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Generation of induced pluripotent stem cells from newborn marmoset skin fibroblasts
Stem Cell Research (2010) 4, 180–188
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REGULAR ARTICLE
Generation of induced pluripotent stem cells from newborn marmoset skin fibroblasts Yuehong Wu a,b , Yong Zhang a , Anuja Mishra b , Suzette D. Tardif b , Peter J. Hornsby b,⁎ a
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China Department of Physiology and Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, TX 78245, USA b
Received 21 December 2009; received in revised form 21 February 2010; accepted 25 February 2010
Abstract Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine. For the application of iPSCs to forms of autologous cell therapy, suitable animal models are required. Among species that could potentially be used for this purpose, nonhuman primates are particularly important, and among these the marmoset offers significant advantages. In order to demonstrate the feasibility of the application of iPSC technology to this species, here we derived lines of marmoset iPSCs. Using retroviral transduction with human Oct4, Sox2, Klf4 and c-Myc, we derived clones that fulfil critical criteria for successful reprogramming: they exhibit typical iPSC morphology; they are alkaline phosphatase positive; they express high levels of NANOG, OCT4 and SOX2 mRNAs, while the corresponding vector genes are silenced; they are immunoreactive for Oct4, TRA-1-81 and SSEA-4; and when implanted into immunodeficient mice they produce teratomas that have derivatives of all three germ layers (endoderm, α-fetoprotein; ectoderm, βIII-tubulin; mesoderm, smooth muscle actin). Starting with a population of 4 × 105 newborn marmoset skin fibroblasts, we obtained ∼ 100 colonies with iPSC-like morphology. Of these, 30 were expanded sufficiently to be cryopreserved, and, of those, 8 were characterized in more detail. These experiments provide proof of principle that iPSC technology can be adapted for use in the marmoset, as a future model of autologous cell therapy. © 2010 Elsevier B.V. All rights reserved.
Introduction Induced pluripotent stem cells (iPSCs) have been derived from somatic cells of mouse (Takahashi and Yamanaka,
⁎ Corresponding author. University of Texas Health Science Center, 15355 Lambda Drive, San Antonio, TX 78245, USA. Fax: +1 281 582 3538. E-mail addresses: [email protected] (Y. Wu), [email protected] (Y. Zhang), [email protected] (A. Mishra), [email protected] (S.D. Tardif), [email protected] (P.J. Hornsby).
2006), human (Takahashi et al., 2007), rhesus monkey (Liu et al., 2008), rat (Liao et al., 2009), pig (Ezashi et al., 2009), and dog (Shimada et al., 2009). It is widely thought that an important future use for iPSC technology is a form of autologous cell therapy in which the donor of the reprogrammed somatic cells is also the recipient of the cells following appropriate differentiation protocols (Yamanaka, 2007). Potentially, any mammalian species could serve as a translational model for this form of autologous cell therapy, but nonhuman primates are particularly suited for such studies because of the aspects of their anatomy and physiology that they share with humans. Among nonhuman primates, the marmoset is especially relevant for biomedical research because of well developed models in this species for
1873-5061/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scr.2010.02.003
Marmoset induced pluripotent stem cells neurodegenerative disorders (Parkinson's, Alzheimer's and Huntington's disease), stroke, and spinal cord injury; reproductive biology, infectious disease, and behavioral research; and drug development and safety assessment (Mansfield, 2003). Forms of experimental cell therapy have been developed for models of neural injury in the marmoset (Virley et al., 1999; Oka et al., 2004) and the marmoset has been proposed as an ideal model for elucidation of safety and efficiency of new technologies in regenerative medicine (Tomioka and Sasaki, 2008). Marmoset embryonic stem cells were originally derived by Thomson (Thomson et al., 1996); more recently, 4 other marmoset ESC lines were reported (Sasaki et al., 2005; Muller et al., 2009). While ESCs have vast potential for future cell therapy and regenerative medicine, iPSCs have the specific advantages that (1) it is widely believed that derivatives of iPSCs could be transplanted back into the original donor from which the cells were derived without immune rejection; and (2) iPSCs can potentially be derived from a wide variety of donor individuals of differing ages, health status and genetic background. Here we report the creation of induced pluripotent stem cells from the marmoset, as a first step toward experimental cell therapy based on use of autologous iPSCs in this species.
Materials and Methods Growth of marmoset skin fibroblasts and retroviral infection Marmoset fibroblasts were prepared from newborn skin by digestion with 5 mg/ml collagenase type I (Sigma) overnight. They were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Cosmic Calf Serum (HyClone Laboratories, Logan, UT), 0.1 mg/ml penicillin and 0.05 mg/ml gentamicin. Retroviral plasmids (pMXs) containing human cDNAs for Oct4, Sox2, Klf4 and c-Myc (Takahashi et al., 2007) were obtained from Addgene Inc.. Virus was prepared in Plat-A cells and was used for coinfection of marmoset fibroblasts as previously described (Wu et al., 2009). Because the marmoset and human reprogramming factors are highly homologous, we used vectors encoding the human reprogramming factors. The marmoset proteins encoded by OCT4, SOX2 and KLF4 cDNAs are highly similar to the human proteins. The marmoset and human OCT4, SOX2, and KLF4 proteins are 98.3%, 99.7% and 99.4% identical by amino acid sequence, while the domains of the proteins thought to be critical for their activity as transcription factors are 99.3%, 100% and 100% identical, respectively.
Conditions for reprogramming and iPSC colony isolation Marmoset fibroblasts were trypsinized and plated in Matrigel-coated 6-well plates. The day following plating, cells were infected with the mixture of four retroviruses (Wu et al., 2009); the infection was repeated two more times at
181 24 hour intervals. Following infection, cells were maintained in DMEM with 10% Cosmic Calf Serum and 1 mM valproic acid (Huangfu et al., 2008). Cells were grown under these conditions for 12 days. Cells were then trypsinized and replated at a 1:2 ratio on Matrigel-coated 35-mm dishes in medium without valproic acid. After a further 7 days, colonies with iPSC morphology were identified and were isolated using Accutase (Bajpai et al., 2008; Li et al., 2009). This enzymatic isolation was designated as passage 1. Following removal from the fibroblast culture, colonies were replated in knockout DMEM with 20% knockout serum replacement (KSR, Invitrogen) and 20 ng/ml bFGF onto mitomycin C-treated mouse embryonic fibroblasts (MEFs, GlobalStem Inc., Rockville, MD).
Growth of iPSC clones After cells were grown on MEFs for two passages, they were removed from the dish with Accutase; and were then transferred to feeder-free culture conditions, comprising Matrigel-coated culture plates, and a medium consisting of 45% knockout DMEM, 20% ES-qualified fetal bovine serum (GlobalStem), 35% medium conditioned by 24 hour incubation with mitomycin C-treated mouse embryonic fibroblasts (medium comprising knockout DMEM with 20% KSR), 0.1 mM β-mercaptoethanol, nonessential amino acids, 10 μM ROCK inhibitor Y-27632 (Li et al., 2009) and 20 ng/ml bFGF. Cells were passaged continuously under these conditions, using Accutase, at a 1:3 split ratio. For assessment of alkaline phosphatase activity, cultures were fixed in 4% paraformaldehyde and were incubated with substrate according to the manufacturer's instructions (SigmaFAST BCIP/NBT kit). Karyotyping was performed using conventional techniques by Cell Line Genetics, Madison, WI.
qPCR for pluripotency markers RNA was prepared from marmoset iPSC clones and was used for qPCR. As a control, we prepared RNA from the human embryonic stem cell line I6 (Amit and Itskovitz-Eldor, 2002). Primers were designed to amplify marmoset NANOG, OCT4 (POU5F1), and SOX2. Primers were selected to fulfil two criteria: (a) they were 100% complementary to both the marmoset and human sequences for these cDNAs; and (b) there was at least one marmoset-specific and one human-specific restriction enzyme site within the amplified sequence. qPCR was performed using SYBR green in an ABI 7900HT instrument.
