WO2019095585A1

WO2019095585A1 - eSOX2 POLYPEPTIDES FOR EFFICIENT CELLULAR REPROGRAMMING, DERBY-SEQ METHOD OF OBTAINING THE SAME AND USE THEREOF

Info

Publication number: WO2019095585A1
Application number: PCT/CN2018/077403
Authority: WO
Inventors: Veeramohan VEERAPANDIAN; Jan Ole ACKERMANN; Ralf Jauch
Original assignee: Guangzhou Institutes Of Biomedicine And Health, Chinese Academy Of Sciences
Priority date: 2017-11-17
Filing date: 2018-02-27
Publication date: 2019-05-23
Also published as: WO2019095584A1; CN107868126B; CN107868126A

Abstract

Provided are an artificial polypeptide, a method of obtaining the polypeptide, an artificial nucleic acid encoding the polypeptide, a construct containing the nucleic acid, and a method of cell fate conversion.

Description

eSOX2 POLYPEPTIDES FOR EFFICIENT CELLULAR REPROGRAMMING, DERBY-SEQ METHOD OF OBTAINING THE SAME AND USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to and benefit of Chinese Patent Application No. 2017111466278 filed with the State Intellectual Property Office of P. R. China on November 17, 2017, the entire content of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with support of CAS-TWAS President's Fellowship Programme, University of Chinese Academy of Sciences (UCAS) , the world academy of Sciences (TWAS) and Guangzhou Institutes of Biomedicine and health.

FIELD

The present disclosure relates to the biology field, in particular to an artificial polypeptide, a DERBY-Seq method of obtaining the polypeptide, an artificial nucleic acid encoding the polypeptide, a construct containing the nucleic acid, and a method of cell fate conversion.

BACKGROUND

The forcible expression of defined transcription factor (TF) cocktails can engage and reprogram the epigenome of somatic cells leading to drastic cell fate conversions. A four factor TF cocktail consisting of Sox2 (Sry-related box 2) , Oct4, Klf4 and c-Myc directs pluripotency reprogramming in mouse and human cells. Alternative TF cocktails have been shown to directly interconvert somatic cell types, a process termed direct lineage reprogramming, bypassing the intermediate step of pluripotency. However, the rate, quantity, reproducibility and quality of cells produced by reprogramming technologies is often poor and poses challenges to translate this method for routine clinical diagnostics or cell-based therapies. For example in pluripotency reprogramming under standard Serum/LIF condition, less than 0.1%of mouse embryonic fibroblasts give rise to induced pluripotent stem cell (iPSC) . Only a select subset of TFs is capable to direct cell fate conversions and the unique molecular properties endowing them with the capacity to reprogram are not understood and highly homologous factors function differently. For example, if Sox17 replaces Sox2 or if Oct6 replaces Oct4 in four factor cocktails reprogramming activity is lost. Interestingly, the uniqueness of reprogramming TFs appears to rely on subtle molecular features as 1-3 point mutations rationally introduced at strategic molecular interfaces mediating DNA dependent dimerization could convert Sox17 and Oct6 into pluripotency reprogramming factor although the wild-type proteins induce endodermal or ectodermal cell lineages, respectively. Apparently, protein engineering of endogenous factors can profoundly switch and enhance the function of reprogramming TFs. As our understanding of sequence function-relationships in transcriptional control is incomplete, rational design approaches suffer from major limitations.

Therefore, there is a need for improving induction efficiency of induced pluripotent stem cells.

SUMMARY

Embodiments of the present disclosure seek to solve at least one of the problems existing in the related art to at least some extent. An object of the present disclosure is to provide an artificial polypeptide, a method of obtaining the polypeptide, an artificial nucleic acid encoding the polypeptide, a construct containing the nucleic acid, and a method of cell fate conversion. The artificial polypeptide is a variant of wild-type Sox2 which is capable of inducing pluripotent stem cells with induction efficiency higher than wild-type Sox2. The method of obtaining the polypeptide is directed-evolution of reprogramming factors by cell selection and sequencing (DERBY-seq) combining cellular reprogramming with pooled libraries and amplicons sequencing for variant detection, by which it is firstly demonstrated that variants of Sox2, reproducibly outperform wild-type Sox2 in inducing pluripotent stem cells.

In a first aspect, the present disclosure in embodiments provides an artificial polypeptide, including an amino acid sequence shown as any one of SEQ ID NOs: 1 to 5, characterized in that the artificial polypeptide is a variant of wild-type Sox2, which is obtained by mutation at

amino acids

46, 53 and 57 in a high mobility group (HMG) box domain of the wild-type Sox2.

