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本帖最后由 niyou 于 2009-8-29 21:26 编辑 $ U, D9 M' j0 o3 T! o R+ |* T
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很好!我们于2007年就想到低氧浓度有可能会提高iPS效率，根据是采用低氧浓度（5％ O2）能够提高核移植胚胎及体外受精胚胎囊胚的效率和ES建系效率。因为种种原因一直没有开始做。很可惜。+ v! ?3 g/ j' }/ t
6 X. Y( s" N; V$ j1 F. ]Brief Report
/ w' J7 _& w9 ?8 ]3 oHypoxia Enhances the Generation of Induced Pluripotent Stem Cells7 O: s) u$ m$ f4 @7 `, H; Z
3 Y, h2 y2 b3 }5 L. ?9 @9 FYoshinori Yoshida1,,,Kazutoshi Takahashi1,Keisuke Okita1,Tomoko Ichisaka2andShinya Yamanaka1,2,3,4,,3 M+ V' B4 g4 t0 h
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1 Center for iPS Cell Research and Application (CiRA), Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan# G5 O3 ^# D/ R6 K" ]0 Q7 q
2 Yamanaka iPS Cell Special Project, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan( g! E- G! F- [/ J- ^+ J# |5 F7 z, k
3 Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan5 w% R& `4 w3 i$ O5 e: M
4 Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA: e* a, U7 o; ]
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Main Text
- W' c, [& ?7 }- s3 d; T3 @2 GMouse and human somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the transduction of four transcription factors, Oct 3/4, Sox2, Klf4, and c-Myc (Maherali etal., 2007,Meissner etal., 2007,Okita etal., 2007,Takahashi etal., 2007,Takahashi and Yamanaka, 2006,Wernig etal., 2007). Patient or disease-specific human iPSCs could be used for studying pathogenesis, or potentially also to treat patients suffering from incurable diseases by transplanting the regenerated grafts derived from their own cells. However, the low induction efficiency and high tumorigenesis rate due to the use of proto-oncogenes, such as c-Myc, continue to hinder the clinical application of iPS technology. Many efforts have been made to find otherfactors or small molecules that facilitate the reprogramming process (Huangfu etal., 2008,Shi etal., 2008b). In this study, we show that conducting reprogramming in hypoxic conditions results in improved efficiency for both mouse and human cells.
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, Q c% f0 J0 v' }5 E& W) A8 FSomatic stem cells reside in specific microenvironments, called niches, and environmental changes, such as stromal cell contacts, extracellular matrix proteins, temperature, and O2 tension, have a great influence on stem cell function and differentiation. Notably, low O2 tension promotes the survival of neural crest cells and hematopoietic stem cells and prevents differentiation of human ESCs (Danet etal., 2003,Ezashi etal., 2005,Morrison etal., 2000). Moreover, mammalian embryonic epiblasts reside in a physiologically hypoxic environment. These observations led us to the hypothesis that hypoxic conditions might promote the reprogramming process and thus iPS cell generation.
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5 q. o! d3 |6 b, {' K' w/ [To quantify the effect of hypoxia on iPS cell generation, we performed comparison experiments on mouse embryonic fibroblasts (MEFs) carrying the Nanog-GFP-Ires-Puror cassette (Okita etal., 2007). Four or three transcription factors (Oct3/4, Sox2, Klf4, +/ c-Myc) were introduced into MEFs with retroviral vectors. Four days after transduction, the cells were trypsinized and seeded onto the feeder layer of mitomycin C-treated STO cells. The cells were cultivated under 21%, 5%, or 1% O2 from day 5 to day 14 after transduction. GFP+ iPS cell colonies could be first detected on days 1014 after viral transduction, and we counted the number of GFP-positive colonies on days 21 and 28 after transduction. Under 5% O2, the GFP-positive colonies derived from four-factor transduced MEFs increased 7.4-fold on day 21 and 3.1-fold on day 28, and those from three-factor transduced MEFs increased 20-fold on day 21 and 7.6-fold on day 28 under 5% O2 (Figures 1Aa and 1Ab). Moreover, hypoxic treatment increased the percentage of GFP-positive colonies in total colonies from four- or three-factor transduced MEFs (Figures 1Ac and 1Ad). The GFP-positive colonies derived after