Continuous expression of reprogramming factors induces and maintains mouse pluripotency without specific growth factors and signaling inhibitors

Abstract Objectives Derivation and maintenance of pluripotent stem cells (PSCs) generally require optimized and complex culture media, which hinders the derivation of PSCs from various species. Expression of Oct4, Sox2, Klf4, and c‐Myc (OSKM) can reprogram somatic cells into induced PSCs (iPSCs), even for species possessing no optimal culture condition. Herein, we explored whether expression of OSKM could induce and maintain pluripotency without PSC‐specific growth factors and signaling inhibitors. Methods The culture medium of Tet‐On‐OSKM/Oct4‐GFP mouse embryonic stem cells (ESCs) was switched from N2B27 with MEK inhibitor, GSK3β inhibitor, and leukemia inhibitory factor (LIF) (2iL) to N2B27 with doxycycline. Tet‐On‐OSKM mouse embryonic fibroblast (MEF) cells were reprogrammed in N2B27 with doxycycline. Cell proliferation was traced. Pluripotency was assessed by expression of ESC marker genes, teratoma, and chimera formation. RNA‐Seq was conducted to analyze gene expression. Results Via continuous expression of OSKM, mouse ESCs (OSKM‐ESCs) and the resulting iPSCs (OSKM‐iPSCs) reprogrammed from MEF cells propagated stably, expressed pluripotency marker genes, and formed three germ layers in teratomas. Transcriptional landscapes of OSKM‐iPSCs resembled those of ESCs cultured in 2iL and were more similar to those of ESCs cultured in serum/LIF. Furthermore, OSKM‐iPSCs contributed to germline transmission. Conclusions Expression of OSKM could induce and maintain mouse pluripotency without specific culturing factors. Importantly, OSKM‐iPSCs could produce gene‐modified animals through germline transmission, with potential applications in other species.


Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs)
and induced pluripotent stem cells (iPSCs), [3][4][5] have revolutionized research on embryonic development, genome function, and disease modeling. Furthermore, PSCs hold unprecedented potential in regenerative medicine. External signaling pathways 6 integrate with the internal core transcriptional network to stabilize PSC state. 7,8 Serum with the added leukemia inhibitory factor (LIF) served as the traditional culture medium for derivation of ESCs from certain mouse strains. Serum supplies the bone morphogenetic protein (BMP), which induces inhibitor-of-differentiation proteins to repress differentiation. 9 BMP can replace serum to maintain mouse ESCs in combination with LIF. 10 LIF activates STAT3 to inhibit ESC differentiation and promote viability. [11][12][13] However, these culture conditions have only succeeded in deriving ESCs from certain mouse strains and have failed in other mouse strains and other species. Subsequently, it was supposed that ESCs were in an intrinsic and self-sufficient cell state once being well protected from differentiation stimuli, including autocrine FGF4 (an activator of the ERK pathway). 14 Based on this assumption, the MEK/ERK inhibitor PD0325901, GSK3β inhibitor ChIR99021 (2i) condition was established to robustly maintain undifferentiated and homogenous mouse ESCs and derive ground state ESCs from mouse embryos. 14 The MEK/ERK inhibitor blocked the differentiation of ESCs. GSK3 inhibition resulted in the activation of β-catenin in canonical WNT pathways, which abrogated the repressive effects of TCF3 on core pluripotency genes including Esrrb. 15,16 Importantly, 2i along with LIF (2iL) overcame the mouse recalcitrant strain barrier and derived ESCs from all mouse strains 17,18 and even the rat. 19,20 The core pluripotency regulatory network guaranteed the selfrenewal and pluripotency state. Transcription factors (TFs) OCT4, SOX2, and NANOG cross-regulate each other and occupy the core of the TF hierarchy that sustains self-renewal and restricts differentiation of PSCs. 21 Oct4 and Sox2 were reported to be indispensable for mouse ESCs. While Nanog 22 and Klf4 23 were individually dispensable, whereas their overexpression could support self-renewal, respectively. A previous report also claimed that Myc could support self-renewal and pluripotency. 24 These factors jointly exerted a critical role in reconstructing the genetic regulatory network of ESCs, as was confirmed by the outbreaking finding that Yamanaka factors Oct4, Sox2, Klf4, and c-Myc (OSKM) were sufficient to reprogram somatic cells into iPSCs under ESC culture conditions, which resets cellular plasticity to a state akin to that of ESCs. 3 Based on studies in rodents, it has been generally thought that the achievement of pluripotency depends on fine adjustments in the growth factors and signaling inhibitors in the culture media. 6,25 Nonetheless, the appropriate culture conditions ensuring rodent pluripotency could not be applied to efficiently derive PSCs from other species such as domestic mammals, and the derivation of ESCs from domestic species has undergone a long and unproductive past. 26 However, more evidences revealed that the evolutionarily conserved TF cocktail OSKM could reprogram somatic cells of nonrodent species such as the pig, 27,28 marmoset, 29 rabbit, 30 and horse 31 into putative iPSCs or iPSC-like cells, under the inappropriate culture conditions "borrowed from" other species such as the human and mouse. These reports demonstrated the importance of reprogramming factors in driving self-renewal and pluripotency state. Thus, we proposed that reprogramming factors may be able to induce and support PSCs even without the support of specific growth factors and signaling inhibitors. Herein, we explore this possibility by using the classical reprogramming factors OSKM in the mouse. We successfully induced and maintained mouse iPSCs from somatic cells via the continuous expression of OSKM without PSC-specific growth factors and signaling inhibitors. The resulting iPSCs could contribute to germline transmission, permitting the generation of gene-edited mice.