It should be noted that SEQ ID NO: 1 is an amino acid sequence of eSox2 NRR variant; SEQ ID NO: 2 is an amino acid sequence of eSox2 AHK variant; SEQ ID NO: 3 is an amino acid sequence of eSox2 DTK variant; SEQ ID NO: 4 is an amino acid sequence of eSox2 FRT variant, and SEQ ID NO: 5 is an amino acid sequence of eSox2 QTI variant.

In a second aspect, the present disclosure in embodiments provides a method of obtaining the artificial polypeptide of the first aspect, including steps as follows:

(1) generating a transcription factor Sox2 library by means of comprehensive randomization mutagenesis at

amino acids

46, 53 and 57 in a high mobility group (HMG) box domain of the wild-type Sox2 using a transgene delivery system;

(2) subjecting the transcription factor Sox2 library to a transfection process, and then infecting donor cells carrying a fluorescent marker as read out, followed by culturing in suitable media;

(3) isolating positive cells based on the fluorescent marker, followed by genotyping on a high-throughput sequencing platform, so as to identify a positive Sox2 variant; and

(4) selecting a target Sox2 variant by comparing induction efficiency of the positive Sox2 variant with wild-type Sox2, thereby obtaining the artificial polypeptide,

wherein the positive Sox2 variant with iPSC generation efficiency more than 1.5 times, preferably 2 times, more preferably 3 times as high as the wild-type Sox2 is selected as the target Sox2 variant.

In an embodiment of the present disclosure, the positive Sox2 variants with 3 times iPSC generation efficiency as high as the wild-type Sox2 are selected as the target Sox2 variants.

In an embodiment of the present disclosure, in step (1) of the above method, the transcription factor Sox2 library consists of 8000 variants of the wild-type Sox2 obtained by comprehensive randomization mutagenesis at

amino acids

46, 53 and 57 in a high mobility group (HMG) box domain of the wild-type Sox2.

In an embodiment of the present disclosure, in step (2) of the above method, the fluorescent marker is a transgenic green fluorescent protein (GFP) driven by an Oct4 promoter.

In an embodiment of the present disclosure, the induction efficiency is measured as the number of GFP positive colonies.

In an embodiment of the present disclosure, each of the

amino acids

46, 53 and 57 is randomized using NNK codons.

In an embodiment of the present disclosure, the transfection process is achieved by transfecting Plat-E cells with the library.

In an embodiment of the present disclosure, in the step (2) of the above method, the donor cells are infected by a retrovirus expressing the library obtained.

In an embodiment of the present disclosure, the donor cells are somatic cells.

In an embodiment of the present disclosure, the somatic cells are mouse embryonic fibroblasts (MEFs) .

In an embodiment of the present disclosure, in the step (2) of the above method, the donor cells are cultured in the suitable media for 12 to 15 days.

In an embodiment of the present disclosure, the transgene delivery system is a vector based on an in vitro reprogramming system selected from the group consisting of a) a retroviral vector, b) a lentiviral vector, c) an AAV safer harbor vector, d) a Piggybac vector, or e) an Adenovirus vector.

In an embodiment of the present disclosure, the transgene delivery system is a retroviral pMX vector.

In an embodiment of the present disclosure, the positive cells in step (3) are selected by phenotypic selection strategies comprising a) a genetic marker, preferably a surface marker; b) a physiological response, such as contractility for cardiocytes or chemical signal for neurons; or c) a molecular barcode.

In a third aspect, the present disclosure in embodiments provides an artificial nucleic acid encoding the polypeptide in the first aspect and having a nucleotide sequence shown as any one of SEQ ID NOs: 6 to 10.

It should be noted that SEQ ID NO: 6 is a nucleic acid sequence encoding the polypeptide of eSox2 NRR variant; SEQ ID NO: 7 is a nucleic acid sequence encoding the polypeptide of eSox2 AHK variant; SEQ ID NO: 8 is a nucleic acid sequence encoding the polypeptide of eSox2 DTK variant; SEQ ID NO: 9 is a nucleic acid sequence encoding the polypeptide of eSox2 FRT variant; and SEQ ID NO: 10 is a nucleic acid sequence encoding the polypeptide of eSox2 QTI variant.

In a fourth aspect, the present disclosure in embodiments provides a construct, including the nucleic acid described in the above aspect.

In a fifth aspect, the present disclosure in embodiments provides a method of cell fate conversion, including:

transducing donor cells with a transcription factor comprising the artificial nucleic acid selected from any one of SEQ ID NOs: 6 to 10, followed by culturing in suitable media, such that the artificial polypeptide in the first aspect is overexpressed and the donor cells are reprogrammed into induced pluripotent stem cells.

In an embodiment of the present disclosure, the donor cells are somatic cells.

In an embodiment of the present disclosure, Oct4, Klf4 and C-Myc are also overexpressed.

