- Email: [email protected]
Rat bone marrow derived mesenchymal progenitor cells support mouse ES cell growth and germ-like cell differentiation
Cell Biology International 33 (2009) 434e441 www.elsevier.com/locate/cellbi
Short communication
Rat bone marrow derived mesenchymal progenitor cells support mouse ES cell growth and germ-like cell differentiation Guanghui Cui, Zhengyu Qi, Xin Guo, Jie Qin, Yaoting Gui, Zhiming Cai* Laboratory of Male Reproduction of Peking University Shenzhen Hospital, Medical Center of Peking University & Hong Kong Science and Technology University, Guangdong 518036, China Received 10 August 2008; revised 4 November 2008; accepted 9 January 2009
Abstract Mouse embryonic fibroblasts (MEFs) have been used as feeder cells to support the growth of mouse embryonic stem cell (mESC) and primordial germ cells (PGC) in culture for many years. However, MEF preparation is a complex and tedious task. Recently, there are reports indicating that the microenvironment provided by bone marrow stromal cells could support the survival of embryonic-like stem cells in bone marrow. In this report, rat bone marrow derived mesenchymal progenitor cells (MPC) were used as feeder cells to culture mouse Oct4-GFP ES cell and ES cell derived germ cells. FACS results show that similar to MEF, rat MPC could efficiently support growth of the mouse Oct4-GFP ES cell line in culture (MPC 85.5 5.1% vs MEF 84.1 6.2%). ES cells could be subcultured for >15 passages without losing morphological characteristics. The cultured cells expressed stem cell marker alkaline phosphatase, Oct4, Sox2, and SSEA-1. Furthermore, rat MPC cells were able to support survival of germ cells isolated from mouse Oct4-GFP ES cell formed embryoid bodies (EB). After induction by retinoic acid for 7 days, some isolated cells differentiated to spermatogonial stem-like cells, expressing Mvh, Stra-8, Hsp90-a, integrinb1 and a6. Compared with traditional MEF culture systems, the rat MPC culture system is effective in supporting ES cell growth and is easy to prepare. Ó 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Bone marrow; Mesenchymal progenitor cell; Embryonic stem cell; Primordial germ cell; Retinoic acid; Differentiation
1. Introduction Mouse embryonic fibroblasts (MEFs) have been used as feeder cells to culture embryonic stem cells (ESCs) since the first mouse ESCs were derived in 1981. MEFs provide a complex unknown mixture of nutrients and substrata for long-term growth and proliferation of undifferentiated ESCs. However, preparation of fresh MEF feeder or MEFconditioned medium includes the steps of mating of mice, plug observation, repeated sacrifice of 12.5 to 14.5-day mouse
Abbreviations: MEF, mouse embryonic fibroblast; ESC, embryonic stem cell; PGC, primordial germ cell; MPC, mesenchymal progenitor cells; EB, embryonic body; RA, retinoic acid; ALP, alkaline phosphatase. * Corresponding author. Tel: þ86 0755 83923333 8704; fax: þ86 0755 83061340. E-mail address: [email protected] (Z. Cai).
embryos and enzyme digestion, which is a complex and tedious task. As a result, there is an intense search for new ES culture systems which are simple, effective and labor saving. Accumulated evidence suggests that in addition to hematopoietic stem cells, bone marrow also harbors endothelial stem cells, MSCs and MPCs (Jiang et al., 2002; Kassem, 2004; Ratajczak et al., 2004; Reyes et al., 2002). MPCs serve as precursors for bone, cartilage, fat, and muscle, and also contribute to the marrow microenvironment by constitutively secreting interleukins and leukemia inhibitory factor (Sun et al., 2003). Recently, Kuica et al. (2007) identified a population of CXCR4þ lin-CD45 cells that express SSEA, Oct4 and Nanog in adult bone marrow. These cells are very small and display several features typical for primary embryonic stem cells. When cultured on an MEF cell layer, these cells can form ES cell-like colonies, and can be induced to differentiate to cells
1065-6995/$ - see front matter Ó 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2009.01.008
G. Cui et al. / Cell Biology International 33 (2009) 434e441
435
including MPC can support ES cell growth and germ-like cell differentiation. We set up a culture system using rat bone marrow derived mesenchymal progenitor cells (MPCs) as feeder cells to culture mouse Oct4-GFP ES cell and ES cell derived germ cells. 2. Materials and methods 2.1. Animals
Fig. 1. After three passages, the adherent cells isolated from bone marrow were of large flat fibroblastic morphology. Magnification 100.
