Short Communication Cell Biology International† 10.1002/cbin.10305 Induction of dedifferentiated male mouse adipose stromal vascular fraction cells to primordial germ cell-like cells (Inducing mouse SVF cells to PGC like-cells) ★
Guanghui Cui Zhengyu Qi, Yanmin Zhang, Xia Long, Jie Qin, Xin Guo Guangdong Key Laboratory of Male Reproductive Medicine and Genetics, Peking University Shenzhen Hospital, Medical Center of Peking University & Hong Kong Science and Technology University, Guangdong 518036, China ★
Correspondence: Dr. Guanghui Cui Tel: +86075583923333-3518 or 13823600241 Fax: +86075583061340 E-mail: [email protected] Address: Laboratory of Male Reproduction, Peking University Shenzhen Hospital, No.1120 Lianhua Street, Futian, Shenzhen, Guangdong 518036, China.
Keywords: dedifferentiation, embryonic stem cell, epiblast cell, primordial germ cell, stromal vascular fraction cell, imprint erasure Abbreviations: SVF stromal vascular fraction MSC mesenchymal stem cell ESCLC embryonic stem cell-like cell EpiLC epiblast-like cell PGCLC primordial germ cell-like cell
†
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/cbin.10305]
This article is protected by copyright. All rights reserved Received 29 September 2013; Revised 25 February 2014; Accepted 20 March 2014
Abstract The adipose stromal vascular fraction (SVF) contains abundant mesenchymal stem cell populations that have a limited ability to self-renew and differentiate. Male mouse adipose SVF cells were dedifferentiated by reprogramming factors (c-Myc, Oct4, Sox2 and Klf4) to form ESCLCs, which upgraded their limited differentiation potential. The ESCLCs were induced to differentiate towards epiblast-like cells (EpiLCs) and primordial germ cell-like cells (PGCLCs) by culturing in media supplied with activin A and BMP-4, respectively. The derived ESCLCs possess embryonic stem cell features and can automatically form embryonic bodies. After culture in EpiLC induction medium for 2-3 days, ESCLCs formed flattened epithelial structures that were different from their original water drop-like colonies, and the expression of pluripotency-related genes decreased. When the cells that had been cultured in EpiLC induction medium for 2 days were isolated and cultured in PGCLC induction medium for 4-6 days, they formed typical water drop-like colonies again. Moreover, expression of the pluripotency-related genes and the primordial germ cell specification-related genes increased. During progression from ESCLCs towards EpiLCs and PGCLCs, the levels of histone methylases H3K9me2 and H3K27me3 kept changing, which resembled those seen in primordial germ cell specification. The derived PGCLCs expressed SSEA-1, Blimp-1 and Stella. Furthermore, methylation of Igf2r and Snrpn was retained, but H19 and Kcnq1ot1 methylation levels were slightly reduced compared to non-PGCLCs, suggesting that the derived PGCLCs may have initiated the process of imprint erasure. 1. Introduction Stem cells are promising sources for developing regenerative therapeutics. However, many problems affect their clinical applications, such as availability, efficiency, safety and ethical issues. In addition to mature adipocytes, adipose tissue contains a relative abundance of progenitor and mesenchymal stem cell (MSC) populations in the stromal vascular fraction (SVF). Fat tissue has attracted increasing attention as a source of mesenchymal stem cells potentially beneficial for regenerative medicine (Zuk et al., 2002; Fraser et al., 2006; Zeve et al., 2009). MSCs from adipose tissue and bone marrow possess many common cell surface markers, although a precise definition of an MSC is not well established. As much as 1% of adipose cells are estimated to be MSCs, compared with the 0.001-0.002% found in bone marrow, which is currently a common source of stem cells. These adipose-residing stem cells have great potential for self-renewal, while maintaining the ability to become a limited repertoire of cell types such as adipocytes, myocytes, osteoblasts, and chondrocytes (Sugii et al., 2011). Because adipose tissue is abundant and relatively easy to obtain, it is a potential source for regenerative therapies The creation of embryonic stem cell-like cells (ESCLCs), or induced pluripotent stem cells (iPSCs), by reprogramming somatic cells dramatically increases the differentiation potential of somatic cells (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Huang et al., 2008; Shi et al., 2008). This technology enables not only the study of cell dedifferentiation and differentiation, but the development of patient-specific cells for disease models and regenerative therapeutics. Unlike embryonic stem cells, which are isolated from the blastocysts of an embryo, ESCLCs can be derived from adult somatic cells, thus avoiding many ethical concerns. Infertility affects 13-18% of couples and growing evidence from clinical and epidemiological studies suggests an increasing incidence of male reproductive problems. There is a male factor involved in up to half of all infertile couples. The pathogenesis of male infertility can be reflected
