Author contributions: K.H.: conception, design, experimentation, data analysis and interpretation, writing, final approval of manuscript; P.Z.: experimentation, data analysis and interpretation, writing, final approval of manuscript; Z.W. and J. E. P.: experimentation, data analysis and interpretation; W.L. and M.F.P.: conception, design, data analysis and interpretation, writing, financial support, final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLS EXPRESS June 22, 2010.
Reprogramming human somatic cells into pluripotent cells opens up new possibilities for transplantation therapy, the study of disease, and drug screening. In addition to somatic cell nuclear transfer, several approaches to reprogramming human cells have been reported: transduction of defined transcription factors to generate induced pluripotent stem cell (iPSC), human embryonic stem cell (hESC)–somatic cell fusion, and hESC cytoplast–somatic cell fusion or exposure to extracts of hESC. Here, we optimized techniques for hESC–human fibroblast fusion and enucleation and cytoplast fusion, and then compared the reprogramming efficiency between iPSC generation, cell-fusion and cytoplast-fusion. When compared with iPSC, hESC-fusion provided much faster and efficient reprogramming of somatic cells. The reprogramming required more than 4 weeks and the efficiency was less than 0.001% in iPSC generation, and it was less than 10 days and more than 0.005% in hESC-fusion. In addition, fusion yielded almost no partially reprogrammed cell colonies. However, the fused cells were tetraploid or aneuploid. hESC cytoplast fusion could initiate reprogramming but was never able to complete reprogramming. These data indicate that in cell fusion, as in nuclear transfer, reprogramming through direct introduction of a somatic nucleus into the environment of a pluripotent cell provides relatively efficient reprogramming. The findings also suggest that the nucleus of the host pluripotent cell may contain components that accelerate the reprogramming process. STEM CELLS 2010;28:1338–1348
The discovery of human embryonic stem cells (hESC) opened up the possibility for the application of human pluripotent stem cells in transplantation therapy, drug screening, and toxicology studies, as well as functional genomics and proteomics research [1, 2]. However, the problem of immune rejection may limit the application of hESC in transplantation, because it would be difficult to obtain the large numbers of embryos required to cover a wide range of human leukocyte antigen (HLA) haplotypes for close tissue matching to patients . It is also difficult to envision how hESC technology could provide models for the study of diseases that have multigenic susceptibility. Reprogramming somatic cells into pluripotent cells could address these limitations .
In mammals, establishment of ES cell lines from embryos developed after somatic cell nuclear transfer (SCNT) into enucleated unfertilized eggs could potentially facilitate complete reprogramming of somatic cells into pluripotent cells [5–7]. However, the SCNT approach may be difficult to apply in humans because there are ethical and practical barriers to obtain sufficient numbers of human unfertilized eggs. There is one report of human SCNT using rabbit oocytes . However, the reprogrammed cells in the method retain rabbit mitochondria and mitochondrial DNA, and therefore might not be useable for future transplantation therapies. Therefore, other reprogramming techniques will be required for reprogramming human somatic cells apart from SCNT.
The most promising reprogramming technique for human cells is the generation of induced pluripotent stem cells (iPSC) by transduction of defined transcription factors such as OCT4, SOX2, and KLF4 [9, 10]. In general, the efficiency of iPSC generation is very low and reprogrammed clones often show differences at the level of the epigenome and transcriptome when compared with stem cells derived from embryos [11, 12]. Another major technique of reprogramming is cell–cell fusion of somatic cells and hESCs [13, 14]. Cell–cell fusion-mediated reprogramming may be faster and more efficient than the iPSC method, because the hESC used for cell fusion provides all the factors required for maintenance of pluripotency. However, the fused and reprogrammed cells retain both the somatic and ES cell genome, an obvious barrier to their application in anything other than mechanistic studies. A less studied technique is the fusion of a somatic cell and a hESC cytoplasm, conceptually similar to SCNT except using enucleated hESCs instead of enucleated unfertilized eggs. There is only one successful report of this approach, however, and the study lacked detailed characterization of the reprogrammed cells . Enucleation of hESC is technically challenging and therefore the technique of cytoplast fusion has not been widely explored.
No previous report has carefully compared these methods for reprogramming. Such a comparison would indicate whether cytoplast fusion is a feasible alternative to iPS cell generation, and would provide some insight into how transduction of a few key pluripotency factors compares with provision of the complete pluripotent cytoplasmic or cytoplasmic plus nuclear environment in cell reprogramming. Here, we optimized an efficient enucleation technique for hESCs, and somatic cell–hESC or somatic cell–hESC cytoplast fusion methods, then compared the reprogramming time course and efficiency of reprogramming between the iPS technique, cell fusion, and cytoplast fusion using the same somatic cell clones and culture systems.