of all three germ layers. These cells were named bone marrow derived very small embryonic-like stem cells (VSEL-SC). Nayernia et al. (2006) reported that precursors of male germ cells could be isolated from mouse bone marrow cells. Germ and blood lineage cells are of common origin, by reason of primordial germ cells (PGCs) arising from the proximal epiblast, a region of the early embryo that also contributes to the first blood lineages of the embryonic yolk sac (Lawson and Hage, 1994; Zhao and Garbers, 2002). Based on these reports that embryonic-like stem and primitive germ cells can survive in a bone marrow microenvironment, we hypothesized that bone marrow stromal cells
Six-week-old male SD rats purchased from the animal center of Zhongshan University were housed at constant temperature (20 C) and humidity (45%) on a 12-h light/dark cycle, and were fed ad libitum on standard laboratory chow and tap water. Chinese laws concerning the protection and control of experiment animals were strictly followed throughout the experiments. 2.2. Bone marrow MPC isolation and culture Bone marrow cells were isolated from femurae and tibiae of SD rats and seeded into plastic flasks at 1 106/cm2 in MPC culture medium (DMEM/F12, 10% FBS Hyclon, Longan, UT, USA; 10 ng/ml bFGF, 2 ng/ml VEGF CytoLab Rehovot, Israel). The medium was completely replaced every 3 days, and non-adherent cells were discarded. When adherent cells reached 70e80% confluence, they were detached with 0.25% trypsin containing 0.02% EDTA, and subcultured at the ratio of 1:2 (Sun et al., 2003; Schrepfer et al., 2007).
Fig. 2. Bone marrow derived adherent cells expressed Sca-1, CD90, CD105 and CD44, and no CD11b expression was detected. (A) Expression of Sca-1. (B) Expression of CD90. (C) Expression of CD105; (D) Expression of CD44. (E) No expression of CD11b. (F) Negative control. Magnification 200.
436
G. Cui et al. / Cell Biology International 33 (2009) 434e441
Fig. 3. Bone marrow derived adherent cells can be induced to differentiate into chondrogenic, adipogenic and osteogenic cells. (A) Chondrogenic differentiation: sulfated proteoglycans can be detected using Alcian Blue staining. (B) Cells without induction. (C) Adipogenic differentiation: intercellular lipid vacuoles could be seen by Oil Red-O staining. (D) Cells without induction. (E) Osteogenic differentiation: substantial calcium deposition can be identified by von Kossa staining. (F) Cells without induction. Magnification 200.
2.3. MPC phenotypic assays Adherent cells of the fifth generation were prepared for immunohistochemical detection of Sca-1, CD90, CD105, CD44 and CD11b expression (primary antibodies: anti-Sca-1 and CD90, Biolegend, San Diego, CA, USA; anti-CD105, CD11b and CD44, Boster, Wuha, China; secondary antibodies:
biotin-coupled anti-mouse or rabbit IgG, and avidin-horseradish peroxidase conjugates, Boster, Wuha, China). 2.4. MPC differentiation assays MPC osteogenic, chondrogenic and adipogenic differentiation induction and the following cytological staining were
G. Cui et al. / Cell Biology International 33 (2009) 434e441
437
Chondrogenic differentiation was induced by culturing cells with chondrogenic medium (DMEM supplemented with 10% FBS, 6.25 mg/ml insulin, 50 nM ascorbate phosphate, 10 ng/ml transforming growth factor-b) for 2 weeks. Sulfated proteoglycans formation was visualized by Alcian Blue stain. 2.5. ES cell culture, alkaline phosphatase and phenotype examination
Fig. 4. Oct4-GFP ES cells cultured on MPC monolayer formed clones and expressed GFP. Observed under a mixture of daylight and UV light. Magnification 100.
conducted as described by Sun et al. (2003). Osteogenic differentiation was induced by culturing cells with osteoinductive medium (DMEM supplemented with 10% FBS, 10 mM glycerol phosphate, 50 mM ascorbate phosphate, 107 M dexamethasone, and 100 ng/ml recombinant human bone morphogenic protein-2) for 2 weeks. Extracellular matrix calcification was detected by von Kossa staining. To induce adipocytic differentiation, cells were cultured as monolayers, allowed to become near confluent, and maintained in adipogenic induction medium (DMEM supplemented with 10% FBS, 107 M dexamethasone and 10 mg/ml insulin) for 2 weeks. The cells were assessed by Oil Red-O stain to indicate intracellular lipid accumulation.