Immunocytochemistry for pluripotency markers Cells were fixed in 4% paraformaldehyde and were processed for fluorescence immunocytochemistry. Antibodies used were: Oct4; (mouse monoclonal, SC-5279, Santa Cruz Biotechnology, at 1:100); TRA-1-81 (phycoerythrin-conjugated mouse monoclonal, 09-0012, Stemgent, at 1:50); SSEA-4 (mouse monoclonal SP2/0, University of Iowa Developmental Hybridoma Bank, at 1:100). Images were obtained using a Zeiss Axiovert fluorescence microscope.
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Teratoma formation and immunohistochemistry for differentiation markers For assessment of differentiation potential, marmoset iPSCs were implanted into immunodeficient mice. We used a highly immunodeficient mouse model (RAG2-/- , γc-/- ). Initially we injected cells in the subrenal capsule based on our previous studies on cancer cell growth in this immunodeficient model and transplant site (Sun et al., 2004). This is also a site often employed for teratoma formation (Przyborski, 2005; Prokhorova et al., 2008; Hentze et al., 2009). Using a transrenal injection, 2 × 106 cells were introduced per animal and mice were sacrificed at 6 weeks following transplantation. Subsequently we also used subcutaneous injection of the same number of cells suspended in 100 μl of 50% Matrigel in culture medium (Prokhorova et al., 2008; Hentze et al., 2009); mice were sacrificed after 8 weeks. Tumors were excised, fixed in 4% paraformaldehyde, and processed for conventional histology. Sections were stained with hematoxylin and eosin or were used for immunohistochemical detection of markers of differentiation of the three germ layers. Following antigen retrieval (0.1 M citrate buffer, 105 °C, 20 minutes) sections were incubated at 4 °C overnight with the following antibodies: (ectodermal) βIII-tubulin (chicken polyclonal, AB9354, Chemicon, at 1:500); (endodermal) α-fetoprotein (rabbit polyclonal, A0008, Dako, at 1:100); (mesodermal) smooth muscle actin (mouse monoclonal 1A4 at 1:50). Antibody localization was developed using biotinylated secondary antibodies and the Vectastain Elite ABC kit (Vector
Y. Wu et al. Laboratories, Burlingame, CA). Sections were lightly counterstained with hematoxylin.
Results Reprogramming of marmoset skin fibroblasts to iPSCs Following the infection of marmoset skin fibroblasts with retroviruses encoding Oct4, Sox2, Klf4 and c-Myc, cultures were maintained in normal fibroblast growth conditions with the addition of valproic acid (Huangfu et al., 2008). After 1421 days, small colonies of altered morphology were noted in the confluent fibroblast cultures. These colonies comprised small rapidly dividing cells with high nuclear/cytoplasmic ratio and prominent nucleoli (Takahashi et al., 2007). When cultures containing such colonies were fixed and stained for alkaline phosphatase activity, most of the small colonies of altered morphology were found to be positive for alkaline phosphatase (Fig. 1), a marker of pluripotency (O'Connor et al., 2008). These colonies expanded rapidly, producing very dense patches of small cells (Fig. 1). These cells have the morphological characteristics previously reported for human iPSCs (Takahashi et al., 2007). Starting with a population of 4 × 105 marmoset fibroblasts, we obtained ∼ 100 colonies of cells with iPSC-like morphology. Colonies were isolated and expanded on MEF feeder layers (Fig. 1). Of those colonies that were isolated from the fibroblast cultures, 30 showed sustained growth and were
Figure 1 Reprogramming marmoset skin fibroblasts. Primary cultures of newborn marmoset skin fibroblasts were transduced with retroviruses encoding Oct4, Sox2, Klf4 and c-Myc and treated with valproic acid as described in the text. (A - C) Colonies of altered morphology developing among the transduced fibroblasts, 21 days after retroviral infection. (A) Low power image of multiple colonies of altered morphology stained for alkaline phosphatase activity. (B) Phase-contrast appearance of a single colony with altered cell morphology. (C) Alkaline phosphatase staining of cells in three adjacent colonies developing among the fibroblasts. (D - F) Expansion of putative iPSC colonies following isolation. D and E shows a colony similar to those in A - C following isolation and growth on MEF feeder layers (D and E, photographs obtained 2 days apart). F shows a similar colony after assay for alkaline phosphatase activity.
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Figure 2 Karyotypes of marmoset iPSC clones. Metaphase chromosomes of two clones (#15 and #88) were analyzed by conventional G banding.
able to be expanded to the point where they could be cryopreserved. Of these, 8 were selected for further study. Karyotypes were studied at an early passage (passage 4). The karyotypes of 3 clones (#15, #88, and #B8) were investigated
by G banding and were found to be normal. Karyotypes for 2 of these clones are shown in Figure 2. Following the initial expansion of marmoset iPSC clones on MEF feeder layers, we investigated if the cells could be grown
Figure 3 Growth of isolated marmoset iPSC clones. Following isolation of putative iPSC clones, cells were expanded under feederfree conditions as described in the text. Observations were performed at passages 6 - 8. The series of phase-contrast images in A and B show two different clones photographed at intervals of one day (A clone #15; B clone #88). The two series of images show the rate of growth of the cells and the patterns of growth of cells under feeder-free conditions. C shows alkaline phosphatase activity of cells of a clone (B8) that have been fixed at low, middle and high cell density (2 day intervals: C, C' and C'') showing the pattern of expansion of alkaline phosphase staining within the cultures as cells achieve high cell density.
184 under feeder-free conditions. Cells were replated on Matrigelcoated dishes in medium containing 20% fetal bovine serum and 35% MEF-conditioned medium and continued to grow rapidly (Fig. 3). After plating in feeder-free conditions, cells were observed in dense patches with more sparse cells intervening. Dense patches expanded, filling the culture dishes (Fig. 3). When cultures were fixed and stained for alkaline phosphatase, we observed that the dense patches of small rapidly dividing cells were strongly alkaline phosphatase positive, while the flat cells among the patches were weakly positive or negative (Fig. 3). The positive dense patches shown in Figure 3 have the same morphology as the colony shown at higher power in Figure 1F. The 8 clones that were selected for detailed studies have been grown continuously in culture without evidence of any reduction in their growth rate for N20 passages in each case. Over this period they maintained the patterns of growth and alkaline phosphatase activity shown in Figure 3.
Markers of pluripotency in marmoset iPSCs Marmoset iPSC clones expressed pluripotency markers at levels that were comparable to that in a human embryonic
Y. Wu et al. stem cell line (I6) or exceeded that level (Fig. 4). In all 8 marmoset iPSC clones, NANOG and SOX2 mRNA levels were higher than those in I6 cells, and levels of OCT4 were comparable to that of I6 cells. Levels of OCT4 mRNA were N 100-fold higher in iPSC clones than in the fibroblasts used for reprogramming, and levels of NANOG and SOX2 were N 50fold higher (Fig. 4). We assessed the relative levels of vector and total mRNAs for OCT4 and SOX2, two of the factors used for reprogramming. We used primer pairs specific for reprogramming vectors (vector sequence 5′ primer and coding region 3′ primer). Vector OCT4 mRNA was present at 0.01% to 0.1% of that of total OCT4 mRNA, while vector SOX2 mRNA was present at 0.1% to 1% of the total SOX2 mRNA. In addition, the PCR products amplified by the primers for endogenous OCT4 and SOX2 mRNAs were digested almost completely by restriction enzymes specific for the marmoset sequence (see Materials and Methods). We conclude that endogenous OCT4 and SOX2 mRNAs comprise 99% of the totals. Additionally, using fluorescence immunocytochemistry, we found nuclear Oct4 in most cells within marmoset iPSC cultures, with a greater intensity in the dense patches of cells that have typical iPSC morphology and are
Figure 4 Markers of pluripotency in marmoset iPSC clones. Levels of mRNA for NANOG, OCT4 (POU5F1) and SOX2 in 8 clones were compared to the levels in a human embryonic stem cell line, I6, and to levels in the marmoset skin fibroblasts (MF) used for reprogramming. qPCR was performed on RNA isolated from triplicate cultures at early passage levels (6 - 8). The relative level ± S.E. is shown for each clone. The level in the I6 cells is indicated by a dashed line. Below, ratios of total/vector mRNA for OCT4 and SOX2 are shown for each clone. Vector RNA levels were determined using vector-specific primers; total mRNA levels were determined using primers that amplify both endogenous and vector sequences (see Materials and Methods).