MATERIALS AND METHODS
The human embryonic stem cell (hESC) lines HES2 and HES3 (established in our laboratory) were maintained using standard cell culture methodology . For enzymatic bulk expansion, the cells were cultured on mitotically arrested mouse embryonic fibroblast (MEF) feeder cell layers in Dulbecco's Modified Eagle Medium (DMEM)/F12 supplement with 20% knockout serum replacement (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), L-glutamine, nonessential amino acid and 4 ng/ml FGF2, as described previously [1, 16]. The human fetal lung fibroblast, MRC5, and the human fetal dermal fibroblasts (HDFf) were obtained from America Type Culture Collection (ATCC, Manassas, VA, http://www.atcc.org/) and ScienCell Research Laboratories (Carlsbad, CA, http://www.sciencellonline.com/), respectively.
Transgenic Cell Lines
The plasmid pCAG-DsRed-mito-Pgk-TK-SV40-Pur was constructed by ligation of the HindIII- SalI CAG-DsRed-mito fragment of pCAG-DsRed-mito  and Sse8387I Pgk-TK fragment of pPgk-TK (a gift from Dr. Suemori, Kyoto University) into PvuII site in pPUR plasmid (Clontech, Palo Alto, CA, http://www.clontech.com). The 5 kb human OCT4 promoter, −5920 to +1 bp from transcription initiation site, was cloned by polymerase chain reaction (PCR). The plasmid pOCT4-EGFP-IRES-Hyg-SV40-Neo was constructed by combination of the OCT4 promoter, the AgeI- NotI EGFP fragment from pEGFP-N1 (Clontech), the SmaI– BstXI internal ribosomal entry site (IRES) fragment from pIRES2-EGFP (Clontech), the BamHI- NotI hygromycin resistance gene fragment from pPgkHygPA (a gift from Dr. Tada, Kyoto University) and the NheI- NotI vector backbone, including SV40-Neo, from pEGFP-N1. The OCT4 promoter used here yielded green fluorescent protein (GFP) fluorescence positive in undifferentiated hESC cells that was extinguished following retinoic acid induced differentiation.
To establish the DsRed-mito-TK-Pur (DTP) transgenic hESC lines, the pCAG-DsRed-mito-Pgk-TK-SV40-Pur plasmid was linearized, transfected into HES2 or HES3 cells using FuGENE HD (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com), and stable cell lines were selected by Puromycin as described previously . The transgene activity in the cell lines was evaluated by DsRed-mito fluorescence and ganciclovir (GCV) sensitivity. The GFP transgenic fibroblasts were generated by transfection of pCAG-EGFP-SV40-Neo. The OCT-GFP-IRES-Hyg-SV40-Neo (OGIH) transgenic fibroblast clones were isolated by transfection of linearized pOCT4-EGFP-IRES-Hyg-SV40-Neo plasmid. After selection with G418, small colonies were picked, reseeded at low density, expanded, and evaluated for the insertion of a full-length transgene with PCR. Finally, transgene activity was assessed by cell fusion with hESCs.
Induced Pluripotent Cells
To generate the doxycline-inducible viral expression vector, the digested rtTA2S-M2 fragment  was inserted into the vector FUIPW, containing an IRES fragment followed by the puromycin resistance gene. The ubiquitin promoter of FUW  was replaced with a tetracycline-responsive element containing a cytomegalovirus (CMV) minimal promoter to construct tetracycline response elements (FTRE). cDNAs encoding OCT4, KLF4, SOX2, and cMYC were subsequently cloned into FTRE between the BamHI and XbaI sites .
To produce lentivirus, 293T cells were transfected at 80% confluence using the polyethylenimine (PEI) reagent. For a 10-cm plate, 500 μl DMEM, 30 μl PEI reagent, and 10 μg DNA (5:3:2 PSPAX2: vector: PMD2G) was used. After about 48–72 hours of culture, virus was harvested and concentrated by 300-fold. For virus infection, 1 × 106 OGIH transgenic fibroblast cells were seeded onto one 10 cm plate. Fifty microliters of lentivirus was applied in an overnight infection. A second round of lentiviral infection was performed 2 days afterwards. The hESC culture medium was used after 4 days culture with 1 μg/ml doxycyline (DOX) addition. The iPSC colonies were picked up starting from 5 weeks after induced transgene expression based on appearance of hESC-like morphology. Five HDFf-derived induced pluripotent stem cell (iPSC) cell lines were used for further characterization.
