Partial Somatic to Stem Cell Transformations Induced By Cell-Permeable Reprogramming Factors

The production of pluripotent stem cells (iPSCs) for therapeutic applications will require practical methods to achieve tight temporal and quantitative control of reprogramming factor (RF) expression, while avoiding the mutagenic potential of gene transfer. Toward this end, we have developed cell-permeable RF proteins (CP-RFs) incorporating newly developed macromolecule transduction domains (MTDs). Treatment of human dermal fibroblasts (HDFs) with combinations of cell-permeable OCT4, SOX2, KLF4, CMYC and either NANOG or LIN28 proteins induced the outgrowth of stem cell-like colonies (iSCs). iSC colonies generated with CP-RFs resembled embryonic stem cells with regard to morphology, biomarker expression, and extended capacity for self-renewal, but failed to expand as iPSC or ES cell lines. Partial reprogramming appears to be a common response to protein-based delivery of programming factors into somatic cells.

membrane 29,31,32 . By contrast, cationic protein transduction domains (PTDs, e.g. those derived from HIV Tat and Antennapedia) enhance protein uptake predominately through absorptive endocytosis and macropinocytosis, which sequester significant amounts of protein into membrane-bound and endosomal compartments 33,34 . In practice, uptake mechanisms are strongly influenced by the cargo, and must be investigated on a case-by-case basis [33][34][35] . In the present study, we experienced difficulties expressing recombinant RF proteins in soluble form. The problem was solved, in part, by testing multiple MTD sequences. The resulting cell-permeable RFs induced colonies with extended capacity for self-renewal that resembled ES cells with regard to morphology and biomarker expression.

Results
Cell-permeable reprogramming factors. Protein sequences derived from the leader peptides of secreted proteins, designated macromolecule transduction domains, have been shown to enhance the cellular uptake of recombinant proteins both in vitro and in vivo. These domains were identified from a screen of 1,500 potential hydrophobic signal peptides for sequences with protein transduction activity as assessed using an EGFP reporter protein 28,29 . However, achieving practical levels of cytosolic protein delivery requires solving problems relating to protein yield, solubility, structure/activity, route of entry, and sequestration. In practice, the process is largely empirical.
To deliver recombinant transcription factors for the purpose of reprogramming differentiated somatic cells, candidate MTDs were modified and analyzed for the ability to promote the uptake of FITC-labeled proteins by cultured cells . The resulting MTDs (MTD47, MTD52,  MTD84, MTD86, MTD132, MTD173 and MTD181; Supplementary  Tables S1 A and B)    greatest solubility (data not shown). A 6-histidine tag (6xHis: H) and a nuclear localization sequence (NLS) derived from SV40 large T-antigen (KKKRK) were also fused to the N-terminus of RFs to facilitate single-step affinity chromatography and cellular nuclear localization, respectively. Each protein was expressed in E. coli, purified under denaturing conditions ( Figure 1 A and B) and refolded as described previously 28,29 . The individual CP-RFs were similarly capable of activating the expression of cognate reporter genes (Supplementary Figure S3 and data not shown). Control proteins (HO, HS, HM, HK, HL and HN) that lacked a MTD sequence were also prepared (data not shown). Finally, plasmids used to express Group A RFs utilized mammalian gene sequences; whereas, coding sequences for Group B reprogramming factors were modified to conform to E. coli codon preferences.
Group A recombinant proteins with MTD13 (LAAAALAVLPL), MTD57 (LIALLAAPLA), MTD85 (LLAAAAALLLA) and MTD108 (ALLAALLAP) in place of MTD84 and MTD86 were also evaluated but the proteins were less soluble, produced lower yields when expressed in E. coli and entered cells less efficiently (data not shown); therefore, these proteins were not evaluated further.
