development of this invention.

Example 1: Materials and Methods

This embodiment illustrates some of the materials and methods used in developing the present invention.

Cell culture

HEK293T/17 cells (female) were cultured in DMEM (Invitrogen) supplemented with 10% FBS.

Mouse embryonic fibroblasts (MEFs) were prepared from E13.5 embryos (mixed sex, combining male and female embryos to generate primary cells), and mouse tail tip fibroblasts (TTFs) (male) were derived from 14-month-old adult male mice. MEFs and TTFs were cultured in MEF medium (DMEM supplemented with 10% FBS and non-essential amino acids (NEAA, Invitrogen)).

Mouse primary neural progenitor cells (NPCs) were prepared from the heads of E13.5 embryos (mixed sexes, combining male and female embryos to generate primary cells) and maintained on plates coated with Matrigel (BD, 356231) in NPC medium (Neuralbasal medium (Invitrogen), 2% B27 (Invitrogen), 1% GlutaMAX ^™ (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 2 μg/ml heparin (Sigma Aldrich), 20 ng/ml bFGF (Thermo Fisher Scientific), and 20 ng/ml EGF (R&D)).

Mice in ESC (male) and iPSC (male) were maintained on a feeder in ESC medium containing 5% ES-FBS (Invitrogen) and 15% Knock Out serum substitute (KSR, Invitrogen), 1% GlutaMAX ^™ , 1% NEAA, 0.1 mM 2-mercaptoethanol (Sigma Aldrich), 10 ng/ml leukemia inhibitory factor (LIF, Millipore), 3 μM CHIR99021 (Selleck), and 1 μM PD0325901 (Selleck).

For microinjection, male iPSCs were maintained under N2B27 conditions without a feeder layer (50% DMEM/F12 (Invitrogen), 50% Neurobasal medium, 0.5% N2 (Invitrogen), 1% B27, 0.1 mM 2-mercaptoethanol, 10 ng/ml LIF, 25 μg/ml BSA (Invitrogen), 3 μM CHIR99021 and 1 μM PD0325901).

mice

OG2 mice (B6; CBA-Tg(Pou5f1-EGFP)2 Mnn/J) (male and female) were obtained from Jackson Laboratory (004654). CD-1 (ICR) mice (male and female) were obtained from Charles River (#022). OG2 mice were crossed to obtain OG2 MEF and NPC in the resulting embryos at day 13.5 of embryonic development. 14-month-old male OG2 mice were used for TTF derivatization.

Superovulated female CD1(ICR) mice were mated with CD1(ICR) males for blastocyst preparation and further microinjection experiments. Tetraploid E13.5 embryos were used for secondary MEF and NPC derivatization.

All animal procedures were approved by the Institutional Animal Care and Use Committee of Tsinghua University (Beijing) and the Institutional Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences (Beijing).

plasmid construction

The plasmids produced in this study are listed in Table 1.

Table 1: Generated plasmids. Related to the STAR method.

TetO-FUW-OSKM (catalog number 20321), TetO-FUW-Oct4 (catalog number 20323), TetO-FUW-Sox2 (catalog number 20326), TetO-FUW-Klf4 (catalog number 20322), TetO-FUW-c-Myc (20324), and FUW-M2rtTA (catalog number 20342) were all derived from Addgene. See also Brambrink et al. Cell Stem Cell 2: 151-159 (Feb. 2008). All plasmids in this study were based on the TetO-FUW backbone. For cloning, the backbone was digested with appropriate enzymes, and each insert (e.g., the coding regions of Sox2, Klf4, and c-Myc) was recovered by gel extraction. All inserts were amplified by PCR using KOD XtremeHS polymerase (Novagen, 71975-3) and ligated into polycistronic expression cassettes using T4 ligase or Gibson assembly master mix (NEB, E2611). All plasmids were identified by enzyme digestion and sequencing.

Virus preparation and transduction

For lentivirus preparation, HEK293T cells were seeded on plates one day in advance to achieve approximately 70% confluence for transfection. VSV-G envelope expression plasmids pMD2.G (Addgene, 12259) and psPAX2 (Addgene, 12260) were used for lentivirus packaging. For each well in a six-well plate, 1.8 μg of the plasmid containing the target gene was mixed with 1.35 μg of psPAX2 and 0.45 μg of pMD2.G, and transfection was performed using 3000 reagent (Thermo Fisher Scientific, L3000). After five to eight hours, the medium was replaced with fresh MEF medium. At 48 hours, the virus-containing supernatant was harvested, passed through a 0.45 μM filter to remove cell debris, and mixed with 1 volume of fresh medium for immediate use.

For infection, mouse embryonic fibroblasts (MEF) or neural progenitor cells (NPC) were incubated with lentiviral supernatant for 8 hours or overnight in the presence of 5 μg/ml Millipore. After infection, the culture medium was changed back to MEF or NPC medium to allow the cells to recover.

Mouse embryonic fibroblast derivatives

E13.5 embryos were used for MEF derivation. After embryo recovery, the head, limbs, and internal organs, especially the gonads, were removed under a dissecting microscope. The remaining embryonic body was finely minced with two blades and digested in 0.05% trypsin-EDTA for 15 minutes. MEF medium was then added to stop trypsin digestion. Further dissection of the tissues was performed by several up-and-down pipetting and aspiration. Cells were then collected by centrifugation and plated onto 15 cm culture dishes for amplification (generation 0, P0). All tests were performed using MEF up to generation 4.

Derivatives of mouse neural progenitor cells

One day prior to the experiment, poly-D-lysine (PDL)/lamin-coated plates were prepared for NPC culture. Briefly, 12-well plates were filled with PDL (10 μg/ml in distilled water) and incubated overnight at 37°C. The next day, the solution was removed from the wells. The wells were then washed three times with distilled water and air-dried. Laminin (5 μg/ml in distilled water) was then added and incubated overnight at 37°C for 4 hours. Laminin was removed from the wells before using the plates.

E13.5 embryos were used for NPC derivatization. The embryos were decapitated using dissecting forceps. The skin and skull were peeled back from the head to expose the brain. The entire brain was removed using curved forceps and placed in cold DPBS. After rinsing twice with DPBS, the brain was placed in a 35 cm dish and finely minced with sharp scissors. The minced tissue was transferred to a 15 ml centrifuge tube and digested with 1 ml of 0.05% trypsin-EDTA at 37°C for 7 minutes. To stop the enzymatic reaction, 5 ml of NPC medium was added to the tube, followed by centrifugation and removal of the supernatant. The tissue particles were further separated by pipetting several times with 1 ml of NPC medium and filtered through a 70 μm cell filter. The cells were then placed in 12-well plates coated with PDL/laminusoids and cultured in NPC medium for several days. During culture, the NPCs proliferated and separated from the plate to form floating neurospheres (P0). The spheres were then collected and digested into individual NPCs using StemPro Accutase (Thermo Fisher Scientific). From then on, NPCs were attached to substrate-coated plates and cultured for subsequent passages. All tests were conducted using NPCs up to generation 4.

