Rapid conversion of human ESCs into mouse ESC-like pluripotent state by optimizing culture conditions

doi:10.1007/s13238-012-2007-8

Prot Cell

2012, Vol. 3

Issue (1) : 71-79 https://doi.org/10.1007/s13238-012-2007-8 PMID: 22271597

RESEARCH ARTICLE

Rapid conversion of human ESCs into mouse ESC-like pluripotent state by optimizing culture conditions

Qi Gu^1,2, Jie Hao¹, Xiao-yang Zhao¹, Wei Li¹, Lei Liu¹, Liu Wang¹, Zhong-hua Liu², Qi Zhou¹(

)

1. State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; 2. College of Life Science, Northeast Agricultural University of China, Harbin 150030, China

Download: PDF(553 KB) HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

Abstract

The pluripotent state between human and mouse embryonic stem cells is different. Pluripotent state of human embryonic stem cells (ESCs) is believed to be primed and is similar with that of mouse epiblast stem cells (EpiSCs), which is different from the na?ve state of mouse ESCs. Human ESCs could be converted into a na?ve state through exogenous expression of defined transcription factors (Hanna et al., 2010). Here we report a rapid conversion of human ESCs to mouse ESC-like na?ve states only by modifying the culture conditions. These converted human ESCs, which we called mhESCs (mouse ESC-like human ESCs), have normal karyotype, allow single cell passage, exhibit domed morphology like mouse ESCs and express some pluripotent markers similar with mouse ESCs. Thus the rapid conversion established a na?ve pluripotency in human ESCs like mouse ESCs, and provided a new model to study the regulation of pluripotency.

Keywords human embryonic stem cells (hESCs) mouse ESCs na?ve pluripotent state

Corresponding Author(s): Zhou Qi,Email:qzhou@ioz.ac.cn

Issue Date: 01 January 2012

Cite this article:

Qi Gu,Jie Hao,Xiao-yang Zhao, et al. Rapid conversion of human ESCs into mouse ESC-like pluripotent state by optimizing culture conditions[J]. Prot Cell, 2012, 3(1): 71-79.

URL:

https://academic.hep.com.cn/pac/EN/10.1007/s13238-012-2007-8
https://academic.hep.com.cn/pac/EN/Y2012/V3/I1/71

Fig.1 Determining the optimized conversion condition.
(A) The P0 mhESCs in different media. H9 cells (P37) with flat colonies were digested to single cells using Tryple (Invitrogen). On the third day (3 days after incubation on feeders, named as D3), the colonies appeared as shown in the figure. (B) The P1 mhESCs on D2 in different media. The P0 mhESCs were digested on D3 of P0. On the second day, the colonies of P1 mhESCs appeared as shown in the figure. (C) Cell morphology. The mhESCs at P20 in BH, LBH and LBX media. The mhESCs in BH, LBH and LBX had been passaged for more than 20 generations. Bars= 500 μm. (D) Colony number per unit area (CNpUA) of mhESCs in different media, LN: LIF+ N2B27; LH: LIF+ KOSR; LBH: LIF+ bFGF+ KOSR; BN: bFGF+ N2B27; LBN: LIF+ bFGF+ N2B27; LX: LIF+ KOSR+ N2B27; BX: bFGF+ KOSR+ N2B27; LBX: LIF+ bFGF+ KOSR+ N2B27. The unit area was 1 mm; the CNpUA of LN, LH, LBH, BN, LBN, LX, BX and LBX was 0.67±0.58, 1.00±0.00, 3.33±0.58, 0.67±0.58, 0.67±0.58, 8.00±1.00, 9.00±1.00, respectively.

Fig.2 Characterization of mESC-like human ESCs (mhESCs).
(A) Immunostaining for pluripotency markers (OCT4, SSEA1, SSEA4, and TRA-1-60) of mhESCs and H9 cells. AP, alkaline phosphatase. mhESCs in LBX medium and H9 cells in KOSR were stained with the same antibodies. Positive AP, Oct4, SSEA4 and Tra-1-60 were observed in H9 and mhESCs and SSEA1 is only positive in mhESCs. PI was used to stain the nucleus. Bars= 150 μm (AP), Bars= 25 μm (Immunostaining). (B) Karyotype of mhESCs. mhESCs in LBX medium were supplied for karyotype analysis. More than 75% of the cells showed normal human karyotype of 46 chromosomes. (C) RT-PCR for pluripotency genes (, 2, , , an) of H9 and mhESCs. Seven pluripotency genes primers were supplied for PCR of H9, mhESCs and HO. (D) Embryoid body (EB) formation of H9 cells and mhESCs. Bars= 500 μm. (E) RT-PCR for three layers markers. The first column was the EB of H9 and the second and third columns were mhESCs’ EBs and HO, respectively.