by defective spermatogenesis due to failure in germ cell proliferation and differentiation (Nayernia et al., 2004). Embryonic stem cells can be induced into germ cells (Toyooka et al., 2003; Geijsen et al., 2004; Nayernia et al., 2006). However, gametes derived from embryonic stem cell lines would be unrelated to the patient in need of fertility treatment. The creation of ESCLCs overcomes this issue as the cells are genetically related to the donor individual. Mouse and human adipose SVF cells highly express self-renewal supporting factors such as basic fibroblast growth factor (bFGF), vitronectin, fibronectin and leukemia inhibitory factor (LIF). Thus, these factors make reprogramming SVF cells over 5-fold and 100-fold more efficient than mouse and human fibroblasts, respectively, which are the most commonly used cell lines for iPSC generation (Sugii et al., 2011). Therefore, we sought to dedifferentiate mouse adipose SVF cells into ESCLCs by transfection of the 4 standard reprogramming factors (c-Myc, Klf4, Oct4, and Sox2), upgrading their limited pluripotency. The cells were induced to differentiate towards epiblast-like cells (EpiLC) and primordial germ cell-like cells (PGCLC) in vitro. 2. Materials and Methods 2.1 Animals The 10-week-old C57BL/6 mice purchased from the animal center of Zhongshan University [Guangzhou, Guangdong 510080, China] were housed at constant temperature (20C) and humidity (45%) on a 12-h light/dark cycle. They were fed ad libitum on standard laboratory chow and tap water. Chinese laws concerning the protection and control of experimental animals were strictly followed throughout all experiments. 2.2 Adipose stromal vascular fraction cell isolation Adipose stromal vascular fraction (SVF) cells were isolated from the epididymal fat pads of 10-week-old C57BL/6 mice by digestion at 37°C for 1 h with 1 mg/ml type IV collagenase [Sigma-Aldrich, St. Loius, MO 63103, USA] in Hank’s buffered salt solution containing 1% BSA [Sigma-Aldrich, St. Loius, MO 63103, USA], 200 nM adenosine [Sigma-Aldrich, St. Loius, MO 63103, USA], and 50 mg/ml glucose. After filtration through 250 μm nylon filters and centrifugation for 10 min at 400 ×g, the floating adipocytes were removed, and the pellet of SVF cells was washed three times. The collected cells were cultured in DMEM [Hyclon, Longan, Utah 84321, USA] containing 10% heat-inactivated FBS [Hyclon, Longan, Utah 84321, USA] and 5 ng/ml bFGF [CytoLab, Rehovot 76703, Israel]. Cells collected after 2-4 passages were used in future inductions (Sugii et al., 2011). 2.3 Retrovirus production and embryonic stem cell-like cell creation Mouse embryonic stem cell-like cells (ESCLCs) were created as previously described [4]. Briefly, pMX-GFP-based retroviral vectors harboring each of the mouse reprogramming genes(c-Myc, Klf4, Oct4, and Sox2) were transfected along with the VSV-G envelope gene into platinum E cells using TurboFectTM [Thermo Scientific, Waltham, MA 02454,USA]. Two days after transfection, the supernatant containing viruses was collected and filtered through a 0.45-μm filter. The SVF cells (after 2-4 passages) were infected with retrovirus mixtures in six-well plates (day 0). On day 2, one-fifth of the cells were passaged onto gelatin-coated plates without MEF feeder layers and cultured in 2i medium (Knockout DMEM [GIBCO，Carlsbad, CA 92008 USA],
0.1 mM NEAA, 1 mM sodiumpyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine, 100 × N2B27 [GIBCO，Carlsbad, CA 92008 USA], 1000 units/ml LIF [Invitrogen, Carlsbad, CA 92008,USA], 0.4 mM PD0325901 [Stemgent， Cambridge, MA 02138, USA], 3 mM CHIR99021[Stemgent，Cambridge, MA 02138, USA], and 15% KSR [Invitrogen，Carlsbad, CA 92008,USA]). The medium was changed every other day. On days 7-10, individual colonies were selected and amplified for storage and downstream analyses. 2.4 Pluripotency analysis of embryonic stem cell-like cells Selected embryonic stem cell-like cells (ESCLCs）were cultured and maintained feeder-free on a dish coated with 0.01% poly-L-ornithine and 10 ng/ml laminin [Sigma-Aldrich, St. Loius, MO 63103, USA] in 2i medium. The pluripotency-related gene expression, alkaline phosphatase expression, and the differentiation of three embryonic germ layers were tested. 2.5 Epiblast-like cell induction Epiblast-like cell (EpiLC) induction involved plating ESCLCs on a well of a 12-well plate coated with 16.7 mg/ml human plasma fibronectin [Sigma-Aldrich, St. Loius, MO 63103, USA] in EpiLC medium (Knockout DMEM, 0.1 mM NEAA, 1 mM sodiumpyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine, 20 ng/ml activin A [Peprotech, Rocky Hill, NJ 08553, USA], 12 ng/ml bFGF [Peprotech, Rocky Hill, NJ 08553, USA], and 1% KSR[Invitrogen, Carlsbad, CA 92008, USA]) for 1-3 days. The medium was changed every day (Hayashi et al., 2011). 2.6 Primordial germ cell-like cell induction Primordial germ cell-like cells (PGCLCs) were induced under floating conditions by plating day 2 EpiLCs in the wells of a low cell-binding U-bottom 96-well plate in PGCLC medium (GMEM [Invitrogen, Carlsbad, CA 92008,USA], 15% KSR, 0.1 mM NEAA, 1 mM sodiumpyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine， 500 ng/ml BMP4, 100 ng/ml SCF [R&D Systems], and 50 ng/ml EGF [R&D Systems, McKinley Place，MN 55413, USA] and 1000 u/ml LIF[Invitrogen, Carlsbad, CA 92008,USA]) for 2-6 days (Hayashi et al., 2011). 2.7 Pluripotency-related gene expression analysis RNA was extracted from ESCLCs, EpiLCs and PGCLCs using a RNA isolation kit and reverse transcribed to first-strand cDNA using a Super Script II kit [Thermo Scientific, Waltham, MA 02454, USA]. The samples were run in triplicate and the expression was normalized to the levels of the house keeping controls GAPDH. The samples were analyzed by qPCR, using SYBR Green dye [Takara, Dalian, Liaoning 116600, China]. 2.8 Western blotting analysis of histone methylases For western blotting analysis, ESCLCs, EpiLCs and PGCLCs were lysed in protein isolation solution, and the isolated proteins were subjected to 10% SDS-PAGE. After transfer to Hybond C nitrocellulose membranes, the blots were treated according to standard procedures. The blots were probed with rabbit anti-histone methylases H3K9me2, H3K27me3 and H3 antibodies [Santa Cruz