Derivatives of mouse tail tip fibroblasts

For the derivation of tail tip fibroblasts (TTF), 14-month-old adults were used. The tails were skinned, cut into 1 mm pieces, and cultured in 60 cm dishes. Half of the culture medium was replaced every 3 days until the fibroblasts migrated from the transplanted pieces. The cells were then passaged and ready for use (P1).

iPSC reprogramming and derivatives

Oct4-GFP (OG2) MEF or TTF were seeded at a density of 10,000 cells/ ^cm² onto gelatin-coated plates. After transduction, cells were allowed to recover in MEF medium for 24 to 36 hours. Unless otherwise specified, cells were then re-seeded at a density of 10,000 cells/ ^cm² . For NPC, cells were seeded at 5,000 cells/ ^cm² onto six-well plates coated with poly-D-lysine (PDL)/laminusoids. After transduction, cells were allowed to recover in NPC medium for 24 to 36 hours. To begin reprogramming, the culture was transferred to reprogramming medium containing 1 μg/ml doxycycline (ESC medium without Chirr99021 and PD0325901). Doxycycline was used to induce protein expression from the polycistronic expression cassette. The introduction of doxycycline is indicated as day 0. The medium was changed every other day for the first 10 days throughout the process, and daily thereafter. From day 10, use ESC medium with 1 μg/ml doxycycline. EGFP-positive colonies are typically counted on day 12 and prepared for iPSC derivatization on day 16.

For iPSC line derivation, the reprogrammed culture was incubated with 1 mg/ml collagenase B (Roche) at 37°C for 20 minutes. Individual colonies were picked under a microscope and digested in 0.05% trypsin for 5 to 10 minutes to obtain a single-cell suspension. The cells were then seeded on a feeder layer in normal ESC medium, and these cells were considered generation 0 (P0) iPSCs.

Evaluation of EGFP-positive colony efficiency

To accurately calculate EGFP-positive colony efficiency, 2° MEF or NPC cells were seeded into 48-well plates. After 24 hours (day 0), half of the wells were stained with Heochest 33342 (Thermo Fisher Scientific), and the exact number of cells in each well was recorded by counting the stained nuclei. The other half of the cells were transferred to reprogrammed medium containing 1 μg/ml doxycycline for further culture. The medium was changed every other day during the experiment, and EGFP-positive colonies were counted on day 12. The final efficiency was calculated by dividing the number of EGFP-positive colonies by the initial cell count recorded on day 0.

Another method was also used. Single cells were seeded into 96-well plates with a feeder layer. On the second day, the MEF medium was converted to reprogramming medium with 1 μg/ml doxycycline (day 0). During the reprogramming process, the medium was changed every 4 days, and the number of EGFP-positive colonies was counted on day 16. Efficiency was calculated by dividing the total number of EGFP-positive colonies by the number of wells.

Blastocyst microinjection

iPSCs were cultured under N2B27 conditions without a feeder layer. On the day of injection, the cells were suspended in blastocyst injection medium (25 mM HEPES-buffered DMEM with 10% FBS, pH 7.4).

To generate chimeric mice, superovulated female CD1(ICR) mice (4 weeks old) were mated with CD1(ICR) males. Morulae (2.5 days post-coitum) were collected and cultured overnight in KSOM (Millipore) medium at 37°C and 5% _CO2 . The following morning, blastocysts were prepared for iPSC injection, with approximately 10 cells injected into each blastocyst. The injected blastocysts were cultured in KSOM medium at 37°C and 5% CO2 for 1 to ₂ hours and then implanted into the uterus of pseudopregnant CD1(ICR) female mice 2.5 days post-coital.

For tetraploid complementation assays, two CD1 (ICR) embryos at different cell stages were electrofused to produce tetraploid embryos, and approximately 10 iPSCs were injected into the reconstructed tetraploid blastocysts. The injected blastocysts were cultured in KSOM medium at 37°C and 5% _CO2 for 1 to 2 hours and then implanted into the uterus of pseudopregnant CD1 (ICR) female mice 0.5 days post-mating. E13.5 embryos were dissected to produce secondary MEFs and NPCs (2° MEF and NPC).

The contribution of the gonads was restored in the injected embryos 13 days post-implantation (E13.5). The gonadal region of each embryo was collected and the EGFP signal was visualized under a microscope.

Inspection of the secondary reprogramming system

To verify the induction of reprogramming factors, 2°MEF and NPC cells were seeded at a density of 20,000 cells/ ^cm² in 24-well plates. After culturing in reprogramming medium containing 1 μg/ml doxycycline for 48 hours, the cells were fixed for immunofluorescence staining to detect the expression of Sox2 and Klf4.

To test the effect of initial cell density on final reprogramming efficiency, 2°MEF and NPC cells were seeded on feeder layers in 12-well plates at densities of 500 cells/well, 1,000 cells/well, 2,000 cells/well, and 4,000 cells/well, respectively. Cells were reprogrammed as previously described. On day 12 of reprogramming, the number of EGFP-positive colonies was counted under a fluorescence microscope.

To validate the doxycycline requirement during reprogramming, 2°MEF and NPC were seeded at a density of 1,000 cells/well on feeder layers in 12-well plates. Doxycycline was removed from the reprogramming medium from day 0 to day 12. EGFP-positive colony counts were performed on day 16.

To test the reprogramming kinetics with small molecules, 2°MEF and NPC were seeded at a density of 1,000 cells/well on feeder layers in 12-well plates. Cells were cultured for 12 days in reprogramming medium containing 1 μg/ml doxycycline, 1 μM A83-01, and 10 μM pharyngin. Cell morphology was recorded for reprogramming kinetics. All conditions were performed in triplicate.

Reprogramming of early EGFP-positive cells

Secondary (2°) MEF and NPC cells were seeded onto feeder layers and reprogrammed as previously described. On day 6 of reprogramming, EGFP-positive and EGFP-negative cells were sorted by flow cytometry, and the same number of cells were re-seeded into new 6-well plates with feeder layers. Cells were cultured for another 6 days in reprogramming medium containing 1 μg/ml doxycycline, and the number of EGFP-positive colonies was counted.

Teratoma formation

To generate teratomas, iPSCs maintained on feeder layers were transferred to a matrix gel-coated plate and cultured in ESC medium without Chirr99021 and PD0325901. The iPSCs were then trypsin-digested and suspended in medium containing 2% matrix gel. 1.0 × ^10⁶ cells were then subcutaneously injected into the hind limbs of SCID mice. Five weeks post-injection, the tumors were dissected and fixed in 4% polyformaldehyde (Sigma Aldrich), followed by paraffin sectioning and hematoxylin-eosin (HE) staining.

Bisulfite sequencing

Bisulfite treatment was performed precisely according to the protocol provided for cell culture using the EpiTect Bisulfite kit (Qiagen, 59104). The recovered DNA was amplified by two rounds of PCR using primers targeting the Oct4 promoter, and the PCR products were ligated into the T-vector pMD20 (Clontech, 3270). Ten randomly selected clones were sequenced. The PCR primers used are listed in Table 2.

Karyotype analysis

Karyotype analysis of iPS cell lines was performed in Cell Line Genetics by analyzing Giemsa binding (Meisner and Johnson, 2008). Briefly, actively dividing iPSCs were blocked in metaphase with 0.1 μg/ml colchicine. The iPSCs were then trypsinized into single cells using 0.05% trypsin-EDTA. The iPSCs were resuspended in a hypotonic KCl solution (0.075 M) and swelled by gentle rotation and incubation at room temperature for 20 min. Subsequently, the iPSCs were fixed in a fixative (methanol to acetic acid v/v ratio 3:1), and slides were prepared for karyotype analysis.