DESCRIPTION OF DRAWINGS

Figure 1 are schematic graphs showing three residues of helix 3 in the high mobility group (HMG) box domain that is variable amongst paralogous mouse Sox genes according to an embodiment of the present disclosure. Multiple sequence alignment of helix 3 of the HMG box of 20 paralogous mouse Sox proteins is shown in panel A. Helix 3 mediates DNA dependent dimerization with Oct4 on canonical and compressed DNA elements with juxtaposed Sox and Oct half-sites (B) . The box marks sites 1 (Glu46) , 2 (Ile53) and 3 (Lys57) are subjected to randomization with NNK codons (C) .

Figure 2 is schematic graphs showing a DERBY-seq workflow according to an embodiment of the present disclosure. A pooled library of 8000 eSox variants is used in three biological replicates to reprogram 90,000 OG2-MEFs (30,000 MEFs plated per well of a 6-well plate) to iPSCs in LIF/Serum/VitaminC medium using 3F (Sox2, Oct4 and Klf4) or 4F (Sox2, Oct4, Klf4 and c-Myc) conditions (A-D) . After FACS sorting, the genomic DNA is isolated and fragments encompassing randomized codons are amplified in a two (eSox17) or three (eSox2) -step PCR procedure and submitted for amplicon sequencing (D-F) .

Figure 3 is schematic graphs showing GFP-positive colonies according to an embodiment of the present disclosure. The upper panel shows the counts of GFP-positive iPSC colonies from three independent biological experiments performed in technical replicates and the black bar indicates the mean. The lower panel shows representative whole well scans (using 12 well plates) of eSox2 and eSox17 libraries compared to wild-type Sox2 and Sox17 controls at day 12 of reprogramming and 4F condition.

Figure 4 is schematic graphs showing GFP-positive cells according to an embodiment of the present disclosure. The upper panel shows the percentages of GFP positive cells after FACS analysis at

day

12 and 4F condition performed in three biological replicates. The black bar is the mean. The lower panel shows representative FACS plots.

Figure 5 is schematic graphs showing GFP-positive cells and GFP-negative cells for eSox2 and eSox17 under 3F and 4F conditions respectively according to an embodiment of the present disclosure. pMX-GFP is used as positive control and Sox17 as a negative control to optimize the gating strategy (A) . FACS plots from the eSox2 screen are compared to the Sox2 wild-type control in 3F and 4F conditions (B) . FACS plots from the eSox17 screen are compared to the Sox2 wild-type control in 3F and 4F conditions (C) .

Figure 6 is a flow chart showing a DERBY-seq method according to an embodiment of the present disclosure.

Figure 7 is schematic graphs showing volcano plots of differential enrichment of variants in eSox2 (A) and eSox17 (B) screens under 3F and 4F conditions according to an embodiment of the present disclosure. Every dot represents an eSox variant.

Figure 8 is schematic graphs showing GFP-positive colonies, and FACS plots and whole well scans of eSox 2 variants and Sox 2 wide type according to an embodiment of the present disclosure. The panel A and B show colony count of each variant identified from the eSox2 screen and Sox2 and Sox17 wild-type controls performed in three independent experiments in technical duplicates in 4F and 3F conditions, and the panel C and D depict images of iPSC colonies of eSox 2 variants and Sox 2 wide type. The panel E shows FACS plots and whole well scans using a GFP fluorescence channel.

Figure 9 is a schematic graph showing hierarchically clustered Pearson correlation heat map of read counts from the three independent eSox experiments under 3F and 4F conditions according to an embodiment of the present disclosure.

Figure 10 is schematic graphs showing immunofluorescence of iPSC colonies generated with eSox2 NRR (A) and eSox17 FNV (B) using antibodies for pluripotency markers Nanog, Sox2 and Oct4 in comparison with that obtained using Sox2 wild-type controls according to an embodiment of the present disclosure. Scale bar: 100μm.

Figure 11 is schematic graphs showing relative expression to actin of each pluripotency marker expressed by pluripotent colonies derived for eSox variants according to an embodiment of the present disclosure.

Figure 12 is schematic graphs showing cell morphologies of eSox variants and Sox2 wild-type according to an embodiment of the present disclosure.

Figure 13 is schematic graphs showing karyotypes of eSox2 NRR (A) and eSox17 FNV (B) according to an embodiment of the present disclosure.

Figure 14 is schematic graphs respectively showing iPSC colony count (panels A and B) , FACS plots (panels C and D) and whole well scans (panels E and F) of eSox 17 variants under 4F (panels A, C and E) and 3F (panels B, D and F) conditions compared to wide type Sox2 and Sox17 according to an embodiment of the present disclosure.

Figure 15 is a schematic graph showing iPSC colony count of each of wide type and variants derived from Sox2, Sox4, Sox17 and Sox18 according to an embodiment of the present disclosure.