hypoxic treatment was comparable in morphology and size to those derived under normoxic conditions (FigureS1 available online). Alkaline phosphatase staining showed that cultivation under 5% O2 increased the number of colonies with a positive alkaline phosphatase activity (FigureS2 ). We also examined whether GFP-positive cells were detected more quickly under hypoxic conditions. The four-factor transduced MEFs were cultivated under 21% O2 or under 5% O2 with or without VPA from day 5 to day 9 after transduction and were subjected to flow cytometric analysis on day 9. Retroviral expression of four factors induced 0.01% of the cells to become GFP-positive on day 9 after transduction. Treating the four-factor transduced MEFs for 4 days with hypoxia or with VPA increased the percentage ofGFP-positive cells to 0.40% and 0.48%, respectively. Moreover, cotreatment with hypoxia and VPA increased the percentage of GFP-positive cells to 2.28%. These data suggest that GFP-positive cells can be detected earlier and that the effect of hypoxic culture synergizes with VPA (Figures 1Ba1Bd).+ Q7 C7 k K$ {6 X1 l$ k% B0 W: g
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8 `7 m! m1 ^4 B; b& {Hypoxia Promotes Reprogramming of Mouse Embryonic Fibroblasts to iPS Cells( R! i: O( P- u2 z
(AaAd) Counts of the Nanog-GFP-positive colonies from four-factor-transduced MEFs on day 21 (white) and on day 28 (black) (Aa), from three-factor transduced MEFs on day 21 (white) and on day 28 (black) (Ab). The percentage of GFP-positive colonies in total colonies from four-factor-transduced MEFs (Ac) and three-factor-transduced MEFs (Ad) on day 21.
3 w. \) Y/ j5 }* ?$ |(Ae) Counts of Nanog-GFP-positive colonies from Oct3/4 and Klf4-transduced MEFs on day 28. The averages and standard deviations of four experiments are shown. p < 0.05 versus 21% O2, p < 0.01 versus 21% O2.5 j; a/ ]# d5 k! i" [1 }) F- B, G' _" T
(B) Percentage of GFP-positive cells from four-factor-transduced MEFs on day 9 cultivated under hypoxic and normoxic conditions with and without VPA. Representative flow cytometric analysis of four-factor-transduced MEFs under 21% O2 (Ba) and 5% (Bb) without VPA, and under 21% O2 (Bc) and under 5% (Bd) with VPA. The signal from the PE channel was used as a control for autofluorescence. The averages and standard deviations of three independent experiments are shown. p < 0.05 versus 21% O2 without VPA, p < 0.0001 versus 21% O2 with VPA. p < 0.0001 versus 5% O2 without VPA.8 Q7 p0 M! |% d
(C) The iPS cells derived from two-factor-transduced MEFs gave rise to chimeric mice with a cinnamon coat color in contrast to wild-type ICR mice. Chimeric mouse from MEF-2F-iPS (Ca) and wild-type ICR mouse (Cb).
! T% m" y( r. w- L [(D) MEFs were transiently transfected with expression plasmids of reprogramming factors and cultivated under hypoxic and normoxic conditions. On day 25, the number of GFP-positive colonies was counted. Counts of the Nanog-GFP-positive colonies from six experiments were plotted. p < 0.05 versus 21% O2.5 t3 C( s& F' e9 w0 m; n* x
(E) MEFs were reprogrammed with doxycycline-inducible transcription factors delivered by PB transposition. The cells were cultivated under hypoxic or normoxic conditions. Counts of the Nanog-GFP-positive colonies on day 12 were shown. The averages and standard deviations of three experiments are shown. p < 0.01 versus 21% O2, p < 0.001 versus 21%O2.
9 a2 @4 P2 M# v4 w$ o5 F8 T- ?Although neural stem cells that express SOX2 endogenously can be reprogrammed to iPS cells with the transduction of Oct3/4 and Klf4 (Kim etal., 2008), the reprogramming of MEFs to iPS cells rarely occurs with two transcription factors of Oct3/4 and Klf4. Recently, small-molecule compounds have been reported to enable the reprogramming of Oct3/4 and Klf4-transduced MEFs to iPS cells (Shi etal., 2008a). We examined whether hypoxic conditions could enhance MEFs to be reprogrammed to iPS cells with Oct3/4 and Klf4 transduction. Figure1Ae shows an increased efficiency of the iPS cell generation derived from MEFs with Oct3/4 and Klf4 (MEF-2F-iPS) under 5% O2 in comparison to 21% O2.