Flow cytometry

Depending on cell density, reprogrammed cells were treated with 1 mg/ml collagenase B for 10 to 30 minutes, followed by trypsin digestion with 0.05% trypsin for 5 minutes. Cells were then resuspended in culture medium and filtered through a 40 μm cell filter. Flow cytometry analysis or sorting was performed on a BD FACS Aria III. Treatment with collagenase B, filtration, and sorting typically resulted in a 30- to 50-fold reduction in the production of EGFP-positive colonies. All data were analyzed using FlowJo v10.

Western Imprint and Quantitative Analysis

Cell lysate samples or immunoprecipitation (IP) samples were loaded onto a 10% SDS-PAGE gel for separation and subsequently transferred to a 0.45 μm nitrocellulose membrane (BioRad, 1620115). The following antibodies were used for immunoblotting (IB): anti-Oct4 (Abcam ab19857), anti-Sox2 (Millipore AB5603 for IP, Abcam ab79351 for IB), anti-Klf4 (Stemgent 09-0021), and anti-actin (Santa Cruzsc-47778).

Immunoprecipitation

Secondary MEF (10,000 cells/ ^cm² ) plates were seeded onto gelatin-coated 10cm dishes and cultured for 2 days in reprogrammed medium containing 1 μg/ml doxycycline. Cells were lysed on ice for 20 min with 500 μL of ice-cold IP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% NP-40, and 1.5 mM EDTA). Protein A dynabead slurry (20 μL, Life Sciences Technologies, 10001D) was used for each IP assay. Target and co-IP proteins were eluted with SDS sample buffer for direct Western blotting.

Immunofluorescence staining and image analysis

Cells were washed three times with DPBS and fixed with 4% PFA at 4°C for 30 minutes. Donkey serum (10% in DPBS) with 1% BSA was used for blocking at 4°C for 1 hour. Triton-X100 (0.3%) was added during blocking to stain for nuclear localization proteins. Antibodies were diluted in DPBS with 1% BSA. The following primary antibodies were used for staining: anti-Sox2 (Millipore, AB5603; Abcam, ab79351), anti-Klf4 (Stemgent, 09-0021; R&D, AF3158), anti-c-Myc (epitomics, 1472-1), anti-Nanog (Abcam, 80892), and anti-SSEA-1 (Stemgent, 09-0095).

Single-field imaging was performed using a fluorescence microscope (IX83, Olympus); images were acquired and analyzed using a CellSens Dimension. For multi-field imaging and analysis, cell culture plates were scanned using an automated microscope (Lionheart FX, BioTek). Images were stitch-together and analyzed using Gen5 software.

RNA extraction

For cultured cells, samples were lysed and total RNA was extracted using the RNeasy Plus mini kit (Qiagen, 74136) and QiaShredder (Qiagen, 79656) according to the manufacturer's instructions. For sorted cells, samples were collected at specified time points and lysed in TRIzol ^™ reagent (Invitrogen, 15596026). Total RNA was extracted following the procedure below. Linear acrylamide (Thermo Fisher Scientific, AM9520) was added to the lysed cell samples to enhance RNA precipitation. Chloroform was then added, and the mixture was vigorously shaken with the lysed samples to extract RNA. After centrifugation, the RNA dissolved in the aqueous phase was carefully transferred to an RNase-free tube and thoroughly mixed with 1 volume of isopropanol (Sigma Aldrich). The sample was then incubated overnight at -20°C to precipitate RNA. On the second day, the isopropanol was carefully removed after centrifugation, allowing the RNA to precipitate at the bottom of the tube. The RNA precipitate was then washed with 75% ethanol to remove any trace amounts of guanidine that may remain. Then, after centrifugation, remove the ethanol through the tip of a pipette and air dry for 10 minutes. Finally, if necessary, dissolve the total RNA in 20 μl of nuclease-free water by several up-and-down pipetting motions.

Quantitative PCR

To assess gene expression levels, total RNA was used for qPCR experiments. Genomic DNA elimination and reverse transcription were performed using the iScript cDNA Synthesis Kit (Bio-Rad), followed by qPCR on a CFX384 real-time PCR system (Bio-Rad) using the iQ ^™ SYBRGreen Supermix (Bio-Rad). All reactions were performed in quadruplicate. All data were statistically analyzed using built-in analysis methods in Prism 7.

RNA sequencing of reprogrammed MEF and NPC

Total RNA from samples taken at specified time points was sequenced. Sequencing libraries were generated using the Ultra ^™ RNA Library Prep Kit (NEB#E7530L) according to the manufacturer's instructions. A total of 2 μg RNA/sample was used as input for library preparation. Library fragments were purified using a QiaQuick PCR kit (Qiagen, 28106), quality-controlled using an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA), and quantified by qPCR. The library was then sequenced using an Illumina HiSeq 2500 platform, yielding 150 bp paired-end (PE150) reads.

Chromatin Immunoprecipitation

All ChIP experiments were performed using the EZ-ChIP Chromatin Immunoprecipitation Kit (Millipore, 17-371) with slight modifications to the protocol provided. Briefly, reprogrammed cells (approximately 1.0 × ^10⁷ ) from day 0 or day 2 were cross-linked in 20 ml of growth medium with 0.55 ml of 37% formaldehyde. 1 ml of 2.5 M glycine (20×) was added to quench any unreacted formaldehyde. Cells from each 15 cm plate were collected and resuspended in 830 μl of lysis buffer. Genomic DNA was then sheared to a length of 100–500 bp on a Covaris S220 sonicator under optimized conditions. For Sox2 or Klf4 ChIP, 1.0 × ^10⁷ reprogrammed cells and 10 μg of antibody were used for each experiment, and for H3K27acChIP, 5.0 × ^10⁶ reprogrammed cells and 2 μg of antibody were used. Finally, DNA fragments were recovered using NucleoSpin gel and a PCR Clean-up kit (MAGHEREY-NAGEL, 740609) and used for qPCR or library preparation. The primary antibodies used were: anti-Sox2 (Millipore, AB5603), anti-Klf4 (R&D, AF3158), and anti-H3K27ac (Abcam, ab4729).

Preparation of DNA libraries for sequencing

Sequencing libraries were generated according to the manufacturer's instructions using the Ultra™ II DNA Library Prep Kit (E7645S) for Illumina. In short, 4 ng of ChIP DNA and 40 ng of Input DNA were used for library preparation. NEBNext Multiplex Oligos (sets 1, 2, and 3) for Illumina were used for PCR amplification of the adaptor-ligated DNA. The library was purified using a Reagent kit (Beckman-Coulter, Inc., B23317), quality-controlled using a Bioanalyzer 2100, and quantified by qPCR. Sequencing was performed on an Illumina NextSeq 550AR using single-end 50 bp reads.

Statistical analysis

Statistical analysis was performed in GraphPad Prism 7. The value of n and significance were calculated using the method indicated in each legend. Data are expressed as mean ± SD. *p < 0.05; **p < 0.01; ns, not significant.

Comparison and processing of RNA-Seq data

Before alignment, low-quality reads and those containing adaptors or poly-N sequences are removed using FastQC. The remaining reads are then mapped to the assembled mm9 genome using the default parameters in the STAR (2.5.1b) aligner.

Clustering of RNA-Seq data

To cluster samples at different reprogramming time points, the Manhattan method was used to calculate the distance, and then hierarchical clustering was applied using hclust.

Differential gene expression analysis

Differentially expressed genes (DEGs) between two groups were identified using the DESeq2 R package (1.10.1). DESeq2 provides a statistical procedure for identifying differentially expressed genes from digital gene expression data using a negative binomial distribution-based model. The resulting p-values were adjusted using the Beniamini and Hochberg methods to control for false discovery rates. Genes identified by DESeq2 with adjusted p-values < 0.05 were designated as differentially expressed.