Figure 16 is a schematic graph showing multiple sequence alignment of helix 3 of the HMG box of wild-type and variants of Sox2 and Sox17 proteins according to an embodiment of the present disclosure.

DETAILED DESCRIPTION

Embodiments of the present disclosure are described in detail below, and exemplary embodiments are shown in the drawings. The embodiments described below with reference to the drawings are exemplary, and they are intended to be illustrative of the disclosure and are not to be construed to limit the disclosure.

The present disclosure is accomplished by the present inventors based on the following discoveries. The present inventors found that mouse embryonic fibroblasts (MEFs) can be reprogrammed into induced pluripotent stem cells effectively by means of directed-evolution of reprogramming factors by cell selection and sequencing (DERBY-seq) combining cellular reprogramming with pooled libraries and amplicons sequencing for variant detection, specifically, firstly a library consisting of 8000 variants of wild-type Sox2 is generated by means of comprehensive randomization mutagenesis at

amino acids

46, 53 and 57 in a high mobility group (HMG) box domain of the wild-type Sox2 using a retroviral-containing vector; secondly the retroviral mixture containing the library is subjected to transducing MEFs carrying an Oct4 promoter-driven transgenic green fluorescent protein (GFP) , along with Oct4, Klf4 and optional c-Myc; and then GFP positive cells are isolated from GFP negative cells by flow cytometry, followed by high-throughput sequencing of genomic DNA of GFP positive cells and genomic DNA of GFP negative cells respectively; finally Sox variants which are capable of inducing pluripotent stem cells are identified by comparing induction efficiency with that of the wild-type Sox2, with the induction efficiency up to about 5 times as high as the wild-type Sox2.

With reference to the accompanying drawings, the method for obtaining variants of wild-type Sox, especially Sox2 or Sox17 by DERBY-seq screening will be described in detail in an embodiment in the following steps.

Step 1: Site selection and library construction

On the basis of structural models and molecular dynamics simulations, three homologous residues of the third helix of the mouse HMG box of Sox2 or Sox17 involved in the DNA dependent heterodimerisation with POU factors were selected for comprehensive randomisation mutagenesis (corresponding to HMG box residues E46/I53/K57 of Sox2 and L46/V53/E57 of Sox17) . Each residue was randomized using NNK codons (where N represents A, C, G or T and K represents G or T) resulting in a library of 20 ³=8000 variants excluding STOP codon containing variants (Figure 1) . Libraries were generated using the retroviral pMX vector backbone. Randomization and library generation was performed by GENEWIZ Shanghai, China. 100 μl of the bacterial suspension was diluted with LB media, plated on 15 cm Luria broth (LB) /ampicillin plates and grown for approximately 18 hrs, then the cells were harvested and cultured in 100 mL liquid culture LB for another 18 hrs. Maxi prep plasmid DNA preparations were performed with the EndoFree Maxi prep kit (Tiangen, #DP117) .

Step 2: Selection of reprogrammed cells

Step 2.1: Library transfection and virus preparation

Plat-E cells were thawed and cultured in Plat-E medium (composed of Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher) supplemented with 10%FBS (Natocor, Argentina) in 10-cm cell culture dishes for at least 36 hours without changing media. Cells were passaged every 2 to 3 days at confluency 70-80%. About 7-8 million cells per 10 cm plate were seeded 12-16 hours prior to transfection. At 70-80%confluency, 10 μg of each pMX plasmid was used to transfect PlatE cells with the transfection reagent polyethyleneimine (Polysciences; #23966) at a concentration of 1 mg/mL dissolved in 1ml Opti-MEM (Thermo Fisher Scientific; #31985070) . After 12 hours transfection, the media changed. Virus-containing media was collected at 48 and 72 hours after transfection and passed through 0.45-μm filters (Millipore) (Figure 2) .