Marmoset induced pluripotent stem cells alkaline phosphatase positive (Fig. 5). Similarly, two plasma membrane markers of pluripotency, TRA-1-81 and SSEA-4 (Takahashi et al., 2007), were also detected in the denser patches (Fig. 5).
Differentiation of marmoset iPSC clones to cells of the three germ layers In order to assess the potential of marmoset iPSC clones to differentiate to cells of all three germ layers, cells were transplanted into immunodeficient mice. Because previous studies showed that the subrenal capsule site is an excellent one for teratoma development (Przyborski, 2005; Prokhorova et al., 2008; Hentze et al., 2009) we first injected cells beneath the kidney capsule by transrenal injection. Subsequently we also tested teratoma formation by subcutaneous injection in 50% Matrigel (Prokhorova et al., 2008; Hentze et al., 2009). Teratomas developed in both the subrenal capsule and subcutaneous sites. Immunohistochemical studies revealed that tissue structures within the teratomas represented derivatives of all three germ layers (Fig. 6). Ectodermal tissue (developing neural tissue) was demonstrated by presence of βIII-tubulin; mesodermal tissue by smooth muscle actin; and endodermal tissue by α-fetoprotein. The structures stained by the anti-α-fetoprotein antibody resembled the structures previously identified by this marker in teratomas (Zhang et al., 2009). Because it has been
185 reported that teratomas derived from incompletely reprogrammed cells formed tissues of ectodermal and mesodermal origin but not of endodermal origin (Chan et al., 2009) we performed histological studies of the development of mature structures of endodermal origin. Teratomas from marmoset iPSCs contained a variety of mature endodermal tissues, including simple columnar and pseudostratified epithelia, epithelia with goblet cells, and exocrine glandular structures (Fig. 7).
Discussion The development of technology to generate induced pluripotent stem cells from potentially any somatic cell has provided an invaluable tool for cell therapy and regenerative medicine. The creation of patient-specific iPSCs by reprogramming of somatic cells would create an inexhaustible source of cells that could be used to derive differentiated cells required for patient-specific therapy, such as cardiomyocytes, neurons, and pancreatic beta cells (Yamanaka, 2007). In order to provide the appropriate translational proof of principle that differentiated cells derived from individualspecific iPSCs will be safe and effective, and will not be subject to rejection by the immune system, appropriate studies are needed in suitable mammalian models. Although rodents are excellent sources of cells for reprogramming, it is generally accepted that primates will serve as the most
Figure 5 Markers of pluripotency in marmoset iPSC clones. Fluorescence immunocytochemistry was performed for three markers of pluripotency on fixed cells of three clones. A, Oct4 (clone #81); B, TRA-1-81 (clone #15); C, SSEA-4 (clone #88).
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Figure 6 Teratoma formation by marmoset iPSC clones formed by transplantation in immunodeficient mice in the subrenal capsule (A, C - E, clone #15; B, clone #81). Hematoxylin/eosin-stained sections are shown in A and B. Immunohistochemical demonstration of differentiation markers for (C) an endodermal marker (α-fetoprotein); (D) a mesodermal marker (smooth muscle actin); (E) an ectodermal marker (βIII tubulin). For each antibody, a stained section (C - E) is shown together with an adjacent section stained with hematoxylin and eosin (C' - E'). Teratomas were formed by transplantation of cells at passages 7 - 8. Size bar = 500 μm.
appropriate mammalian models for autologous cell therapy and other aspects of regenerative medicine (Wolf et al., 2004). Among primates, the marmoset stands out as especially relevant for biomedical research because of well developed models in this species for several human disease states. Here we produced several lines of iPSCs from newborn marmoset fibroblasts. While the methodology we used followed many aspects of those previously used, we also provided significant modifications of the methodology. It is generally assumed that feeder-free conditions for iPSC and ES cell growth offer advantages for subsequent studies of directed differentiation and cell transplantation. In most prior reprogramming studies, iPSCs have been developed and grown on feeder layers. In some cases cells were later adapted to feeder-free conditions. In the experiments reported here, we transferred marmoset iPSC clones to feeder-free conditions at an early stage. Many clones grew well under these conditions, were expanded, and were cryopreserved. It has been noted that under some circumstances the adaptation of human ES cells to feeder-free conditions of cell lines that have
been grown routinely on feeders is associated with the expansion of altered subclones that may have abnormal karyotypes (Hasegawa et al., 2006). Marmoset iPSCs transferred to feeder-free conditions exhibited a normal karyotype. Moreover they maintained a constant pattern of growth, with expanding patches of small cells with typical iPSC/ES cell morphology (high nuclear/cytoplasmic ratio and prominent nucleoli). Cells between patches tended to differentiate, and were weakly alkaline phosphatase positive or were negative. As cells achieved high density, patches of iPSCs expanded while the differentiated cells grew more slowly or not at all; thus cultures always reached high percentages of undifferentiated cells at high density. These cells also had markers of pluripotency (nuclear Oct4, and plasma membrane TRA-1-81 and SSEA-4). A quantitative assessment of the major pluripotency genes (NANOG, OCT4, SOX2) showed that mRNAs for these genes were maintained at a level either comparable to that of the I6 human ESC line (OCT4), or above that level (NANOG and SOX2) in the case of 8 marmoset iPSC clones selected for detailed study. Importantly, we showed that
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Figure 7 Histological analysis of differentiation within subcutaneous teratomas (marmoset iPSC clone B8; passage 7). Hematoxylin and eosin stain. A Low power view of subcutaneous teratomas; sections showing teratoma in subdermal tissue. (Bar = 2.5 mm). B Higher power view showing varieties of differentiated tissues. Boxed areas are also shown at high power in C (lower box) and D (upper box). (Bar = 500 μm) C - F High power views of epithelium with goblet cells (C), exocrine glandular structure (D), simple columnar epithelium (E) and pseudostratified epithelium (F). (Bar = 150 μm).
the levels of vector RNA for OCT4 and SOX2 were at levels much lower than those of the corresponding endogenous genes. Recent studies show that reprogramming by viral vectors is associated with complete silencing of the viral genome when reprogramming is complete, and that partially reprogrammed cells still express the viral genome at a high level (Chan et al., 2009). In the marmoset iPSCs, viral OCT4 mRNA was present at a level of only 0.01% to 0.1% of the level of the endogenous gene, and viral SOX2 mRNA was present at a level of 0.1% to 1% of the level of the endogenous gene. In contrast, in partially reprogrammed clones, the viral mRNAs exceed the levels of the endogenous mRNAs (Chan et al., 2009). Moreover, the same study showed that teratomas formed by partially reprogrammed cells lacked differentiated tissues of endodermal origin, while this was present in teratomas formed from fully reprogrammed cells. In the teratomas formed from marmoset iPSCs we found differentiated endoderm, as evidenced by immunohistochemistry and by conventional histological analysis, that showed welldeveloped features of tissues of endodermal lineage. For example, goblet cells are characteristic of several epithelia of endodermal origin. The goblet cells detected in the marmoset iPSC teratomas closely resemble those of the small intestine of the marmoset (Miraglia et al., 1967). We conclude that the marmoset iPSC clones described here are pluripotent and fully reprogrammed. While retroviral silencing is a feature of full reprogramming, it is also important for suppressing the potential adverse consequences of transgene expression, particularly c-Myc (Nakagawa et al., 2008). While retrovirally generated iPSCs are highly valuable for basic and translational experiments in animals, for clinical use it is likely that reprogramming methods will be employed in which
the reprogramming vectors are excised from the cells before therapeutic use (Soldner et al., 2009; Chang et al., 2009) or methods that do not employ genetic modification at all (Zhou et al., 2009; Kim et al., 2009). Nevertheless, the relative efficiency of reprogramming using retroviral vectors makes this method attractive for solving basic issues. Once these are solved, safer methods can and should be developed, and compared with methods that use genetic modification. The presently reported characterization of iPSCs from the marmoset provides the proof of principle of iPSC generation in this species, and paves the way for the generation of indvidual-specific iPSCs for proof of principle experiments on autologous cell therapy in nonhuman primates.