The Oct4-GFP ES cell line was kindly provided by Professor Bing Huang of the Ophthalmology Center of Zhongshan University. Cell line background: Oct4 promoter region fragments were derived by amplified mouse genome DNA with specific designed primers. The fragment was inserted into GFP-expressing retroviral vector to construct Oct4 controlled GFP-expressing recombinant vector. Male D3 ES cell line was transfected by this recombinant vector. After being selected by G418 for 2 months, stably transfected Male D3 ES cell line was obtained. When ES cells start differentiation, the Oct4 controlled GFP expression within cell is extinguish. Male D3 Oct4-GFP ES cells were maintained on rat MPC formed monolayer in MPC culture medium without application of LIF. After being subcultured 15 times, ES cells were prepared for alkaline phosphatase (ALP) test according to the protocol provided by the manufacturer (Alkaline Phosphatase Detection Kit, Boster, Wuha, China). For immunofluorescence detection of Sox2 and SSEA-1 expression in the cultured ES cell, primary antibodies (antiSox2 and SSEA-1; Santa Cruz Biotech, CA, USA), and red fluorescence-conjugated secondary antibodies (anti-mouse IgG or IgM; Boster) were used. Fluorescence was examined with a Carl Zeiss confocal microscope. 2.6. FACS analysis of GFP-expressing ES cell To evaluate the effectiveness of MPC feeler layer in supporting ES cell growth, 1 105 Oct4-GFP ES cells were inoculated on to MPC and MEF feeder layers. Two days later, the cultured ES cells were collected and fluorescence of GFPexpressing cells was analyzed by FACS Caliber (BectonDickinson). Each analysis was run three times. 2.7. EB differentiation and single cell solution preparation
Fig. 5. Oct4-GFP ES cells cultured on MPC monolayer expressed ALP. Magnification 100.
EB formation and EB derived single cell solution preparation was carried out followed the methods described by Geijsen et al. (2004). For EB differentiation, ES cells were digested with trypsin, collected in EB medium (DMEM with 15% FCS, 0.1 mM non-essential amino acids, 2 mM glutamine, penicillin/streptomycin, 0.1 mM mercaptoethanol) and plated for 45 min to allow MPC cells to adhere. Non-adherent cells were collected and plated in hanging drops at 300 cells per 30-ml droplet in an inverted bacterial Petri dish. EBs were collected from the hanging drops at day 3 and transferred into 10 ml EB medium in slowly rotating 10-cm Petri dishes. On
438
G. Cui et al. / Cell Biology International 33 (2009) 434e441
Fig. 6. Oct4-GFP ES cells cultured on MPC monolayer expressed Sox2 and SSEA-1. (A) Sox2 expression. (B) SSEA-1 expression. (C) Negative control. Magnification 600.
day 6, the cultured EBs were collected from the dishes, and washed twice with PBS. A single cell suspension was obtained after the EBs were digested with 0.1% collagenase for 20 min and 0.25% trypsin for 30 min. The collected single cells were washed with PBS twice, and prepared for further use. 2.8. SSEA-1þ cell isolation from EB derived cells Immunomagnetic isolation of SSEA-1þ cells from EB derived cells was performed using a monoclonal antibody (IgM) against SSEA-1 (Santa Cruz), and immunomagnetic rat anti-mouse IgM beads ((Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer’s protocol. 2.9. RA induced germ-like cell differentiation Retinoic acid acts rapidly to differentiate ES cells while stimulating proliferation of PGCs, and can therefore be used to eliminate the remaining ES-like populations existing in the isolated SSEA-1þ cells (Geijsen et al., 2004). EB derived SSEA-1þ cells were plated onto a rat MPC feeder cell layer in MSC medium and cultured for 7 days in the presence of 2 mM RA. Germ cell differentiation of the induced cells was examined by immunofluorescence staining for Mvh, Stra-8, Hsp-90a, integrin-b1 and a6 expression. Three days before staining, 500 mg/ml G418 was added to the culture medium, in order to kill the feeder MPC, thus
gradually reducing the amount of secreted cytokines. The ES cell line used in this research is G418-resistant. Primary antibodies used in detection: anti-Mvh, anti-Stra-8, Santa Cruz; anti-Hsp-90a, anti-integrin b1 and a6 (Boster). Secondary antibodies: red fluorescence-conjugated anti-rat or rabbit IgG (Boster). Results were analyzed using a Carl Zeiss confocal microscope.
3. Results 3.1. MPC morphology The initial adherent spindle-shaped cells appeared as individual cells or clusters of a few cells. After three passages, most of the adherent cells had a large flat fibroblastic morphology (Fig. 1).
3.2. Phenotypic characterization of MPC After being cultured for five passages, MPCs were analyzed for the expression of a panel of antigens. Results show that >90% of the cells expressed Sca-1, CD90, CD105 and CD44 (the positively stained cells in each 200 cells were counted to evaluate the positive cell percentage) and no CD11b expression was detected (Fig. 2).