Step 2.2: Pluripotency reprogramming

Mouse embryonic fibroblasts (OG2-MEF from mouse embryos collected at embryonic day 13.5 carrying a transgenic GFP reporter driven by an Oct4 promoter were obtained from the GIBH animal facility and seeded at ～ 30,000 cells per well of a 6-well plate and cultured in MEF medium composed of high glucose DMEM containing (4.5 g/l D-glucose) supplemented with 10%fetal bovine serum (FBS, Natocor, #SFBE) , 1x GlutaMAX (Thermo Fisher Scientific; #35050061) , 1x nonessential amino acids (NEAA, Thermo Fisher Scientific; #11140050) and 0.5 X penicillin/streptomycin (Hyclone; #SV30010) for 8-10 hours prior viral transduction. 1 mL of filtered retroviral supernatant containing polybrene at a concentration of 8mg/mL (Sigma-Aldrich; #40804ES76) of each factor (OSKM) was added twice in a 24 hour interval. After 48 hours of viral infection, MEF medium was replaced with mES medium (high glucose DMEM containing 4.5 g/l D-glucose supplemented with 10%FBS, 1%NEAA, 1%GlutaMAX, 1%Sodium Pyruvate, 0.5%penicillin/streptomycin, 1000 U/mL Leukemia inhibiting factor (LIF) , 0.055 mM β-mercaptoethanol and 50 μg/mL Vitamin C. The day of media change is considered as reprogramming day 0. The reprogramming cells were maintained at 37 ℃ and 5%CO ₂ (BB15 incubator Thermo Fisher Scientific) and monitored using a phase contrast microscope (Zeiss Axio Vert. A1) . Every 24 hours mES media was changed. For whole scanning the media was removed and 1 x Dulbecco’s phosphate buffered saline (DPBS Thermo Fisher Scientific #14190144) was added (at day 10 for 4F and day12 for 3F eSox2 variants and at day10 for eSox17 variants) and whole well scans were taken from 12 well plate using an ImageXpress Micro XLS confocal High-Content Analysis System (Molecular Devices) .

Step 2.3: Fluorescence-assisted cell sorting (FACS)

To separate reprogramming from non-reprogramming populations in pooled library screens, OG2-MEFs were reprogrammed in 6-well tissue culture plates for 12 to 15 days, mES medium was removed and cells were washed twice with 1x DPBS. The cells in each well were then dissociated with 1 mL 0.25%Trypsin/1mM EDTA (Thermo Fisher Scientific; #25300054) , passed through a 40-μm BD cell strainer and diluted in FACS buffer (1xDPBS + 2mM EDTA + 0.1%BSA) to 6-7 million cells/ml. For each sample, cells from three replicate wells were combined in one tube and used for two-way cell sorting. Cells were sorted by using the 488 nm GFP laser channel of a Beckman Coulter-MoFlo ^TM Astrios. Approximately, 20,000 -100,000 GFP positive and GFP negative cells were collected for each sample. To compare eSox variants analytical cell sorting was performed at day 10 using a BD Accuri ^TM C6 device with FlowJo 7.6 software analyzing ～30,000 life cells per variant.

Step 3: Next generation library preparation and sequencing

Genomic DNA was isolated from GFP positive and GFP negative cells using Quick gDNA micro prep Kit (Zymo research #D3020) . As a control, gDNA was also extracted from unsorted, transduced cells 60 hours after transfection (for eSox17 library only) . As a further control, the maxi-prepped library in the pMX backbone was sequenced for both eSox libraries. For the eSox2 input library control, the plasmid library was serially diluted prior to the PCR starting from 5.6810 to 5.686 molecules (Figure 2E) . For eSox17 experiment, the plasmid library was diluted to 1 million molecules per PCR reaction (～approximately 0. 625 pg) .

Amplicon libraries were produced in a 3 step (eSox2 library) or 2 step (eSox17 library) PCR scheme (Figures 2E and 2F) . First pMX transgenes were amplified by a 15 cycle PCR using DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, #K1082) and products were purified using a PCR purification kit (Tiangen, #DP209) . Second, a 6 cycle PCR was performed with primers flanking the randomised portion and overhangs encoding barcodes and parts of the adapters required for Illumina sequencing (Table 1) . Third, in a last 6 cycle PCR, the remainder of the Illumina adapters were added. The resulting ～250 base-pair PCR products were electrophoresed and purified using Midi Gel Purification kit (Tiangen, #DP209) . In the case of the eSox17 library, the exon-intron gene structure allows for the discrimination of endogenous and exogenous Sox17 and primers flanking the randomised portion and overhangs with adapters were used in the first 15-cycle PCR reaction. In the second 12 cycle PCR, the full bardcoded Illumina adapters were added. Samples were quantified with aQubit (Thermo Fisher Scientific) and 50 ng of DNA was submitted to WuxiApptech for sequencing with an Illumina HiSeq 2500 with cluster generation (concentration 8pM ²) by using 150 bp paired-end reads. The sample was run using Illumina standard procedures, with 15%genomic PhiX DNA (Illumina) added to increase sequence diversity. Primer sequences used to extract exogenous genes and Illumina adaptors with barcodes used for multiplexing are listed in Table 1.

Table 1. Genotyping and NGS library primers

Step 4: Colony picking and passaging

iPS colonies with compact dome-shaped morphology, were picked between

day

10 and 12 using a sterile glass rod, then the cells were trypisinized by 0.25%trypsin EDTA and seeded on feeders MEF treated with mitomycin-C and grown for 4 to 5 days. Then colonies were selected and picked based on the dome-shaped morphology and bright GFP florescence and seeded on gelatine-coated 24 well plates. The cells were maintained in feeder-free condition supplemented with the chemically defined 2i medium.