The DTP transgenic hESCs were enzymatically passaged at high density as small clumps on Matrigel-coated 6-well culture plates. The OGIH transgenic fibroblasts were harvested, bound with Sendai virus (HVJ) envelope twice and plated at 2–2.5 × 105 fibroblasts per well on 6-well plates containing hESCs in culture. The fibroblast on hESC culture were then centrifuged and incubated to induce cell fusion according to the manufacturer's protocol (Genomone-CF; Ishihara Sangyo, Osaka, Japan, http://www.iskweb.co.jp). Immediately after fusion, the cells were cultured in MEF-conditioned medium . After day 1 culture, drug-resistant inactivated MEF feeder cells were seeded onto the plates, or the fused cells were reseeded onto the MEF feeder layer, and then selection of fused cells was started by addition of 100 μg/ml G418 after 2 days. In some experiments, further selection was performed using 40 μg/ml hygromycin and/or 1 μg/ml puromycin after 1 week following cell fusion. The colonies were picked after 10–14 days, transferred onto new feeder layers, expanded according to standard hESC culture methods, and then 12 HDFf-hESC-fused cell lines and 12 MRC5-hESC-fused cell lines were used for further characterization. G-banding karyotype analysis was performed as described previously .
For transient transfection of reprogramming factors prior to cell fusion, nuclear localized CFP (CFP-nuc; Clontech), human OCT4, SOX2, c-MYC, KLF4, NANOG, and LIN-28 were cloned into a pCS2+ expression vector (gift from Dr. Turner, University of Michigan) and 1 μg of each plasmid was simultaneously transfected into OGIH fibroblasts in 60-mm culture dishes with Lipofectamine 2000 (Invitrogen) two times at day 2 and day 1 before cell fusion. For treatment with epigenetic modifiers, the OGIH fibroblast were cultured with 2 mM valproic acid (VA; Sigma-Aldrich, St Louis, MO, http://www.sigmaaldrich.com/) and 2 μM 5-azacytidine (5AZC; Sigma-Aldrich) from day 2 before through 1 week after cell fusion. Ten transfected HDFf-hESC-fused cell lines and four transfected MRC5-hESC fused cell lines were used for further characterization.
Enucleation and Cytoplasm Fusion
The enucleation procedure for hESCs was developed based on a previous report [23, 24]. The DTP hESCs were enzymatically passaged at high density as small clumps on Matrigel-coated round glass plates, 25 mm diameter and 0.5 mm thickness (custom made by Thermo Fisher Scientific, Waltham, MA, http://www.thermofisher) in 6-well culture plates. After 24 hours, the confluent ES cells were treated for 15 minutes with 50 μg/ml Cytochalasin B. Then the glass plates were inverted, transferred into 50-ml conical tubes containing 25 ml Cytochalasin B medium, centrifuged at 15,000g, at 37°C for 30 minutes in a fixed angle rotor. The glass plates were then transferred into new 6-well plates with the cells facing up into new culture medium and incubated 30 minutes to allow recovery of the cytoplasts. Enucleation efficiency was evaluated by Hoechst 33,342 staining. Then the enucleated cells were fused with OGIH fibroblasts following the same procedure described for cell fusion above. The first phase of selection of reprogrammed cells was as described for the cell–cell fusion experiments. After 1 week of selection, further selection was performed with 2 μg/ml GCV.
For transient transfection of reprogramming factors prior to cytoplast fusion, CFP-nuc, OCT4, SOX2, c-MYC, KLF4, NANOG, and LIN-28 expression vectors were simultaneously transfected into OGIH HDFf two times at day 2 and day 1 before cytoplast fusion. For treatment with epigenetic modifiers, the OGIH HDFf was cultured with 2 mM VA and 2 μM 5AZC from day 2 before enucleation through 1-week after cytoplast fusion.
Characterization, Differentiation Assays, and Immunostaining
The reprogrammed cell colonies were fixed with 4% paraformaldehyde and alkaline phosphatase (ALP) activity was determined with a Vector Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Immunostaining was carried out with the following primary antibodies: TRA-1-60 and TRA-1-80 (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com/), anti-stage specific embryonic antigen (SSEA)-3 and -4 (Developmental Studies Hybridoma Bank at the University of Iowa, Iowa city, IA, http://dshb.biology. uiowa.edu/), anti-OCT4 (clone C-10, Santa Cruz Biotechnology), rabbit anti-SOX2 (AB5603, Chemicon, Millipore, Billerica, MA, http://www.millipore.com/), rabbit anti-KLF4 (sc-20691, Santa Cruz Biotechnology), rabbit anti-cMYC (sc-764, Santa Cruz Biotechnology) or goat anti-NANOG (AF1997, R&D systems, Minneapolis, MN, http://www.rndsystems.com/). Then, samples were incubated with Alexa Fluor 594-conjugated (Molecular Probes, Invitrogen) or HRP-conjugated secondary antibodies (Santa Cruz Biotechnology Inc.), then detected with indirect immunofluorscence microscopy or staining with HRP substrates. Western blotting for OCT4, SOX2, KLF4, or cMYC were carried out with same primary antibodies and secondary HRP-conjugated antibodies for immunostaining, and detected with WesternBreeze chemiluminescence detection system (Invitrogen).
