Reprogramming of T Cells from Human Peripheral Blood
2010; Elsevier BV; Volume: 7; Issue: 1 Linguagem: Inglês
10.1016/j.stem.2010.06.004
ISSN1934-5909
AutoresYuin‐Han Loh, Odelya Hartung, Li Hu, Chunguang Guo, Julie M. Sahalie, Philip D. Manos, Achia Urbach, Garrett C. Heffner, Marica Grs̆ković, François Vigneault, M. William Lensch, In-Hyun Park, Suneet Agarwal, George M. Church, James J. Collins, Stefan Irion, George Q. Daley,
Tópico(s)Biomedical Ethics and Regulation
ResumoHuman induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for modeling human diseases. Dermal fibroblasts are frequently used for reprogramming, but require an invasive skin biopsy and a prolonged period of expansion in cell culture prior to use. Here, we report the derivation of iPSCs from multiple human blood sources including peripheral blood mononuclear cells (PBMCs) harvested by routine venipuncture. Peripheral blood-derived human iPSC lines are comparable to human embryonic stem cells (ESCs) with respect to morphology, expression of surface antigens, activation of endogenous pluripotency genes, DNA methylation, and differentiation potential. Analysis of immunoglobulin and T cell receptor gene rearrangement revealed that some of the PBMC iPSCs were derived from T cells, documenting derivation of iPSCs from terminally differentiated cell types. Importantly, peripheral blood cells can be isolated with minimal risk to the donor and can be obtained in sufficient numbers to enable reprogramming without the need for prolonged expansion in culture. Reprogramming from blood cells thus represents a fast, safe, and efficient way of generating patient-specific iPSCs. Somatic cells can be induced to the pluripotent state by the enforced expression of several transcription factors including OCT4, SOX2, KLF4, MYC, NANOG, and LIN28 (Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (13487) Google Scholar, Yu et al., 2007Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. et al.Science. 2007; 318: 1917-1920Crossref PubMed Scopus (7585) Google Scholar, Park et al., 2008aPark I.H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Nature. 2008; 451: 141-146Crossref PubMed Scopus (2280) Google Scholar). Human iPSCs are commonly generated from dermal fibroblasts harvested by surgical skin biopsy (Park et al., 2008bPark I.H. Lerou P.H. Zhao R. Huo H. Daley G.Q. Nat. Protoc. 2008; 3: 1180-1186Crossref PubMed Scopus (304) Google Scholar). Exposure of the dermis to ultraviolet light increases the risk for chromosomal aberrations (Ikehata et al., 2003Ikehata H. Masuda T. Sakata H. Ono T. Environ. Mol. Mutagen. 2003; 41: 280-292Crossref PubMed Scopus (36) Google Scholar), raising concerns for whether iPSCs will reflect the patient's constitutional genotype. For routine clinical application, it would be desirable to reprogram cell types that are safe and can be collected noninvasively in large numbers. Blood is a cell source that can be easily obtained from patients. Mouse B and T cells are amenable to reprogramming by overexpressing Oct4, Sox2, Klf4, and Myc with the ectopic expression of Cepbα and p53 knockdown, respectively (Hanna et al., 2008Hanna J. Markoulaki S. Schorderet P. Carey B.W. Beard C. Wernig M. Creyghton M.P. Steine E.J. Cassady J.P. Foreman R. et al.Cell. 2008; 133: 250-264Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, Hong et al., 2009Hong H. Takahashi K. Ichisaka T. Aoi T. Kanagawa O. Nakagawa M. Okita K. Yamanaka S. Nature. 2009; 460: 1132-1135Crossref PubMed Scopus (1008) Google Scholar). iPSC lines have also been generated from mouse bone marrow progenitor cells (Okabe et al., 2009Okabe M. Otsu M. Ahn D.H. Kobayashi T. Morita Y. Wakiyama Y. Onodera M. Eto K. Ema H. Nakauchi H. Blood. 