Step 5: Karyotyping

iPSC cells were cultured in 2i medium (a 1: 1 mix of high glucose DMEM/F12 (Thermo Fisher Scientific; #C11320500BT) and Neurobasal medium (Thermo Fisher Scientific; #21103049) containing 1x N2 (Thermo Fisher Scientific; #17502048) , 1x B27 (Thermo Fisher Scientific; #17504044) , 1x GlutaMax (Thermo Fisher Scientific) , 1x NEAA (Thermo Fisher Scientific) , 1 mM sodium pyruvate (Thermo Fisher Scientific) , 0.055 mM β-mercaptoethanol (MP Biomedicals) , 0.5x penicillin/streptomycin, 1,000 U/ml LIF, 3 μM CHIR99021 (Selleck; #S2924-25mg ) , 1 μM PD0325901 (Selleck; #S1036-25mg) ) on 6 cm plates. At 70%confluency demecolcine (Aladdin; #477305) was added to a concentration of 20 μg/mL. After 1 h cells were trypsinized, collected by centrifugation at 200 × g for 3 min, resuspended in 8 mL of 0.075 M KCl, and incubated at 37 ℃ for 20 min. 2 mL fixative solution (acetic acid (Merck millipore, #100062) and methanol (Merck millipore #822283) at 1: 3) were added, mixed gently, and incubated at 37℃ for 10 min. The supernatant was removed by centrifugation and the pre-cooled fixative solution was added to 10 ml. Cells were distributed on a cold cover slide and incubated at 75 ℃ for 3 hr. After trypsin treatment and Giemsa staining (Sigma-Aldrich; #48900) , metaphase spreads were analyzed on a microscope (Olympus BX51) .

Step 6: Immunofluorescence

After passage 4 to 5, the iPS cells were counted and 1-2 million cells were seeded on 0.1%24 well cell culture dishes pre-coated with gelatine and grown 24-48 hours in 2i media until 80%confluency. Cells were washed three times with DPBS (1X) and fixed with 4%paraformaldehyde at room temperature for 15 min. Cells were permeabilized by incubation with 0.2%Triton X-100 (Sigma-Aldrich; #T8787) and dissolved in 10%BSA (MPBIO; #0218054991) in 1X DPBS (1X) at room temperature for 30 min. Afterwards, the permeabilized cells were washed twice with 1X DPBS and incubated with primary antibodies NANOG (Novus; #NB100-58842, 1: 500) , Oct4 (Santa Cruz; #sc-5279, 1: 500) and Sox2 (Santa Cruz; #sc-17320, 1: 500) at 4℃ overnight. Then the cells were washed three times for 5 min with DPBS (1X) and incubated with secondary antibodies required for the respective primary antibody (donkey-anti-rabbit: Thermo Fisher Scientific #A24870, 1: 250; rabbit-anti-mouse: Thermo Fisher Scientific #A21063, 1: 500; donkey-anti-goat: Abcam #ab6949, 1: 500) in dark at room temperature for 2 h. Cells were washed three times with DPBS (1X) for 5 min. The cells were further stained with 1 x 4', 6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific; #R37606) and imaged with an Axio Vert. A1 (ZEISS) .

Step 7: mRNA isolation and quantitative real time-PCR

The iPS cells after 4-5 passages were cultured in 6-well plates and at 80%confluency the media was removed, cells were washed with DPBS and the RNA was extracted using the Trizol method. The total RNA was purified using the PureLink RNA MiniKit (#12183025, Ambion) and quantified using a Nanodrop spectrophotometer. 5 μg total mRNA was used to synthesize the cDNA with a ReverTra Ace qPCR RT master mix (Toybo. FSQ-201s) . Quantitative PCR was performed with ITaq Universal SYBR Green Supermix on a CFX-96 thermocycler (Bio-Rad) . Samples were run in technical triplicates. Relative gene expression was calculated using the 2 ^-Δ Δ CT method (Livak and Schmittgen, 2001) with Actin as endogenous control and the MEF samples as calibrator. Primers are listed in Table 2.

Table 2. qPCR primers

Step 8: Estimation of cell proliferation

OG2 MEF cells were transduced with OKS (3F) retroviral supernatant and at reprogramming day 1 cells were trypsinzed. The cells from single cells suspension were counted using a ScepterTM 2.0 cell counter (Millipore) and 10,000 cells were seeded on 12 well cell culture plates and cultured for 48 hours in mES medium and counted again.