Principal component analysis

Principal component analysis (PCA) is performed in R using the R package gmodels (2.16.2). Fast.prcomp is used for efficient computation of principal components and singular value decomposition.

Ontology annotation

The DAVID 6.8 functional annotation bioinformatics tool was used to compute gene ontology (GO) enrichment of the DEG during reprogramming (see david.ncifcrf.gov). Items with a p-value < 0.05 were defined as significantly enriched.

Correlation analysis of SKM samples

The correlation of all RNA sequencing data between MEF or NPC samples at different reprogramming times was analyzed using pheatmap (1.0.10) in R. Corrplot (0.84) was used to analyze the correlation of 112 pluripotency-related genes between reprogrammed NPC and MEF.

Comparison and processing of ChIP-Seq data

ChIP-seq reads were aligned using Bowtie2 to construct mm9 from the mouse genome, and then filtered using smtools with a MAPQ (0.1.19) score to retain only reads with a MAPQ greater than 10 (Langmead et al., 2009). To identify ChIP-Seq enriched regions relative to the background, peak calling was performed using MACS2 (2.1.0) with the corresponding input DNA as a control for each sample (Zhang et al., 2008). Default parameters in MACS were used. The number of reads per million mapped reads (RPM) was calculated for each peak and its corresponding input control.

Sequence analysis

Collect FASTA sequences from peak regions identified from MACS and use them as input to the MEME-Chip motif-finding algorithm (maximum motif width = 30, assuming each sequence has an arbitrary number of motifs) (Machanick and Bailey, 2011).

Peak distribution analysis

Peak distribution was analyzed using the Genomic Regions Enrichment of Annotations Tool (GREAT) (McLean et al., 2010). For each peak, the minimum distance between the peak and a nearby transcription start site (TSS) was calculated (the negative distance of the upstream peak to the TSS). Distance distributions of peaks from different samples were compared. Bedtools was used to intersect peaks from Sox2 and Klf4 to identify co-localized (Sox_Klf) peaks, Sox_solo, and Klf_solo peaks.

Comparison of spectra

The Sox2, Klf4, and H3K27 acetylated ChIP-seq signals of the Sox_Klf, Sox_solo, and Klf_solo peaks were analyzed and quantitatively measured, sorted by the intensity of Sox2 in Sox_Klf and Sox_solo and by the intensity of Klf4 in Klf_solo. Ngsplots were used to create heatmaps and average profile plots around the centers of the three sets of peaks in Figures 6E and 6F (Shen et al., 2014).

Sox2 target gene analysis

Genes with TSS values within +/- 5 kb of the Sox_Klf, Sox_solo, and Klf_solo peaks were identified. The Mann-Whitney U test was performed to measure the statistical significance of differences in normalized reads among the gene groups.

Combined with spectral analysis

In IGV (2.4.10), the acetylation binding peaks of Sox2, Klf4, and H3K27 in the pluripotency-related region were enriched and visualized. ChIP-qPCR was further performed to detect the Sox2 and Klf4 binding characteristics from the first exon of Oct4 to the distal enhancer. Primers used for qPCR are listed in Table 2.

Table 2: PCR primer sequences

Data and code availability

The accession numbers for RNA-seq and ChIP-seq data are NCBI GEO: GSE98280.

Example 2: S _2A K _2A M reprograms fibroblasts into iPSCs

This example describes the precise and convenient control of stoichiometry of multiple factors at the single-cell level using a polycistronic expression cassette.

Polycistronic boxes were constructed between segments encoding reprogramming factors (e.g., Oct4, Klf4, Sox2, and/or c-Myc) using 2A peptide cleavage sequences (de Felipe et al., 2006). Multiple combinations of the two precursor factors were initially tested, and c-Myc (M) was included in all combinations because it is alleged to play a role in enhancing reprogramming efficiency through transcriptional amplification (Linet et al., 2012; Nie et al., 2012).

Therefore, the polycistronic Oct4, Sox2, and c-Myc ( _O2AS2AM ), Oct4, Klf4 _, and c-Myc ( _O2AK2AM ) _, and Sox2, Klf4 _, and c _- Myc ( _S2AK2AM ) were all derived from the previous _O2AS2AK2AM plasmid ( _Carey et al., 2009). These cassettes were transduced into mouse embryonic fibroblasts (MEFs) (Fig. 1A and 1J), and protein expression was assessed by Western blotting, confirming the high efficiency of polycistronic peptide processing (Fig. 1K to 1L).

Following widely used methods, the ability of three combinations to induce reprogramming in OG2 MEFs was first tested (Takahashi and Yamanaka, 2006). OG2 MEFs contain an EGFP reporter under the control of an endogenous Oct4 promoter, thus the EGFP signal can be used as a marker of reprogramming efficiency (Szabo et al., 2002). During the 2-week reprogramming period, EGFP-positive colonies were counted on days 4, 7, 10, and 14. Surprisingly, EGFP-positive colonies were observed on day 7 under the S _2A K _2A M condition, and by day 14, approximately 60 EGFP-positive colonies (0.06%) were generated per 100,000 starting MEFs (Figures 1B and 1M). This efficiency is higher than that observed under the O _2A S _2A M and O _2A K _2A M conditions, although still 10-fold lower than the efficiency under the O _2A S _2A K _2A M condition (Figure 1M).

_S2AK2A M _cells produced typical iPSC-like colonies, and iPSC lines originated from these colonies. When these cell lines were passaged in ESC medium, they formed ESC-like, domed colonies that remained positive for Oct4-EGFP even after 20 passages (Fig. 1C). Consistent with this stable marker expression, bisulfite sequencing showed that the Oct4 promoter was completely demethylated in these cells (Fig. 1D). Immunofluorescence analysis showed that these cells were positive for Nanog, Sox2, and SSEA1, and that overall gene expression was very similar to that of the R1 mouse ESC line (Figs. 1E, 1F _{, and 1N). These data indicate that a pluripotency network has been established in S2AK2A M} iPSCs.

The functional pluripotency of these cell lines was then tested by examining their ability to form teratomas and chimeras. _{The S2A K2A} M iPSCs were able to form teratomas containing all three germ layers and were successfully used to generate chimeric mice (Fig. 1G). The pluripotency of these cell lines was then tested using the most stringent method, tetraploid complementation assay (4N). For E13.5, normal viable embryos were restored, indicating that the iPSCs correctly differentiated into all tissues in vivo. EGFP signaling was observed in the gonadal regions of the embryos (Figs. 1H and 1I), indicating successful transmission to the germline.

Example 3: _{S2A K2A} M reprograms multiple differentiated cell types into pluripotent cells.

This embodiment describes an experiment illustrating the ability of _{S2A K2A} M to reprogram different cell types to form pluripotent stem cells.

OG2 neural progenitor cells (NPCs) expressing the NPC markers Nestin, Sox2, and Pax6 and forming neurospheres were transduced using _{S2A K2A} M and exposed to a similar reprogramming protocol. OG2 MEFs contain an EGFP reporter under the control of the endogenous Oct4 promoter, thus the EGFP signal can be used as a marker of reprogramming efficiency (Szabo et al., 2002). EGFP-positive colonies were obtained after 2 weeks, and stable iPSC lines were established (Figs. 10–1P), demonstrating that cells derived from the ectoderm can also be reprogrammed via _{S2A K2A} M.