Fig. 7. After being cultured for 2 days, fluorescence of GFP-expressing ES cell cultured on MPC feeder layer was compared with that of ES cell on MEF. Fluorescence was measured in arbitrary units on a log scale. The average percent of GFP-expressing cells in the two different culture systems was very similar in three experiments: (A) MPC culture system 85.5 5.1%. (B) MEF culture system 84.1 6.2%. Values are mean SEM. p > 0.05.
G. Cui et al. / Cell Biology International 33 (2009) 434e441
439
Fig. 8. Oct4-GFP ES cells formed EB by hanging drop culture. (A) Three-day EB collected from hanging drop. (B) Three-day EB under UV light. (C) Six-day EB under UV light. GFP expression in some parts of the EB has decreased indicating the cells in these parts of EB had differentiated. Magnification 100.
3.3. Osteogenic, chondrogenic and adipogenic differentiation assays of MPC
decreased, indicating that the cells in these parts of EB had differentiated (Fig. 8).
After being cultured with osteo-inductive medium for 2 weeks, substantial calcium deposition could be identified by von Kossa staining in the cultured cells. When cells were cultured in adipo-inductive medium, intercellular lipid vacuoles could be seen from day 10 by Oil Red-O staining. By culture with chondrogenic differentiation medium for 2 weeks, sulfated proteoglycans produced by cell plaque were detected by Alcian Blue staining (Fig. 3).
3.7. Germ-like cell differentiation
3.4. Characteristic of ES cells cultured on an MPC monolayer After being cultured for 15 passages with MPCs as the feeder cells, the ES cell clones were similar to those formed by MEF supported ES cells, and expressed GFP (Fig. 4) and ALP (Fig. 5). In this cell line, GFP expression was controlled by the Oct4 promoter; when ES cells start differentiation, GFP expression is diminished. Immunofluorescence examination showed that these cells were positive for Sox2 and SSEA-1 (Fig. 6). The results indicate that the cultured ES cells kept their undifferentiated characteristics.
SSEA-1þ cells isolated from 6-day EBs expressed GFP, indicating that the Oct4 promoter was functioning (Fig. 9). After being treated with RA for 7 days, some of the treated cells expressed Mvh, Stra-8, Hsp90-a, integrinb1 and a6 (Fig. 10). The expression of these markers indicated that some RA-treated SSEA-1þ cells had started differentiating towards spermatogonial stem cells. 4. Discussion Recently, two groups have reported that embryonic stemlike cells could be isolated from bone marrow (Kucia et al., 2007), and bone marrow derived adherent cells could be induced to form male germ cells (Nayernia et al., 2006). To a certain degree, these results demonstrate that bone marrow
3.5. FACS analysis of GFP-expressing ES cells After being cultured for 2 days, fluorescence of GFPexpressing ES cells cultured on an MPC feeder layer was compared with that on MEF. Fluorescence was measured in arbitrary units on a log scale. Results showed the average percent of GFP-expressing cells in the two different culture systems was very similar in three experiments: MPC culture system 85.5 5.1% vs MEF culture system 84.1 6.2%. Values are mean SEM. p > 0.05 (Fig. 7). This result indicates that an MPC feeder layer is as effective as an MEF feeder layer in supporting ES cell growth. 3.6. EB differentiation ES cells cultured in hanging drops formed EBs. By the end of the sixth day, GFP expression in some parts of the EB had
Fig. 9. SSEA-1þ cells isolated form 6-day EBs expressed GFP indicating the working of Oct4 promoter. Magnification 100.
440
G. Cui et al. / Cell Biology International 33 (2009) 434e441
Fig. 10. After being treated by RA for 7 days, some cells isolated from EB expressed spermatogonial stem cell marker. (A) Expression of Mvh. (B) Expression of Stra-8. (C) Expression of Hsp90-a. (D) Expression of integrin b1. (E) Expression of integrin a6. (F) Negative control. Magnification 600.