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To further evaluate the pluripotency ofthe iPS cells generated with hypoxic treatment, we randomly picked up and established multiple iPS cell lines from two-, three-, and four-factor-infected MEFs. These iPS cells exhibited typical ES cell morphology. We examined the karyotype of iPS cell lines derived after hypoxic treatment (521AH5-1 and 527CH5-1), and these cell lines showed normal karyotypes (FigureS3 ). We investigated the mRNA expression of pluripotency-related genes in the iPS cells generated after hypoxic treatments. The mRNA expression patterns of these iPS cells were comparable to those of ESCs (FigureS4 ). When injected into nude mice subcutaneously, all of the established iPS cell lines gave rise to teratomas with histologic evidence of cells differentiating into all three germ layers (FigureS5 ). Moreover, MEF-2F-iPS cells derived under hypoxic conditions contributed to the formation of somatic tissue in chimeric mice (Figures 1Ca and 1Cb), but we have not yet obtained germline transmission with these mice, so the extent of reprogramming is not entirely clear. Previous studies have shown that iPSCs generated with the same three or four factors are capable of germline transmission (Nakagawa etal., 2008,Okita etal., 2007).
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* _% u, N! P9 {7 P; KRecently, it was reported that iPS cellscould be established by other methods than retroviruses or lentiviruses. We reported that transient transfection ofexpression plasmid vectors of four reprogramming factors could reprogram MEFs to iPS cells (Okita etal., 2008). It was also reported that MEFs could be reprogrammed by transcription factors delivered by piggyBac (PB) transposition system (Kaji etal., 2009,Woltjen etal., 2009). The PB insertions can beremoved from established iPS cells. These methods minimize the potential for insertional mutagenesis. We examined whether hypoxia could improve the efficiency of iPS cell generation with plasmid vectors and with PB transposition system. Figure1D shows that hypoxic cultivation significantly increased the number of GFP-positive colonies with transient transfection of plasmid vectors, and Figure1E shows that hypoxic treatment for 5 and 10 days increased the number of GFP-positive colonies with the piggyBac transposition system by 2.9-fold and 4.0-fold, respectively. These data suggest that hypoxia can increase the efficiency of iPS cell generation by nonviral vectors such as plasmid expression vectors or the piggyBac transposition system.$ H7 V _8 Z/ k y1 W* _
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We next examined the effect of hypoxic culture on proliferation, survival, and gene expression. Flow cytometric analysis with annexin V demonstrated that hypoxic culture had no protective effect on mouse ESCs or on four-factor transduced MEFs (FigureS6 ). Furthermore, hypoxic cultivation showed no effect on proliferation ofmouse ESCs (FigureS7 ). Although hypoxic incubation from day 1 to day 4 after transduction had no significant effect on proliferation of mock-transduced MEFs, it had significant effect on four-factor-transduced MEFs (FigureS8 ). To investigate the expression profile of cells in reprogramming process, we performed microarray analysis and quantitative real-time RT-PCR. We examined the expression profile of MEFs cultivated under hypoxic and normoxic conditions for 10 days. Microarray analysis shows that 57.2% of ESC-specific genes were upregulated and 67.5% of MEF-specific genes were downregulated in the MEFs cultivated under 5%O2 (Figures S9 A and S9B). In Figures S9 C and S9D, microarray analysis of four-factor-transduced MEFs cultivated under hypoxic and normoxic conditions from day 1 to day 4 showed that 73.2% of ESC-specific genes (765 genes out of 1045 total genes) were upregulated and 85.8% of MEF-specific genes (980 genes out of 1142 total genes) were downregulated in the cells treated with hypoxia. Moreover, quantitative real-time RT-PCR analysis demonstrated that expression of endogenous Oct3/4 and Nanog increased 3.4-fold and 2.1-fold, respectively, in four-factor-transduced MEFs after 3 days of hypoxic treatment (Figures S9 E and S9F).1 I" `! n% | y/ P+ f* d* O