Fig.3 The pluripotent state of mhESCs.
(A) Quantitative RT-PCR for gene ( ( exons 1, 2), ( exons 5, 6)) and . The expression of and was relative to the expression of . (B) H9 cells and mhESCs converted from H9 cells were stained with polyclonal antibodies against H3K27me3. Red arrow points to the H3K27me3 ‘spot’. PI was used to stain the nucleus. Bars=20 μm.

Fig.4 Conversion of other human ESC lines.
(A) Cell morphology. P-TJ cells that were digested by collagenase IV. Bars= 500 μm. (B) Cell morphology. The mhESCs converted from P-TJ cells. Bars= 500 μm. (C) Karyotype analysis of m_P-TJ cells. More than 75% of the cells showed normal human karyotype of 46 chromosomes. (D) P-TJ cells and m_P-TJ cells were stained with polyclonal antibodies against H3K27me3. Red arrow points to the H3K27me3 ‘spot’. PI was used to stain the nucleus. Bars= 20 μm.

Tab.1 Primer sequence for PCR

1	Bao, L., He, L., Chen, J., Wu, Z., Liao, J., Rao, L., Ren, J., Li, H., Zhu, H., Qian, L., (2011). Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors. Cell Res 21, 600–608 .
2	Bao, S., Tang, F., Li, X., Hayashi, K., Gillich, A., Lao, K., and Surani, M.A. (2009). Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells. Nature 461, 1292–1295 pmid:19816418.
3	Bendall, S.C., Stewart, M.H., Menendez, P., George, D., Vijayaragavan, K., Werbowetski-Ogilvie, T., Ramos-Mejia, V., Rouleau, A., Yang, J., Bossé, M., (2007). IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature 448, 1015–1021 pmid:17625568.
4	Brons, I.G., Smithers, L.E., Trotter, M.W., Rugg-Gunn, P., Sun, B., Chuva de Sousa Lopes, S.M., Howlett, S.K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R.A., (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 pmid:17597762.
5	Buehr, M., Meek, S., Blair, K., Yang, J., Ure, J., Silva, J., McLay, R., Hall, J., Ying, Q.L., and Smith, A. (2008). Capture of authentic embryonic stem cells from rat blastocysts. Cell 135, 1287–1298 pmid:19109897.
6	Buryanov, Y.I., and Shevchuk, T.V. (2005). DNA methyltransferases and structural-functional specificity of eukaryotic DNA modification. Biochemistry (Mosc) 70, 730–742 pmid:16097936.
7	Chen, G., Gulbranson, D.R., Hou, Z., Bolin, J.M., Ruotti, V., Probasco, M.D., Smuga-Otto, K., Howden, S.E., Diol, N.R., Propson, N.E., (2011). Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8, 424–429 .
8	Dvorak, P., Dvorakova, D., Koskova, S., Vodinska, M., Najvirtova, M., Krekac, D., and Hampl, A. (2005). Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells. Stem Cells 23, 1200–1211 pmid:15955829.
9	Eiges, R., Schuldiner, M., Drukker, M., Yanuka, O., Itskovitz-Eldor, J., and Benvenisty, N. (2001). Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr Biol 11, 514–518 pmid:11413002.
10	Esteban, M.A., Wang, T., Qin, B., Yang, J., Qin, D., Cai, J., Li, W., Weng, Z., Chen, J., Ni, S., (2010). Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell 6, 71–79 pmid:20036631.
11	Evans, M.J., and Kaufman, M.H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 pmid:7242681.
12	Ezashi, T., Telugu, B.P., Alexenko, A.P., Sachdev, S., Sinha, S., and Roberts, R.M. (2009). Derivation of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci U S A 106, 10993–10998 pmid:19541600.
13	Folch, J., Cocero, M.J., Chesné, P., Alabart, J.L., Domínguez, V., Cognié, Y., Roche, A., Ferníndez-Arias, A., Martí, J.I., Sánchez, P., (2009). First birth of an animal from an extinct subspecies (Capra pyrenaica pyrenaica) by cloning. Theriogenology 71, 1026–1034 pmid:19167744.