stromal cells provide a suitable environment for maintenance of ES-like cell and primitive male germ cells. MPCs isolated from mouse bone marrow is difficult to amplify in the long term. As a result, we isolated MPCs from rat bone marrow, and amplified the cells by at least 10 doublings. The isolated cells expressed CD44, CD105, CD90 and Sca-1, and were able to differentiate into osteogenic, chondrogenic and adipogenic cells. With these properties, we have the tendency to define these cells as bone marrow derived mesenchymal stem cells. After being cultured on MPC monolayer for 15 generations without the addition of LIF, the cultured ES cells kept expressing Oct4 controlled GFP, ALP, Sox2 and SSEA-1. Oct4, ALP, Sox2 and SSEA-1 expression have long been regarded as the markers of undifferentiated ES cells. EB formation is a widely accepted way to induce ES cell differentiation to various kinds of somatic cells and PGCs (Geijsen et al., 2004; Shiota et al., 2007). Without interaction with feeder cells and their secreted cytokines, the isolated PGCs quickly enter apoptosis (Farini et al., 2005). We isolated SSEA1þ cells from EB, and cultured these cells on MPC monolayer in the presence of 2 mM RA for 4 days. RA acts rapidly to aid differentiation into ES cells while stimulating proliferation of PGCs (Geijsen et al., 2004). After being treated by RA for 4 days, large colonies of cells positive for ALP could still be found in EB derived SSEA-1þ culture, indicating the existence of RA-resistant PGCs, which express ALP. However, in RAtreated ES cell culture, no ALP-positive cell colonies were found (data not shown). This result indicates that this culture system successfully protected the PGCs existing in the isolated SSEA-1þ cells from becoming apoptotic. At present, the commonly used feeder cells for ES and PGC culture are MEFs, which are widely accepted as being
complicated and time-consuming to prepare, as explained in Section 1. Additionally, there are other inconveniences in using MEF as feeder cells; (i) MEF is liable to undergo senescence (only 4e6 passages can be achieved, after which cells become senescent). (ii) MEF needs mitomycin treatment before being used as a feeder layer. (iii) When MEF serves as the feeder layer for culturing Oct4-GFP ES cells, LIF cannot be omitted from the medium. As a result, different media for the culture of MEF and ES cells have to be prepared. Compared with the preparation of MEF, obtaining MPC from rat bone marrow is easier, and time saving, the process only involving bone marrow cell collection and adherent cell amplification. MPC can be amplified to at least 10 generations without obvious senescence. MPC grows at a very low rate, and mitomycin treatment is not necessary when it is made a feeder cell layer. Because of Oct4-GFP, ES cells grow well in MPC culture medium, and therefore no special ES culture medium containing LIF is needed. Acknowledgements This work was supported by grants from Shen Zhen and Hong Kong Creative Circle Plan [2008] 121 and China Post doctor fund 20080430012. We thank Professor Bing Huang at the Ophthalmology Center of Zhongshan University for kindly providing the Oct4-GFP ES cell line. References Farini D, Scaldaferri ML, Iona S, Sala GL, Felici MD. Growth factors sustain primordial germ cell survival, proliferation and entering into
G. Cui et al. / Cell Biology International 33 (2009) 434e441 meiosis in the absence of somatic cells. Developmental Biology 2005; 285:49e56. Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 2004;427:148e154. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, OrtizGonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41e49. Kassem M. Mesenchymal stem cells: biological characteristics and potential clinical applications. Cloning and Stem Cells 2004;6:369e374. Kucia M, Zuba-Surma EK, Wysoczynski M, Wu W, Ratajczak J, Machalinski B, et al. Adult marrow-derived very small embryonic-like stem cells and tissue engineering. Expert Opinion on Biological Therapy 2007;7(10):1499e 1514. Lawson KA, Hage WJ. Clonal analysis of the origin of primordial germ cells in the mouse. Ciba Foundation Symposium 1994;182:68e91. Nayernia K, Lee JH, Drusenheimer N, Nolte J, Wulf G, Dressel R, et al. Derivation of male germ cells from bone marrow stem cells. Laboratory Investigation 2006;86:654e663.
441
Ratajczak MZ, Kucia M, Majka M, Reca R, Ratajcak J. Heterogeneous populations of bone marrow stem cells: are we spotting on the same cells from the different angles? Folia Histochemica et Cytobiologica 2004;42:139e 146. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. Journal of Clinical Investigation 2002;109:337e346. Schrepfer S, Deuse T, Lange C, Katzenberg R, Reichenspurner H, Robbins R, et al. Simplified protocol to isolate, purify, and culture expand mesenchymal stem cells. Stem Cells and Development 2007;16:105e107. Shiota M, Heike T, Haruyama M, Baba S, Tsuchiya A, Fujino H, et al. Isolation and characterization of bone marrow-derived mesenchymal progenitor cells with myogenic and neuronal properties. Experimental Cell Research 2007;313(5):1008e1023. Sun S, Guo ZK, Xiao XR, Liu B, Liu X, Tang PH, et al. Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method. Stem Cells 2003;21:527e535. Zhao GQ, Garbers DL. Male germ cell specification and differentiation. Developmental Cell 2002;5:537e547.