| Animals
The Tet-On-OSKM mice were described in previous studies. 32,33 They carry a doxycycline (DOX)-inducible reverse tetracycline trans-activator (M2rtTA) in the Rosa26 locus, and a single polycistronic OSKM transgene in the Col1a1 locus. Oct4-GFP mice carried a GFP under control of the endogenous Oct4 distal promoter.
Tet-On-OSKM mice and Oct4-GFP mice were both obtained from the Jackson Laboratory. The SCID mice used for teratoma formation were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All experiments involving animals were approved by the Institutional Animal Use Committee of the Institute of Zoology, Chinese Academy of Sciences in Beijing.

| Embryonic stem cell derivation and culture
The Tet-On-OSKM/Oct4-GFP mouse ESC line was derived from the blastocysts obtained from crossbreeding of the above DOX-OSKM mice and Oct4-GFP mice according to standard procedures.
The cells were derived and further cultured in 2iL medium on the mitomycin-c treated mouse embryonic fibroblast (MEF) cells (feeder cells). The detail components of slightly modified N2B27 medium 14 were listed in Table S1. The 2iL medium 14 contained N2B27 medium with the addition of PD0325901 (Stemgent, 04-0006), CHIR99021 (Stemgent, 04-0004), and LIF (Millipore, ESG1007). ESCs cultured in 2iL medium were switched into three different types of medium: N2B27 with 2i and LIF as the 2iL group, N2B27 with 2 μg/mL DOX (Sigma-Aldrich) as the OSKM group, and N2B27 as the N2B27 group.

| Induced pluripotent stem cell induction and culture
To generate induced pluripotent stem cells (iPSCs), Tet-On-OSKM MEFs were seeded onto the feeder cells at a density of 20000 cells per well in 6-well-plates. There were two types of induction media.
The induction medium of the control group comprised 2iL with DOX.
Once iPS-like clones were picked up to culture in new dishes, DOX was withdrawn. The induction medium of OSKM group consisted of N2B27 with DOX, and the DOX was continued to be supplied throughout daily culture.

| Generation of GFP transgenic OSKM-iPSCs
The PiggyBac (PB) transposon system was used. A PB transposase enzyme (PBase) vector and a PB-GFP vector were constructed. The PBase vector contained the EF1a promoter and the coding sequence of the PBase. The CAG promoter, 3 × HA, GFP, and polyA sequences were cloned into the PiggyBac backbone to form the PB-GFP vector ( Figure S4A). These two vectors were transfected into OSKM-iPSCs by using the Neon transfection system (Invitrogen, MPK5000).

| Growth curves
To generate growth curves for ESCs and iPSCs, the Cell Counting Kit-8 (CCK-8, Sigma-Aldrich, 96992) was used. After seeding 2500 cells/well in a 48-well dish, a 1/10 volume of CCK-8 solution was added to the medium for a two-hour-incubation at days 1, 2, 3, 4, and 5. The absorbance of each well at 450 nm was measured using a microplate reader. All the experiments were performed in quadruplicate.
DNA was stained with Hoechst 33342 (Thermo Fisher Scientific) for 10 minutes. Images were captured using a two-photon confocal laser scanning microscope (Leica, TCS Sp8). The BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, C3206) was applied to perform alkaline phosphatase staining according to the manufacturer's instructions.

| PCR genotyping
KOD One™ PCR Master Mix -Blue (TOYOBO, KMM-201) was used for PCR. The sequences of the primer pair were listed in Table S2.