Next, the more differentiated cell type, OG2 adult mouse tail tip fibroblasts (TTF), was examined. Similarly, iPSC lines were obtained from EGFP-positive colonies following the S _2A K _2A M transduction and reprogramming protocol, and their pluripotency gene expression was not different from that of R1 ESCs.

Example 4: S _2A K _2A M 2°MEF and NPC can be effectively reprogrammed to have pluripotency.

This embodiment describes _{S2A K2A} M-mediated reprogramming of MEFs and NPCs from embryos generated by _{S2A K2A} M iPSC 4N assay. These embryo-derived MEFs and NPCs are referred to as secondary _{S2A K2A} M cells (or _{S2A K2A} M 2°MEF and NPC) because these cells are 100% iPSC derived (Figure 2A).

These 2°MEF and NPC cells responded robustly to doxycycline. Sox2 and Klf4 proteins were readily detected 12 hours after induction (Fig. 2B). Immunostaining showed widespread expression of Sox2 and Klf4 in either 2°MEF or NPC cells after 24 hours (Fig. 2C), confirming that all cells originated from _{S2A K2A} MiPSCs.

The inventors then evaluated whether 2°MEF cells could be reprogrammed. After 2 days, all cells underwent dramatic morphological changes simultaneously, which became more pronounced on day 3 (Fig. 2D). As shown in Fig. 2E, upregulation of mesenchymal-to-epithelial transition (MET) genes, including Cdh1, EpCAM, Krt8, and Ocln, was observed. EGFP-positive cell clusters were observed on day 4, and iPSC-like colonies could be easily identified by day 10 (Figs. 2F and 2G), consistent with the upregulation of Oct4 and Nanog, although Nanog levels were relatively low on day 12, indicating that these EGFP-positive cells were not yet fully reprogrammed (Fig. 2H). With further culture, 2°NPC lines were established from these colonies (Figs. 2O to 2P). Reprogramming with 2°NPC occurred with similar kinetics, except that EGFP signaling was not observed until after 2 days, i.e., on day 6.

During MEF reprogramming, approximately 3% of cells were reprogrammed to form EGFP-positive colonies (Fig. 2I). This is comparable to the efficiency (2% to 4%) observed in another study of OSKM 2° reprogramming (Wernig et al., 2008). The inventors also tested whether higher efficiency could be achieved by optimizing culture conditions. First, two small molecules (trichosamine and A83-01) were used to promote reprogramming by activating cAMP production while inhibiting the TGF-β pathway. When trichosamine and A83-01 were added to the culture medium, a three-fold increase in EGFP-positive colonies was observed (Fig. 2I), while the general reprogramming kinetics remained unchanged (Fig. 2F). Second, the effect of cell density was tested. Higher cell densities in the culture were observed to significantly reduce reprogramming efficiency (Fig. 2J).

Optimized conditions were used, and the efficiency of generating EGFP-positive colonies was then precisely calculated. The exact cell counts were performed before reprogramming and after 12 days. As shown in Figure 2K, 15% of the cell population resulted in EGFP-positive colonies. Importantly, nearly 100% of these EGFP-positive colonies were positive for Nanog after further culture (Figure 2M), indicating the establishment of a pluripotent network. As an alternative method, flow cytometry was used, and single cells were seeded into individual wells; from 288 seeded cells, 44 colonies (15.28%) were obtained, and of these, 41 (14.24%) were EGFP-positive (Figure 2L).

Finally, the time requirement for MEF reprogramming due to exogenous factors was examined. Doxycycline was removed from day 1 to day 12 (Figure 2N). A minimum of 4 days of induction was required for EGFP-positive colony formation, consistent with observations of the earliest EGFP-positive clusters. After day 10, the number of colonies obtained no further increased. This indicates that 10 days of induction reached the maximum colony count.

Similar results were observed for the 2°NPC (Figures 2Q to 2S). In summary, these data demonstrate that the 2°S _2A K _2A M MEF and the 2°S _2A K _2A M NPC can be easily reprogrammed in an efficient manner.

Example 5: S _2A K _2A M optimizes the stoichiometry of Sox2 and Klf4 for reprogramming

This example illustrates that, in addition to providing simultaneous expression of Sox2, Klf4, and c-Myc, another advantage of S _2A K _2A M is that the stoichiometry of Sox2, Klf4, and c-Myc from the polycistronic cassette is stable at the single-cell level.

The optimal chemometry of Sox2, Klf4, and c-Myc was validated by observing the signal intensity of Sox2 and Klf4, as analyzed by immunostaining. In single cells transduced with S _2A K _2A M, the expression signals of Sox2 and Klf4 were generally equivalent, which contrasted sharply with the mosaic pattern observed in cells transduced with the three vectors (S+K+M) expressing Sox2, Klf4, and c-Myc separately (Figs. 3B to 3C).

The effect of disrupting factor stoichiometry was then tested by moving one factor to a monocistronic cassette, resulting in combinations of monocistronic Sox2 plus polycistronic Klf4 and c-Myc (S+K _2AM ), monocistronic Klf4 plus polycistronic Sox2 and c-Myc (K+S _2AM ), and monocistronic c-Myc plus polycistronic Sox2 and Klf4 (M+S _2AM K) (Figure 3A). Figures 3N-1 to 3N-3 illustrate the loss of co-expression of Sox2 and Klf4 in S+K _2AM and K+S _2AM cell types.

The inventors then tested how disruption of Sox2 and Klf4 chemometry would affect reprogramming results. To facilitate comparison of reprogramming efficiency, viral titrations were adjusted to achieve a comparable percentage of cells co-expressing Sox2 and Klf4 under all conditions (Figs. S3C and S3D). Sixteen days after reprogramming, colony numbers were significantly reduced under the conditions of Sox2 and Klf4 isolation, decreasing by 90% and 80% respectively in the S+ _K2AM and K+ _S2AM combinations compared to _{the S2AK2AM} conditions, while in the M+ _S2AK combination, colony numbers were only 30% lower than the control (Figs. 3N to 3O). These results indicate that factor chemometry, particularly the chemometry of Sox2 and Klf4 _, is crucial for _S2AK2AM reprogramming.

The inventors also investigated how the stoichiometry of Sox2 and Klf4 affects _{S2A K2A} M reprogramming by manipulating the ratio of these two factors. Sox2(+Sox2) or Klf4(+Klf4) was overexpressed individually in 2°MEF (Fig. 3E). Since _{S2A K2A} M was already expressed in these cells, overexpression of Sox2 or Klf4 resulted in an increased Sox2/Klf4 ratio in +Sox2 cells and a decreased Sox2/Klf4 ratio in +Klf4 cells, as verified by single-cell fluorescence analysis (Fig. 3F) and qPCR (Fig. 3G). At the end of reprogramming, the number of EGFP-positive colonies was lower for +Sox2 conditions and higher for +Klf4 conditions (Fig. 3I). Consistent with these results, on day 4, Oct4 activation was decreased when Sox2 was overexpressed, and increased when Klf4 was overexpressed (Fig. 3H). These data suggest that a higher Klf4/Sox2 ratio promotes more effective reprogramming.