Short communication
Rat bone marrow derived mesenchymal progenitor cells support mouse ES cell growth and germ-like cell differentiation Guanghui Cui, Zhengyu Qi, Xin Guo, Jie Qin, Yaoting Gui, Zhiming Cai* Laboratory of Male Reproduction of Peking University Shenzhen Hospital, Medical Center of Peking University & Hong Kong Science and Technology University, Guangdong 518036, China Received 10 August 2008; revised 4 November 2008; accepted 9 January 2009
Abstract Mouse embryonic fibroblasts (MEFs) have been used as feeder cells to support the growth of mouse embryonic stem cell (mESC) and primordial germ cells (PGC) in culture for many years. However, MEF preparation is a complex and tedious task. Recently, there are reports indicating that the microenvironment provided by bone marrow stromal cells could support the survival of embryonic-like stem cells in bone marrow. In this report, rat bone marrow derived mesenchymal progenitor cells (MPC) were used as feeder cells to culture mouse Oct4-GFP ES cell and ES cell derived germ cells. FACS results show that similar to MEF, rat MPC could efficiently support growth of the mouse Oct4-GFP ES cell line in culture (MPC 85.5 5.1% vs MEF 84.1 6.2%). ES cells could be subcultured for >15 passages without losing morphological characteristics. The cultured cells expressed stem cell marker alkaline phosphatase, Oct4, Sox2, and SSEA-1. Furthermore, rat MPC cells were able to support survival of germ cells isolated from mouse Oct4-GFP ES cell formed embryoid bodies (EB). After induction by retinoic acid for 7 days, some isolated cells differentiated to spermatogonial stem-like cells, expressing Mvh, Stra-8, Hsp90-a, integrinb1 and a6. Compared with traditional MEF culture systems, the rat MPC culture system is effective in supporting ES cell growth and is easy to prepare. Ó 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Bone marrow; Mesenchymal progenitor cell; Embryonic stem cell; Primordial germ cell; Retinoic acid; Differentiation
1. Introduction Mouse embryonic fibroblasts (MEFs) have been used as feeder cells to culture embryonic stem cells (ESCs) since the first mouse ESCs were derived in 1981. MEFs provide a complex unknown mixture of nutrients and substrata for long-term growth and proliferation of undifferentiated ESCs. However, preparation of fresh MEF feeder or MEFconditioned medium includes the steps of mating of mice, plug observation, repeated sacrifice of 12.5 to 14.5-day mouse
Abbreviations: MEF, mouse embryonic fibroblast; ESC, embryonic stem cell; PGC, primordial germ cell; MPC, mesenchymal progenitor cells; EB, embryonic body; RA, retinoic acid; ALP, alkaline phosphatase. * Corresponding author. Tel: þ86 0755 83923333 8704; fax: þ86 0755 83061340. E-mail address: [email protected] (Z. Cai).
embryos and enzyme digestion, which is a complex and tedious task. As a result, there is an intense search for new ES culture systems which are simple, effective and labor saving. Accumulated evidence suggests that in addition to hematopoietic stem cells, bone marrow also harbors endothelial stem cells, MSCs and MPCs (Jiang et al., 2002; Kassem, 2004; Ratajczak et al., 2004; Reyes et al., 2002). MPCs serve as precursors for bone, cartilage, fat, and muscle, and also contribute to the marrow microenvironment by constitutively secreting interleukins and leukemia inhibitory factor (Sun et al., 2003). Recently, Kuica et al. (2007) identified a population of CXCR4þ lin-CD45 cells that express SSEA, Oct4 and Nanog in adult bone marrow. These cells are very small and display several features typical for primary embryonic stem cells. When cultured on an MEF cell layer, these cells can form ES cell-like colonies, and can be induced to differentiate to cells
1065-6995/$ - see front matter Ó 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2009.01.008
G. Cui et al. / Cell Biology International 33 (2009) 434e441
435
including MPC can support ES cell growth and germ-like cell differentiation. We set up a culture system using rat bone marrow derived mesenchymal progenitor cells (MPCs) as feeder cells to culture mouse Oct4-GFP ES cell and ES cell derived germ cells. 2. Materials and methods 2.1. Animals
Fig. 1. After three passages, the adherent cells isolated from bone marrow were of large flat fibroblastic morphology. Magnification 100.