| Real-time quantitative PCR
Total RNA was extracted using the TRIzol reagent (Thermo Fisher Scientific, 15596018). RNA was reverse transcribed using a ReverTra  Table S3. All reactions were conducted in triplicate.

| RNA-Seq library preparation and data analysis
Total RNA was extracted from cells with the TRIzol reagent (Thermo Fisher Scientific, 15596018). The Illumina platform was applied for RNA-Seq. Reads were aligned to the mouse reference genome assembly (GRCm38/mm10) using STAR (version 2.7.1a) 34 36 Heatmaps were generated using log-transformed gene FPKM with the R "pheatmap" function (https://cran.r-proje ct.org/web/packa ges/pheat map/index.html). After the log transformation, gene FPKM values (larger than 1 in at least one sample) were used in principal component analysis (PCA) with the R "prcomp" function and in the hierarchical clustering analysis with the R "hclust" function. Published RNA-Seq data were downloaded from NCBI GEO GSE97954, 37 and the R "sva" function was used to eliminate the batch effect.

| Blastocyst injection
Blastocyst injection was performed according to the procedures given in a previous report. 39 Briefly, diploid blastocysts were collected from the uterus of 3.5 days post coitum (dpc) super-ovulated female CD-1 mice after mating with male CD-1 mice. Cells were harvested by trypsinization and 12-15 cells were microinjected into each blastocyst. After 1-4 hours of culture, these processed embryos were transferred into the oviduct of pseudo-pregnant CD-1 mice at 0.5 dpc. Chimeras were identified by GFP expression or coat colors.

| Induced expression of OSKM maintained self-renewal and pluripotency of mouse ESCs after withdrawal of 2i and LIF
To test whether continuous OSKM expression could maintain the cell identity of mouse ESCs, we used an ESC line stably expressing the pluripotency reporter Oct4-DE-GFP (GFP under control of the endogenous Oct4 distal promoter) and the single polycistronic OSKM transgene upon the addition of DOX ( Figure 1A). This cell line was derived and cultured in N2B27 medium supplemented with the MEK inhibitor PD0325901, GSK3 inhibitor ChIR99021, and LIF (2iL medium). Next, the culture medium was switched from 2iL to N2B27 with DOX (OSKM medium) and the OSKM transgene was continuously activated (Figure 1A), whereas N2B27 medium alone was used as the negative control condition, and 2iL medium acted as

| Induction of pluripotency from somatic cells without specific growth factors and signaling inhibitors
Next, we determined whether this system could reconstruct pluripotency de novo from MEF cells obtained from the above Tet-On-OSKM mice. MEF cells were reprogrammed by OSKM medium (N2B27 supplemented with DOX) and 2iL (N2B27 supplemented with 2iL and DOX, and DOX was withdrawn after reprogramming), respectively.
As shown in the heatmap, naïve pluripotency genes, such as Klf2, Esrrb, Nanog, and Rex1 were consistently expressed to a lower degree in OSKM-iPSCs compared to 2iL-iPSCs, while expression levels were higher than those in somatic cells ( Figure 3G). Real-time quantitative PCR confirmed these results ( Figure 3H), which were also observed in OSKM-ESCs when compared to those of 2iL-ESCs ( Figure S3A). The RNA levels of Sox2 and Klf4 were comparable to those of 2iL-iPSCs, although Oct4 expression was slightly lower than that in 2iL-iPSCs; and endogenous Oct4, Sox2, and Klf4 were activated, albeit endogenous Klf4 to a lower degree ( Figure S3B).
The PCA analysis and hierarchical clustering analysis ( Figure 3I and Figure S3C

| Application of OSKM-iPSCs in producing gene-edited animals
One major application of PSCs is to produce gene-edited animal models for genome function research, disease modeling and drug screening. For this purpose, we developed a route to produce F I G U R E 4 Potential of obtaining gene-edited animals using OSKM-iPSCs. A, Schematic of process for obtaining gene-edited animals using OSKM-iPSCs. B, Morphology of GFP-transgenic OSKM-iPSCs. Scale bar, 50 μm. C, Images of chimeric mouse embryo (days post coitum 12.5) with contribution of OSKM-iPSCs generated by blastocyst injection (marked by an asterisk). The other served as the negative control. Genital ridges of the chimeric embryo were shown on the right. Scale bars, 500 μm. D, Chimeric mouse at postnatal day 1 (PND 1) with contribution of OSKM-iPSCs (marked by an asterisk). The other served as the negative control. (E) Adult chimeric mouse with contribution of OSKM-iPSCs chimera mice with germline transmission using transgenic OSKM-iPSCs ( Figure 4A). We randomly selected two OSKM-iPSC lines and inserted the GFP transgene into their genome using the PiggyBac transposon vector ( Figure S4A) to examine their ability to produce chimeras. The resulting GFP-transgenic subclones were collected ( Figure 4B) and were further injected into mouse blastocysts to form chimeras. We dissected embryos at 12.5 days post coitum (dpc), and a chimera embryo with germline chimerism was observed ( Figure 4C).
Genotyping PCR results in further showed that OSKM-iPSCs contributed to various organs and tissues ( Figure S4B). Chimerism in the newborn and adult mouse was also observed, and genotyping PCR results confirmed the contribution of transgenic OSKM-iPSCs ( Figure 4D,E, and Figure S4C).