4 F3 s, z! t) |. c* G1 m% d% kTo rule out the possibility that hypoxia enhances iPS cell generation by stimulating STO cells, we examined growth situation of iPS cells under hypoxic cultivation without the feeder layer of STO cells. FigureS10 shows that cultivation under 5%O2 increased the number of GFP-positive colonies, suggesting that hypoxic enhancement of reprogramming was not mediated by STO cells.4 h. R& G" l. l$ |
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We next examined whether the exposure to hypoxia increases the efficiency of iPS cell generation from human somatic cells. The four transcription factors were introduced into adult human dermal fibroblasts (HDFs) by retroviral vectors. At six days after transduction, the cells were trypsinized and seeded onto the feeder layer of mitomycin C-treated STO cells. The cells were cultivated under 5% O2 for 7 (1w), 14 (2w), 21 (3w), or 33 days (Long), and the number of human ESC-like colonies was counted on day 24, 32, and 40 after transduction (Figure2A). Figure2B shows that 14 day and 21 day hypoxic cultivation increased the efficiency of iPS cell generation by 4.2-fold and 3.6-fold on day 24, by 2.8-fold and3.0-fold on day 32, and by 2.6-fold and 2.5-fold on day 40, respectively. We randomly selected and established several clones of human iPS cells derived under hypoxic conditions. All of the humaniPS cell lines had a typical ESC morphology and were strongly positive for alkaline phosphatase while also expressing pluripotent-related gene markers (Figures 2Ca, 2Cb, and 2D). Moreover, immunocytological staining showed that all of the cell lines expressed SSEA3, SSEA4, and Nanog (Figures 2Cc2Ce).+ P' S7 l! i* T" e
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Figure2) Q, w' s* E( H4 y1 r" {
Hypoxia Increases the Efficiency of iPS Cell Generation from Human Dermal Fibroblasts
$ f9 C' H! n% G; o$ l1 J(A) Time schedules of human iPS cell generation from HDFs.
+ v4 [' W% Z2 E5 i(B) The number of human ESC-like colonies on day 24 (Ba), on day 32 (Bb), and on day 40 (Bc). The averages and standard deviations of three experiments are shown. p < 0.01 versus 21% O2; p < 0.05 versus 21% O2.
% g7 S& C, x+ s; c1 P3 [(C) Representative phase contrast image of human ESC-like colonies (Ca) and alkaline phosphatase staining of the established iPS clone generated under 5% O2 (Cb). Immunohistochemical staining of undifferentiated human iPS cells generated under hypoxic conditions is shown in the following subpanels: Nanog (Cc), SSEA3 (Cd), and SSEA4 (Ce). Immunohistochemical staining of three germ layer markers in the differentiated human iPS cells generated under hypoxic conditions is shown in the following subpanels: Alpha-fetoprotein (Cf), alpha-smooth muscle actin (Cg), glial fibrillary acidic protein (Ch), and beta-3 tubulin (Ci). Scale bars represent 100 m.
9 n! k1 U1 M3 v" y9 p% J& l6 k& n(D) Semiquantative RT-PCR analysis of ESC-marker genes in human iPS cells generated under normoxic and hypoxic conditions and human ESCs (hES3).
; I) [0 Q; K2 u(E) Teratoma formation of human iPS cells generated under hypoxic conditions. Neural epithelium (Ea), pigmented retinal epithelium (Eb), bone-like structure (Ec), smooth muscle cells (Ed), endodermal epithelium (Ee) are shown. Scale bars represent 100 m.
6 d$ L( q5 d' P- [8 P' w$ m! `9 H/ ?To investigate the differentiation ability of the human iPS cells derived under hypoxic conditions, we used floating cultivation to form embryoid bodies (EBs). After 8 days, the iPS cells formed round embryoid bodies and we then transferred the EBs to gelatin-coated plates and cultivated them for another 8 days. Immunocytochemical analysis showed that for all the iPS cell lines, attached cells derived from the EBs were positive for alpha-fetoprotein (endoderm), alpha-smooth muscle actin (mesoderm), glial fibrillary acidic protein (ectoderm), and beta-3 tubulin (ectoderm) (Figures 2Cf2Ci). To evaluate pluripotency invivo, we transplanted the human iPS cells into the testes of SCID mice. All of the established human iPS cell lines derived after hypoxic treatment developed teratomas and the histological study showed the cells in the teratomas to differentiate into tissues representing all three germ layers (Figure2E).