Cellular reprogramming by group A reprogramming factors. We first assessed the ability of human dermal fibroblasts (HDFs) to be reprogrammed by a cocktail of Group A RFs (OSKMN). 2 3 10 5 HDFs, maintained in HDF media, were treated with Group A CP-RFs (1 mM each) for 16 days. Media and recombinant proteins were replaced daily and the cultures were switched to human embryonic stem (hES) cell media after 7 days (Protocol 1, Figure 2A). Twenty-three granulated colonies emerged in cultures treated with MTD-containing RFs between day 5 and day 8; whereas, no colonies were observed in cultures treated with control proteins that lacked MTD sequence. The colonies morphologically resembled mouse embryonic stem (mES) cells ( Figure 2B), and expressed alkaline phosphatase, as assessed by TRA-2-49/6E antibody staining 21 days into the experiment ( Figure 2C). The 21-day colonies also expressed higher levels of stem cell-specific biomarkers 36 as compared to cells from untreated controls, including OCT4 and NANOG as assessed by indirect immunofluorescence ( Figure 3A) and OCT4, NANOG, KLF4 CMYC, LEFTB, EBAF and UTF1 as determined by reverse transcriptase PCR ( Figure 3B). By contrast, expression of all non-stem cell differentiation markers examined (CD9, ESG1, IFITM1, GAL, BRIX, REX1 and GAPDH) was unchanged ( Figure 3B).
These results indicate that cell-permeable Group A RFs can induce colonies with enhanced capacity for self-renewal and these colonies display morphologies and patterns of biomarker expression consistent with somatic to stem cell reprogramming. While the reprogramming was inefficient (0.01%), no colonies were observed (0%) following treatment with RF proteins lacking a MTD sequence. Since the colonies were selected based on enhanced self-renewal, unselected cells in the cultures were also tested for evidence of cellular reprogramming after treatment with CP-RF. For this we monitored the expression of stem cell-specific surface markers, SSEA-3 and SSEA-4, by flow cytometry as shown with a representative plots ( Figure 3C) and analyzed statistically ( Figure 3D, p , 0.001). Levels of SSEA-3 (and to a lesser extent SSEA-4) expression suggest a greater level of cellular reprogramming in the population as a whole than was inferred by colony outgrowth.
Group A CP-RFs were further tested using a variety of conditions, as illustrated by Protocol 2 ( Figure 4A). Maintaining cells in hES media from the start had no effect (data not shown); whereas, increasing the concentration of CP-RFs (4 mM each) increased the frequency of colony formation by ,2.5 fold (0.025%) compared to Protocol 1 (0.01%) which used lower concentration of CP-RFs (1 mM each). As before, the colonies emerged between day 5 and 8. Prolonging the treatment interval had no effect on colony formation (data not shown), nor did a second application of 8 mM CP-RFs administered between days 18 and 23 ( Figure 4A). The colonies morphologically resembled ES cells, although this was less apparent at day 23 ( Figure 4B), when the colonies had become over-grown. As before, the colonies expressed alkaline phosphatase (data not shown), OCT4 and NANOG ( Figure 5A) and the cell surface markers TRA-1-60 and SSEA-3 detected by flow cytometry as shown with a representative plots ( Figure 5B) and analyzed statistically ( Figure 5C, * p , 0.05 or ** p , 0.01). The latter were expressed at levels 9 to 12 and 8 to 33 fold higher than in untreated HDFs ( Figure 5 B and C, and data not shown). Since colonies induced by Group A RFs resembled iPSCs with regard to enhanced capacity for self renewal, colony morphology, and biomarker expression, we repeatedly tried to expand the cells in order to assess their differentiation potential. However, we were unable to expand the colonies either by picking individual colonies or by passaging all cells on plates that contained multiple colonies. These unsuccessful efforts employed a variety of growth conditions including those (lot selected FBS, LIF, mouse embryo feeder layers and collagen-treated culture dishes) routinely used to establish murine ES cell lines from pre-implantation embryos.