The inventors then examined whether polycistronic Sox2 and Klf4 were sufficient to generate iPSCs in the absence of co-expression of c-Myc. The two-factor combination _S2AK , _S2AM , and _K2AM was used for reprogramming (Fig. 3J). Interestingly, EGFP-positive colonies were obtained under _S2AK conditions alone, and iPSC lines were established (Figs. 3K to 3M). However, when Sox2 and Klf4 were expressed separately from monocistronic plasmids, no EGFP-positive colonies were generated. These results further confirm that the stoichiometry of Sox2 and Klf4 is a factor in reprogramming cells to pluripotency.

Example 6: Transcriptional conversion marker shifts at day 0/day 2 and day 12/iPSC during 2°MEF and NPC reprogramming

This embodiment describes an experiment aimed at understanding how transcriptional networks transition from different differentiation lineages to pluripotency, in order to gain insights into _{S2AK2AM reprogramming} .

Due to Oct4's well-characterized function in pluripotency induction and its early detection in MEF and NPC reprogramming, the inventors used the activation of endogenous Oct4 to monitor _{S2A K2A} M reprogramming into pluripotency. As shown in Figure 4I, the EGFP-positive cell population was significantly more efficient at generating iPSC-like colonies than its EGFP-negative counterpart. RNA sequencing (RNA-seq) was performed on cells at days 0, 2, 4, 8, and 12 (Figure 4A).

Compared to MEF on day 0, the number of differentially expressed genes (DEGs) detected in the reprogramming intermediates and iPSCs on days 2, 4, 8, and 12 were 1941, 3523, 3910, 2972, and 3969, respectively. Figure 4B illustrates the reprogramming process from MEF to iPSCs as provided by principal component analysis (PCA). The cells at different time points showed clear separation, indicating that these populations were transcriptionally distinct. In particular, the cells on day 2 were far removed from the MEF on day 0, indicating a robust transcriptional shift within the two days prior to reprogramming.

Hierarchical clustering brought the reprogramming intermediates from day 2 to day 12 closer together, indicating two major transcriptional transitions occurring between day 0 and day 2 (day 0/day 2) and between day 12 and mature iPSCs (day 12/iPSC) (Fig. 4C). To validate this, correlation analysis and DEGs were used (Figs. 4D–4E). A larger number of DEGs were observed during the day 0/day 2 and day 12/iPSC transitions, and this was reflected in the low correlation between day 0/day 2 samples and between day 12 samples and iPSCs. These data support the existence of day 0/day 2 and day 12/iPSC transcriptional transitions.

Next, the inventors evaluated whether a similar transition occurred during NPC reprogramming. Since EGFP-positive cells were not visible on day 4, sorting was performed only on days 8 and 12 (Figure 4A). RNA-seq revealed that the number of DEGs in reprogrammed NPCs was similar to the number of DEGs observed in MEFs at all time points except day 4. Interestingly, transcriptional transitions at day 0/day 2 and day 12/iPSC were also identified during NPC reprogramming.

Example 7: The molecular trajectories of MEF and NPC reprogrammed cells are convergent.

In MEF reprogramming, 699 genes were upregulated during the day 0/day 2 transition. GO analysis revealed overexpression of epithelial genes, indicating involvement of mesenchymal-to-epithelial transition (MET). Interestingly, epithelial genes were also highly enriched among the 880 genes upregulated during the day 0/day 2 transition in NPC reprogramming. This suggests _{that by day 2, both MEF and NPC are reprogramming into intermediate cells with epithelial cell characteristics. These analyses suggest that S2AK2AM reprogramming} can lead to convergent molecular trajectories following the day 0/day 2 transcriptional transition in both cell types.

The inventors compared the transcriptional profiles of MEF and NPC at day 0. Figure 4G shows differential expression of 2165 genes, of which 1066 and 1099 genes were highly expressed in MEF and NPC, respectively. Biological processes associated with embryonic fibroblasts were enriched in MEF, while genes enriched in NPC included those related to nervous system development, thus identifying the primitive identities of these two cell types.

Surprisingly, on day 2, the number of DEGs between reprogrammed MEF and NPC decreased sharply by 93.8% to 174, indicating transcriptional similarity between MEF and NPC intermediates. Cell types continued to converge during reprogramming, with no detectable differences in gene expression on day 12 (Figure 4G).

PCA and correlation analysis clearly supported the disappearance of transcriptional differences between cell types (Figure 4F). Starting from day 2, MEF and NPC reprogramming intermediates clustered together and were indistinguishable based on the top three major components, covering 55% of the total genes. These data suggest that the molecular trajectories of MEF and NPC reprogramming converge after day 0/day 2 transcriptional conversion through dominant activation of similar genes (e.g., epithelial genes) (Figure 4H).

Example 8: Day 0/Day 2 conversion to remove cell type identity markers

This example describes the key molecular events that control two transcriptional transitions.

For the day 0/day 2 transition, many genes were differentially expressed, with 699 upregulated versus 1242 downregulated in MEF and 880 upregulated versus 1245 downregulated in NPC (Figure 5A). Of the downregulated genes, 71.33% (886 out of 1242) and 72.93% (908 out of 1245) were silenced in the remaining reprogramming processes in MEF and NPC, respectively, indicating that this suppression is a key first step in induced pluripotency.

In the MEF gene set, gene ontology (GO) analysis showed that downregulated genes were primarily responsible for tissue development, and tissue expression analysis revealed enrichment of genes associated with fibroblasts and mesenchymal stem cells (Tables 3A to 3B).

Table 3A: GO analysis of biological processes in the 886 downregulated genes shown in Figure 5A

GO project p-value Phylogeny 1.20E-43 Multicellular organism development 5.70E-42 Anatomical morphogenesis 5.90E-41 Vascular development 9.10E-37 Regulation of development in multicellular organisms 2.40E-34 Tissue development 2.60E-34 Regulation of cell motility 2.00E-33 Regulation of cellular component movement 2.30E-33

Table 3B: Gene enrichment in tissues with 886 downregulated genes as shown in Figure 5A.

These analyses indicated that the MEF program was silenced during the day 0/day 2 transition. Downregulation of fibroblast markers was identified by qPCR (Figure 5B).

Similarly, in NPC reprogramming, 908 downregulated genes, primarily associated with neural development, including Nestin, Lhx2, and Nlgn1, were overexpressed in the brain, hypothalamus, and cerebellum. Therefore, through reprogramming of both MEF and NPC, our data suggest that the removal of primitive cell identity marks the transcriptional transition on day 0/day 2.

Example 9: Gradual activation of multipotential networks driving MEF and NPC reprogramming

This embodiment demonstrates how a pluripotent network is established during _S2AK2AM reprogramming by showing a significant upregulation of pluripotent _gene expression.

During MEF reprogramming into iPSCs, 1615 genes were upregulated. These genes were grouped based on the time it took for them to reach a two-fold upregulation threshold, and a pattern of progressive gene activation was established (Figure 5C). As shown in Figure 5D, Lin28a, Lin28b, Zfp296, Sox21, and Cdh1 were upregulated as early as day 2, and by day 4, the expression of three other pluripotency factors, Oct4, Utf1, and Zsacn10, was increased. These results were confirmed by qPCR analysis (Figure 5E). By day 8, a large group of pluripotency factors were elevated, including Nanog, Sall4, Zfp42, Fgf4, Nr5a2, Dppa5/4/3, Esrrb, Tcl1, Tdgf1, Gdf3, Tex19.1, and Fbxo15, and by day 12, several other genes were also activated (e.g., Nodal, Dppa2, Eras, Tet1, and Dnmt3l). These genes showed a gradual activation flow (Figure 5D).

