Endogenous KLF4 Expression in Human Fetal Endothelial Cells Allows for Reprogramming to Pluripotency With Just OCT3/4 and SOX2—Brief Report
Objective—The introduction of 4 transcription factors—c-MYC, OCT3/4, SOX2, and KLF4—can reprogram somatic cells back to pluripotency. However, some of the factors used are oncogenic, making therapeutic application unfeasible. Although the use of adult stem cells expressing high endogenous levels of some of these factors allows for reprogramming with fewer exogenous genes, such cells are rare and may have accumulated genetic mutations. Our goal was to reprogram human somatic cells without oncogenic factors. We found that high endogenous expression of KLF4 in human umbilical vein endothelial cells (HUVECs) allows for generation of induced pluripotent stem cells (iPSCs) with just 2 nononcogenic factors, OCT3/4 and SOX2.
Methods and Results—HUVECs were infected with lentivirus containing OCT4 and SOX2 for generation of iPSCs. These 2-factor HUVEC iPSCs were morphologically similar to embryonic stem cells, express endogenous pluripotency markers postreprogramming, and can differentiate toward lineages of all 3 germ layers both in vitro and in vivo.
Conclusion—iPSCs can be generated from HUVECs with only 2 nononcogenic factors. The use of fetal cells for reprogramming without oncogenic factors may provide an efficient in vitro model for human iPSC research, as well as a novel source for possible therapeutic use.
The discovery that somatic cells can be reprogrammed to pluripotency with the addition of 4 transcription factors—c-Myc, Oct3/4, Sox2, and Klf4—is revolutionary.1,2 These induced pluripotent stem cells (iPSCs) are similar to embryonic stem cells but can be generated without ethical concern and transplanted without immune rejection. Although this method of reprogramming is straightforward, efficiency is still quite low even in the best system.3 Subsequent reports reveal that oncogenic factors increase efficiency,4 but in anticipation of clinical use, exclusion of such factors is necessary.
See accompanying article on page 1880
To date, the generation of human iPSCs from adult somatic cells has been reported5,6 but generally with 3 or more factors, because reprogramming of human cells is less efficient than reprogramming of mouse cells3 and may require immortalization in some instances.6 Recent reports have shown that some human adult stem cells can be reprogrammed with relatively few factors because of endogenous expression of SOX2.7 However, the rarity of adult stem cells makes this a difficult option. Because fetal-derived cells (FDCs) are more proliferative and at an earlier stage of development than adult cells, we hypothesized that reprogramming of FDCs may require fewer factors and obviate oncogenes. As proof of principle, we used an easily obtainable source of FDCs for iPSC generation: human umbilical vein endothelial cell (HUVECs). We found that HUVECs endogenously express high levels of KLF4 and can be reprogrammed with just OCT3/4 and SOX2. Our data may have important implications in terms of providing an efficient in vitro model for human iPSCs research and possibly open up a new cell source for reprogramming.
HUVECs (Bioresource Collection and Research Center, Hsinchu, Taiwan) were infected with lentivirus containing OCT3/4 and SOX2 and monitored for iPSC formation as previously reported.1 Reprogrammed HUVECs were assayed for in vitro and in vivo differentiation capacity and characterized. For detailed methodology, please see the Supplemental Materials and Methods, available online at http://atvb.ahajournals.org.
Before reprogramming, FDCs including HUVECs and mesenchymal stromal cells from Wharton’s jelly and placenta do not express the pluripotency genes OCT3/4, SOX2, REX1, NANOG, or telomerase (Figure 1A). However, FDCs do express KLF4, with HUVECs having the highest levels of all. Based on the high levels of KLF4 in HUVECs, we attempted to generate iPSCs by introducing only 2 factors, OCT3/4 and SOX2. HUVECs infected with lentiviruses containing human OCT3/4 and SOX2 showed embryonic stem cell–like colonies after 2 weeks (Figure 1B), and the efficiency can reach 0.024%, which is comparable to previous reports of reprogramming with 4 factors.1 The 2-factor HUVEC iPSCs (iHUV2F) cells were characterized for pluripotency by ALP, SSEA-4, and OCT3/4 (Figure 1C and 1D) staining, along with expression of pluripotency genes (Figure 1E). Transgene expression of OCT4 and SOX2 was found in virally infected HUVECs at day 6 (HUVEC +2F) but not in iHUV2F clones (Figure 1F), indicating transgene silencing and initiation of endogenous gene expression. Further characterization of HUVEC before reprogramming and postreprogramming as iHUV2Fs in terms of proliferative potential and karyotype analysis can be found in the Supplemental Material.