Cellular reprogramming by group B reprogramming factors. The Group A RFs tended to precipitate after being added to cells maintained either in human dermal fibroblast (HDF) or human embryonic stem cell (hESC) media, and CP-NANOG (HM 86 N) had the worst solubility problems. We therefore developed a second group of CP-RFs, designated Group B, in which NANOG was replaced with LIN28. LIN28 has been reported to promote cell proliferation by regulating mRNA stability of various cell cycle regulators in ESCs. Each Group B RF also employed a different MTD (Supplementary Table S1B), empirically selected to produce fusion proteins with the greatest relative solubility.
In Protocol 3 ( Figure 6A), 1 3 10 5 HDFs maintained in HDF media including LIF were plated on irradiated mouse embryo fibroblast feeder layers and treated daily for seven days with 0.5 mM of Group B RFs for 3 hrs. Colonies emerged between days 3 to 8 (7 colonies at day 3, 9 at day 5 and 3 at day 8) at an overall frequency of 0.02% and were tested for the ability to be expanded in mass culture on day 8. After adding fresh feeder cells, the colonies were maintained for another 7 days in the absence of RFs and stained with alkaline phosphatase ( Figure 6B). Control cultures (i.e. feeder cells alone) treated with RFs did not produce colonies, indicating the colonies were derived from HDFs, which based on cell morphology, enhanced capacity for self-renewal and alkaline phosphatase staining had undergone somatic to stem-cell reprogramming.
It has been reported that vitamin C could improve the reprogramming efficiency and promote the transition of partially reprogrammed iPSCs to a fully reprogrammed state 37 . The effect was attributed to anti-oxidant properties of ascorbic acid and suppression of replicative senescence. Therefore, we decided to use vitamin C as an adjuvant for reprogramming. Increasing the exposure to Group B RFs from 3 to 6 hours per day had no effect (data not shown) on colony formation (Protocol 4, Figure 7A). However, the addition of 1 and 10 mg/ml vitamin C to the media increased the number of colonies by 3.7-(0.074%) and 17-fold (0.34%), respectively  Figure 7B, p , 0.001). These results may suggest that the combination treatment of optimized CP-RFs and an adjuvant molecule could increase the efficiency of iSC generation. However, we were again unable to expand these colonies either individually or in mass culture using a variety of growth conditions including those routinely used to establish murine ES cell lines.

Discussion
The reprogramming of somatic cells into stem cells has tremendous medical potential as a way to generate patient-specific stem cells for tissue regeneration and repair. In the present study we used macromolecular intracellular transduction technology to develop cell-permeable OCT4, SOX2, KLF4, CMYC, NANOG and LIN28 transcription factors and tested mixtures of these proteins for the ability to reprogram primary human dermal fibroblasts. Protein cocktails consisting of cell-permeable OCT4, SOX2, KLF4, CMYC and either NANOG or LIN28 induced stem cell-like colonies (iSCs) with frequencies ranging from 0.01 to 0.34% as summarized in Table 1. The earliest colonies emerged within three days after starting protein treatment (0.5 or 4 mM each; added daily for 3 or 24 hours over 7 days) and resembled stem cells in terms of morphology, extended capacity of self-renewal, and expression of stem cell-specific markers, notably OCT4, NANOG, KLF4, CMYC, LEFTB, EBAF, UTF1, SSEA3 and alkaline phosphatase. By contrast, control proteins lacking MTD sequences did not induce stem cell colonies.
In principle, protein transduction technologies provide a direct way to manipulate biochemical processes in living cells under non steady-state conditions. The approach exploits the ability of specific basic, amphipathic and hydrophobic (or amphipathic depending on cargo) protein sequences to enhance the uptake of proteins and other macromolecules by mammalian cells 33,34 . The 7 MTDs described in the present study add to a growing list of signal peptide-derived sequences that enhance protein uptake by cultured cells. Features of the MTD responsible for protein transduction activity are clearly shared by a variety of peptide sequences raging from 7 to 12 amino acids in size. We are currently trying to identify optimal sequence and/or structural determinants for cytosolic and tissue delivery/ uptake and to assess potential contributions by cargo sequences. Our efforts to develop cell-permeable reprogramming factors were hampered by poor solubility of the recombinant proteins in culture media. The problem was improved, but not completely eliminated by extensively testing a variety of MTD sequences with each cargo protein.