Acknowledgments This work was supported by grants from the National Institute on Aging, from the Owens Medical Research Foundation, and from the Glenn Foundation for Medical Research.
References Amit, M., Itskovitz-Eldor, J., 2002. Derivation and spontaneous differentiation of human embryonic stem cells. J. Anat. 200, 225–232. Bajpai, R., Lesperance, J., Kim, M., Terskikh, A.V., 2008. Efficient propagation of single cells Accutase-dissociated human embryonic stem cells. Mol. Reprod. Dev. 75, 818–827. Chan, 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., Daley, G.Q.,
188 Schlaeger, T.M., 2009. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27, 1033–1037. Chang, C.W., Lai, Y.S., Pawlik, K.M., Liu, K., Sun, C.W., Li, C., Schoeb, T.R., Townes, T.M., 2009. Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells. Stem Cells 27, 1042–1049. Ezashi, T., Telugu, B.P., Alexenko, A.P., Sachdev, S., Sinha, S., Roberts, R.M., 2009. Derivation of induced pluripotent stem cells from pig somatic cells. Proc. Natl Acad. Sci. U. S. A. 106, 10993–10998. Hasegawa, K., Fujioka, T., Nakamura, Y., Nakatsuji, N., Suemori, H., 2006. A method for the selection of human embryonic stem cell sublines with high replating efficiency after single-cell dissociation. Stem Cells 24, 2649–2660. Hentze, H., Soong, P.L., Wang, S.T., Phillips, B.W., Putti, T.C., Dunn, N.R., 2009. Teratoma formation by human embryonic stem cells: Evaluation of essential parameters for future safety studies. Stem Cell Res. 2, 198–210. Huangfu, D., Osafune, K., Maehr, R., Guo, W., Eijkelenboom, A., Chen, S., Muhlestein, W., Melton, D.A., 2008. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat. Biotechnol. 26, 1269–1275. Kim, D., Kim, C.H., Moon, J.I., Chung, Y.G., Chang, M.Y., Han, B.S., Ko, S., Yang, E., Cha, K.Y., Lanza, R., Kim, K.S., 2009. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4, 472–476. Li, X., Krawetz, R., Liu, S., Meng, G., Rancourt, D.E., 2009. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum. Reprod. 24, 580–589. Liao, J., Cui, C., Chen, S., Ren, J., Chen, J., Gao, Y., Li, H., Jia, N., Cheng, L., Xiao, H., Xiao, L., 2009. Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 4, 11–15. Liu, H., Zhu, F., Yong, J., Zhang, P., Hou, P., Li, H., Jiang, W., Cai, J., Liu, M., Cui, K., Qu, X., Xiang, T., Lu, D., Chi, X., Gao, G., Ji, W., Ding, M., Deng, H., 2008. Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell 3, 587–590. Mansfield, K., 2003. Marmoset models commonly used in biomedical research. Comp. Med. 53, 383–392. Miraglia, T., Ledoux, L., Branco, A.L., 1967. Histological and histochemical data on the intestinal tract of the marmoset (Callithrix jacchus). Acta Anat. (Basel) 68, 459–472. Muller, T., Fleischmann, G., Eildermann, K., Matz-Rensing, K., Horn, P.A., Sasaki, E., Behr, R., 2009. A novel embryonic stem cell line derived from the common marmoset monkey (Callithrix jacchus) exhibiting germ cell-like characteristics. Hum. Reprod. 24, 1359–1372. Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Okita, K., Mochiduki, Y., Takizawa, N., Yamanaka, S., 2008. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol. 26, 101–106. O'Connor, M.D., Kardel, M.D., Iosfina, I., Youssef, D., Lu, M., Li, M.M., Vercauteren, S., Nagy, A., Eaves, C.J., 2008. Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells. Stem Cells 26, 1109–1116. Oka, S., Honmou, O., Akiyama, Y., Sasaki, M., Houkin, K., Hashi, K., Kocsis, J.D., 2004. Autologous transplantation of expanded neural precursor cells into the demyelinated monkey spinal cord. Brain Res. 1030, 94–102. Prokhorova, T.A., Harkness, L.M., Frandsen, U., Ditzel, N., Burns, J.S., Schroeder, H.D., Kassem, M., 2008. Teratoma formation by human
Y. Wu et al. embryonic stem cells is site-dependent and enhanced by the presence of Matrigel. Stem Cells Dev. 200, 225–232. Przyborski, S.A., 2005. Differentiation of human embryonic stem cells after transplantation in immune-deficient mice. Stem Cells 23, 1242–1250. Sasaki, E., Hanazawa, K., Kurita, R., Akatsuka, A., Yoshizaki, T., Ishii, H., Tanioka, Y., Ohnishi, Y., Suemizu, H., Sugawara, A., Tamaoki, N., Izawa, K., Nakazaki, Y., Hamada, H., Suemori, H., Asano, S., Nakatsuji, N., Okano, H., Tani, K., 2005. Establishment of novel embryonic stem cell lines derived from the common marmoset (Callithrix jacchus). Stem Cells 23, 1304–1313. Shimada, H., Nakada, A., Hashimoto, Y., Shigeno, K., Shionoya, Y., Nakamura, T., 2009. Generation of canine induced pluripotent stem cells by retroviral transduction and chemical inhibitors. Mol. Reprod. Dev. 77, 2. Soldner, F., Hockemeyer, D., Beard, C., Gao, Q., Bell, G.W., Cook, E.G., Hargus, G., Blak, A., Cooper, O., Mitalipova, M., Isacson, O., Jaenisch, R., 2009. Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964–977. Sun, B., Huang, Q., Liu, S., Chen, M., Hawks, C.L., Wang, L., Zhang, C., Hornsby, P.J., 2004. Progressive loss of malignant behavior in telomerase-negative tumorigenic adrenocortical cells and restoration of tumorigenicity by human telomerase reverse transcriptase. Cancer Res. 64, 6144–6151. Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S., 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872. Thomson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, C.P., Hearn, J.P., 1996. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol. Reprod. 55, 254–259. Tomioka, I., Sasaki, E., 2008. Recent progress in reproductive technologies based on the common marmoset (Callithrix jacchus). J. Mamm. Ova Res. 25, 143–149. Virley, D., Ridley, R.M., Sinden, J.D., Kershaw, T.R., Harland, S., Rashid, T., French, S., Sowinski, P., Gray, J.A., Lantos, P.L., Hodges, H., 1999. Primary CA1 and conditionally immortal MHP36 cell grafts restore conditional discrimination learning and recall in marmosets after excitotoxic lesions of the hippocampal CA1 field. Brain 122, 2321–2335. Wolf, D.P., Kuo, H.C., Pau, K.Y., Lester, L., 2004. Progress with nonhuman primate embryonic stem cells. Biol. Reprod. 71, 1766–1771. Wu, Y., Melton, D.W., Zhang, Y., Hornsby, P.J., 2009. Improved coinfection with amphotropic pseudotyped retroviral vectors. J. Biomed. Biotechnol. 2009, 901079. Yamanaka, S., 2007. Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 1, 39–49. Zhang, X., Neganova, I., Przyborski, S., Yang, C., Cooke, M., Atkinson, S.P., Anyfantis, G., Fenyk, S., Keith, W.N., Hoare, S.F., Hughes, O., Strachan, T., Stojkovic, M., Hinds, P.W., Armstrong, L., Lako, M., 2009. A role for NANOG in G1 to S transition in human embryonic stem cells through direct binding of CDK6 and CDC25A. J. Cell Biol. 184, 67–82. Zhou, H., Wu, S., Joo, J.Y., Zhu, S., Han, D.W., Lin, T., Trauger, S., Bien, G., Yao, S., Zhu, Y., Siuzdak, G., Scholer, H.R., Duan, L., Ding, S., 2009. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4, 381–384.