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& |2 ^! t9 Z; _3 h5 E9 f d; j* ^3 FAlthough hypoxic conditions promote reprogramming, hypoxia also induces cytotoxicity. There are significant differences between cell types in terms of their susceptibility to hypoxia. In our experiments, HDFs were more susceptible to hypoxic cytotoxicity than MEFs. Cultivation under 1% O2 inhibited the proliferation of HDFs and even led cell death withinseveral days, whereas cultivation under 1% O2 had little effect on the proliferation of MEFs. In our experiments, hypoxic cultivation showed no significant effects on the survival of mouse ESCs and four-factor-transduced cells or on the proliferation of mouse ESCs and mock-transduced MEFs. However, in four-factor-transduced MEFs, hypoxia showed a significant proliferative effect and increased the expression level of Oct3/4 and Nanog. In addition, exposure of MEFs to hypoxic conditions shifted the overall gene expression pattern toward that of ESCs. Although there may be several explanations for the positive effect of hypoxia on the efficiency of reprogramming, these results suggest that hypoxic conditions may contribute to the reprogramming process itself.
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8 I" I e/ X9 }7 q+ k" EIn this study, we created hypoxic conditions by flushing hypoxic gas mixture, byusing Forma Series II Universal CO2 incubators (Thermo Scientific), in which mild hypoxia (5%6% O2) in a gas phase can be achieved within 10 min after opening and closing of the door. However, because we changed the medium in a laminar flow hood under normoxic atmosphere, there must have been some fluctuation in O2 content after medium change. More strict control of hypoxia, with a closed hypoxia workstation or medium pre-equilibrated under hypoxic conditions, might further increase the efficiency of iPS cell generation.9 ^" W& M. G- N# d
9 T4 o9 S7 z; ?In summary, by comparing the efficiency of iPS cell induction under normoxic and hypoxic conditions, we showed that hypoxic conditions can improve the efficiency of iPS cell generation from mouse and human somatic cells. We have found that cultivation under 5% O2 favors more efficient iPSC generation, but further characterization is needed to determine the optimal O2 concentration and duration of hypoxic culture for promoting reprogramming process. Ultimately, we hope that understanding the basis of this effect of hypoxia will contribute to ongoing efforts to devise a method for efficient iPSC that does not require genetic modification.* h# W3 r; p9 y7 l( E5 X m$ c; l4 J3 n
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Acknowledgments% ?( z$ b6 N9 A7 E6 E3 e
We thank Drs. Masato Nakagawa, Takashi Aoi, Michiyo Koyanagi, and Koji Tanabe and other members of our laboratory for their valuable scientific comments and fruitful discussions; Tetsuya Ishii and Kanon Takeda for their critical reading of the manuscript; and Drs. Jun K. Yamashita and Masataka Fujiwara for assistance in the cultivation of human ESCs. We are also grateful to Aki Okada, Nanako Takizawa, Misato Nishikawa, and Megumi Kumazaki for technical support and Rie Kato, Ryoko Iyama, Noriyo Maruhashi, and Eri Nishikawa for administrative support. We also thank Dr. Robert Farese, Jr for RF8 ESCs, Dr. Toshio Kitamura for the Plat-E cells and pMX retroviral vectors, Dr.Andras Nagy for piggyBac vectors, and Dr.Malcolm J. Fraser for providing pBSII-IFP2-orf. This study was supported in part by a grant from the Program for Promotion of Fundamental Studies in Health Sciences of NIBIO, a grant from the Leading Project of MEXT, and Grants-in-Aid for Scientific Research of JSPS and MEXT (to S.Y. and Y.Y.).
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Supplemental Data/ p% [* I- ~. ~5 p E' I: k( _* I
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Document S1. Ten Figures and Supplemental Experimental Procedures (PDF 518 kb)
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# f; m( G( ?7 c: W. ?$ x( j( BPublication Information
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Received: January 15, 2009, q9 b# c# h$ P, b3 |
Revised: July 6, 2009+ u T% i/ q" A. z O
Accepted: August 11, 2009
" K( K6 @3 ^0 ~) G8 ZPublished online: August 27, 2009 |
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发表于 2009-8-29 19:02
发表于 2009-8-29 21:25
发表于 2009-8-30 09:20
