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Leukemia Inhibitory Factor (LIF)-dependent, Pluripotent Stem Cells Established from Inner Cell Mass of Porcine Embryos*

Open AccessPublished:June 24, 2011DOI:https://doi.org/10.1074/jbc.M111.229468
      The pig is important for agriculture and as an animal model in human and veterinary medicine, yet despite over 20 years of effort, there has been a failure to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of leukemia inhibitory factor-dependent, so-called naive type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. The alkaline phosphatase-positive colonies resulting from reprogramming resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile, and expression of pluripotent markers, such as POU5F1, SOX2, and surface marker SSEA1. They are dependent on leukemia inhibitory factor signaling for maintenance of pluripotency, can be cultured over extended passage, and have the ability to form teratomas. These cells derived from the inner cell mass of pig blastocysts are clearly distinct from the FGF2-dependent “primed” induced pluripotent stem cells described recently from porcine mesenchymal cells. The data are consistent with the hypothesis that the up-regulation of KLF4, as well as POU5F1, is required to create and stabilize the naive pluripotent state and may explain why the derivation of embryonic stem cells from pigs and other ungulates has proved so difficult.

      Introduction

      Pluripotent stem cell lines from the inner cell mass (ICM)
      The abbreviations used are: ICM
      inner cell mass
      ESC
      embryonic stem cells
      iPSC
      induced pluripotent stem cells
      LIF
      leukemia inhibitory factor
      CH
      CHIR99021
      KP
      kenpaullone
      DOX
      doxycycline
      PFF
      porcine fetal fibroblast(s)
      h
      human
      p
      pig
      m
      mouse.
      of the embryo, the so-called embryonic stem cells (ESC), were first established over 30 years ago from day 3.5 mouse (m) blastocysts (
      • Evans M.J.
      • Kaufman M.H.
      ,
      • Martin G.R.
      ) and more recently from totipotent blastomeres (
      • Eistetter H.R.
      ,
      • Delhaise F.
      • Bralion V.
      • Schuurbiers N.
      • Dessy F.
      ). These authentic mESC lines are dependent on LIF/STAT3 signaling for maintenance of pluripotency (
      • Nichols J.
      • Smith A.
      ), tolerate complete enzymatic dispersal, and fulfill the stringent criteria of pluripotency, such as the lack of senescence when cultured in vitro, the ability to differentiate into multiple cell types representing the three germ layers both in vitro and in vivo, and finally, contribution to the germ line in chimeric offspring (
      • Wobus A.M.
      • Boheler K.R.
      ). These attributes of mESC formed the basis for the transformation of the field of mammalian genetics and developmental biology and propelled the mouse as the prime biomedical model for studying the genetic basis of disease. Until now, attempts to isolate equivalent kinds of ESC from the blastocysts of all but a few “permissive” mouse strains without a major pharmacological intervention have met with failure. ESC established from the human (
      • Thomson J.A.