Similar analyses were performed on NPC reprogramming. By day 4, Lin28a, Lin28b, Zfp296, Cdh1, Oct4, and Zscan10 were upregulated. Following that, Nanog, Sall4, Tcl1, Fgf4, Zpf42, Gdf3, Utf1, Fbxo15, Esrrb, Dppa4/5, and Nodal were activated by day 8. By day 12, several genes were found to be activated, including Tdgf1, Dppa3, Eras, and Tex19.1. This list is similar to that of MEF reprogramming, where Oct4, Lin28a/b, Zfp296, and Chd1 are primarily activated, followed by a group of other key pluripotency factors. These observations suggest that, regardless of the original cell identity, the pluripotency network gradually establishes itself in a similar manner during MEF and NPC reprogramming.

To further validate the similar dynamics of pluripotency activation in MEF and NPC, 112 pluripotency-related genes were selected, and their expression levels in MEF and NPC reprogramming intermediates were compared in parallel. This correlation analysis revealed that the intermediates at each time point were highly similar (Fig. 5F), indicating a shared mechanism for pluripotency establishment in MEF and NPC reprogramming (Fig. 5G).

Regarding the day 12/iPSC transition, Figure 5F shows that most key pluripotency genes are further upregulated in MEF and NPC reprogramming at this time point. These data validate that the pluripotency network is stable and mature during the day 12/iPS transcriptional transition.

Example 10: Sox2 and Klf4 synergistically bind and activate their targets

This example illustrates the genome binding pattern of Sox2 and Klf4, demonstrating how _S2AK2AM facilitates _{reprogramming} .

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed on MEFs reprogrammed on day 2. Overexpressed proteins tend to bind indiscriminately throughout the genome; therefore, two separate experiments were performed to capture true binding events, and only those peaks consistently observed in this study were used (31236 for Sox2 and 1175 for Klf4). De novo motif discovery showed that Sox2 and Klf4 motifs were highly enriched in the immunoprecipitated DNA fragments, validating the validity of our experiments (Figure 6A). Although the genomic distribution of Sox2 and Klf4 binding sites in reprogrammed cells was similar to that in ESCs, there was little overlap between the occupied sites, indicating that overexpressed Sox2 and Klf4 had little access to their ESC targets during early reprogramming.

Interestingly, the Klf4 motif is overexpressed in the Sox2 peak and vice versa (Fig. 6A). The Klf4 motif appears in approximately half of the Sox2 peak, while the Sox2 motif appears in 20% of the Klf4 peak, as shown in the figure. The inventors found that a hybrid motif appears in the binding region of both Sox2 and Klf4, containing at least one Sox2 and one Klf4 motif within 30 base pairs. Furthermore, the Sox2 and Klf4 motifs tend to be close to each other (Fig. 6B). In summary, these data indicate that Sox2 and Klf4 bind synergistically to their targets. In fact, the inventors determined the direct interaction between Sox2 and Klf4 by immunoprecipitation (Fig. 6C).

To further investigate their synergy, the global colocalization of Sox2 and Klf4 in the genome was analyzed. Approximately 80% of the Klf4 peaks were bound to Sox2 (Sox_Klf peaks) (Figure 6D). For peaks designated as Sox2- or Klf4-only binding (Sox_solo or Klf_solo peaks), we still observed low levels of Klf4 or Sox2 enrichment, respectively (Figure 6E). This was determined by quantification of signal intensity (Figure 6F). This phenomenon indicates that Sox2 and Klf4 synergistically bind their targets in the genome with slightly different preferences.

The inventors then examined whether this synergistic binding promoted the activation of its target genes. Sox2 binding (Sox2_Klf and Sox2_solo) led to increased H3K27 acetylation on day 2, but no similar effect was observed for Klf4 (Klf4_solo) (Figs. 6E and 6F). This is likely because the Klf4 binding region is already highly acetylated. Consistently, by day 2, the expression of Sox2 target genes was also significantly upregulated (Fig. 6G).

Example 11: Klf4 overexpression leads to Sox2 binding shift

The inventors then investigated whether Sox2 and Klf4 binding was identical under the S _2A K _2A M condition and between Sox2 or Klf4 overexpression alone. Samples used for Sox2 or Klf4 overexpression alone (Sox2_tetO or Klf4_tetO) were derived from previous data (Chronis et al., 2017). Despite similar binding motifs (Fig. 6I), the Sox2 binding region underwent fundamental changes under the S _2A K _2A M and Sox2_tetO conditions, with only about 10% overlap (Fig. 6H), while the Klf4 binding region showed high similarity (77% overlap) between the S _2A K _2A M and Klf4_tetO conditions. Due to the overpresentation of the Klf4 motif at the Sox2 binding locus under the S _2A K _2A M condition, the inventors inferred that the higher Klf4 level might be the cause of the Sox2 binding translocation. Furthermore, H3K27 acetylation was increased in the _{S2A K2A} M-related peaks (Sox_co and Sox_SKM), but not in the Sox2 peak specific to the Sox2_tetO condition (Sox_tetO) (Figure 6J). However, no binding shift of the Klf4 peak was observed.

Example 12: Sox2 and Klf4 synergistically bind and activate pluripotency-related regions

This embodiment illustrates how Sox2 and Klf4 collaborate in binding to and activating pluripotency loci. Previously, the inventors have shown that Oct4, Lin28a/b, Zfp296, and Sox21 are upregulated early during MEF reprogramming. In this embodiment, the inventors investigated whether Sox2 and Klf4 co-occupy these genes.

Figure 6K shows Sox2 and Klf4 binding peaks observed at the promoter and some distal elements near these loci, with corresponding increases in H3K27 acetylation levels.

Because of Oct4's crucial role in pluripotency induction and maintenance, the inventors investigated this situation separately using ChIP-qPCR. Primers were designed to cover a large region along the Oct4 regulatory region of chromosome 17, from the first exon of Oct4 to the distal enhancer (Fig. 6K). Similar to the ChIP-seq data, binding of Sox2 and Klf4 at the distal enhancer was observed as early as day 2, while binding of Sox2 and Klf4 was much less observed in the proximal enhancer and promoter regions (Fig. 6L). These bindings became more significant by day 5 (Fig. 6M). Consequently, the H3K27 acetylation level in this region was significantly increased. Therefore, Sox2 and Klf4 were already bound to the Oct4 locus before Oct4 transcription was detected.

More interestingly, we noted that the co-binding of Sox2 and Klf4 occurs at one of the 231 ESC-specific superenhancers upstream of Oct4. These superenhancers were reported by Whyte et al. in 2013 and are associated with high expression of nearby pluripotency genes (Whyte et al., 2013). We searched for other ESC-specific superenhancers that also bind to Sox2 or Klf4. Interestingly, Sox2 binding also occurs at four superenhancers close to Nanog and Sox2, and these superenhancers have been shown to be crucial for the expression of Nanog and Sox2 in the ESC (Blinka et al., 2016; Li et al., 2014; Zhou et al., 2014). The Fgf4 superenhancer also binds to Sox2. These results indicate that on day 2 of _{S2A K2A} M reprogramming, Sox2 and Klf4 synergistically bind and reshape some pluripotency loci, even before their transcriptional activation, suggesting their role in early pluripotency initiation.

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All patents and publications cited or referenced herein represent the skill level of a person skilled in the art to which this invention pertains, and each such cited patent or publication is expressly incorporated herein by reference to the extent that it would be individually incorporated by reference in its entirety or stated herein as such. The applicant reserves the right to actually incorporate any and all material and information from any such cited patent or publication into this specification.