2009; 114: 1764-1767Crossref PubMed Scopus (36) Google Scholar). We have previously reprogrammed cytokine-mobilized human CD34+ peripheral blood cells to pluripotency, but such harvests are cumbersome, expensive, and time consuming (Loh et al., 2009Loh Y.H. Agarwal S. Park I.H. Urbach A. Huo H. Heffner G.C. Kim K. Miller J.D. Ng K. Daley G.Q. Blood. 2009; 113: 5476-5479Crossref PubMed Scopus (465) Google Scholar). Several recent studies reported the generation of iPSCs from human bone marrow and cord blood (Ye et al., 2009Ye Z. Zhan H. Mali P. Dowey S. Williams D.M. Jang Y.Y. Dang C.V. Spivak J.L. Moliterno A.R. Cheng L. Blood. 2009; 114: 5473-5480Crossref PubMed Scopus (292) Google Scholar, Giorgetti et al., 2009Giorgetti A. Montserrat N. Aasen T. Gonzalez F. Rodríguez-Pizà I. Vassena R. Raya A. Boué S. Barrero M.J. Corbella B.A. et al.Cell Stem Cell. 2009; 5: 353-357Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, Haase et al., 2009Haase A. Olmer R. Schwanke K. Wunderlich S. Merkert S. Hess C. Zweigerdt R. Gruh I. Meyer J. Wagner S. et al.Cell Stem Cell. 2009; 5: 434-441Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar), but bone marrow harvesting is an invasive procedure, and cord blood is available for only a minority of individuals who have their samples banked at birth. A recent study with peripheral blood from donors with myeloproliferative disorder (MPD) isolated iPSC colonies that contain the JAK2-V617F mutation (Ye et al., 2009Ye Z. Zhan H. Mali P. Dowey S. Williams D.M. Jang Y.Y. Dang C.V. Spivak J.L. Moliterno A.R. Cheng L. Blood. 2009; 114: 5473-5480Crossref PubMed Scopus (292) Google Scholar), but MPD is characterized by abnormally high numbers of circulating CD34+ cells from the bone marrow. These previous studies demonstrating successful reprogramming of blood cells into iPSCs have relied on specialized blood cell sources with high proliferative potential. CD34+ hematopoietic stem/progenitor cells mobilized into the donor's peripheral blood by pretreatment with granulocyte colony-stimulating factor (G-CSF) can be successfully reprogrammed to pluripotency (Loh et al., 2009Loh Y.H. Agarwal S. Park I.H. Urbach A. Huo H. Heffner G.C. Kim K. Miller J.D. Ng K. Daley G.Q. Blood. 2009; 113: 5476-5479Crossref PubMed Scopus (465) Google Scholar). To test whether we can reprogram cells from routine peripheral blood (PB) sources, we obtained CD34+ purified blood samples from a healthy 49-year-old male donor who had undergone simple apheresis without cytokine priming. We also isolated mononuclear cells (PBMCs) from the peripheral blood samples collected by venipuncture of four healthy donors (28- to 49-years-old) via Ficoll density centrifugation. To induce reprogramming of enriched CD34+ blood cells, we infected with lentiviruses expressing OCT4, SOX2, KLF4, and MYC reprogramming factors (Figure 1A ). Colonies with well-defined hESC-like morphology were first observed 21 days after transduction (Figure 1B). For reprogramming of fresh peripheral blood mononuclear cells (PBMCs), we employed two rounds of lentiviral infection (day 0 and day 8) and isolated colonies with distinct flat and compact morphology with clear-cut round edges reminiscent of hESCs after a slightly longer latency of around 35 days (Figure 1C). Interestingly, a previous study with a single round of lentiviral infection of PBMCs failed to observe iPSC colony formation (Haase et al., 2009Haase A. Olmer R. Schwanke K. Wunderlich S. Merkert S. Hess C. Zweigerdt R. Gruh I. Meyer J. Wagner S. et al.Cell Stem Cell. 2009; 5: 434-441Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). In a separate set of experiments, we tested the ability of retroviruses encoding the human reprogramming factors to generate iPSCs