| D ISCUSS I ON
Originally, self-renewal and pluripotency were thought to be supported by interaction between external signaling pathways and intracellular core pluripotency transcription regulatory networks.
Obtaining authentic pluripotency was generally considered to de- and support PSCs in the absence of "essential" PSC-specific culturing factors. It would also be of interest to determine how the expression of these TFs maintained authentic pluripotency with the naïve pluripotency genes consistently downregulated, which were thought to be positively associated with developmental potential.
As previously reported, the stoichiometry of reprogramming factors applied during reprogramming significantly influenced the resulting pluripotency of iPSCs. 43 Our present system harbors a F I G U R E 5 Schematic of several pluripotency states established in mouse. An alternative pluripotency state was established in the OSKM system of this study. This schematic was adapted from Conrad Waddington's model. On the top of the "developmental potential" mountain, the 2iL maintained the cells in a naïve pluripotency state, serum with LIF supported another metastable pluripotency state, activin A (low) with XAV939 (A lo XR) 41 maintained formative pluripotency state, and activin A with bFGF supported the primed pluripotency state. 42 An alternative pluripotency state was established by continuous expression of OSKM, while bypassing the requirement of specific growth factors and signaling inhibitors. All these PSCs in different states could differentiate into somatic cells. Somatic cells could be reprogrammed into PSCs in our proposed OSKM system single copy transgene of OSKM. In future studies, the comparison among the cell states obtained via different gene combinations and dosages of TF cocktails in parallel can help us to further understand the regulatory effects of TFs on pluripotency. Furthermore, considering the potential risk of tumor formation upon c-Myc activation, 44 although the Tet-On-OSKM mice did not develop tumors as reported, 43 we aim to attempt other combinations, bypassing c-Myc altogether. 45,46 Due to differences in transcriptional regulatory networks and signaling stimuli among species, and other unclear reasons, 26,47 the appropriate culture conditions ensuring rodent pluripotency could not be used to efficiently derive authentic PSCs from other species such as valuable domestic mammals. Numerous efforts have been made to optimize culture media in order to obtain authentic PSCs from livestock species, as summarized in several reviews. 26,48 However, to date, there are no repeatable and reliable culture conditions to derive authentic pluripotency of domestic animals. This largely hinders the advancement of producing geneedited animals. Based on our study, we hypothesized that although differences exist among various animal species, there might be a possibility of conserved reprogramming factors establishing a so-called "common" expandable and pluripotency state across different species. A similar method may be used to create PSCs of other species while bypassing the requirement of complex and specific culture conditions, and to produce gene-edited animal models. This route might serve as a so-called "universal" approach to obtain gene-edited animal models from various species, which requires further study.

ACK N OWLED G EM ENTS
This study was supported by grants from the National Key

CO N FLI C T O F I NTE R E S T
The Editor-in-Chief of the journal, Professor Qi Zhou, is a co-author of this article. The Editor-in-Chief was blinded to the peer review process. An Associate Editor handled the peer review process for this article and made the final decision as to its suitability for publication.

AUTH O R CO NTR I B UTI O N S
Yihuan Mao: investigation, methodology, formal analysis, resources, validation, visualization and writing-original draft; Libin Wang: investigation, methodology, formal analysis, resources, validation and writing-original draft; Bei Zhong: investigation, formal analysis, resources and visualization; Ning Yang: formal analysis, software and visualization; Zhikun Li: investigation and writing-review and editing; Tongtong Cui: formal analysis and writing-review and editing; Guihai Feng: formal analysis and writing-review and editing; Wei Li: funding acquisition and supervision; Ying Zhang: conceptualization, supervision and writing-review and editing; Qi Zhou: conceptualization, funding acquisition, supervision and writing-review and editing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The accession number for the RNA-Seq data reported in this paper is GEO: GSE17 3471.