Biotech, Santa Cruz, CA 95060, USA] (1:100) for 3 h at room temperature. After a second 1 h incubation with anti-rabbit peroxidase-conjugated antibody [Keygene, Rockville, MD 20850, USA]. The blots were developed for the peroxidase reaction. 2.9 SSEA-1+ cell isolation After culture in PGCLC medium for 6 days, the immunomagnetic isolation of SSEA-1+ cells from the induced cells was done with a monoclonal antibody (IgM) against SSEA-1 [Santa Cruz Biotech, Santa Cruz, CA 95060, USA] and immunomagnetic rat anti-mouse IgM beads [Invitrogen，Carlsbad, CA 92008,USA]. 2.10 Immunohistochemistry analysis of cell surface markers Immuno-isolated SSEA-1+ cells were fixed and blocked for immunohistochemical detection. Primary antibodies (anti Blimp-1 and Stella, [Biolegend, San Diego, CA 92121, USA]) and secondary antibodies (biotin-coupled anti-mouse IgG and avidin-horseradish peroxidase conjugates [Boster, Wuhan, Hubei 430074, China]) were applied, respectively, following a standard immunohistochemistry procedure. 2.11 DNA isolation and bisulfite sequencing Genomic DNA was prepared from the immuno-isolated SSEA-1+ cells. The DNA was digested using Hind III, according to standard protocols and treated with sodium bisulfite using a DNA Methylation Kit [Thermo Scientific, Waltham, MA 02454, USA]. The aliquots of bisulfite-treated DNA solution were used for 2 rounds of PCR with nested primer pairs, except for Igf2r, which was semi-nested. The PCR products were subcloned into the pGEM-T vector and transformed into JM109 cells. Colonies were picked and the colony DNA was amplified. The DNA samples were sent to BGI Biotech Co. Ltd [Shenzhen,Guagndong 518083, China] for sequencing. Sequence characterization and processing were performed using a BiQ Analyzer 2.0 (Hayashi et al., 2011). 3. Results 3.1 Characteristics of the SVF cell derived embryonic stem cell-like cells The initial isolated SVF cells appeared as adherent spindle-shaped individual cells or small clusters. After 3 passages, most of the adherent cells exhibited large flat fibroblastic morphology. Four days after virus transfection, small embryonic stem cell-like colonies appeared among the cultured SVF cells. On days 7-10, representative cell colonies expressing GFP were selected and cultured for further tests. GFP expression in the selected ESCLCs gradually decreased after several passages, which is a typical phenomenon observed in retrovirus transfection-created iPSCs cells. The selected cells expressed pluripotency-related genes (Oct3, Sox2, Klf4, Nanog, and Nanos3), as well as alkaline phosphatase, and could form embryonic bodies (EBs) automatically in vitro. (Figures 1, 2) 3.2 Epiblast-like cell and primordial germ cell-like cell differentiation Upon stimulation with EpiLC medium (containing actin A, bFGF, and 1% KSR), the embryonic stem cell-like cells grew rapidly for the first 2 days, but thereafter underwent significant cell death, making it difficult to maintain the cells over 3 days. Expression of cell
pluripotency-related genes (Oct3, Sox2, Klf4, and Nanog) decreased compared with those of the ESCLCs. (Figure 3A) When day 2 EpiLCs were cultured with PGCLC medium (containing BMP4, LIF, SCF and EGF), robust cell proliferation was observed, however, the cells from day 1 and day 3 EpiLCs grew much slower compared with those from day 2 EpiLCs. In the induction period from EpiLCs to PGCLCs (2-6 days), the expression levels of pluripotency-related genes (Oct3, Sox2, Klf4, and Nanog) and primordial germ cell specification-related genes (Blimp-1 and Stella) of the induced cells were increased in comparison with those of the initial day 2 EpiLCs. (Figure 3B) 3.3 Cell growth model transformation and analysis of histone methylases during the process of induction After being induced by EpiLC medium for 2-3 days, the ESCLCs formed flattened epithelial structures, different from their original water drop-like cell colonies. When the cells that had been induced by the EpiLC medium for 2 days were isolated and cultured in PGCLC medium for 4-6 days, they formed typical water drop-like colonies again. In the differentiation from ESCLC to EpiLC and PGCLC, H3K9me2 levels decreased. Instead, in ESCLC to EpiLC differentiation, H3K27me3 level decreased, but in EpiLC to PGCLC differentiation, it slightly increased. (Fig 4) 3.4 Specific gene imprinting states analysis of the primordial germ cell-like cells Blimp-1, Stella and SSEA-1+ are typically regarded as the primordial germ cell specification markers. Therefore, we isolated SSEA-1+ cells from day 6 PGCLCs and found that these cells were Blimp-1 and Stella positive (Figure 5). The maternally (Snrpn and Kcnq1ot1) and paternally (Igf2r and H19) imprinted genes in the isolated cells were analyzed by bisulfite sequencing. In comparison with SVF cells and ESCLCs, PGCLCs retained methylation of Igf2r and Snrpn, but the methylation levels of H19 and Kcnq1ot1 were slightly reduced, suggesting that PGCLCs may have initiated the process of imprint erasure. (Figure 6) 4. Discussion Considerable optimism has been generated for the use of stem cells for the treatment of human diseases. Much of the interest centers on embryonic stem cells, but this approach remains controversial for ethical reasons. In contrast, mesenchymal stem cells from bone marrow and adipose tissue are well characterized and have long been used therapeutically (Lee et al., 2004; Cai et al., 2009; Gonzalez et al., 2009; Nakao et al., 2010). Bone marrow MSCs can differentiate into early germ cells, primordial germ cells (PGCs) and even spermatogonia in vitro and in vivo(; Nayernia et al., 2006; Lue et al., 2007), but no systematic induction has been used. The cells were either induced by retinoic acid alone or directly injected into recipient seminiferous tubules without and treatment. Although the induced cells expressed PGC and spermatogonia surface makers, in depth information regarding the specification of the PGCs has not been presented. In mammals, PGCs that originate from a population of pluripotent epiblast cells give rise to functional gametes. During development, the number of PGCs increases while migrating through the developing hindgut and mesentery to reach the urogenital ridge (UGR), and PGCs show different features at different developmental stages (Chuma et al., 2004; Ohat et al., 2004).