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REGULAR ARTICLE
Generation of induced pluripotent stem cells from newborn marmoset skin fibroblasts Yuehong Wu a,b , Yong Zhang a , Anuja Mishra b , Suzette D. Tardif b , Peter J. Hornsby b,⁎ a
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China Department of Physiology and Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, TX 78245, USA b
Received 21 December 2009; received in revised form 21 February 2010; accepted 25 February 2010
Abstract Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine. For the application of iPSCs to forms of autologous cell therapy, suitable animal models are required. Among species that could potentially be used for this purpose, nonhuman primates are particularly important, and among these the marmoset offers significant advantages. In order to demonstrate the feasibility of the application of iPSC technology to this species, here we derived lines of marmoset iPSCs. Using retroviral transduction with human Oct4, Sox2, Klf4 and c-Myc, we derived clones that fulfil critical criteria for successful reprogramming: they exhibit typical iPSC morphology; they are alkaline phosphatase positive; they express high levels of NANOG, OCT4 and SOX2 mRNAs, while the corresponding vector genes are silenced; they are immunoreactive for Oct4, TRA-1-81 and SSEA-4; and when implanted into immunodeficient mice they produce teratomas that have derivatives of all three germ layers (endoderm, α-fetoprotein; ectoderm, βIII-tubulin; mesoderm, smooth muscle actin). Starting with a population of 4 × 105 newborn marmoset skin fibroblasts, we obtained ∼ 100 colonies with iPSC-like morphology. Of these, 30 were expanded sufficiently to be cryopreserved, and, of those, 8 were characterized in more detail. These experiments provide proof of principle that iPSC technology can be adapted for use in the marmoset, as a future model of autologous cell therapy. © 2010 Elsevier B.V. All rights reserved.
Introduction Induced pluripotent stem cells (iPSCs) have been derived from somatic cells of mouse (Takahashi and Yamanaka,
⁎ Corresponding author. University of Texas Health Science Center, 15355 Lambda Drive, San Antonio, TX 78245, USA. Fax: +1 281 582 3538. E-mail addresses: [email protected] (Y. Wu), [email protected] (Y. Zhang), [email protected] (A. Mishra), [email protected] (S.D. Tardif), [email protected] (P.J. Hornsby).
2006), human (Takahashi et al., 2007), rhesus monkey (Liu et al., 2008), rat (Liao et al., 2009), pig (Ezashi et al., 2009), and dog (Shimada et al., 2009). It is widely thought that an important future use for iPSC technology is a form of autologous cell therapy in which the donor of the reprogrammed somatic cells is also the recipient of the cells following appropriate differentiation protocols (Yamanaka, 2007). Potentially, any mammalian species could serve as a translational model for this form of autologous cell therapy, but nonhuman primates are particularly suited for such studies because of the aspects of their anatomy and physiology that they share with humans. Among nonhuman primates, the marmoset is especially relevant for biomedical research because of well developed models in this species for
1873-5061/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scr.2010.02.003
Marmoset induced pluripotent stem cells neurodegenerative disorders (Parkinson's, Alzheimer's and Huntington's disease), stroke, and spinal cord injury; reproductive biology, infectious disease, and behavioral research; and drug development and safety assessment (Mansfield, 2003). Forms of experimental cell therapy have been developed for models of neural injury in the marmoset (Virley et al., 1999; Oka et al., 2004) and the marmoset has been proposed as an ideal model for elucidation of safety and efficiency of new technologies in regenerative medicine (Tomioka and Sasaki, 2008). Marmoset embryonic stem cells were originally derived by Thomson (Thomson et al., 1996); more recently, 4 other marmoset ESC lines were reported (Sasaki et al., 2005; Muller et al., 2009). While ESCs have vast potential for future cell therapy and regenerative medicine, iPSCs have the specific advantages that (1) it is widely believed that derivatives of iPSCs could be transplanted back into the original donor from which the cells were derived without immune rejection; and (2) iPSCs can potentially be derived from a wide variety of donor individuals of differing ages, health status and genetic background. Here we report the creation of induced pluripotent stem cells from the marmoset, as a first step toward experimental cell therapy based on use of autologous iPSCs in this species.
Materials and Methods Growth of marmoset skin fibroblasts and retroviral infection Marmoset fibroblasts were prepared from newborn skin by digestion with 5 mg/ml collagenase type I (Sigma) overnight. They were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Cosmic Calf Serum (HyClone Laboratories, Logan, UT), 0.1 mg/ml penicillin and 0.05 mg/ml gentamicin. Retroviral plasmids (pMXs) containing human cDNAs for Oct4, Sox2, Klf4 and c-Myc (Takahashi et al., 2007) were obtained from Addgene Inc.. Virus was prepared in Plat-A cells and was used for coinfection of marmoset fibroblasts as previously described (Wu et al., 2009). Because the marmoset and human reprogramming factors are highly homologous, we used vectors encoding the human reprogramming factors. The marmoset proteins encoded by OCT4, SOX2 and KLF4 cDNAs are highly similar to the human proteins. The marmoset and human OCT4, SOX2, and KLF4 proteins are 98.3%, 99.7% and 99.4% identical by amino acid sequence, while the domains of the proteins thought to be critical for their activity as transcription factors are 99.3%, 100% and 100% identical, respectively.
Conditions for reprogramming and iPSC colony isolation Marmoset fibroblasts were trypsinized and plated in Matrigel-coated 6-well plates. The day following plating, cells were infected with the mixture of four retroviruses (Wu et al., 2009); the infection was repeated two more times at
181 24 hour intervals. Following infection, cells were maintained in DMEM with 10% Cosmic Calf Serum and 1 mM valproic acid (Huangfu et al., 2008). Cells were grown under these conditions for 12 days. Cells were then trypsinized and replated at a 1:2 ratio on Matrigel-coated 35-mm dishes in medium without valproic acid. After a further 7 days, colonies with iPSC morphology were identified and were isolated using Accutase (Bajpai et al., 2008; Li et al., 2009). This enzymatic isolation was designated as passage 1. Following removal from the fibroblast culture, colonies were replated in knockout DMEM with 20% knockout serum replacement (KSR, Invitrogen) and 20 ng/ml bFGF onto mitomycin C-treated mouse embryonic fibroblasts (MEFs, GlobalStem Inc., Rockville, MD).
Growth of iPSC clones After cells were grown on MEFs for two passages, they were removed from the dish with Accutase; and were then transferred to feeder-free culture conditions, comprising Matrigel-coated culture plates, and a medium consisting of 45% knockout DMEM, 20% ES-qualified fetal bovine serum (GlobalStem), 35% medium conditioned by 24 hour incubation with mitomycin C-treated mouse embryonic fibroblasts (medium comprising knockout DMEM with 20% KSR), 0.1 mM β-mercaptoethanol, nonessential amino acids, 10 μM ROCK inhibitor Y-27632 (Li et al., 2009) and 20 ng/ml bFGF. Cells were passaged continuously under these conditions, using Accutase, at a 1:3 split ratio. For assessment of alkaline phosphatase activity, cultures were fixed in 4% paraformaldehyde and were incubated with substrate according to the manufacturer's instructions (SigmaFAST BCIP/NBT kit). Karyotyping was performed using conventional techniques by Cell Line Genetics, Madison, WI.
qPCR for pluripotency markers RNA was prepared from marmoset iPSC clones and was used for qPCR. As a control, we prepared RNA from the human embryonic stem cell line I6 (Amit and Itskovitz-Eldor, 2002). Primers were designed to amplify marmoset NANOG, OCT4 (POU5F1), and SOX2. Primers were selected to fulfil two criteria: (a) they were 100% complementary to both the marmoset and human sequences for these cDNAs; and (b) there was at least one marmoset-specific and one human-specific restriction enzyme site within the amplified sequence. qPCR was performed using SYBR green in an ABI 7900HT instrument.