For differentiation assays, embryoid bodies (EB) were formed for 2 weeks in low-attachment culture plates and then plated onto glass culture slides as described previously . After the cells grew out from attached EBs, they were fixed with 4% paraformaldehyde (PFA), incubated with anti-N-tubulin (clone TU-20, Chemicon, Millipore), -glial fibrillary acidic protein (clone 4A11, BD biosciences, San Jose, CA, http://www.bdbiosciences.com/), -alpha smooth muscle actin (clone 1A4, Dako Cytomation, Glostrup, Denmark, http://www.dako.com/), -Desmin (clone 1A4, Thermo Fisher Scientific), -GATA6 (clone 222228, R&D systems), or -alpha-fetoprotein antibody (clone C3, Sigma-Aldrich) as the primary antibodies and then detected using Alexa Fluor 596- or Alexa Fluor 488-conjugated secondary antibodies (Molecular Probes, Invitrogen) and fluorescence microscopy.
For quantitative PCR analysis, total RNA was isolated from cells cultured in feeder-free conditions using RNeasy micro kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). Reverse transcription was performed with Omniscript (Qiagen) and quantitative reverse transcription PCR was then performed using each gene-specific primer/probe mix (TaqMan Gene Expression Assays: OCT4; Hs01895061_u1. SOX2; Hs01053049_s1. KLF4; Hs00358836_m1. cMYC; Hs00153408_m1. NANOG; Hs02387400_g1. LIN28; Hs00702808_s1. PPIA; Hs99999904_m1. DNMT3B; Hs00171876_m1.), TaqMan ×2 master mix and ABI PRISM 7,900 Sequence Detection System (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) according to manufacture's protocol. The PCR data were analyzed by the Δ/Δ CT method and normalized to levels of PPIA expression.
For DNA methylation analysis, 1 μg genomic DNA was treated with EpiTect Bisulfite Kit (Qiagen), according to the manufacturer's recommendations. The promoter regions of the NANOG genes were amplified from the bisulfite converted DNA by PCR. Primer sequences used for PCR amplification were described previously . The PCR products were subcloned into pGEM-T Easy vector (Promega, Madison, WI, http://www.promega.com/). Randomly 8–10 clones of each sample were picked and verified by sequencing with the T7 or SP6 primer.
Reprogramming by Cell Fusion with hESC Is Much More Efficient and Faster Than Virus Transduction of Reprogramming Factors
Human fetal lung fibroblasts (MRC5) or human fetal dermal fibroblasts (HDFf), capable of supporting human embryonic stem cell (hESC) as a feeder cell layer (data not shown), were transfected with an OCT4 promoter-driven GFP-IRES-hygromycin-resistance gene (OGIH) to visualize and select reprogrammed cells. To avoid complication of results from use of a mixed cell population, the transgenic fibroblasts were cloned, expanded, and used between passage 4 and 7 after cloning for comparison of reprogramming efficiency between gene transduction and cell fusion.
We first generated induced pluripotent stem cell (iPSC) lines from the OGIH transgenic fibroblasts. The OGIH transgenic fibroblasts were transduced by lentivirus expressing KLF4, OCT4, SOX2, and cMYC whose expression is controlled by doxycycline (DOX) induction. These cells were also transduced with lentivirus expressing doxycycline coactivator rtTA. To evaluate our transduction system, protein expression of the transgenes were examined by western blotting and immunostaining 2 and 7 days postinfection (Supporting Information Fig. 1). Following induction with DOX, over 95% of the cells expressed transgene protein. After 4 weeks of induction in hESC culture medium in the presence of DOX, DOX was then removed as many colonies emerged. Less than 1% of colonies resembled hESC-like morphologically. As many non-hESC-like cell colonies displayed strong autofluorescence (Supporting Information Fig. 2), reprogrammed cell colonies were selected on the basis of hESC-like morphology and these gave rise to iPSC lines (Fig. 1A). As expected, a GFP signal was detected in the iPS cell lines, indicating that the OCT4 promoter is activated on the reprogrammed cells (Fig. 1B and Supporting Information Fig. 2). This result was also further confirmed by immunocytochemistry of OCT4 (Fig. 1C). Besides OCT4, the iPSCs also expressed other pluripotency markers including SOX2, NANOG, alkaline phosphatase (ALP), SSEA-3, SSEA-4, TRA-1-60, and TRA-1-80 (Fig. 1C). DNA methylation analysis of cytosine guanine dinucleotides (CpG) in the promoter regions of NANOG revealed a highly unmethylated state in the iPS cells, similar to hESCs, whereas the NANOG promoter region was more methylated in parental HDFf (Supporting Information Fig. 3). To further confirm that the colonies are pluripotent iPS cells, these cells were induced to form embryoid bodies (EBs). Immunocytochemistry of lineage markers suggested that embryoid body cells contained cells of ectodermal, mesodermal, and endodermal lineage as shown by the expression of appropriate markers (Fig. 1D). In agreement with other reports [9, 10, 25], the reprogramming process of iPSC generation required more than 4 weeks for emergence of hESC-like colonies and the efficiency was less than 0.001% (Table 1).