of all three germ layers. These cells were named bone marrow derived very small embryonic-like stem cells (VSEL-SC). Nayernia et al. (2006) reported that precursors of male germ cells could be isolated from mouse bone marrow cells. Germ and blood lineage cells are of common origin, by reason of primordial germ cells (PGCs) arising from the proximal epiblast, a region of the early embryo that also contributes to the first blood lineages of the embryonic yolk sac (Lawson and Hage, 1994; Zhao and Garbers, 2002). Based on these reports that embryonic-like stem and primitive germ cells can survive in a bone marrow microenvironment, we hypothesized that bone marrow stromal cells
Six-week-old male SD rats purchased from the animal center of Zhongshan University were housed at constant temperature (20 C) and humidity (45%) on a 12-h light/dark cycle, and were fed ad libitum on standard laboratory chow and tap water. Chinese laws concerning the protection and control of experiment animals were strictly followed throughout the experiments. 2.2. Bone marrow MPC isolation and culture Bone marrow cells were isolated from femurae and tibiae of SD rats and seeded into plastic flasks at 1 106/cm2 in MPC culture medium (DMEM/F12, 10% FBS Hyclon, Longan, UT, USA; 10 ng/ml bFGF, 2 ng/ml VEGF CytoLab Rehovot, Israel). The medium was completely replaced every 3 days, and non-adherent cells were discarded. When adherent cells reached 70e80% confluence, they were detached with 0.25% trypsin containing 0.02% EDTA, and subcultured at the ratio of 1:2 (Sun et al., 2003; Schrepfer et al., 2007).
Fig. 2. Bone marrow derived adherent cells expressed Sca-1, CD90, CD105 and CD44, and no CD11b expression was detected. (A) Expression of Sca-1. (B) Expression of CD90. (C) Expression of CD105; (D) Expression of CD44. (E) No expression of CD11b. (F) Negative control. Magnification 200.
436
G. Cui et al. / Cell Biology International 33 (2009) 434e441
Fig. 3. Bone marrow derived adherent cells can be induced to differentiate into chondrogenic, adipogenic and osteogenic cells. (A) Chondrogenic differentiation: sulfated proteoglycans can be detected using Alcian Blue staining. (B) Cells without induction. (C) Adipogenic differentiation: intercellular lipid vacuoles could be seen by Oil Red-O staining. (D) Cells without induction. (E) Osteogenic differentiation: substantial calcium deposition can be identified by von Kossa staining. (F) Cells without induction. Magnification 200.
2.3. MPC phenotypic assays Adherent cells of the fifth generation were prepared for immunohistochemical detection of Sca-1, CD90, CD105, CD44 and CD11b expression (primary antibodies: anti-Sca-1 and CD90, Biolegend, San Diego, CA, USA; anti-CD105, CD11b and CD44, Boster, Wuha, China; secondary antibodies:
biotin-coupled anti-mouse or rabbit IgG, and avidin-horseradish peroxidase conjugates, Boster, Wuha, China). 2.4. MPC differentiation assays MPC osteogenic, chondrogenic and adipogenic differentiation induction and the following cytological staining were
G. Cui et al. / Cell Biology International 33 (2009) 434e441
437
Chondrogenic differentiation was induced by culturing cells with chondrogenic medium (DMEM supplemented with 10% FBS, 6.25 mg/ml insulin, 50 nM ascorbate phosphate, 10 ng/ml transforming growth factor-b) for 2 weeks. Sulfated proteoglycans formation was visualized by Alcian Blue stain. 2.5. ES cell culture, alkaline phosphatase and phenotype examination
Fig. 4. Oct4-GFP ES cells cultured on MPC monolayer formed clones and expressed GFP. Observed under a mixture of daylight and UV light. Magnification 100.
conducted as described by Sun et al. (2003). Osteogenic differentiation was induced by culturing cells with osteoinductive medium (DMEM supplemented with 10% FBS, 10 mM glycerol phosphate, 50 mM ascorbate phosphate, 107 M dexamethasone, and 100 ng/ml recombinant human bone morphogenic protein-2) for 2 weeks. Extracellular matrix calcification was detected by von Kossa staining. To induce adipocytic differentiation, cells were cultured as monolayers, allowed to become near confluent, and maintained in adipogenic induction medium (DMEM supplemented with 10% FBS, 107 M dexamethasone and 10 mg/ml insulin) for 2 weeks. The cells were assessed by Oil Red-O stain to indicate intracellular lipid accumulation.