from human PBMCs, and despite low infection efficiency, we observed iPSC colonies after 25–35 days (Figure 1D). With immunohistochemistry and flow cytometry, we analyzed the iPSC lines for expression of markers shared with hESCs. Consistent with their hESC-like morphology, both PB34 iPSCs and PBMC iPSCs stained positive for Tra-1-81, NANOG, OCT4, Tra-1-60, SSEA4, and alkaline phosphatase (AP) staining (Figures 1B–1D; Figures S1A–S1C available online; Chan et al., 2009Chan E.M. Ratanasirintrawoot S. Park I.H. Manos P.D. Loh Y.H. Huo H. Miller J.D. Hartung O. Rho J. Ince T.A. et al.Nat. Biotechnol. 2009; 27: 1033-1037Crossref PubMed Scopus (386) Google Scholar). We routinely observed a reprogramming efficiency of 0.002% for PB CD34+ cells (Table S1), comparable to prior experience with primary fibroblasts, mobilized PBMCs, and cord blood cell reprogramming (Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (13487) Google Scholar, Park et al., 2008aPark I.H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Nature. 2008; 451: 141-146Crossref PubMed Scopus (2280) Google Scholar, Loh et al., 2009Loh Y.H. Agarwal S. Park I.H. Urbach A. Huo H. Heffner G.C. Kim K. Miller J.D. Ng K. Daley G.Q. Blood. 2009; 113: 5476-5479Crossref PubMed Scopus (465) Google Scholar, Haase et al., 2009Haase A. Olmer R. Schwanke K. Wunderlich S. Merkert S. Hess C. Zweigerdt R. Gruh I. Meyer J. Wagner S. et al.Cell Stem Cell. 2009; 5: 434-441Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). For PBMCs, we obtained hESC-like colonies at the lower efficiency of 0.0008%–0.001% (Table S1). We further characterized the PB34 iPSC and PBMC iPSC lines for properties specific to hESCs. Efficient transgene silencing is essential for the derivation of pluripotent iPSC lines (Brambrink et al., 2008Brambrink T. Foreman R. Welstead G.G. Lengner C.J. Wernig M. Suh H. Jaenisch R. Cell Stem Cell. 2008; 2: 151-159Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). qRT-PCR via primers specific for endogenous and total transcripts of the reprogramming factors confirmed that OCT4, SOX2, KLF4, and MYC transgenes were efficiently silenced in the blood-derived iPSCs (Figure S1D). Additional analysis via quantitative PCR revealed the activation of pluripotency markers NANOG, hTERT, REX1, and GDF3 to a level similar to the expression in H1 hESCs (Figure 1E). We next performed global gene expression analysis of the peripheral blood-derived iPSCs comparing it to hESCs, fibroblast iPSCs, and somatic parental cells. Clustering analysis revealed a high degree of similarity among the reprogrammed iPSCs (dH1F-iPS, PBMC iPS1, PB34 iPS1, PB34 iPS2), which clustered together with the H1 and H9 ESCs and were distant from the parental somatic cells, as determined by a Euclidean distance metric (Figure S1E). Analysis of scatter plots similarly shows a tighter correlation among reprogrammed iPSCs (PB34 iPSCs, PBMC iPSCs) and human ESCs (H1 ESCs) than between differentiated parental cells and their reprogrammed derivatives (Figure 1F). Consistent with the activation of endogenous pluripotency-associated gene expression, reprogramming of the blood cells was accompanied by the demethylation of CpG dinucleotides at the NANOG promoters (Figure 1G). Moreover, cytogenetic analysis showed normal karyotypes for the iPSC lines (Figure S1F). Next, we evaluated the developmental potential of the iPSC lines by in vitro embryoid body differentiation, hematopoietic colony forming assays, and in vivo teratoma induction. The iPSCs readily formed embryoid bodies upon induction (Figure S2A). qRT-PCR of the differentiated cells showed strong suppression of the pluripotency genes and activation of lineage-specific genes representing the three germ layers (Figures S2B and S2C). Hematopoietic differentiation of iPSC lines resulted in erythroid, myeloid, and granulocytic colony formation (Figures 2A and 2B ). Interestingly, all PB CD34+-derived iPS lines we tested show greater hematopoietic colony forming activity than PBMC iPSCs (Figure 2A). The most rigorous test for pluripotency of human ESCs is the formation of teratomas in immunodeficient mouse hosts (Lensch et al., 2007Lensch M.W. Schlaeger T.M. Zon L.I. Daley G.Q. Cell Stem Cell. 2007; 1: 253-258Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Upon subcutaneous injection into immunodeficient Rag2−/−γc−/− mice, the iPSC lines generated well-differentiated cystic teratomas representing all three embryonic germ layers (Figures 2C and 2D). DNA fingerprinting analysis verified that these cells were indeed derived from the parental blood cells and not a result of contamination from existing hESC or iPSC lines (Table S2). The iPSC clones have been propagated for at least 20 passages as of this submission. Because peripheral blood mononuclear cells consist of both myeloid and lymphoid elements (Figure S2D), we were interested in determining the lineage of origin of the reprogrammed cells. We tested the iPSC clones for the presence of functionally rearranged immunoglobulin and T cell receptor genes by using probes specific for IgH, TCR-δ, and TCR-β2. Among 12 independent clones from 3 separate individuals, we failed to detect IgH recombination, indicating that none of our lines arose from B lymphocytes (Figure S2E). As reported for the mouse, reprogramming human B lymphocytes may require additional factors like CEBPα (Hanna et al., 2008Hanna J. Markoulaki S. Schorderet P. Carey B.W. Beard C. Wernig M. Creyghton M.P. Steine E.J. Cassady J.P. Foreman R. et al.Cell. 2008; 133: 250-264Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar). Next, we analyzed the iPSC lines for TCR-δ and TCR-β2 recombination (Figures 2E and 2F; Figure S2F). No PBMC iPSC lines demonstrated TCR-β2 recombination, whereas six of seven PBMC iPSC lines isolated from a single donor sample exhibited rearrangement of the TCR-δ locus, indicative of derivation from cells of the T lineage (Figure 2F). In contrast, PBMC iPSC lines from donors 34 and 76 lacked rearrangement of IgH, TCR-δ, and TCR-β2, indicating derivation from nonlymphoid lineages (Figure 2F; Figures S2E and S2F). Isolation of iPSCs from T lymphocytes represents definitive proof that even terminally differentiated human cells are susceptible to reprogramming to pluripotency. Distinct protocols of cytokine stimulation and viral infection of the PBMC cells may predispose to derivation from lymphoid versus nonlymphoid hematopoietic cells from peripheral blood sources, as can preselection of lymphoid target cells prior to reprogramming (Hong et al., 2009Hong H. Takahashi K. Ichisaka T. Aoi T. Kanagawa O. Nakagawa M. Okita K. Yamanaka S. Nature. 2009; 460: 1132-1135Crossref PubMed Scopus (1008) Google Scholar). PBMCs from donor GH were grown in medium containing IL-3, which is known to stimulate the growth of subsets of CD4+ T cells (Figure S2D; Mueller et al., 1994Mueller D.L. Chen Z.M. Schwartz R.H. Gorman D.M. Kennedy M.K. J. Immunol. 1994; 153: 3014-3027PubMed Google Scholar). In contrast, PBMCs from donors 34 and 76 were cultured in medium promoting expansion of dendritic cells and yielded iPSCs with germline IgH and TCR alleles. For applications in regenerative medicine, iPSCs containing antibody or T cell receptor gene rearrangement may be undesirable (Serwold et al., 2007Serwold T. Hochedlinger K. Inlay M.A. Jaenisch R. Weissman I.L. J. Immunol. 