The Yamanaka group reported a gradual reprogramming process of fibroblasts, in which granulated colonies appeared approximately two weeks after retroviral transduction of 4 RFs, and distinct types of colonies that were flat and resembled hESCs were observed around day 25 38,39 . In Protocol 1, we used Group A-5 CP-RFs and observed many granulated colonies (efficiency: 0.01%) within a week, and the colonies maintained in ES cell media displayed ESC-like morphology and robust expression of stem cell-specific markers (ex, SSEA-3, p , 0.001). The efficiency was increased to 0.025% with expression of biomarkers (ex, TRA-1-60, p , 0.05; SSEA-3, p , 0.01) when the cells were treated with a higher concentration of CP-RFs in Protocol 2, and in both cases colony formation was not observed with RFs without MTD sequences. These results suggest that the reprogramming process depended on intracellular levels of RF proteins.
In Protocols 3 and 4, we reduced the duration of daily treatment of CP-RFs from 24 hrs/day to 3 hrs/day for a week, and we reduced the concentration of protein in cells from 1-4 mM of Group A CP-RFs (OSKMN) to less than 0.5 mM of Group B (OSKML). Reprogramming efficiencies maintained (0.02%) even though the duration and the concentration were significantly reduced (both 8fold) compared with Protocols 1 and 2.
Compared to previous reports using CP-RFs, we observed higher reprogramming frequencies (0.025% for 3 days to 0.001% for 56 days 21 and 0.002% for 35 days 22 , respectively) using human dermal fibroblasts as reported with newborn human fibroblasts 21 and mouse embryo fibroblasts 22 . Moreover, these efficiencies were achieved using purified recombinant RF proteins, unlike previous studies that used unpurified RFs present in cell lysates 21 or RFs supplemented with valproic acid, a histone deacetylase inhibitor, to enhance reprogramming 22 . Thus, our study is the first to achieve stem cell reprogramming with purified CP-RFs alone.
However, we were unable to expand iSC colonies as expected for iPSC or ES cell lines and therefore were unable to assess cellular differentiation potentials. Our experience differs from previous reports using gene transfer to induce pluripotent stem cell reprogramming. This suggests cell permeable reprogramming factors have a greater propensity as compared to gene transfer approaches toward non-productive or partial reprogramming, even when similar cell culture conditions were used. Future studies will test whether protein-based approaches will benefit from changes in culture conditions known to enhance iPSC reprogramming following gene transfer. Furthermore, testing different protein formulations and optimizing the levels and timing of their application may be required to completely reprogram human somatic cells into pluripotent stem cells with the expected capacity for self-renewal.
Vitamin C has been reported to stimulate iPSC reprogramming of human dermal fibroblasts 40 . Similarly, we observed 3.7 to 17 more iSCs induced by CP-RFs with the addition of 1 and 10 mg/ml vitamin C, respectively. The effect has been attributed to anti-oxidant properties of ascorbic acid and suppression of replicative senescence; however, the other anti-oxidants tested did not enhance iPSC formation 40 . Alternatively, the effects of vitamin C--which has long been known to stimulate the proliferation, lifespan and multi-cellular depth of human dermal fibroblasts--are as readily attributed to enhanced collagen production [41][42][43][44] .