The following statements are intended to describe and summarize various embodiments of the invention based on the foregoing description in the specification.

statement:

1. A polycistronic expression cassette comprising a promoter operatively linked to a nucleic acid segment encoding a Sox2 polypeptide, a Klf4 polypeptide, and optionally a c-Myc polypeptide.

2. The polycistronic expression cassette described in statement 1, wherein the nucleic acid segment encodes a Sox2 polypeptide in the same frame as the Klf4 polypeptide, and optionally in the same frame as the c-Myc polypeptide, as a single continuous open reading frame.

3. The polycistronic expression cassette of statement 1 or 2, wherein the nucleic acid segment further encodes one or more cleavable peptide linkers between the Sox2 peptide, the Klf4 peptide, and/or the optional c-Myc peptide.

4. The polycistronic expression cassette described in statement 1, 2, or 3, wherein the promoter is heterologous to the nucleic acid segment encoding the Sox2 polypeptide, the Klf4 polypeptide, and the optional Myc polypeptide.

5. The polycistronic expression cassette described in statements 1 to 3 or 4, wherein the promoter is an inductive promoter.

6. The polycistronic expression cassette described in statements 1 to 3 or 4, wherein the promoter is a constitutive promoter.

7. Host cell containing polycistronic expression cassettes stating statements 1 to 5 or 6.

8. The host cell described in statement 7 is an adult cell.

9. The host cells described in statement 7 or 8, which are autologous to the selected patient or animal.

10. The host cell described in statement 9, wherein the animal is an experimental (e.g., laboratory) animal, a domesticated animal, an endangered animal, or a zoo animal.

11. The host cell described in statement 9 or 10, wherein the selected patient suffers from a disease or medical condition.

12. The host cell described in statements 7 to 10 or 11, which is located within a cell population.

13. A method comprising contacting selected cells with a polycistronic expression cassette stated in statements 1 to 4 or 5, thereby generating a host cell containing the polycistronic expression cassette.

14. The method of statement 13, further comprising incubating host cells in a reprogramming culture medium to generate reprogrammed cells.

15. The method of statement 13 or 14, wherein the host cells are incubated in a reprogramming medium to reprogram the host cells to cross cell lineage boundaries, such that the reprogrammed cells have a phenotype different from that of the host cells.

16. The method of statement 14 or 15, wherein the reprogrammed culture medium does not contain Chirr99021, PD0325901 or a combination of Chirr99021 and PD0325901.

17. The method of statement 14, 15 or 16, wherein the reprogrammed culture medium contains an inducer.

18. The method described in statements 14 to 16 or 17, wherein the reprogrammed culture medium comprises doxycycline.

19. The method described in statements 14 to 17 or 18, wherein the reprogrammed culture medium comprises doxycycline, A83-01, salvia miltiorrhiza, or a combination thereof.

20. The method described in statements 14 to 18 or 19, further comprising incubating the reprogrammed cells in a culture medium for a time sufficient to produce a population of reprogrammed cells.

21. The method described in statements 14 to 19 or 20, wherein the host cell population is incubated with the reprogrammed medium.

22. The method of statement 21, wherein at least 1%, at least 3%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15% of the host cell population are reprogrammed to reprogrammed cells.

23. The method described in statements 14 to 21 or 22, wherein one or more of the reprogrammed cells are stem cells.

24. The method described in statements 14 to 22 or 23, wherein the reprogrammed cell is a pluripotent stem cell.

25. The method described in statement 23 or 24, further comprising differentiating the stem cell or pluripotent stem cell into ectoderm cells, mesoderm cells or endoderm cells.

26. The method described in statement 23, 24, or 25, further comprising differentiating the said stem cell or pluripotent stem cell into neurons, cardiomyocytes, pancreatic cells, hepatocytes, dermal cells, chondrocytes, or their progenitor cells.

27. The method of statement 24, further comprising generating an animal embryo from the pluripotent stem cells.

28. The method described in statements 14 to 24 or 25, further comprising administering the reprogrammed cells or the stem cells or the cells to a patient or animal.

29. The method of statement 26, further comprising administering the said neuronal cells, cardiomyocytes, pancreatic cells, hepatocytes, dermal cells, chondrocytes, or their progenitor cells to a patient or animal.

The specific methods and compositions described herein represent some preferred embodiments and are exemplary and not intended to limit the scope of the invention. Other objects, aspects, and embodiments will arise in those skilled in the art upon consideration of this specification, and all such other objects, aspects, and embodiments are covered within the spirit of the invention as defined by the claims. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention described illustratively herein may be suitably practiced without any or more elements or limitations not specifically disclosed herein as essential. The methods and processes described illustratively herein may be practiced in different sequences of steps, and are not necessarily limited to the sequence of steps indicated herein or in the claims.

The terms and expressions used herein are descriptive rather than restrictive, and are not intended to exclude any equivalents or portions thereof of the features shown and described, but it is recognized that various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that while the invention has been specifically disclosed by way of preferred embodiments and optional features, modifications and variations of the concepts disclosed herein can be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims and statements. In no event should this patent be construed as limited to the specific embodiments, implementations, or methods specifically disclosed herein. In no event should this patent be construed as being limited by any statement made by any examiner or any other officer or employee of the Patent and Trademark Office, unless such statement is expressly and unconditionally or reservedly adopted in the applicant's responsive written statement.

Claims (17)

1. A polycistronic expression cassette comprising a nucleic acid segment and a promoter operatively linked to the nucleic acid segment, the nucleic acid segment encoding a Klf4 peptide and a Sox2 peptide in a single consecutive open reading frame. 2. The polycistronic expression cassette of claim 1, wherein the nucleic acid segment further encodes one or more cleavable peptide linkers between the Sox2 peptide and the Klf4 peptide. 3. The polycistronic expression cassette of claim 1, wherein the promoter is heterologous to the nucleic acid segments encoding the Sox2 polypeptide and the Klf4 polypeptide. 4. The polycistronic expression cassette of claim 1, wherein the promoter is an inducible promoter. 5. A host cell comprising the polycistronic expression cassette of claim 1. 6. The host cell of claim 5, wherein the polycistronic expression cassette is contained within a vector. 7. The host cell of claim 6, wherein the carrier is freely maintained in the host cell. 8. The host cell of claim 5, wherein the host cell is an adult cell. 9. The host cell of claim 5, wherein the selected patient or animal is autologous. 10. The host cell of claim 9, wherein it has a mutation associated with a disease or condition. 11. A method comprising contacting selected cells with the polycistronic expression cassette of claim 1 to generate a host cell containing the polycistronic expression cassette. 12. The method of claim 11, further comprising incubating the host cells in a reprogramming culture medium to generate reprogrammed pluripotent stem cells. 13. The method of claim 12, wherein the reprogrammed culture medium comprises saliva extract and A83-01. 14. The method of claim 11, wherein the nucleic acid segment further encodes one or more cleavable peptide linkers between the Sox2 polypeptide and the Klf4 polypeptide. 15. The method of claim 11, wherein the promoter is heterologous to the nucleic acid segments encoding the Sox2 polypeptide and the Klf4 polypeptide. 16. The method of claim 11, wherein the promoter is an inducible promoter. 17. The method of claim 12, further comprising differentiating the reprogrammed pluripotent stem cells into fractionated cells.