14	Guo, G., Yang, J., Nichols, J., Hall, J.S., Eyres, I., Mansfield, W., and Smith, A. (2009). Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136, 1063–1069 pmid:19224983.
15	Han, X., Han, J., Ding, F., Cao, S., Lim, S.S., Dai, Y., Zhang, R., Zhang, Y., Lim, B., and Li, N. (2011). Generation of induced pluripotent stem cells from bovine embryonic fibroblast cells. Cell Res 21, 1509–1512 .
16	Hanna, J., Cheng, A.W., Saha, K., Kim, J., Lengner, C.J., Soldner, F., Cassady, J.P., Muffat, J., Carey, B.W., and Jaenisch, R. (2010). Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc Natl Acad Sci U S A .
17	Hanna, J., Markoulaki, S., Mitalipova, M., Cheng, A.W., Cassady, J.P., Staerk, J., Carey, B.W., Lengner, C.J., Foreman, R., Love, J., (2009). Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell 4, 513–524 pmid:19427283.
18	Hao, J., Zhu, W., Sheng, C., Yu, Y., and Zhou, Q. (2009). Human parthenogenetic embryonic stem cells: one potential resource for cell therapy. Sci China C Life Sci 52, 599–602 pmid:19641863.
19	Honda, A., Hirose, M., Hatori, M., Matoba, S., Miyoshi, H., Inoue, K., and Ogura, A. (2010). Generation of induced pluripotent stem cells in rabbits: potential experimental models for human regenerative medicine. J Biol Chem 285, 31362–31369 pmid:20670936.
20	Lengner, C.J., Gimelbrant, A.A., Erwin, J.A., Cheng, A.W., Guenther, M.G., Welstead, G.G., Alagappan, R., Frampton, G.M., Xu, P., Muffat, J., (2010). Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell 141, 872–883 pmid:20471072.
21	Li, P., Tong, C., Mehrian-Shai, R., Jia, L., Wu, N., Yan, Y., Maxson, R.E., Schulze, E.N., Song, H., Hsieh, C.L., (2008). Germline competent embryonic stem cells derived from rat blastocysts. Cell 135, 1299–1310 pmid:19109898.
22	Li, Z.K., and Zhou, Q. (2010). Cellular models for disease exploring and drug screening. Protein Cell 1, 355–362 pmid:21203947.
23	Liao, J., Cui, C., Chen, S., Ren, J., Chen, J., Gao, Y., Li, H., Jia, N., Cheng, L., Xiao, H., (2009). Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 4, 11–15 pmid:19097959.
24	Liu, H., Zhu, F., Yong, J., Zhang, P., Hou, P., Li, H., Jiang, W., Cai, J., Liu, M., Cui, K., (2008). Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell 3, 587–590 pmid:19041774.
25	Liu, Y., Song, Z., Zhao, Y., Qin, H., Cai, J., Zhang, H., Yu, T., Jiang, S., Wang, G., Ding, M., (2006). A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells. Biochem Biophys Res Commun 346, 131–139 pmid:16753134.
26	Lu, Z., Zhu, W., Yu, Y., Jin, D., Guan, Y., Yao, R., Zhang, Y.A., Zhang, Y., and Zhou, Q. (2010). Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders. J Assist Reprod Genet 27, 285–291 pmid:20393797.
27	Luo, J., Suhr, S.T., Chang, E.A., Wang, K., Ross, P.J., Nelson, L.L., Venta, P.J., Knott, J.G., and Cibelli, J.B. (2011). Generation of leukemia inhibitory factor and basic fibroblast growth factor-dependent induced pluripotent stem cells from canine adult somatic cells. Stem Cells Dev 20, 1669–1678 .
28	Martin, G.R. (1981). Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 78, 7634–7638 pmid:6950406.
29	Matsuda, T., Nakamura, T., Nakao, K., Arai, T., Katsuki, M., Heike, T., and Yokota, T. (1999). STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 18, 4261–4269 pmid:10428964.
30	Najm, F.J., Chenoweth, J.G., Anderson, P.D., Nadeau, J.H., Redline, R.W., McKay, R.D., and Tesar, P.J. (2011). Isolation of epiblast stem cells from preimplantation mouse embryos. Cell Stem Cell 8, 318–325 pmid:21362571.