Mouse epiblast cells and iPSC-derived epiblast-like cells can differentiate into PGCs and then into spermatogonial stem cells (Chuma et al., 2004; Ohat et al., 2004). In the development of male gametes, epiblast cells and PGCs are key players in the entire process, and epiblast cells seemed to serve as the starting material for the induction of other lineages. As a result, we chose to dedifferentiate mouse adipose SVF cells into ES cell-like conditions. We induced the dedifferentiated cells into epiblast-like cells and PGC-like cells by a 2-step procedure, which mimicked the process of germ cell development. Adipose SVF cells could be efficiently dedifferentiated into ES cell-like cells. During the process from ESCLCs to EpiLCs and PGCLCs, the cell growth models altered, the pluripotency-related gene expression levels and histone methylase levels fluctuated, and the harvested PGCLCs expressed PGC specification markers. Epigenetic changes characteristic of germline cells also occurred in PGCs. The erasure of parental genomic imprints on both the paternal and maternal alleles in the PGCs commences near the time of their settlement in the UGR, and new imprints are imposed in pro-spermatogonia before birth (Lee et al., 2002; Lucifero et al., 2002; Chuma et al., 2004; Ohat et al., 2004). In our immuno-selected PGCLCs, methylation of Igf2r and Snrpn was retained, but the methylation levels of H19 and Kcnq1ot1 were slightly reduced. This reduction suggests that these PGCLCs have initiated the process of imprint erasure, which happens during the PGC migration stage. These dynamic changes are a recapitulation of what had been observed during PGC formation. Acknowledgments This work was supported by grants [81070530] and [81270748] from the China National Natural Science Foundation. References Cai, L., Johnstone, B.H., Cook, T.G., Tan, J., Fishbein, M.C., Chen, P.S., March, K.L. (2009). IFATS collection: human adipose tissue-derived stem cells induce angiogenesis and nerve sprouting following myocardial infarction, in conjunction with potent preservation of cardiac function. Stem Cells;27:230–237. Chuma, S., Shinohara, K., Inoue, K., Ogonuki, N., Miki, H., Toyokuni, S., Hosokawa, M., Nakatsuji, N., Ogura, A., Shinohara, T. (2004). Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis. Development;132;117-122. Fraser, JK., Wulur, I., Alfonso, Z., Hedrick, M.H. (2006). Fat tissue: An under appreciated source of stem cells for biotechnology. Trends Biotechnol;24:150-154. Geijsen, N., Horoschak, M., Kim, K., Gribnau, J., Eggan, K., Daley, G. (2004). Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature;427;148-157. Gonzalez, MA., Gonzalez-Rey, E., Rico, L., Buscher, D., Delgado, M. (2009). Treatment of experimental arthritis by inducing immune tolerance with human adipose-derived mesenchymal stem cells. Arthritis Rheum;60:1006–1019.
Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S., Saitou, M. (2011). Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell;146:1-4. Huang, F.D., Osafune, K., Maehr, R. (2008). Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat Biotechnol; 26(11):1269-1275. Lee, J., Inoue, K., Ono, R., Ogonuki, N., Kohda, T., Kaneko-Ishino, T., Ogura. A., Ishino, F. (2002). Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells. Development;129:1807-1817. Lee, RH, Kim, B., Choi, I., Kim, H., Choi, H.S., Suh, K., Bae, Y.C., Jun, J.S. (2004). Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adiposetissue. Cell Physiol Biochem;14:311-324. Lucifero, F., Mertineit, C., Clarke, H.J., Bestor, T.H., Trasler, J.M. (2002). Methylation dynamics of imprinted genes in mouse germ cells. Genomics; 79(4);530-8. Lue, Y., Erkkila, K., Liu, Y.P., Ma, K., Wang, C., Hikim, A.S., Swerdloff, R.S. (2007). Fate of bone marrow stem cells transplanted into the testis: potential implication for men with testicular failure. Am J Pathol;170(3):899-908. Nakao, N., Nakayama, T., Yahata, T., Muguruma, Y., Saito, S., Miyata, Y., Yamamoto, K., Naoe, T. (2010). Adipose tissue-derived mesenchymal stem cells facilitate hematopoiesis in Vitro and in Vivo. Am J Pathol;177(8):1-8. Nayernia, K., Li, M., Jaroszynski, L., Khusainov, R., Wulf, G., Schwandt, I., Korabiowska, M., Michelmann, H.W., Meinhardt, A., Engel, W. (2004). Stem cell based therapeutical approach of male infertility by teratocarcinoma derived germ cells. Hum Mol Genet;13(14):1451-60. Nayernia, K., Lee, J.H., Drusenheimer, N., Nolte, J., Wulf, G., Dressel, R., Gromoll, J., Engel, W. (2006). Derivation of male germ cells from bone marrow stem cells. Laboratory Investigation;86: 654–663. Nayernia, K., Nolte, J., Michelmann, H.W., Lee, J.H., Rathsack, K., Drusenheimer, N., Dev, A., Wulf, G., Ehrmann, I.E., Elliott, D.J., Okpanyi, V., Zechner, U., Haaf, T., Meinhardt, A., Engel, W. (2006). In Vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Developmental Cell; 11:125–132. Ohat, H., Wakayama, T., Nishimune, Y. (2004). Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development. Biol Reprod;70(5):1286-1291. Shi, Y., Desponts, C., Jeong, T.D., Do, J.T., Hahm, H.S., Schöler, H.R., Ding, S. (2008). Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with
small-molecule compounds. Cell Stem Cell;3(5):568-574. Sugii,S., Kida, Y., Berggren, WT., Evans, RM. (2011). Feeder-dependent and feeder-independent iPS cell derivation from human and mouse adipose stem cells. Nature Protocols;6:346-358. Takahashi, K., Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell;126 (4): 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 (5): 861–872. Toyooka, Y., Tsunekawa, N., Aksu, R.，Noce, T. (2003). Embryonic stem cells can form germ cells in Vitro. Pro Natl Acad Sci. USA;100:11457-11462. Zeve, D., Tang, W., Graff, J. (2009). Fighting fat with fat: The expanding field of adipose stem cells. Cell Stem Cell;5: 472-481. Zuk, P.A., Zhu, M., Ashjian, P.D., Ugarte, D.A., Huang, J.I., Mizuno, H., Alfonso, Z.C., Fraser, J.K., Benhaim, P., Hedrick, M.H. (2002). Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell;13:4279-4295.