2007; 179: 928-938PubMed Google Scholar). In conclusion, we have successfully reprogrammed cells from peripheral blood sources including samples obtained through routine venipuncture. Our study provides a strategy for the reliable generation of induced pluripotent stem cells from peripheral blood mononuclear cells. Although the per-cell derivation efficiency is low, peripheral blood is an accessible source of a large number of primary cells (easily 105–106), thus enabling reliable iPSC isolation from only a few milliliters of whole blood. 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Zhu Y. et al.Cell Stem Cell. 2009; 4: 381-384Abstract Full Text Full Text PDF PubMed Scopus (1446) Google Scholar) to peripheral blood reprogramming will greatly facilitate the development of efficient and safe ways of generating patient-specific pluripotent stem cells. This research was funded by grants from the National Institutes of Health (NIH) and the Howard Hughes Medical Institute to G.Q.D. J.J.C. is supported by SysCODE (Systems-based Consortium for Organ Design & Engineering), NIH grant # RL1DE019021. Y.-H.L. is supported by the overseas fellowship from Agency of Science, Technology, and Research (A∗Star) and the Institute of Medical Biology, Singapore. We are grateful to Ann M. Mullally, Anupama Narla, Benjamin L. Ebert, Lars U.W. Müller, Axel Schambach, and Stelios Andreadis for technical assistances. We acknowledge Sabine Loewer for helpful discussions and critical comments on the manuscript. G.Q.D. is a member of the Scientific Advisory Board of iPierian, Inc. M.G. and S.I. are employed by iPierian, Inc., a biotechnology company using iPSCs for drug discovery. The array data have been deposited in the GEO database under the accession number GSE22167. Download .pdf (.5 MB) Help with pdf files Document S1. Supplemental Experimental Procedures, Two Figures, and Two Tables A manuscript has appeared online demonstrating isolation of iPSCs from peripheral blood, including a single line that showed evidence for both TCR-β and TCR-γ rearrangement by PCR (Kunisato, A., Wakatsuki, M., Shinba, H., Ota, T., Ishida, I., and Nagao, K. [2010]. Direct generation of induced pluripotent stem cells from human non-mobilized blood. Stem Cells Dev., in press. Published online May 24, 2010. 10.1089/scd.2010.0063). Generation of Induced Pluripotent Stem Cells from Human Terminally Differentiated Circulating T CellsSeki et al.Cell Stem CellJuly 02, 2010In BriefThe direct reprogramming of somatic cells to produce induced pluripotent stem cells (iPSCs) is a prominent recent advance in stem cell biology (Takahashi and Yamanaka, 2006). Generation of iPSCs without genomic integration of extrinsic genes is highly desirable. Initially, human dermal fibroblasts were used to derive human iPSCs (hiPSCs) (Takahashi et al., 2007; Yu et al., 2007). However, recent studies have shown that other human somatic stem cells can be used (Aasen et al., 2008; Eminli et al., 2009; Kim et al., 2009; Ye et al., 2009). Full-Text PDF Open ArchiveReprogramming of Human Peripheral Blood Cells to Induced Pluripotent Stem CellsStaerk et al.Cell Stem CellJuly 02, 2010In BriefEmbryonic stem cells are pluripotent cells derived from the inner cell mass of the developing embryo that have the capacity to differentiate into every cell type of the adult (Evans and Kaufman, 1981; Martin, 1981; Martin and Evans, 1975; Thomson et al., 1998). The generation of patient-specific pluripotent cells is therefore an important goal of regenerative medicine. A major step to achieve this was the recent discovery that ectopic expression of defined transcription factors induces pluripotency in somatic cells (Lowry et al., 2008; Park et al., 2008b; Takahashi et al., 2007; Yu et al., 2007). Full-Text PDF Open Archive
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