31	Nichols, J., and Smith, A. (2009). Naive and primed pluripotent states. Cell Stem Cell 4, 487–492 pmid:19497275.
32	Plath, K., Fang, J., Mlynarczyk-Evans, S.K., Cao, R., Worringer, K.A., Wang, H., de la Cruz, C.C., Otte, A.P., Panning, B., and Zhang, Y. (2003). Role of histone H3 lysine 27 methylation in X inactivation. Science 300, 131–135 pmid:12649488.
33	Rossant, J. (2008). Stem cells and early lineage development. Cell 132, 527–531 pmid:18295568.
34	Smith, A.G., Heath, J.K., Donaldson, D.D., Wong, G.G., Moreau, J., Stahl, M., and Rogers, D. (1988). Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336, 688–690 pmid:3143917.
35	Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 pmid:18035408.
36	Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 pmid:16904174.
37	Tesar, P.J., Chenoweth, J.G., Brook, F.A., Davies, T.J., Evans, E.P., Mack, D.L., Gardner, R.L., and McKay, R.D. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 pmid:17597760.
38	Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 pmid:9804556.
39	Thomson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, C.P., Becker, R.A., and Hearn, J.P. (1995). Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A 92, 7844–7848 pmid:7544005.
40	Vallier, L., Alexander, M., and Pedersen, R.A. (2005). Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J Cell Sci 118, 4495–4509 pmid:16179608.
41	Wang, S., Tang, X., Niu, Y., Chen, H., Li, B., Li, T., Zhang, X., Hu, Z., Zhou, Q., and Ji, W. (2007). Generation and characterization of rabbit embryonic stem cells. Stem Cells 25, 481–489 pmid:17038672.
42	Williams, R.L., Hilton, D.J., Pease, S., Willson, T.A., Stewart, C.L., Gearing, D.P., Wagner, E.F., Metcalf, D., Nicola, N.A., and Gough, N.M. (1988). Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684–687 pmid:3143916.
43	Xu, R.H., Sampsell-Barron, T.L., Gu, F., Root, S., Peck, R.M., Pan, G., Yu, J., Antosiewicz-Bourget, J., Tian, S., Stewart, R., (2008). NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs. Cell Stem Cell 3, 196–206 pmid:18682241.
44	Xu, Y., Zhu, X., Hahm, H.S., Wei, W., Hao, E., Hayek, A., and Ding, S. (2010). Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc Natl Acad Sci U S A 107, 8129–8134 pmid:20406903.
45	Ying, Q.L., Nichols, J., Chambers, I., and Smith, A. (2003). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281–292 pmid:14636556.
46	Ying, Q.L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. (2008). The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 pmid:18497825.
47	Zhao, X.Y., Li, W., Lv, Z., Liu, L., Tong, M., Hai, T., Hao, J., Guo, C.L., Ma, Q.W., Wang, L., (2009). iPS cells produce viable mice through tetraploid complementation. Nature 461, 86–90 .
48	Zhao, X.Y., Lv, Z., Li, W., Zeng, F., and Zhou, Q. (2010). Production of mice using iPS cells and tetraploid complementation. Nat Protoc 5, 963–971 pmid:20431542.
49	Zwaka, T.P., and Thomson, J.A. (2003). Homologous recombination in human embryonic stem cells. Nat Biotechnol 21, 319–321 pmid:12577066.

[1]	Sun-Ku Chung,Shengyun Zhu,Yang Xu,Xuemei Fu. Functional analysis of the acetylation of human p53 in DNA damage responses[J]. Protein Cell, 2014, 5(7): 544-551.
[2]	Rui Li, Ye Bai, Tongtong Liu, Xiaoqun Wang, Qian Wu. Induced pluripotency and direct reprogramming: a new window for treatment of neurodegenerative diseases[J]. Prot Cell, 2013, 4(6): 415-424.
[3]	Jolene Ooi, Pentao Liu. Delineating nuclear reprogramming[J]. Prot Cell, 2012, 3(5): 329-345.

Viewed

Full text

Abstract

Cited

Shared

Discussed