Legends
Figure 1. Pluripotency-related genes expression of ESCLCs. 1. Marker 2. Oct4 3. Sox2 3.Nanog 4. Klf4 5. Prdm14
6. Nanos3
7. GAPDH
Figure 2. Characteristics of SVF cell-derived ESCLCs A. Adherent SVF cells B. ESCLCs express GFP C. ESCLCs express alkaline phosphatase D. Embryonic body formation of ESCLCs (200×) Figure 3A. Expression levels of cell pluripotency-related genes in the progression from ESCLC to EpiLC. Figure 3B. Expression levels of cell pluripotency-related genes and PGC specification-related genes in the progression from EpiLC to PGCLC. Figure 4. Cell growth model transformation and western blotting analysis of histone methylases in the progression from ESCLC to EpiLC and PGCLC.
A. Growth model of ESCLCs B. Growth model of EpiLCs C. Growth model of PGCLCs (200×) Figure 5. Immunohistochemistry detection of primodium germ cell specification markers A. Negative control B. Blimp-1 expression C. Stella expression (200×) Figure 6. Specific gene imprinting state analysis of SVF cells (SVFCs), ESCLCs and PGCLCs.
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Guanghui Cui Zhengyu Qi, Yanmin Zhang, Xia Long, Jie Qin, Xin Guo Guangdong Key Laboratory of Male Reproductive Medicine and Genetics, Peking University Shenzhen Hospital, Medical Center of Peking University & Hong Kong Science and Technology University, Guangdong 518036, China ★
Correspondence: Dr. Guanghui Cui Tel: +86075583923333-3518 or 13823600241 Fax: +86075583061340 E-mail: [email protected] Address: Laboratory of Male Reproduction, Peking University Shenzhen Hospital, No.1120 Lianhua Street, Futian, Shenzhen, Guangdong 518036, China.
Keywords: dedifferentiation, embryonic stem cell, epiblast cell, primordial germ cell, stromal vascular fraction cell, imprint erasure Abbreviations: SVF stromal vascular fraction MSC mesenchymal stem cell ESCLC embryonic stem cell-like cell EpiLC epiblast-like cell PGCLC primordial germ cell-like cell
†
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/cbin.10305]
This article is protected by copyright. All rights reserved Received 29 September 2013; Revised 25 February 2014; Accepted 20 March 2014
Abstract The adipose stromal vascular fraction (SVF) contains abundant mesenchymal stem cell populations that have a limited ability to self-renew and differentiate. Male mouse adipose SVF cells were dedifferentiated by reprogramming factors (c-Myc, Oct4, Sox2 and Klf4) to form ESCLCs, which upgraded their limited differentiation potential. The ESCLCs were induced to differentiate towards epiblast-like cells (EpiLCs) and primordial germ cell-like cells (PGCLCs) by culturing in media supplied with activin A and BMP-4, respectively. The derived ESCLCs possess embryonic stem cell features and can automatically form embryonic bodies. After culture in EpiLC induction medium for 2-3 days, ESCLCs formed flattened epithelial structures that were different from their original water drop-like colonies, and the expression of pluripotency-related genes decreased. When the cells that had been cultured in EpiLC induction medium for 2 days were isolated and cultured in PGCLC induction medium for 4-6 days, they formed typical water drop-like colonies again. Moreover, expression of the pluripotency-related genes and the primordial germ cell specification-related genes increased. During progression from ESCLCs towards EpiLCs and PGCLCs, the levels of histone methylases H3K9me2 and H3K27me3 kept changing, which resembled those seen in primordial germ cell specification. The derived PGCLCs expressed SSEA-1, Blimp-1 and Stella. Furthermore, methylation of Igf2r and Snrpn was retained, but H19 and Kcnq1ot1 methylation levels were slightly reduced compared to non-PGCLCs, suggesting that the derived PGCLCs may have initiated the process of imprint erasure. 1. Introduction Stem cells are promising sources for developing regenerative therapeutics. However, many problems affect their clinical applications, such as availability, efficiency, safety and ethical issues. In addition to mature adipocytes, adipose tissue contains a relative abundance of progenitor and mesenchymal stem cell (MSC) populations in the stromal vascular fraction (SVF). Fat tissue has attracted increasing attention as a source of mesenchymal stem cells potentially beneficial for regenerative medicine (Zuk et al., 2002; Fraser et al., 2006; Zeve et al., 2009). MSCs from adipose tissue and bone marrow possess many common cell surface markers, although a precise definition of an MSC is not well established. As much as 1% of adipose cells are estimated to be MSCs, compared with the 0.001-0.002% found in bone marrow, which is currently a common source of stem cells. These adipose-residing stem cells have great potential for self-renewal, while maintaining the ability to become a limited repertoire of cell types such as adipocytes, myocytes, osteoblasts, and chondrocytes (Sugii et al., 2011). Because adipose tissue is abundant and relatively easy to obtain, it is a potential source for regenerative therapies The creation of embryonic stem cell-like cells (ESCLCs), or induced pluripotent stem cells (iPSCs), by reprogramming somatic cells dramatically increases the differentiation potential of somatic cells (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Huang et al., 2008; Shi et al., 2008). This technology enables not only the study of cell dedifferentiation and differentiation, but the development of patient-specific cells for disease models and regenerative therapeutics. Unlike embryonic stem cells, which are isolated from the blastocysts of an embryo, ESCLCs can be derived from adult somatic cells, thus avoiding many ethical concerns. Infertility affects 13-18% of couples and growing evidence from clinical and epidemiological studies suggests an increasing incidence of male reproductive problems. There is a male factor involved in up to half of all infertile couples. The pathogenesis of male infertility can be reflected