Immunocytochemistry for pluripotency markers Cells were fixed in 4% paraformaldehyde and were processed for fluorescence immunocytochemistry. Antibodies used were: Oct4; (mouse monoclonal, SC-5279, Santa Cruz Biotechnology, at 1:100); TRA-1-81 (phycoerythrin-conjugated mouse monoclonal, 09-0012, Stemgent, at 1:50); SSEA-4 (mouse monoclonal SP2/0, University of Iowa Developmental Hybridoma Bank, at 1:100). Images were obtained using a Zeiss Axiovert fluorescence microscope.
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Teratoma formation and immunohistochemistry for differentiation markers For assessment of differentiation potential, marmoset iPSCs were implanted into immunodeficient mice. We used a highly immunodeficient mouse model (RAG2-/- , γc-/- ). Initially we injected cells in the subrenal capsule based on our previous studies on cancer cell growth in this immunodeficient model and transplant site (Sun et al., 2004). This is also a site often employed for teratoma formation (Przyborski, 2005; Prokhorova et al., 2008; Hentze et al., 2009). Using a transrenal injection, 2 × 106 cells were introduced per animal and mice were sacrificed at 6 weeks following transplantation. Subsequently we also used subcutaneous injection of the same number of cells suspended in 100 μl of 50% Matrigel in culture medium (Prokhorova et al., 2008; Hentze et al., 2009); mice were sacrificed after 8 weeks. Tumors were excised, fixed in 4% paraformaldehyde, and processed for conventional histology. Sections were stained with hematoxylin and eosin or were used for immunohistochemical detection of markers of differentiation of the three germ layers. Following antigen retrieval (0.1 M citrate buffer, 105 °C, 20 minutes) sections were incubated at 4 °C overnight with the following antibodies: (ectodermal) βIII-tubulin (chicken polyclonal, AB9354, Chemicon, at 1:500); (endodermal) α-fetoprotein (rabbit polyclonal, A0008, Dako, at 1:100); (mesodermal) smooth muscle actin (mouse monoclonal 1A4 at 1:50). Antibody localization was developed using biotinylated secondary antibodies and the Vectastain Elite ABC kit (Vector
Y. Wu et al. Laboratories, Burlingame, CA). Sections were lightly counterstained with hematoxylin.
Results Reprogramming of marmoset skin fibroblasts to iPSCs Following the infection of marmoset skin fibroblasts with retroviruses encoding Oct4, Sox2, Klf4 and c-Myc, cultures were maintained in normal fibroblast growth conditions with the addition of valproic acid (Huangfu et al., 2008). After 1421 days, small colonies of altered morphology were noted in the confluent fibroblast cultures. These colonies comprised small rapidly dividing cells with high nuclear/cytoplasmic ratio and prominent nucleoli (Takahashi et al., 2007). When cultures containing such colonies were fixed and stained for alkaline phosphatase activity, most of the small colonies of altered morphology were found to be positive for alkaline phosphatase (Fig. 1), a marker of pluripotency (O'Connor et al., 2008). These colonies expanded rapidly, producing very dense patches of small cells (Fig. 1). These cells have the morphological characteristics previously reported for human iPSCs (Takahashi et al., 2007). Starting with a population of 4 × 105 marmoset fibroblasts, we obtained ∼ 100 colonies of cells with iPSC-like morphology. Colonies were isolated and expanded on MEF feeder layers (Fig. 1). Of those colonies that were isolated from the fibroblast cultures, 30 showed sustained growth and were
Figure 1 Reprogramming marmoset skin fibroblasts. Primary cultures of newborn marmoset skin fibroblasts were transduced with retroviruses encoding Oct4, Sox2, Klf4 and c-Myc and treated with valproic acid as described in the text. (A - C) Colonies of altered morphology developing among the transduced fibroblasts, 21 days after retroviral infection. (A) Low power image of multiple colonies of altered morphology stained for alkaline phosphatase activity. (B) Phase-contrast appearance of a single colony with altered cell morphology. (C) Alkaline phosphatase staining of cells in three adjacent colonies developing among the fibroblasts. (D - F) Expansion of putative iPSC colonies following isolation. D and E shows a colony similar to those in A - C following isolation and growth on MEF feeder layers (D and E, photographs obtained 2 days apart). F shows a similar colony after assay for alkaline phosphatase activity.
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Figure 2 Karyotypes of marmoset iPSC clones. Metaphase chromosomes of two clones (#15 and #88) were analyzed by conventional G banding.
able to be expanded to the point where they could be cryopreserved. Of these, 8 were selected for further study. Karyotypes were studied at an early passage (passage 4). The karyotypes of 3 clones (#15, #88, and #B8) were investigated
by G banding and were found to be normal. Karyotypes for 2 of these clones are shown in Figure 2. Following the initial expansion of marmoset iPSC clones on MEF feeder layers, we investigated if the cells could be grown
Figure 3 Growth of isolated marmoset iPSC clones. Following isolation of putative iPSC clones, cells were expanded under feederfree conditions as described in the text. Observations were performed at passages 6 - 8. The series of phase-contrast images in A and B show two different clones photographed at intervals of one day (A clone #15; B clone #88). The two series of images show the rate of growth of the cells and the patterns of growth of cells under feeder-free conditions. C shows alkaline phosphatase activity of cells of a clone (B8) that have been fixed at low, middle and high cell density (2 day intervals: C, C' and C'') showing the pattern of expansion of alkaline phosphase staining within the cultures as cells achieve high cell density.
184 under feeder-free conditions. Cells were replated on Matrigelcoated dishes in medium containing 20% fetal bovine serum and 35% MEF-conditioned medium and continued to grow rapidly (Fig. 3). After plating in feeder-free conditions, cells were observed in dense patches with more sparse cells intervening. Dense patches expanded, filling the culture dishes (Fig. 3). When cultures were fixed and stained for alkaline phosphatase, we observed that the dense patches of small rapidly dividing cells were strongly alkaline phosphatase positive, while the flat cells among the patches were weakly positive or negative (Fig. 3). The positive dense patches shown in Figure 3 have the same morphology as the colony shown at higher power in Figure 1F. The 8 clones that were selected for detailed studies have been grown continuously in culture without evidence of any reduction in their growth rate for N20 passages in each case. Over this period they maintained the patterns of growth and alkaline phosphatase activity shown in Figure 3.
Markers of pluripotency in marmoset iPSCs Marmoset iPSC clones expressed pluripotency markers at levels that were comparable to that in a human embryonic
Y. Wu et al. stem cell line (I6) or exceeded that level (Fig. 4). In all 8 marmoset iPSC clones, NANOG and SOX2 mRNA levels were higher than those in I6 cells, and levels of OCT4 were comparable to that of I6 cells. Levels of OCT4 mRNA were N 100-fold higher in iPSC clones than in the fibroblasts used for reprogramming, and levels of NANOG and SOX2 were N 50fold higher (Fig. 4). We assessed the relative levels of vector and total mRNAs for OCT4 and SOX2, two of the factors used for reprogramming. We used primer pairs specific for reprogramming vectors (vector sequence 5′ primer and coding region 3′ primer). Vector OCT4 mRNA was present at 0.01% to 0.1% of that of total OCT4 mRNA, while vector SOX2 mRNA was present at 0.1% to 1% of the total SOX2 mRNA. In addition, the PCR products amplified by the primers for endogenous OCT4 and SOX2 mRNAs were digested almost completely by restriction enzymes specific for the marmoset sequence (see Materials and Methods). We conclude that endogenous OCT4 and SOX2 mRNAs comprise 99% of the totals. Additionally, using fluorescence immunocytochemistry, we found nuclear Oct4 in most cells within marmoset iPSC cultures, with a greater intensity in the dense patches of cells that have typical iPSC morphology and are
Figure 4 Markers of pluripotency in marmoset iPSC clones. Levels of mRNA for NANOG, OCT4 (POU5F1) and SOX2 in 8 clones were compared to the levels in a human embryonic stem cell line, I6, and to levels in the marmoset skin fibroblasts (MF) used for reprogramming. qPCR was performed on RNA isolated from triplicate cultures at early passage levels (6 - 8). The relative level ± S.E. is shown for each clone. The level in the I6 cells is indicated by a dashed line. Below, ratios of total/vector mRNA for OCT4 and SOX2 are shown for each clone. Vector RNA levels were determined using vector-specific primers; total mRNA levels were determined using primers that amplify both endogenous and vector sequences (see Materials and Methods).