Table 1. Reprogramming efficiency
We next examined the reprogramming efficiency in somatic cell and hESC fusion. To select and visualize fused cells, the ubiquitous promoter (CAG)-driven mitochondrial-localized DsRed-thymidine kinase-puromycin resistance gene (DTP) was transfected into HES2 or HES3 hESC lines. To investigate efficient fibroblast and hESC fusion conditions, CAG-GFP transgenic fibroblasts were established and fused with the DTP hESCs. Protocols using polyethylene glycol as a fusogen resulted in large numbers of dead and floating DsRed positive cells (data not shown), perhaps due to extensive hESC death following dissociation to single cells in suspension. To limit physical damage to hESCs, we next examined Sendai virus envelope-mediated cell–cell fusion. Single cell suspensions of the fibroblasts were bound with the virus envelope, and then seeded on intact hESC cultures to fuse. There was no toxicity, fewer multiple fused cells, and around 50% of GFP-positive cells were DsRed positive. Therefore, the virus envelope-mediated cell fusion was used for further experiments.
When OGIH transgenic HDFf or MRC5 were fused with the DTP transgenic hESCs (scheme in Fig. 2A), OCT4 promoter-GFP positive and hESC-like cells appeared from 7 days after selection with G418 (Fig. 2B). Ten days after fusion, the hESC-like reprogrammed cell colonies were large enough to pick up and expand (Fig. 2C). The reprogramming efficiency of cell-fusion was much higher than iPSC generation (Table 1 and Fig. 2D); the difference was mainly attributable to the higher proportion of hESC-like colonies appearing after fusion compared with the iPSC protocol, since colonies with hESC-like morphology gave rise to reprogrammed lines with similar efficiency in either technique. Compared with transduction of reprogramming factors, there were far fewer non-hESC-like and nonfibroblast-like cell colonies (Supporting Information Fig. 4). The proportion of partially reprogrammed colonies was much lower than that of hESC-like reprogrammed colonies and few partially reprogrammed cells grew (Table 1). Thus, there was no significant difference between colony numbers after hygromycin selection versus G418 selection (Fig. 2E), a finding that suggests that the OCT4 promoter was active in almost all growing colonies after fusion. In addition, there was no difference in reprogramming efficiency whether MEF-feeder cells were seeded onto fused cells 1 day postfusion, or the fused cells were seeded onto mouse embryonic fibroblast (MEF)-feeders. After harvest, all colonies that attached to the feeder layer retained hESC-like morphology and grew well. Nevertheless, 20%–30% of fusion-derived colonies failed to attach, much like iPSCs and hESC, (Table 1), which may illustrate a common susceptibility to physical damage among human pluripotent stem cells. Except for a slightly slower rate of proliferation (data not shown), the reprogrammed cells were almost indistinguishable from hESCs or iPSCs. Such cells expressed all examined hESC markers and were also able to differentiate into cells expressing markers of all three germ layers (Fig. 2F and 2G). In addition, the NANOG promoter region was highly unmethylated in the fused cells, resembling that of hESCs (Supporting Information Fig. 3). After passage 7, we examined chromosome numbers in four cell lines. Two cell lines retained a tetraploid complement of 92 chromosomes, however, the other two cell lines lost some chromosomes and showed varying degrees of aneuploidy; one cell line consisted of cells retaining 50–74 chromosomes and the other cell line consisted of cells retaining 48–92 chromosomes (data not shown).