by defective spermatogenesis due to failure in germ cell proliferation and differentiation (Nayernia et al., 2004). Embryonic stem cells can be induced into germ cells (Toyooka et al., 2003; Geijsen et al., 2004; Nayernia et al., 2006). However, gametes derived from embryonic stem cell lines would be unrelated to the patient in need of fertility treatment. The creation of ESCLCs overcomes this issue as the cells are genetically related to the donor individual. Mouse and human adipose SVF cells highly express self-renewal supporting factors such as basic fibroblast growth factor (bFGF), vitronectin, fibronectin and leukemia inhibitory factor (LIF). Thus, these factors make reprogramming SVF cells over 5-fold and 100-fold more efficient than mouse and human fibroblasts, respectively, which are the most commonly used cell lines for iPSC generation (Sugii et al., 2011). Therefore, we sought to dedifferentiate mouse adipose SVF cells into ESCLCs by transfection of the 4 standard reprogramming factors (c-Myc, Klf4, Oct4, and Sox2), upgrading their limited pluripotency. The cells were induced to differentiate towards epiblast-like cells (EpiLC) and primordial germ cell-like cells (PGCLC) in vitro. 2. Materials and Methods 2.1 Animals The 10-week-old C57BL/6 mice purchased from the animal center of Zhongshan University [Guangzhou, Guangdong 510080, China] were housed at constant temperature (20C) and humidity (45%) on a 12-h light/dark cycle. They were fed ad libitum on standard laboratory chow and tap water. Chinese laws concerning the protection and control of experimental animals were strictly followed throughout all experiments. 2.2 Adipose stromal vascular fraction cell isolation Adipose stromal vascular fraction (SVF) cells were isolated from the epididymal fat pads of 10-week-old C57BL/6 mice by digestion at 37°C for 1 h with 1 mg/ml type IV collagenase [Sigma-Aldrich, St. Loius, MO 63103, USA] in Hank’s buffered salt solution containing 1% BSA [Sigma-Aldrich, St. Loius, MO 63103, USA], 200 nM adenosine [Sigma-Aldrich, St. Loius, MO 63103, USA], and 50 mg/ml glucose. After filtration through 250 μm nylon filters and centrifugation for 10 min at 400 ×g, the floating adipocytes were removed, and the pellet of SVF cells was washed three times. The collected cells were cultured in DMEM [Hyclon, Longan, Utah 84321, USA] containing 10% heat-inactivated FBS [Hyclon, Longan, Utah 84321, USA] and 5 ng/ml bFGF [CytoLab, Rehovot 76703, Israel]. Cells collected after 2-4 passages were used in future inductions (Sugii et al., 2011). 2.3 Retrovirus production and embryonic stem cell-like cell creation Mouse embryonic stem cell-like cells (ESCLCs) were created as previously described [4]. Briefly, pMX-GFP-based retroviral vectors harboring each of the mouse reprogramming genes(c-Myc, Klf4, Oct4, and Sox2) were transfected along with the VSV-G envelope gene into platinum E cells using TurboFectTM [Thermo Scientific, Waltham, MA 02454,USA]. Two days after transfection, the supernatant containing viruses was collected and filtered through a 0.45-μm filter. The SVF cells (after 2-4 passages) were infected with retrovirus mixtures in six-well plates (day 0). On day 2, one-fifth of the cells were passaged onto gelatin-coated plates without MEF feeder layers and cultured in 2i medium (Knockout DMEM [GIBCO，Carlsbad, CA 92008 USA],
0.1 mM NEAA, 1 mM sodiumpyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine, 100 × N2B27 [GIBCO，Carlsbad, CA 92008 USA], 1000 units/ml LIF [Invitrogen, Carlsbad, CA 92008,USA], 0.4 mM PD0325901 [Stemgent， Cambridge, MA 02138, USA], 3 mM CHIR99021[Stemgent，Cambridge, MA 02138, USA], and 15% KSR [Invitrogen，Carlsbad, CA 92008,USA]). The medium was changed every other day. On days 7-10, individual colonies were selected and amplified for storage and downstream analyses. 2.4 Pluripotency analysis of embryonic stem cell-like cells Selected embryonic stem cell-like cells (ESCLCs）were cultured and maintained feeder-free on a dish coated with 0.01% poly-L-ornithine and 10 ng/ml laminin [Sigma-Aldrich, St. Loius, MO 63103, USA] in 2i medium. The pluripotency-related gene expression, alkaline phosphatase expression, and the differentiation of three embryonic germ layers were tested. 2.5 Epiblast-like cell induction Epiblast-like cell (EpiLC) induction involved plating ESCLCs on a well of a 12-well plate coated with 16.7 mg/ml human plasma fibronectin [Sigma-Aldrich, St. Loius, MO 63103, USA] in EpiLC medium (Knockout DMEM, 0.1 mM NEAA, 1 mM sodiumpyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine, 20 ng/ml activin A [Peprotech, Rocky Hill, NJ 08553, USA], 12 ng/ml bFGF [Peprotech, Rocky Hill, NJ 08553, USA], and 1% KSR[Invitrogen, Carlsbad, CA 92008, USA]) for 1-3 days. The medium was changed every day (Hayashi et al., 2011). 2.6 Primordial germ cell-like cell induction Primordial germ cell-like cells (PGCLCs) were induced under floating conditions by plating day 2 EpiLCs in the wells of a low cell-binding U-bottom 96-well plate in PGCLC medium (GMEM [Invitrogen, Carlsbad, CA 92008,USA], 15% KSR, 0.1 mM NEAA, 1 mM sodiumpyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine， 500 ng/ml BMP4, 100 ng/ml SCF [R&D Systems], and 50 ng/ml EGF [R&D Systems, McKinley Place，MN 55413, USA] and 1000 u/ml LIF[Invitrogen, Carlsbad, CA 92008,USA]) for 2-6 days (Hayashi et al., 2011). 