      • Itskovitz-Eldor J.
      • Shapiro S.S.
      • Waknitz M.A.
      • Swiergiel J.J.
      • Marshall V.S.
      • Jones J.M.
      ), monkey (
      • Thomson J.A.
      • Kalishman J.
      • Golos T.G.
      • Durning M.
      • Harris C.P.
      • Becker R.A.
      • Hearn J.P.
      ), and more recently from the pig (
      • Alberio R.
      • Croxall N.
      • Allegrucci C.
      ) and those lines established from the epiblast of non-permissive mouse strains, the epiblast stem cells (
      • Tesar P.J.
      • Chenoweth J.G.
      • Brook F.A.
      • Davies T.J.
      • Evans E.P.
      • Mack D.L.
      • Gardner R.L.
      • McKay R.D.
      ,
      • 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.
      • Vallier L.
      ), demonstrate a stark contrast in phenotype and gene expression profile relative to the mESC. These ESC were characterized by their flattened morphology, dependence on FGF2 and TGFB/activin A signaling for maintenance of their pluripotency (
      • Xu R.H.
      • Peck R.M.
      • Li D.S.
      • Feng X.
      • Ludwig T.
      • Thomson J.A.
      ), inactivation of one of the X chromosomes in female cell lines (
      • Hanna J.
      • Cheng A.W.
      • Saha K.
      • Kim J.
      • Lengner C.J.
      • Soldner F.
      • Cassady J.P.
      • Muffat J.
      • Carey B.W.
      • Jaenisch R.
      ), intolerance to passage as single cells, and lack of competence for producing germ line chimeras (
      • Tesar P.J.
      • Chenoweth J.G.
      • Brook F.A.
      • Davies T.J.
      • Evans E.P.
      • Mack D.L.
      • Gardner R.L.
      • McKay R.D.
      ,
      • 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.
      • Vallier L.
      ). Also, they are more susceptible to spontaneous differentiation, making the standard practice of culture and manipulation much more demanding. Nichols and Smith (
      • Nichols J.
      • Smith A.
      ) have suggested that the two types of ESC differ fundamentally in the gene networks that maintain their pluripotency and named the LIF-dependent ESC “naive” and the FGF2-dependent type “primed.”
      Efforts to establish naive ESC from pig (p) embryos began over two decades ago, soon after the first studies describing mESC from blastocysts (
      • Notarianni E.
      • Laurie S.
      • Moor R.M.
      • Evans M.J.
      ,
      • Piedrahita J.A.
      • Anderson G.B.
      • Bondurant R.H.
      ), but the resulting lines bore only a limited resemblance to mESC and failed to meet the full criteria for pluripotency. Four recent studies (
      • Wu Z.
      • Chen J.
      • Ren J.
      • Bao L.
      • Liao J.
      • Cui C.
      • Rao L.
      • Li H.
      • Gu Y.
      • Dai H.
      • Zhu H.
      • Teng X.
      • Cheng L.
      • Xiao L.
      ,
      • Ezashi T.
      • Telugu B.P.
      • Alexenko A.P.
      • Sachdev S.
      • Sinha S.
      • Roberts R.M.
      ,
      • Esteban M.A.
      • Xu J.
      • Yang J.
      • Peng M.
      • Qin D.
      • Li W.
      • Jiang Z.
      • Chen J.
      • Deng K.
      • Zhong M.
      • Cai J.
      • Lai L.
      • Pei D.
      ,
      • West F.D.
      • Terlouw S.L.
      • Kwon D.J.
      • Mumaw J.L.
      • Dhara S.K.
      • Hasneen K.
      • Dobrinsky J.R.
      • Stice S.L.