Analysis and conclusion:

(1) Pooled screening, cell selection and sequencing

To generate our artificially evolving Sox (eSox) libraries we selected three residues of helix 3 in the high mobility group (HMG) box domain that are variable amongst paralogous Sox genes and play a role in the DNA dependent dimerization with Oct4 (Figures 1A and 1B, Figure 16) . NNK sequence diversification was used to cover all 20 amino acids with 32 codons (Figure 1C) . This way we randomized E46, I53 and K57 in Sox2 and the homologous L46, V53 and E57 in Sox17 (HMG box numbering convention) leading to libraries with 20 ³ = 8000 variants excluding truncations caused by STOP codons. To benchmark our pooled library screens, we used the reprogramming of MEFs carrying a GFP transgene controlled by regulatory sequences of Oct4 permitting the identification of pluripotent cells (Figure 2A) . Libraries were prepared as retroviral mixtures and used to transduce MEFs in 4-factor combination (4F: Oct4, Klf4 and c-Myc (OKM) + eSox) or three-factor combinations (3F: Oct4 and Klf4 (OK) + eSox) under LIF/Serum/Vitamin C conditions (Figures 2A to 2D) . Under these conditions, Sox2 containing cocktails can direct pluripotency reprogramming and typically yield 50-100 GFP positive colonies per well of a six-well plate by day 12 whilst the replacement of Sox2 with Sox17 impairs the capacity of 3F and 4F cocktail to generate iPSCs (Figure 3) . However, both eSox2 and eSox17 libraries containing cocktails yielded a high quantity of GFP positive colonies and cells in excess of control cocktails with wild-type Sox2 demonstrating that pooled screens with randomized factors are feasible (Figures 3 and 4) .

(2) Selection of artificially evolved candidates from Sox2 and Sox17 libraries

We screened eSox2 and eSox17 under 3F and 4F conditions and performed reprogramming experiments in three independent experiments with three technical replicates each in six-well plates. At reprogramming days 12-14, cells were trypsinized and single cell suspensions containing heterogeneous populations of GFP positive and GFP negative cells were separated by fluorescence-activated cell sorting (FACS) (Figure 5) . The yield of GFP-positive cells was substantially higher for the eSox17 library than for the eSox2 library or wild-type Sox2 in particular under 3F conditions whilst wild-type Sox17 does not produce pluripotent cells (Figures 5B and 5C) . To genotype candidates from eSox libraries, trans-genes were amplified from genomic DNA in a first round of PCR with primer pairs specifically amplifying exogenously provided factors. In the subsequent PCR cycles, Illumina adaptors and barcodes were added (Figures 1E and 1F) . Each library was sequenced in technical duplicates. Deep sequencing generated ～0.5 million raw reads per sample. Randomized codons were translated and tri-peptide occurrences were counted. To access PCR biases, we sequenced the input library in technical duplicates, used two different numbers of PCR cycles and four dilutions of the library (Figure 1E) . We scored for differentially enriched eSox variants between GFP positive and negative cells using DESeq2 (Figures 6 and 7) . Overall biological replicates correlate better for 3F than for 4F experiments presumably because of proliferative and transcriptional noise introduced by c-Myc (Figure 9) . Similarly, cell proliferation assay showed an increased proliferation rate of eSox libraries and Sox17 as compared to Sox2. We selected candidates for validation with different significance and enrichment levels and also took biophysical parameters and the identity of affected amino acids into account (Figure 7) .

(3) DERBY-seq identifies functionally enhanced reprogramming TFs

We next prepared retroviruses of individual Sox2 and Sox17 mutants identified in our screen and tested their performance in pluripotency reprogramming in comparison to their wild-type counterparts. Sox2 NRR variant reproducibly outperformed wild-type Sox2 in particular under 4F conditions and introducing the NRR trip-peptide into Sox17 converts this factor into an inducer of pluripotency (Figures 8 and 14C) . Candidates from the eSox2 3F screen also outperformed wild-type Sox2 similar to 4F candidates (Figures 8 and 14D) . We next tested candidates derived from the library of the otherwise reprogramming incompetent Sox17 as a scaffold. We could verify a surprisingly large number of high-performance factors from both the 3F as well as the 4F eSox17 experiment (Figure 14) . Pluripotency promoting tripeptides derived from the Sox17 library are highly diverse and include WHC, FNV, SLQ, DYC or HQK variants, and so on respectively. A pattern we observed is that the acidic glutamate at position 57 (the last residue of the LVE tri-peptide) has to be removed in order to allow for iPSC generation. Pluripotent colonies derived from eSox variants expressed pluripotency markers (Figures 10 and 11) , maintained good cell morphologies (Figure 12) , and maintained a normal karyotype (Figure 13) indicating that artificial factor evolution does not compromise the quality of the reprogrammed cells.