2.7 Pluripotency-related gene expression analysis RNA was extracted from ESCLCs, EpiLCs and PGCLCs using a RNA isolation kit and reverse transcribed to first-strand cDNA using a Super Script II kit [Thermo Scientific, Waltham, MA 02454, USA]. The samples were run in triplicate and the expression was normalized to the levels of the house keeping controls GAPDH. The samples were analyzed by qPCR, using SYBR Green dye [Takara, Dalian, Liaoning 116600, China]. 2.8 Western blotting analysis of histone methylases For western blotting analysis, ESCLCs, EpiLCs and PGCLCs were lysed in protein isolation solution, and the isolated proteins were subjected to 10% SDS-PAGE. After transfer to Hybond C nitrocellulose membranes, the blots were treated according to standard procedures. The blots were probed with rabbit anti-histone methylases H3K9me2, H3K27me3 and H3 antibodies [Santa Cruz
Biotech, Santa Cruz, CA 95060, USA] (1:100) for 3 h at room temperature. After a second 1 h incubation with anti-rabbit peroxidase-conjugated antibody [Keygene, Rockville, MD 20850, USA]. The blots were developed for the peroxidase reaction. 2.9 SSEA-1+ cell isolation After culture in PGCLC medium for 6 days, the immunomagnetic isolation of SSEA-1+ cells from the induced cells was done with a monoclonal antibody (IgM) against SSEA-1 [Santa Cruz Biotech, Santa Cruz, CA 95060, USA] and immunomagnetic rat anti-mouse IgM beads [Invitrogen，Carlsbad, CA 92008,USA]. 2.10 Immunohistochemistry analysis of cell surface markers Immuno-isolated SSEA-1+ cells were fixed and blocked for immunohistochemical detection. Primary antibodies (anti Blimp-1 and Stella, [Biolegend, San Diego, CA 92121, USA]) and secondary antibodies (biotin-coupled anti-mouse IgG and avidin-horseradish peroxidase conjugates [Boster, Wuhan, Hubei 430074, China]) were applied, respectively, following a standard immunohistochemistry procedure. 2.11 DNA isolation and bisulfite sequencing Genomic DNA was prepared from the immuno-isolated SSEA-1+ cells. The DNA was digested using Hind III, according to standard protocols and treated with sodium bisulfite using a DNA Methylation Kit [Thermo Scientific, Waltham, MA 02454, USA]. The aliquots of bisulfite-treated DNA solution were used for 2 rounds of PCR with nested primer pairs, except for Igf2r, which was semi-nested. The PCR products were subcloned into the pGEM-T vector and transformed into JM109 cells. Colonies were picked and the colony DNA was amplified. The DNA samples were sent to BGI Biotech Co. Ltd [Shenzhen,Guagndong 518083, China] for sequencing. Sequence characterization and processing were performed using a BiQ Analyzer 2.0 (Hayashi et al., 2011). 3. Results 3.1 Characteristics of the SVF cell derived embryonic stem cell-like cells The initial isolated SVF cells appeared as adherent spindle-shaped individual cells or small clusters. After 3 passages, most of the adherent cells exhibited large flat fibroblastic morphology. Four days after virus transfection, small embryonic stem cell-like colonies appeared among the cultured SVF cells. On days 7-10, representative cell colonies expressing GFP were selected and cultured for further tests. GFP expression in the selected ESCLCs gradually decreased after several passages, which is a typical phenomenon observed in retrovirus transfection-created iPSCs cells. The selected cells expressed pluripotency-related genes (Oct3, Sox2, Klf4, Nanog, and Nanos3), as well as alkaline phosphatase, and could form embryonic bodies (EBs) automatically in vitro. (Figures 1, 2) 3.2 Epiblast-like cell and primordial germ cell-like cell differentiation Upon stimulation with EpiLC medium (containing actin A, bFGF, and 1% KSR), the embryonic stem cell-like cells grew rapidly for the first 2 days, but thereafter underwent significant cell death, making it difficult to maintain the cells over 3 days. Expression of cell
pluripotency-related genes (Oct3, Sox2, Klf4, and Nanog) decreased compared with those of the ESCLCs. (Figure 3A) When day 2 EpiLCs were cultured with PGCLC medium (containing BMP4, LIF, SCF and EGF), robust cell proliferation was observed, however, the cells from day 1 and day 3 EpiLCs grew much slower compared with those from day 2 EpiLCs. In the induction period from EpiLCs to PGCLCs (2-6 days), the expression levels of pluripotency-related genes (Oct3, Sox2, Klf4, and Nanog) and primordial germ cell specification-related genes (Blimp-1 and Stella) of the induced cells were increased in comparison with those of the initial day 2 EpiLCs. (Figure 3B) 3.3 Cell growth model transformation and analysis of histone methylases during the process of induction After being induced by EpiLC medium for 2-3 days, the ESCLCs formed flattened epithelial structures, different from their original water drop-like cell colonies. When the cells that had been induced by the EpiLC medium for 2 days were isolated and cultured in PGCLC medium for 4-6 days, they formed typical water drop-like colonies again. In the differentiation from ESCLC to EpiLC and PGCLC, H3K9me2 levels decreased. Instead, in ESCLC to EpiLC differentiation, H3K27me3 level decreased, but in EpiLC to PGCLC differentiation, it slightly increased. (Fig 4) 3.4 Specific gene imprinting states analysis of the primordial germ cell-like cells Blimp-1, Stella and SSEA-1+ are typically regarded as the primordial germ cell specification markers. Therefore, we isolated SSEA-1+ cells from day 6 PGCLCs and found that these cells were Blimp-1 and Stella positive (Figure 5). The maternally (Snrpn and Kcnq1ot1) and paternally (Igf2r and H19) imprinted