Although we showed that iSC colonies resembled embryonic stem cells with regard to morphology, biomarker expression, and extended capacity for self-renewal, we could not expand the colonies to assess their differentiation potential in vivo. The iSC colonies formed more rapidly (3 to 5 days) than previous studies describing successful iPSC reprogramming. Therefore, the speed of initial colony outgrowth appears to be a negative indication with regard to iPSC reprogramming. This experience is expected to help other groups interested in using CP-RFs to generate functional iPSCs.
While we could not confirm the pluripotency of the colonies generated from the terminally differentiated cells treated with CP-RFs, CP-RFs incorporating macromolecule transduction domains should result in a better quantitative and temporal control of the reprogramming. After somatic cells are induced to form iSC colonies, the transcription factors turnover seamlessly, with no trace left behind.
This study is the first to achieve partial stem cell reprogramming with purified CP-RFs alone through MITT. The approach circumvents safety concerns that preclude clinical applications of retroviral and other gene-based delivery systems. However, we were unable to expand iSC colonies as expected for iPSC or ES cell lines and therefore were unable to assess cellular multilineage differentiation potentials. This experience is expected to help other groups interested in using CP-RFs to generate functional iPSCs.

Methods
Construction of expression vectors for recombinant proteins. MTDs were identified by screening candidate signal peptides for hydrophobic sequences with protein transduction activity (Jo et al., manuscript in preparation). MTD84 and MTD86 for Group A recombinant proteins, and MTD47, MTD52, MTD132, MTD173 and MTD181 for Group B proteins were derived from signal sequences from AAK63068 and NP_629842 from Phytophthora cactorum and Streptomyces coelicolor, respectively, and NP_627512, NP_775628, NP_628377, NP_624384 and CAB84257 from StrePtomyces coelicolor, Homo sapiens, Streptomyces coelicolor, Streptomyces coelicolor and Neisseria meningitides, respectively.
For Group A recombinant RF proteins, the 2 MTDs were used for development of 35 recombinant proteins by attaching to the N-terminus, to the C-terminus or to both ends of each factor (2 MTDs 3 5 RFs 3 3 different structures 5 30 MTD-fused proteins 1 5 MTD-free control proteins). Among them, we chose CMYC and NANOG with N-terminal fused to MTD86, OCT4 with C-terminal fused to MTD84 and SOX2 and KLF4 with both ends fused to MTD86 because these proteins were the most soluble and produced the highest when expressed in E. coli.  plated at one day before (D-1) human dermal fibroblasts (HDF) were plated (day zero, D0). Cultures were treated as described in Figure 6, except the cultures were treated with proteins for 6 hours each day in HDF media containing 0, 1 or 10 mg/ml L-ascorbic acid. (B) Vitamin C enhances iSC colony formation. The percent of treated cells that gave rise to iSCs is tabulated based on the means of three separate experiments 6 s.d. (n 5 3/group). * p , 0.001 as determined by a Student's unpaired t-test. Cell permeability and intracellular localization. Recombinant proteins were purified from E. coli BL21-CodonPlus (DE3) cells, purified under denaturing conditions and refolded as described previously [28][29][30] .  Reverse transcriptase polymerase chain reaction (RT-PCR). RNA was extracted from cells using Trizol Reagent (Invitrogen, Carlsbad, CA), and 5 mg of RNA was used for reverse transcription reaction with SuperScript II (Invitrogen, Carlsbad, CA) and oligo-dT primer, according to the manufacturer's instructions. PCR cycles (30)(31)(32)(33)(34)(35) were performed with a temperature profile consisting of 95uC for 1 min, 55uC for 1 min, and 72uC for 1 min.
Flow Cytometry. Cells and colonies were treated with trypsin/EDTA (Invitrogen, Carlsbad, CA) at 37uC for 3 minutes to detach and dissociate, washed twice with PBS supplemented with 0.1% BSA, treated with Fc Blocker CD16/32 (Fc Blocker,  Statistical analysis. All experimental data obtained are expressed as the means 6 S.D. Statistical significance was evaluated using a one-tailed Student's t-test. Statistical significance was established at p , 0.05.