      ) have reported the derivation of porcine induced pluripotent stem cells (piPSC) from fibroblasts by employing the classic combination of reprogramming factors POU5F1 (OCT4), SOX2, KLF4, and c-MYC (OSKM) originally developed to reprogram fibroblasts from the mouse (
      • Takahashi K.
      • Yamanaka S.
      ). The resulting pluripotent cell lines resembled human cells rather than mESC in terms of their general morphological features and in their requirement for FGF2 rather than LIF (
      • Roberts R.M.
      • Telugu B.P.
      • Ezashi T.
      ). Naive ESC have recently been generated successfully from the embryos of rat, but only with major modifications to standard culture conditions (
      • Buehr M.
      • Meek S.
      • Blair K.
      • Yang J.
      • Ure J.
      • Silva J.
      • McLay R.
      • Hall J.
      • Ying Q.L.
      • Smith A.
      ,
      • Li P.
      • Tong C.
      • Mehrian-Shai R.
      • Jia L.
      • Wu N.
      • Yan Y.
      • Maxson R.E.
      • Schulze E.N.
      • Song H.
      • Hsieh C.L.
      • Pera M.F.
      • Ying Q.L.
      ). In particular, protein kinase inhibitors, CHIR99021 (CH) and PD0325901, were added to the medium to activate the WNT signaling pathway (CH) and to inhibit the ERK-mediated differentiation pathway (PD0325901), respectively. A slightly different strategy was employed to generate naive pluripotent stem cells from the NOD strain of mouse, which previously had failed to yield ESC (
      • Hanna J.
      • Markoulaki S.
      • Mitalipova M.
      • Cheng A.W.
      • Cassady J.P.
      • Staerk J.
      • Carey B.W.
      • Lengner C.J.
      • Foreman R.
      • Love J.
      • Gao Q.
      • Kim J.
      • Jaenisch R.
      ). Here two approaches proved successful. One was to transduce the founder cells with lentiviral vectors designed to overexpress KLF4 and c-MYC, whereas the other was to use pharmacological inhibitors, namely CH to bypass c-MYC function (
      • Marson A.
      • Foreman R.
      • Chevalier B.
      • Bilodeau S.
      • Kahn M.
      • Young R.A.
      • Jaenisch R.
      ) and kenpaullone (KP) to substitute for KLF4 (
      • Lyssiotis C.A.
      • Foreman R.K.
      • Staerk J.
      • Garcia M.
      • Mathur D.
      • Markoulaki S.
      • Hanna J.
      • Lairson L.L.
      • Charette B.D.
      • Bouchez L.C.
      • Bollong M.
      • Kunick C.
      • Brinker A.
      • Cho C.Y.
      • Schultz P.G.
      • Jaenisch R.
      ). We rationalized that the previous failures to establish ESC from porcine embryos might have been due to low endogenous levels of c-MYC and KLF4 in ICM cells (
      • Telugu B.P.
      • Ezashi T.
      • Roberts R.M.
      ). In addition, we had noted that concentrations of mRNA for endogenous c-MYC and KLF4 in porcine piPSC reprogrammed from fetal fibroblasts were extremely low, possibly accounting for the primed phenotype of these cells (
      • Ezashi T.
      • Telugu B.P.
      • Alexenko A.P.
      • Sachdev S.
      • Sinha S.
      • Roberts R.M.
      ). Accordingly, we explored strategies to ectopically overexpress KLF4 in porcine ICM and culture in medium containing KP and CH medium during the reprogramming steps (
      • Telugu B.P.
      • Ezashi T.
      • Roberts R.M.
      ).