Marmoset induced pluripotent stem cells alkaline phosphatase positive (Fig. 5). Similarly, two plasma membrane markers of pluripotency, TRA-1-81 and SSEA-4 (Takahashi et al., 2007), were also detected in the denser patches (Fig. 5).
Differentiation of marmoset iPSC clones to cells of the three germ layers In order to assess the potential of marmoset iPSC clones to differentiate to cells of all three germ layers, cells were transplanted into immunodeficient mice. Because previous studies showed that the subrenal capsule site is an excellent one for teratoma development (Przyborski, 2005; Prokhorova et al., 2008; Hentze et al., 2009) we first injected cells beneath the kidney capsule by transrenal injection. Subsequently we also tested teratoma formation by subcutaneous injection in 50% Matrigel (Prokhorova et al., 2008; Hentze et al., 2009). Teratomas developed in both the subrenal capsule and subcutaneous sites. Immunohistochemical studies revealed that tissue structures within the teratomas represented derivatives of all three germ layers (Fig. 6). Ectodermal tissue (developing neural tissue) was demonstrated by presence of βIII-tubulin; mesodermal tissue by smooth muscle actin; and endodermal tissue by α-fetoprotein. The structures stained by the anti-α-fetoprotein antibody resembled the structures previously identified by this marker in teratomas (Zhang et al., 2009). Because it has been
185 reported that teratomas derived from incompletely reprogrammed cells formed tissues of ectodermal and mesodermal origin but not of endodermal origin (Chan et al., 2009) we performed histological studies of the development of mature structures of endodermal origin. Teratomas from marmoset iPSCs contained a variety of mature endodermal tissues, including simple columnar and pseudostratified epithelia, epithelia with goblet cells, and exocrine glandular structures (Fig. 7).
Discussion The development of technology to generate induced pluripotent stem cells from potentially any somatic cell has provided an invaluable tool for cell therapy and regenerative medicine. The creation of patient-specific iPSCs by reprogramming of somatic cells would create an inexhaustible source of cells that could be used to derive differentiated cells required for patient-specific therapy, such as cardiomyocytes, neurons, and pancreatic beta cells (Yamanaka, 2007). In order to provide the appropriate translational proof of principle that differentiated cells derived from individualspecific iPSCs will be safe and effective, and will not be subject to rejection by the immune system, appropriate studies are needed in suitable mammalian models. Although rodents are excellent sources of cells for reprogramming, it is generally accepted that primates will serve as the most
Figure 5 Markers of pluripotency in marmoset iPSC clones. Fluorescence immunocytochemistry was performed for three markers of pluripotency on fixed cells of three clones. A, Oct4 (clone #81); B, TRA-1-81 (clone #15); C, SSEA-4 (clone #88).
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Figure 6 Teratoma formation by marmoset iPSC clones formed by transplantation in immunodeficient mice in the subrenal capsule (A, C - E, clone #15; B, clone #81). Hematoxylin/eosin-stained sections are shown in A and B. Immunohistochemical demonstration of differentiation markers for (C) an endodermal marker (α-fetoprotein); (D) a mesodermal marker (smooth muscle actin); (E) an ectodermal marker (βIII tubulin). For each antibody, a stained section (C - E) is shown together with an adjacent section stained with hematoxylin and eosin (C' - E'). Teratomas were formed by transplantation of cells at passages 7 - 8. Size bar = 500 μm.
appropriate mammalian models for autologous cell therapy and other aspects of regenerative medicine (Wolf et al., 2004). Among primates, the marmoset stands out as especially relevant for biomedical research because of well developed models in this species for several human disease states. Here we produced several lines of iPSCs from newborn marmoset fibroblasts. While the methodology we used followed many aspects of those previously used, we also provided significant modifications of the methodology. It is generally assumed that feeder-free conditions for iPSC and ES cell growth offer advantages for subsequent studies of directed differentiation and cell transplantation. In most prior reprogramming studies, iPSCs have been developed and grown on feeder layers. In some cases cells were later adapted to feeder-free conditions. In the experiments reported here, we transferred marmoset iPSC clones to feeder-free conditions at an early stage. Many clones grew well under these conditions, were expanded, and were cryopreserved. It has been noted that under some circumstances the adaptation of human ES cells to feeder-free conditions of cell lines that have
been grown routinely on feeders is associated with the expansion of altered subclones that may have abnormal karyotypes (Hasegawa et al., 2006). Marmoset iPSCs transferred to feeder-free conditions exhibited a normal karyotype. Moreover they maintained a constant pattern of growth, with expanding patches of small cells with typical iPSC/ES cell morphology (high nuclear/cytoplasmic ratio and prominent nucleoli). Cells between patches tended to differentiate, and were weakly alkaline phosphatase positive or were negative. As cells achieved high density, patches of iPSCs expanded while the differentiated cells grew more slowly or not at all; thus cultures always reached high percentages of undifferentiated cells at high density. These cells also had markers of pluripotency (nuclear Oct4, and plasma membrane TRA-1-81 and SSEA-4). A quantitative assessment of the major pluripotency genes (NANOG, OCT4, SOX2) showed that mRNAs for these genes were maintained at a level either comparable to that of the I6 human ESC line (OCT4), or above that level (NANOG and SOX2) in the case of 8 marmoset iPSC clones selected for detailed study. Importantly, we showed that
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Figure 7 Histological analysis of differentiation within subcutaneous teratomas (marmoset iPSC clone B8; passage 7). Hematoxylin and eosin stain. A Low power view of subcutaneous teratomas; sections showing teratoma in subdermal tissue. (Bar = 2.5 mm). B Higher power view showing varieties of differentiated tissues. Boxed areas are also shown at high power in C (lower box) and D (upper box). (Bar = 500 μm) C - F High power views of epithelium with goblet cells (C), exocrine glandular structure (D), simple columnar epithelium (E) and pseudostratified epithelium (F). (Bar = 150 μm).
the levels of vector RNA for OCT4 and SOX2 were at levels much lower than those of the corresponding endogenous genes. Recent studies show that reprogramming by viral vectors is associated with complete silencing of the viral genome when reprogramming is complete, and that partially reprogrammed cells still express the viral genome at a high level (Chan et al., 2009). In the marmoset iPSCs, viral OCT4 mRNA was present at a level of only 0.01% to 0.1% of the level of the endogenous gene, and viral SOX2 mRNA was present at a level of 0.1% to 1% of the level of the endogenous gene. In contrast, in partially reprogrammed clones, the viral mRNAs exceed the levels of the endogenous mRNAs (Chan et al., 2009). Moreover, the same study showed that teratomas formed by partially reprogrammed cells lacked differentiated tissues of endodermal origin, while this was present in teratomas formed from fully reprogrammed cells. In the teratomas formed from marmoset iPSCs we found differentiated endoderm, as evidenced by immunohistochemistry and by conventional histological analysis, that showed welldeveloped features of tissues of endodermal lineage. For example, goblet cells are characteristic of several epithelia of endodermal origin. The goblet cells detected in the marmoset iPSC teratomas closely resemble those of the small intestine of the marmoset (Miraglia et al., 1967). We conclude that the marmoset iPSC clones described here are pluripotent and fully reprogrammed. While retroviral silencing is a feature of full reprogramming, it is also important for suppressing the potential adverse consequences of transgene expression, particularly c-Myc (Nakagawa et al., 2008). While retrovirally generated iPSCs are highly valuable for basic and translational experiments in animals, for clinical use it is likely that reprogramming methods will be employed in which
the reprogramming vectors are excised from the cells before therapeutic use (Soldner et al., 2009; Chang et al., 2009) or methods that do not employ genetic modification at all (Zhou et al., 2009; Kim et al., 2009). Nevertheless, the relative efficiency of reprogramming using retroviral vectors makes this method attractive for solving basic issues. Once these are solved, safer methods can and should be developed, and compared with methods that use genetic modification. The presently reported characterization of iPSCs from the marmoset provides the proof of principle of iPSC generation in this species, and paves the way for the generation of indvidual-specific iPSCs for proof of principle experiments on autologous cell therapy in nonhuman primates.