hESC Cytoplasm Partially but Not Fully Reprograms Somatic Cells
To investigate whether hESC cytoplasm can reprogram somatic cells, we first optimized an efficient enucleation method for hESC based on early reports for other mammalian cells [23, 24], using a combination of partial destruction of cytoskeleton and centrifugation to release the nuclei and produce cytoplasts. Using this method, almost all hESCs remained attached to the culture cover slip while few contaminating feeder cells remained attached. Enucleation efficiency was evaluated by live cell nuclear staining of DNA and 90%–95% cells were enucleated (average 91.8%, 10 randomly selected colonies counted) (Fig. 3 B). The cytoplast retained surface hESC markers after enucleation, however, they lost almost all nuclear markers of hESCs, except weak cytoplasmic signals of KLF4, as visualized by immunostaining (Supporting Information Fig. 5). To monitor and remove reprogrammed cells from nonenucleated cells, we used DsRed expression and ganciclovir (GCV) selection to eliminate thymidine kinase (TK) expressing cells (scheme in Fig. 3A). After fusion of enucleated DTP hESCs and the OGIH HDFf or MRC5, the cells were selected for 5 days with G418 followed by GCV and G418 double selection for another 5 days. Fourteen days after cytoplast fusion, only a few small colonies appeared. All the colonies were DsRed-negative. Some of the colonies contained faintly GFP-positive cells, however, these cells soon displayed flat morphology, stopped growing after 14 days, and could not be further expanded (Fig. 3C and Table 1). The GFP-positive colonies expressed ALP, TRA-1-60, and TRA-1-80 at the same levels as hESCs, however, expression levels of SSEA4 were weaker than expression in hESCs. OCT4 and SOX2 were weakly and mosaically expressed, and neither SSEA-3 nor NANOG expression were detectable by immunocytochemistry (Fig. 3D). The mRNA expression levels of reprogramming factors and hESC markers were much weaker than that of hESCs or reprogrammed hybrid cells (Fig. 3E). We used the same protocol for hESC-somatic cell fusion and hESC cytoplast-somatic cell fusion except for the enucleation process (Cytochalasin B-treatment and centrifugation) and GCV-selection. Methodological factors might have caused the differences in reprogramming efficiency between the two protocols. However, the appearance of DsRed- and GFP-positive nonenucleated hESC-fused reprogrammed cell colonies without selection after exposure of Cytochalasin B and centrifugation (data not shown) indicated that these treatments did not affect the reprogramming process. Performing the cell–cell fusion with a hESC line not containing the TK-transgene in the presence of GCV did not affect the reprogramming efficiency of the fusion process (data not shown). These results suggest that the methodological differences are not critical and that hESC cytoplasm may not be enough to complete the reprogramming.
To enhance cytoplast-mediated reprogramming, we attempted transient transfection of reprogramming factors into the fibroblasts prior to enucleation, as well as treatment with small molecule epigenetic modifiers. We first examined these strategies in the cell–cell fusion assay. Expression vectors encoding known reprogramming factors, OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN-28 were transiently transfected simultaneously with the nuclear-localized ECFP (ECFP-nuc) expression vector into the OGIH HDFf or MRC5 (scheme in Fig. 4A). When around 75% of the cells showed ECFP-nuc expression (data not shown) and the transgene expression was peaking at day 1 post-transfection (Supporting Information Fig. 6), the transfected cells were fused with the hESCs. At 7 days postfusion, reprogrammed cell colonies appeared with around three times higher efficiency; the colonies were also much larger than nontransfected controls (Fig. 4B and 4C). All of the cells in the colonies retained ES cell-like morphology, as well as EGFP and ALP expression, although ECFP-nuc expression was undetectable at that time (data not shown). The cells were able to expand similar to nontransfected cell hybrids (Table 1). These findings suggested that the transient expression of reprogramming factors could not only increase reprogramming efficiency but may also accelerate the reprogramming process. It has been reported that small molecule epigenetic modulators, such as histone deacetylase (HDAC) inhibitor; valproic acid (VA), and DNA methyltransferase (DNMT) inhibbitor; 5-azacytidine (5AZC), enhanced efficiency of iPS generation , therefore, the epigenetic modulators were examined in cell–cell-fusion assays. Various dosages, up to those inducing toxicity, of VA (up to 5 mM) and 5AZC (up to 10 μM), and various treatment periods ranging from 2 days before to 7 days after fusion, were examined. However, none of these treatment regimens increased reprogramming efficiency in the cell–cell fusion assay (data not shown).