To evaluate in vitro differentiation capacity, iHUV2Fs were cultured either as embryoid bodies before differentiation (iHUV2F-EBs) or differentiated directly (iHUV2F-DD). RT-PCR showed that endothelial markers were expressed highly in HUVECs but minimally or not at all after reprogramming in iHUV2Fs (Figure 2A). Postdifferentiation, endodermal markers (α-fetoprotein [AFP], cytokeratin-8, and cytokeratin-18) and ectodermal markers (MAP2, NG2, and nestin) were expressed by all clones of iHUV2F-EB and iHUV2F-DD, but mesodermal markers brachyury and PPARγ were expressed by all clones except iHUV2F-EB clone 3 (C3). Protein expression of AFP, vimentin, and nestin can be seen in all iHUV2F-DD clones (Figure 2B).
In vivo differentiation capacity of iHUV2Fs by teratoma formation was evaluated. All clones except iHUV2F-C3 formed teratomas; these results correspond to the lower expression of mesodermal markers for the in vitro differentiation of the same clone (Figure 2A, lane 8). Sections of teratomas stain positive for AFP, vimentin, and nestin (Figure 2C), indicating development of all 3 germ layers.
Generation of iPSCs by transcription factor reprogramming is currently widely investigated for regenerative medicine. To create less manipulated iPSCs, one approach is to decrease the number of reprogramming factors used, especially oncogenes.8 With the exception of adult stem cells, there have been no reports of human cells reprogrammed with 1 or 2 factors. Indeed, we were unable to reprogram adult mesenchymal stromal cells with only OCT3/4 and SOX2 (data not shown). We hypothesized that human FDCs would be efficient sources for reprogramming, being more proliferative than adult cells,9 which is a critical issue for reprogramming of human cells.5 Two recent studies show iPSC generation from amniotic fluid–derived cells10 and HUVECs,11 but with 4 factors. We demonstrate that only 2 nononcogenic factors, OCT3/4 and SOX2, were required to reprogram HUVECs, likely because of the high endogenous expression of KLF4 in these cells,12 as well as the proliferative advantage inherent to fetal cells.
We show for the first time that human somatic cells can be reprogrammed with the use of only 2 nononcogenic factors without compromising efficiency. Although using FDCs such as HUVECs may appear to obviate the attractiveness of iPSCs in terms of donor specificity, adult cells are more likely to accumulate mutations with known tumorigenic consequences after reprogramming.13 The relative lack of mutational damage in FDCs, along with the clinical experience of fewer immunologic events with fetal compared with adult hematopoietic stem cell transplantation, lends support to the possibility of using FDC-derived iPSCs in clinical settings.14 Further investigation on the tumorigenicity and immunogenicity of FDC-derived iPSCs and differentiated progeny will be crucial in facilitating possible therapeutic use.
Sources of Funding
This work was supported by grants from the National Science Council of Taiwan (NSC 97-3111-B-002-009, NSC97-3111-B-400-005, and NSC97-3111-B-400-001).
Received on: March 19, 2010; final version accepted on: July 20, 2010.
Galende E, Karakikes I, Edelmann L, Desnick RJ, Kerenyi T, Khoueiry G, Lafferty J, McGinn JT, Brodman M, Fuster V, Hajjar RJ, Polgar K. Amniotic fluid cells are more efficiently reprogrammed to pluripotency than adult cells. Cloning Stem Cells. 2009.
Yet SF, McA'Nulty MM, Folta SC, Yen HW, Yoshizumi M, Hsieh CM, Layne MD, Chin MT, Wang H, Perrella MA, Jain MK, Lee ME. Human EZF, a Kruppel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains. J Biol Chem. 1998; 273: 1026–1031.
Grewal SS, Barker JN, Davies SM, Wagner JE. Unrelated donor hematopoietic cell transplantation: marrow or umbilical cord blood? Blood. 2003; 101: 4233–4244.