genes in the isolated cells were analyzed by bisulfite sequencing. In comparison with SVF cells and ESCLCs, PGCLCs retained methylation of Igf2r and Snrpn, but the methylation levels of H19 and Kcnq1ot1 were slightly reduced, suggesting that PGCLCs may have initiated the process of imprint erasure. (Figure 6) 4. Discussion Considerable optimism has been generated for the use of stem cells for the treatment of human diseases. Much of the interest centers on embryonic stem cells, but this approach remains controversial for ethical reasons. In contrast, mesenchymal stem cells from bone marrow and adipose tissue are well characterized and have long been used therapeutically (Lee et al., 2004; Cai et al., 2009; Gonzalez et al., 2009; Nakao et al., 2010). Bone marrow MSCs can differentiate into early germ cells, primordial germ cells (PGCs) and even spermatogonia in vitro and in vivo(; Nayernia et al., 2006; Lue et al., 2007), but no systematic induction has been used. The cells were either induced by retinoic acid alone or directly injected into recipient seminiferous tubules without and treatment. Although the induced cells expressed PGC and spermatogonia surface makers, in depth information regarding the specification of the PGCs has not been presented. In mammals, PGCs that originate from a population of pluripotent epiblast cells give rise to functional gametes. During development, the number of PGCs increases while migrating through the developing hindgut and mesentery to reach the urogenital ridge (UGR), and PGCs show different features at different developmental stages (Chuma et al., 2004; Ohat et al., 2004).
Mouse epiblast cells and iPSC-derived epiblast-like cells can differentiate into PGCs and then into spermatogonial stem cells (Chuma et al., 2004; Ohat et al., 2004). In the development of male gametes, epiblast cells and PGCs are key players in the entire process, and epiblast cells seemed to serve as the starting material for the induction of other lineages. As a result, we chose to dedifferentiate mouse adipose SVF cells into ES cell-like conditions. We induced the dedifferentiated cells into epiblast-like cells and PGC-like cells by a 2-step procedure, which mimicked the process of germ cell development. Adipose SVF cells could be efficiently dedifferentiated into ES cell-like cells. During the process from ESCLCs to EpiLCs and PGCLCs, the cell growth models altered, the pluripotency-related gene expression levels and histone methylase levels fluctuated, and the harvested PGCLCs expressed PGC specification markers. Epigenetic changes characteristic of germline cells also occurred in PGCs. The erasure of parental genomic imprints on both the paternal and maternal alleles in the PGCs commences near the time of their settlement in the UGR, and new imprints are imposed in pro-spermatogonia before birth (Lee et al., 2002; Lucifero et al., 2002; Chuma et al., 2004; Ohat et al., 2004). In our immuno-selected PGCLCs, methylation of Igf2r and Snrpn was retained, but the methylation levels of H19 and Kcnq1ot1 were slightly reduced. This reduction suggests that these PGCLCs have initiated the process of imprint erasure, which happens during the PGC migration stage. These dynamic changes are a recapitulation of what had been observed during PGC formation. Acknowledgments This work was supported by grants [81070530] and [81270748] from the China National Natural Science Foundation. References Cai, L., Johnstone, B.H., Cook, T.G., Tan, J., Fishbein, M.C., Chen, P.S., March, K.L. (2009). IFATS collection: human adipose tissue-derived stem cells induce angiogenesis and nerve sprouting following myocardial infarction, in conjunction with potent preservation of cardiac function. Stem Cells;27:230–237. Chuma, S., Shinohara, K., Inoue, K., Ogonuki, N., Miki, H., Toyokuni, S., Hosokawa, M., Nakatsuji, N., Ogura, A., Shinohara, T. (2004). Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis. Development;132;117-122. Fraser, JK., Wulur, I., Alfonso, Z., Hedrick, M.H. (2006). Fat tissue: An under appreciated source of stem cells for biotechnology. Trends Biotechnol;24:150-154. Geijsen, N., Horoschak, M., Kim, K., Gribnau, J., Eggan, K., Daley, G. (2004). Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature;427;148-157. Gonzalez, MA., Gonzalez-Rey, E., Rico, L., Buscher, D., Delgado, M. (2009). Treatment of experimental arthritis by inducing immune tolerance with human adipose-derived mesenchymal stem cells. Arthritis Rheum;60:1006–1019.
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Legends
Figure 1. Pluripotency-related genes expression of ESCLCs. 1. Marker 2. Oct4 3. Sox2 3.Nanog 4. Klf4 5. Prdm14
6. Nanos3
7. GAPDH
Figure 2. Characteristics of SVF cell-derived ESCLCs A. Adherent SVF cells B. ESCLCs express GFP C. ESCLCs express alkaline phosphatase D. Embryonic body formation of ESCLCs (200×) Figure 3A. Expression levels of cell pluripotency-related genes in the progression from ESCLC to EpiLC. Figure 3B. Expression levels of cell pluripotency-related genes and PGC specification-related genes in the progression from EpiLC to PGCLC. Figure 4. Cell growth model transformation and western blotting analysis of histone methylases in the progression from ESCLC to EpiLC and PGCLC.
A. Growth model of ESCLCs B. Growth model of EpiLCs C. Growth model of PGCLCs (200×) Figure 5. Immunohistochemistry detection of primodium germ cell specification markers A. Negative control B. Blimp-1 expression C. Stella expression (200×) Figure 6. Specific gene imprinting state analysis of SVF cells (SVFCs), ESCLCs and PGCLCs.
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