Next, combinations of the transient transfection and maximum nontoxic dosages of the epigenetic modifier were applied in the cytoplast fusion assays with HDFf, which had higher reprogramming efficiency in cell fusion and iPSC generation than MRC5 (scheme in Fig. 4D). Though there were no significant differences between control and VA or 5AZC-treatment, the GFP-positive colony numbers were slightly increased using a combination of VA and 5AZC (Fig. 4E). The transient transfection enhanced the GFP-positive colony number over fivefold, and combination of transfection and VA increased the colony numbers more than sevenfold (Fig. 4E and Table 1). The cells in the colonies formed by transfected cells retained more hESC-like morphology and higher intensity GFP expression than those formed by nontransfected cells, however, such cells and colonies still could not be expanded (Fig. 4F and Table 1). The cells expressed OCT4, SOX2, SSEA4, TRA-1-60, and TRA-1-80, however, NANOG was faintly and mosaically expressed, and SSEA3 was undetectable (Fig. 4G).
Human somatic cell reprogramming has been achieved using hESC-somatic cell fusion [13, 14] or induction of reprogramming factors (iPSC generation) [9, 10]. In these reports, reprogramming efficiencies (established reprogrammed cell lines or colonies with hESC like appearance from somatic cells fused or transduced) of human fibroblasts were ∼0.001% by PEG-mediated cell fusion, 0.0001%–0.05% by conventional OCT4, SOX2, KLF4, and cMYC lentivirus infection [9, 10, 25] and up to1% by combination of the reprogramming factors and small molecules, such as VA , or other factors, such as p53 siRNA [27–29]. However, it is difficult to compare the efficiencies directly, because fibroblast cell types and passage numbers were different in the various reports, primary human fibroblast cultures may be heterogeneous, and virus infection efficiency might differ. Additionally, hESC survival might be low after single cell-suspension-based PEG-mediated cell fusion since hESCs are very sensitive to physical damage.
It is also difficult to directly compare reprogramming efficiency between lentivirus infection and cell fusion because they require quite different procedures, however, the use of uniform fibroblast cultures helps to reduce one source of variability. Therefore in this report, we cloned human fibroblasts to simplify the comparison of reprogramming efficiency between iPSC generation and cell fusion-mediated reprogramming. We also developed mild cell fusion conditions with HVJ-envelope applicable to intact hESC colonies. This fusion procedure did not disturb the hESC niche and did not cause hESC cell death, unlike harvesting cells into suspension. This protocol resulted in ∼0.005% reprogramming efficiency in cell fusion. Although the somatic cell type used was different from earlier reports, making direct comparison difficult, the reprogramming efficiency was five times higher than previous reports [13, 14]. In comparison, reprogramming efficiency in lentivirus-mediated iPSC was 0.00025% and lower than cell fusion significantly (p = .018 in t-test). Our slightly lower efficiency of reprogramming by transduction may relate to our use of a higher passage of the fibroblast cells, rather than virus infection efficiency, because cloning and expansion of the transgenic fibroblast line required more than 1 month culture period.
Besides reprogramming efficiency, there were several differences between cell fusion and the four factor induced iPSC generation. First, fully reprogrammed colonies appeared in cell fusion-mediated reprogramming much faster than in four factor-mediated iPSC generation. The hESC-like OCT4-GFP positive colonies appeared at 7 days postfusion and the colonies were large enough to pick up at day 10–14 in cell fusion-mediated reprogramming, whereas the colonies did not appear until 15–18 days postinfection in virus-mediated iPSC production and took ∼30 days to grow to a sufficient size to pick up. The time course differences between cell fusion and virus induction of iPSC were similar to previously reported results [9, 10, 13, 14, 25]. As reprogramming is an epigenetic change from somatic state to the pluripotent state, requiring establishment of pluripotent cell-specific transcriptional networks and signaling cascades, reprogramming may proceed more efficiently in cell fusion, where the hESC partner provides a fully functional network of pluripotency factors and cell signaling molecules. By contrast, introduction of just a few reprogramming factors may initiate a process that takes some time to fully reconstitute the pluripotent state. Indeed, Hanna et al.  demonstrated that reprogramming by defined factors was a continuous stochastic process, and was accelerated by overexpression of additional factors such as Nanog or Lin28. In addition, even in ESC-somatic cell fusion, it was reported Oct4-GFP expression was initiated much faster and reprogramming efficiency was increased when pluripotent a state-specific transcription factor, such as NANOG, SOX2, or SALL4, was overexpressed in somatic cells , and we demonstrated that transient transfection of the reprogramming factors enabled faster and more efficient reprogramming. Recently, it was reported that demethylation of NANOG and OCT4 promoter region was initiated from day 1 after ESC-somatic cell fusion via activation-induced cytidine deaminase, which is present in ESCs, without cell division or DNA replication . These reports suggest the time course difference due to transcription/epigenetic factors in initiation of reprogramming.