      DISCUSSION

      Here we describe the generation of a putative naive class of porcine pluripotent cells using ICM of blastocysts as the founder population. The rationale for the experiments was that such cells would likely have many of the desirable features of mESC, including the rapid growth rate, resistance to spontaneous differentiation, ease of genetic manipulation, and ability to incorporate into chimeras that have made mESC such a valuable experimental tool for studies on the mouse model. We also hypothesized that if such porcine cells could be derived from a pluripotent source, namely the ICM, they would lack the epigenetic memory that might be carried from more differentiated somatic cells (
      • Kim K.
      • Doi A.
      • Wen B.
      • Ng K.
      • Zhao R.
      • Cahan P.
      • Kim J.
      • Aryee M.J.
      • Ji H.
      • Ehrlich L.I.
      • Yabuuchi A.
      • Takeuchi A.
      • Cunniff K.C.
      • Hongguang H.
      • McKinney-Freeman S.
      • Naveiras O.
      • Yoon T.J.
      • Irizarry R.A.
      • Jung N.
      • Seita J.
      • Hanna J.
      • Murakami P.
      • Jaenisch R.
      • Weissleder R.
      • Orkin S.H.
      • Weissman I.L.
      • Feinberg A.P.
      • Daley G.Q.
      ).
      The experiments clearly demonstrate that it is possible to derive pluripotent stem cells, seemingly of the naive class, directly and relatively efficiently from the ICM of the pig, an important advance because swine are valuable alternative models to the mouse in biomedical research (
      • Roberts R.M.
      • Telugu B.P.
      • Ezashi T.
      ), and naive cells are much more amenable to physical and genetic manipulation than pluripotent cells of the primed type. The studies also demonstrate an evolutionarily shared requirement for KLF4, a key transcription factor that is downstream of the LIF signaling cascade, in achieving pluripotency and LIF dependence. Low expression of KLF4 and c-MYC probably underpins the long history of failure to establish naive pluripotent stem cells from pig and other domestic species, as well as several strains of mouse (
      • Hanna J.
      • Markoulaki S.
      • Mitalipova M.
      • Cheng A.W.
      • Cassady J.P.
      • Staerk J.
      • Carey B.W.
      • Lengner C.J.
      • Foreman R.
      • Love J.
      • Gao Q.
      • Kim J.
      • Jaenisch R.
      ). An examination of deep sequencing data obtained on cDNA from porcine blastocysts confirmed a relatively low abundance of these two crucial transcripts, although POU5F1 mRNA was relatively abundant (
      • Bauer B.K.
      • Isom S.C.
      • Spate L.D.
      • Whitworth K.M.
      • Spollen W.G.
      • Blake S.M.
      • Springer G.K.
      • Murphy C.N.
      • Prather R.S.
      ). Although POU5F1 was represented by 344 reads, KLF4 had 49 and c-MYC only had three. Our choice of culture in a 4% O2 environment, conditions that favor glycolytic metabolism in human embryonic stem cells (
      • Westfall S.D.
      • Sachdev S.
      • Das P.
      • Hearne L.B.
      • Hannink M.
      • Roberts R.M.
      • Ezashi T.
      ), may have facilitated the derivation of pluripotent lines by compensating for low levels of endogenous c-MYC expression. Among its many pleiotropic roles, c-MYC promotes glycolysis (
      • Vander Heiden M.G.
      • Cantley L.C.
      • Thompson C.B.
      ). Additionally, our laboratory has previously shown that maintenance of hESC in an undifferentiated state is favored by culture in low oxygen environment (
      • Ezashi T.
      • Das P.
      • Roberts R.M.
      ), whereas others have demonstrated that such physiological O2 conditions enhance reprogramming efficiency (
      • Yoshida Y.
      • Takahashi K.
      • Okita K.
      • Ichisaka T.
      • Yamanaka S.
      ,
      • Zhu S.
      • Li W.
      • Zhou H.
      • Wei W.
      • Ambasudhan R.
      • Lin T.
      • Kim J.
      • Zhang K.
      • Ding S.
      ) and maintain the active chromatin state of pluripotent cells (
      • 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.
      • Santagata S.
      • Powers D.
      • Barrett C.B.
      • Young R.A.
      • Lee J.T.
      • Jaenisch R.
      • Mitalipova M.
      ), again functions ascribed to c-MYC. On the other hand, although culturing in low O2 and transducing the pig ICM cells with KLF4 generated colonies bearing a superficial resemblance to naive cells, they were not pluripotent by conventional standards. As creation of LIF-dependent human ESC required additional, supplementary expression of either POU5F1 or KLF2 to achieve a “true” pluripotent state (
      • Hanna J.
      • Cheng A.W.
      • Saha K.
      • Kim J.
      • Lengner C.J.
      • Soldner F.
      • Cassady J.P.
      • Muffat J.
      • Carey B.W.
      • Jaenisch R.
      ), we added such an extra step and showed that the requirement for an additional factor also held true for pigs. Our choice of POU5F1 over KLF2 was based on the fact that KLF2 mRNA levels seem to increase concomitantly with POU5F1 expression (
      • Hall J.
      • Guo G.
      • Wray J.
      • Eyres I.
      • Nichols J.
      • Grotewold L.
      • Morfopoulou S.
      • Humphreys P.
      • Mansfield W.
      • Walker R.
      • Tomlinson S.
      • Smith A.
      ). Taken together, we conclude that culturing under physiological O2 conditions is ideal for derivation and propagation of pig pluripotent stem cells, KLF4 is a major player distinguishing naive and primed pluripotent stemness states, and ectopic POU5F1 overexpression is required in the pig and potentially other domestic animals to achieve and maintain that state.

      Acknowledgments

      We thank Dr. Kirk of the Department of Biology for providing the nucleoside solution.

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