Compared to the existing methods of inducing pluripotent stem cells, the present disclosure achieves many advantages as follows:

(1) Several artificially evolved and enhanced Sox variants (eSox2) are identified with outperformance over wild-type Sox2 in three or four factor cocktails, by performing a directed evolution screen in mammalian cells, therefore improving the rate, quantity, reproducibility and quality of cells produced by reprogramming technologies, and further benefiting for routine clinical diagnostics, individualized treatment or cell-based therapies using induced pluripotent stem cells.

(2) DERBY-seq is an easily adaptable high throughput method allowing the robust identification of performance improving mutations in biomolecule driven cell fate conversions, therefore providing a broadly applicable approach for enhancing mammalian cell fate conversion including the direct lineage reprogramming in vitro and in vivo.

Throughout this specification, reference to “an embodiment” , “some embodiments” , “one embodiment” , “another example” , “an example” , “a specific example” or “some examples” means that a particular feature, structure, material or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as “in some embodiments” , “in one embodiment” , “in an embodiment” , “in another example” , “in an example” , “in a specific example” or “in some examples” in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials or characteristics may be combined in any suitable manner in one or more embodiments or examples. In addition, it will be apparent to those skilled in the art that different embodiments or examples as well as features of the different embodiments or examples described in this specification may be combined without contradictory.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims

An artificial polypeptide comprising an amino acid sequence shown as any one of SEQ ID NOs: 1 to 5, characterized in that the artificial polypeptides are the variants of wild-type Sox2, which is obtained by mutation at amino acids 46, 53 and 57 in the high mobility group (HMG) box domain of wild-type Sox2.
A method of obtaining the artificial polypeptides in claim 1, comprising steps as follows:

(1) generating a transcription factor Sox2 library by means of comprehensive sequence randomization by mutagenesis at amino acids 46, 53 and 57 in a high mobility group (HMG) box domain of the wild-type Sox2 using a transgene delivery system;

(2) subjecting the library to a transfection process, and then infecting donor cells carrying a fluorescent marker as read out, followed by culturing in suitable media;

(3) isolating positive cells based on the fluorescent marker, followed by genotyping on a high-throughput sequencing platform, so as to identify a positive Sox2 variant; and

(4) selecting a target Sox2 variant by comparing induction efficiency of the positive Sox2 variant with wild-type Sox2, thereby obtaining the artificial polypeptide of claim 1,

wherein the positive Sox2 variant with iPSC generation efficiency more than 1.5 times as high as the wild-type Sox2 is selected as the target Sox2 variant.
The method according to claim 2, wherein the positive Sox2 variants with 3 times iPSC generation efficiency as high as the wild-type Sox2 are selected as the target Sox2 variants.
The method according to claim 2 or 3, wherein in step (2) , the fluorescent marker is a transgenic green fluorescent protein (GFP) driven by an Oct4 promoter.
The method according to any one of claims 2 to 4, wherein the iPSC generation efficiency is measured as the number of GFP positive colonies.
The method according to any one of claims 2 to 5, wherein each of the amino acids 46, 53 and 57 is randomized using NNK codons.
The method according to any one of claims 2 to 6, wherein the transfection process is achieved by transfecting Plat-E cells with the library.
The method according to any one of claims 2 to 7, wherein in the step (2) , the donor cells are infected by a retrovirus expressing the library obtained.
The method according to any one of claims 2 to 8, wherein the donor cells are somatic cells.
The method according to claim 9, wherein the somatic cells are mouse embryonic fibroblasts (MEFs) .
The method according to any one of claims 2 to 10, wherein in the step (2) , the donor cells are cultured in the suitable media for 12 to 15 days.
The method according to any one of claims 2 to 11, wherein the transgene delivery system is a vector based on an in vitro reprogramming system selected from the group comprising a) a retroviral vector, b) a lentiviral vector, c) an AAV safer harbor vector, d) a Piggybac vector, or e) an Adenovirus vector.
The method according to any one of claims 2 to 12, wherein the positive cells in step (3) are selected by phenotypic selection strategies comprising a) a genetic marker, preferably a surface marker; b) a physiological response; or c) a molecular barcode.
An artificial nucleic acid encoding the polypeptide of claim 1 and having a nucleotide sequence shown as any one of SEQ ID NOs: 6 to 10.
A construct, comprising the nucleic acid of claim 14.
A method of cell fate conversion, comprising:

transducing donor cells with a transcription factor comprising the artificial nucleic acid selected from any one of SEQ ID NOs: 6 to 10 followed by culturing in suitable media, such that the artificial polypeptide of claim 1 or the artificial polypeptide obtained in claim 2 is over expressed and the donor cells are reprogrammed into induced pluripotent stem cells.
The method according to claim 16, wherein the donor cells are somatic cells.
The method according to claim 17, wherein the somatic cells are mouse embryonic fibroblasts (MEFs) .
The method according to claim 17 or 18, wherein Oct4, Klf4 and C-Myc are also overexpressed.