Another difference was the very low frequency of partially reprogrammed cells in cell fusion compared to iPSC generation. In cell fusion-mediated reprogramming, there were almost no growing non-hESC-like, partially reprogrammed, colonies even under G418 selection, which selects fused cells and unfused fibroblasts. By contrast, many partially reprogrammed cell colonies appeared in iPSC generation and hESC-like colonies represented less than 1% of the total. The fully/partially reprogrammed cell ratios we observed were similar to previous cell fusion and iPSC studies [10, 14, 33]. The basis of partial reprogramming is unknown, but it may be due to inadequate levels of reprogramming factors or improper balance in the levels of factors. In addition, eventual silencing of exogenous factors and expression of pluripotency genes from endogenous loci at appropriate levels may be necessary to complete reprogramming to the pluripotent state. It is possible that variability in the expression levels of pluripotency factors is less significant in cell fusion, though the presence of a tetraploid genome might lead to dosage errors. The other possibility for the different ratio of fully/partially reprogrammed cells could be due to the difference between reprogramming initiation with few transcription factors in iPS generation and full pluripotent transcriptional/epigenetic network in cell fusion. Indeed, it was reported that somatic cells closer to hESCs, such as neural stem cells [34, 35], were more efficiently reprogrammed, and epigenetic modulators, such as HDAC inhibitor and/or DNMT inhibitor , or overexpression of pluripotent state-specific transcription factors, such as NANOG [9, 36] or UTF1 , could enhance reprogramming efficiency in both cell fusion and iPSCs.
A third obvious difference was the genetic abnormality of fused cells. By its very nature cell fusion gives rise to a wide range of tetraploid and aneuploid clones. Because of these genetic abnormalities, even though the cell-fusion mediated reprogramming is much easer, faster and efficient, the use of cell–cell hybrids in most applications, such as drug screening of specific cell genotypes or transplantation therapy, may be problematic. In the mouse, tetraploid cells can still contribute extensively to chimeras ; however, it is conceivable, though not likely, that some modification of the technique might still find use for emergency applications requiring urgent treatment. It is possible to envision fusion of patient cells with HLA gene-modified hESCs bearing markers for negative selection in vivo.
In this study, we also optimized an efficient method for enucleation of hESCs, enabling cytoplast fusion without hESC dissociation. This enabled us to test whether the hESC niche, extrinsic signals from feeder-layer and culture medium, and cytoplasmic factors, were sufficient to reprogram somatic cell nuclei. The hESC-cytoplast fusion method demonstrated induction of several pluripotent markers and reprogramming factors, however, the expression levels of these factors were lower than those of hESC-somatic cell fusion derived completely reprogrammed cells or hESCs, resulting in no completely reprogrammed cells obtained by hESC-cytoplast fusion. The combination of hESC cytoplast fusion, transient transfection of reprogramming factors and supplementation with HDAC and DNMT inhibitors was able to induce more colonies retaining a more hESC-like morphology and expression of additional pluripotency markers, such as NANOG. However, all the cells were still partially reprogrammed cells and the colonies stopped growing or differentiated. This suggests that hESC cytoplasm and hESC culture conditions can initiate partial, but not complete reprogramming of human fibroblasts. This is consistent with several reports indicating that some pluripotent-specific genes can be induced in somatic cells by pluripotent cell extracts [38, 39] and a recent report that continuous transient transfection resulted in iPSCs . Therefore, continuous expression of transcription factors may be required to achieve complete reprogramming even though some of the reprogramming process could be initiated by hESC cytoplasm. As these studies and our own work used cells that are relatively difficult to reprogram, it may be worth using other cell types, such as neural stem cells, that are more amenable to reprogramming in the hESC cytoplast fusion-mediated method.
Our results demonstrate that hESC-fusion provides for much faster and efficient reprogramming of somatic cells compared to iPSC generation via transduction of 4 reprogramming factors. Enucleated hESC-fusion initiates reprogramming but does not yield completely reprogrammed cells. These data indicate that reprogramming through direct introduction of a somatic nucleus into the environment of a pluripotent cell provides for efficient reprogramming. The findings also suggest that the nucleus of the host pluripotent cell may contain components that accelerate the reprogramming process. Identifying of the critical nuclear components would facilitate understanding of reprogramming mechanisms.
We thank Dr. David Turner, University of Michigan, Dr. Jun-ichi Miyazaki, Osaka University, Dr. Mirella Dottori, The University of Melbourne, Dr. Takashi Tada and Dr. Hirofumi Suemori, Kyoto University for providing plasmids. This research was supported by California Institute for Regenerative Medicine (CIRM), New Cell Lines Grant #RL1-00667-1 New Technology for the Derivation of Human Pluripotent Stem Cell Lines for Clinical Use.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.