Acknowledgments This work was supported by grants from the National Institute on Aging, from the Owens Medical Research Foundation, and from the Glenn Foundation for Medical Research.
References Amit, M., Itskovitz-Eldor, J., 2002. Derivation and spontaneous differentiation of human embryonic stem cells. J. Anat. 200, 225–232. Bajpai, R., Lesperance, J., Kim, M., Terskikh, A.V., 2008. Efficient propagation of single cells Accutase-dissociated human embryonic stem cells. Mol. Reprod. Dev. 75, 818–827. Chan, 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., Daley, G.Q.,
188 Schlaeger, T.M., 2009. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27, 1033–1037. Chang, C.W., Lai, Y.S., Pawlik, K.M., Liu, K., Sun, C.W., Li, C., Schoeb, T.R., Townes, T.M., 2009. Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells. Stem Cells 27, 1042–1049. Ezashi, T., Telugu, B.P., Alexenko, A.P., Sachdev, S., Sinha, S., Roberts, R.M., 2009. Derivation of induced pluripotent stem cells from pig somatic cells. Proc. Natl Acad. Sci. U. S. A. 106, 10993–10998. Hasegawa, K., Fujioka, T., Nakamura, Y., Nakatsuji, N., Suemori, H., 2006. A method for the selection of human embryonic stem cell sublines with high replating efficiency after single-cell dissociation. Stem Cells 24, 2649–2660. Hentze, H., Soong, P.L., Wang, S.T., Phillips, B.W., Putti, T.C., Dunn, N.R., 2009. Teratoma formation by human embryonic stem cells: Evaluation of essential parameters for future safety studies. Stem Cell Res. 2, 198–210. Huangfu, D., Osafune, K., Maehr, R., Guo, W., Eijkelenboom, A., Chen, S., Muhlestein, W., Melton, D.A., 2008. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat. Biotechnol. 26, 1269–1275. Kim, D., Kim, C.H., Moon, J.I., Chung, Y.G., Chang, M.Y., Han, B.S., Ko, S., Yang, E., Cha, K.Y., Lanza, R., Kim, K.S., 2009. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4, 472–476. Li, X., Krawetz, R., Liu, S., Meng, G., Rancourt, D.E., 2009. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum. Reprod. 24, 580–589. Liao, J., Cui, C., Chen, S., Ren, J., Chen, J., Gao, Y., Li, H., Jia, N., Cheng, L., Xiao, H., Xiao, L., 2009. Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 4, 11–15. Liu, H., Zhu, F., Yong, J., Zhang, P., Hou, P., Li, H., Jiang, W., Cai, J., Liu, M., Cui, K., Qu, X., Xiang, T., Lu, D., Chi, X., Gao, G., Ji, W., Ding, M., Deng, H., 2008. Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell 3, 587–590. Mansfield, K., 2003. Marmoset models commonly used in biomedical research. Comp. Med. 53, 383–392. Miraglia, T., Ledoux, L., Branco, A.L., 1967. Histological and histochemical data on the intestinal tract of the marmoset (Callithrix jacchus). Acta Anat. (Basel) 68, 459–472. Muller, T., Fleischmann, G., Eildermann, K., Matz-Rensing, K., Horn, P.A., Sasaki, E., Behr, R., 2009. A novel embryonic stem cell line derived from the common marmoset monkey (Callithrix jacchus) exhibiting germ cell-like characteristics. Hum. Reprod. 24, 1359–1372. Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Okita, K., Mochiduki, Y., Takizawa, N., Yamanaka, S., 2008. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol. 26, 101–106. O'Connor, M.D., Kardel, M.D., Iosfina, I., Youssef, D., Lu, M., Li, M.M., Vercauteren, S., Nagy, A., Eaves, C.J., 2008. Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells. Stem Cells 26, 1109–1116. Oka, S., Honmou, O., Akiyama, Y., Sasaki, M., Houkin, K., Hashi, K., Kocsis, J.D., 2004. Autologous transplantation of expanded neural precursor cells into the demyelinated monkey spinal cord. Brain Res. 1030, 94–102. Prokhorova, T.A., Harkness, L.M., Frandsen, U., Ditzel, N., Burns, J.S., Schroeder, H.D., Kassem, M., 2008. Teratoma formation by human
Y. Wu et al. embryonic stem cells is site-dependent and enhanced by the presence of Matrigel. Stem Cells Dev. 200, 225–232. Przyborski, S.A., 2005. Differentiation of human embryonic stem cells after transplantation in immune-deficient mice. Stem Cells 23, 1242–1250. Sasaki, E., Hanazawa, K., Kurita, R., Akatsuka, A., Yoshizaki, T., Ishii, H., Tanioka, Y., Ohnishi, Y., Suemizu, H., Sugawara, A., Tamaoki, N., Izawa, K., Nakazaki, Y., Hamada, H., Suemori, H., Asano, S., Nakatsuji, N., Okano, H., Tani, K., 2005. Establishment of novel embryonic stem cell lines derived from the common marmoset (Callithrix jacchus). Stem Cells 23, 1304–1313. Shimada, H., Nakada, A., Hashimoto, Y., Shigeno, K., Shionoya, Y., Nakamura, T., 2009. Generation of canine induced pluripotent stem cells by retroviral transduction and chemical inhibitors. Mol. Reprod. Dev. 77, 2. Soldner, F., Hockemeyer, D., Beard, C., Gao, Q., Bell, G.W., Cook, E.G., Hargus, G., Blak, A., Cooper, O., Mitalipova, M., Isacson, O., Jaenisch, R., 2009. Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964–977. Sun, B., Huang, Q., Liu, S., Chen, M., Hawks, C.L., Wang, L., Zhang, C., Hornsby, P.J., 2004. Progressive loss of malignant behavior in telomerase-negative tumorigenic adrenocortical cells and restoration of tumorigenicity by human telomerase reverse transcriptase. Cancer Res. 64, 6144–6151. Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S., 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872. Thomson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, C.P., Hearn, J.P., 1996. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol. Reprod. 55, 254–259. Tomioka, I., Sasaki, E., 2008. Recent progress in reproductive technologies based on the common marmoset (Callithrix jacchus). J. Mamm. Ova Res. 25, 143–149. Virley, D., Ridley, R.M., Sinden, J.D., Kershaw, T.R., Harland, S., Rashid, T., French, S., Sowinski, P., Gray, J.A., Lantos, P.L., Hodges, H., 1999. Primary CA1 and conditionally immortal MHP36 cell grafts restore conditional discrimination learning and recall in marmosets after excitotoxic lesions of the hippocampal CA1 field. Brain 122, 2321–2335. Wolf, D.P., Kuo, H.C., Pau, K.Y., Lester, L., 2004. Progress with nonhuman primate embryonic stem cells. Biol. Reprod. 71, 1766–1771. Wu, Y., Melton, D.W., Zhang, Y., Hornsby, P.J., 2009. Improved coinfection with amphotropic pseudotyped retroviral vectors. J. Biomed. Biotechnol. 2009, 901079. Yamanaka, S., 2007. Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 1, 39–49. Zhang, X., Neganova, I., Przyborski, S., Yang, C., Cooke, M., Atkinson, S.P., Anyfantis, G., Fenyk, S., Keith, W.N., Hoare, S.F., Hughes, O., Strachan, T., Stojkovic, M., Hinds, P.W., Armstrong, L., Lako, M., 2009. A role for NANOG in G1 to S transition in human embryonic stem cells through direct binding of CDK6 and CDC25A. J. Cell Biol. 184, 67–82. Zhou, H., Wu, S., Joo, J.Y., Zhu, S., Han, D.W., Lin, T., Trauger, S., Bien, G., Yao, S., Zhu, Y., Siuzdak, G., Scholer, H.R., Duan, L., Ding, S., 2009. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4, 381–384.