The Oct4-GFP ES cell line was kindly provided by Professor Bing Huang of the Ophthalmology Center of Zhongshan University. Cell line background: Oct4 promoter region fragments were derived by amplified mouse genome DNA with specific designed primers. The fragment was inserted into GFP-expressing retroviral vector to construct Oct4 controlled GFP-expressing recombinant vector. Male D3 ES cell line was transfected by this recombinant vector. After being selected by G418 for 2 months, stably transfected Male D3 ES cell line was obtained. When ES cells start differentiation, the Oct4 controlled GFP expression within cell is extinguish. Male D3 Oct4-GFP ES cells were maintained on rat MPC formed monolayer in MPC culture medium without application of LIF. After being subcultured 15 times, ES cells were prepared for alkaline phosphatase (ALP) test according to the protocol provided by the manufacturer (Alkaline Phosphatase Detection Kit, Boster, Wuha, China). For immunofluorescence detection of Sox2 and SSEA-1 expression in the cultured ES cell, primary antibodies (antiSox2 and SSEA-1; Santa Cruz Biotech, CA, USA), and red fluorescence-conjugated secondary antibodies (anti-mouse IgG or IgM; Boster) were used. Fluorescence was examined with a Carl Zeiss confocal microscope. 2.6. FACS analysis of GFP-expressing ES cell To evaluate the effectiveness of MPC feeler layer in supporting ES cell growth, 1 105 Oct4-GFP ES cells were inoculated on to MPC and MEF feeder layers. Two days later, the cultured ES cells were collected and fluorescence of GFPexpressing cells was analyzed by FACS Caliber (BectonDickinson). Each analysis was run three times. 2.7. EB differentiation and single cell solution preparation
Fig. 5. Oct4-GFP ES cells cultured on MPC monolayer expressed ALP. Magnification 100.
EB formation and EB derived single cell solution preparation was carried out followed the methods described by Geijsen et al. (2004). For EB differentiation, ES cells were digested with trypsin, collected in EB medium (DMEM with 15% FCS, 0.1 mM non-essential amino acids, 2 mM glutamine, penicillin/streptomycin, 0.1 mM mercaptoethanol) and plated for 45 min to allow MPC cells to adhere. Non-adherent cells were collected and plated in hanging drops at 300 cells per 30-ml droplet in an inverted bacterial Petri dish. EBs were collected from the hanging drops at day 3 and transferred into 10 ml EB medium in slowly rotating 10-cm Petri dishes. On
438
G. Cui et al. / Cell Biology International 33 (2009) 434e441
Fig. 6. Oct4-GFP ES cells cultured on MPC monolayer expressed Sox2 and SSEA-1. (A) Sox2 expression. (B) SSEA-1 expression. (C) Negative control. Magnification 600.
day 6, the cultured EBs were collected from the dishes, and washed twice with PBS. A single cell suspension was obtained after the EBs were digested with 0.1% collagenase for 20 min and 0.25% trypsin for 30 min. The collected single cells were washed with PBS twice, and prepared for further use. 2.8. SSEA-1þ cell isolation from EB derived cells Immunomagnetic isolation of SSEA-1þ cells from EB derived cells was performed using a monoclonal antibody (IgM) against SSEA-1 (Santa Cruz), and immunomagnetic rat anti-mouse IgM beads ((Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer’s protocol. 2.9. RA induced germ-like cell differentiation Retinoic acid acts rapidly to differentiate ES cells while stimulating proliferation of PGCs, and can therefore be used to eliminate the remaining ES-like populations existing in the isolated SSEA-1þ cells (Geijsen et al., 2004). EB derived SSEA-1þ cells were plated onto a rat MPC feeder cell layer in MSC medium and cultured for 7 days in the presence of 2 mM RA. Germ cell differentiation of the induced cells was examined by immunofluorescence staining for Mvh, Stra-8, Hsp-90a, integrin-b1 and a6 expression. Three days before staining, 500 mg/ml G418 was added to the culture medium, in order to kill the feeder MPC, thus
gradually reducing the amount of secreted cytokines. The ES cell line used in this research is G418-resistant. Primary antibodies used in detection: anti-Mvh, anti-Stra-8, Santa Cruz; anti-Hsp-90a, anti-integrin b1 and a6 (Boster). Secondary antibodies: red fluorescence-conjugated anti-rat or rabbit IgG (Boster). Results were analyzed using a Carl Zeiss confocal microscope.
3. Results 3.1. MPC morphology The initial adherent spindle-shaped cells appeared as individual cells or clusters of a few cells. After three passages, most of the adherent cells had a large flat fibroblastic morphology (Fig. 1).
3.2. Phenotypic characterization of MPC After being cultured for five passages, MPCs were analyzed for the expression of a panel of antigens. Results show that >90% of the cells expressed Sca-1, CD90, CD105 and CD44 (the positively stained cells in each 200 cells were counted to evaluate the positive cell percentage) and no CD11b expression was detected (Fig. 2).
Fig. 7. After being cultured for 2 days, fluorescence of GFP-expressing ES cell cultured on MPC feeder layer was compared with that of ES cell on MEF. Fluorescence was measured in arbitrary units on a log scale. The average percent of GFP-expressing cells in the two different culture systems was very similar in three experiments: (A) MPC culture system 85.5 5.1%. (B) MEF culture system 84.1 6.2%. Values are mean SEM. p > 0.05.
G. Cui et al. / Cell Biology International 33 (2009) 434e441
439
Fig. 8. Oct4-GFP ES cells formed EB by hanging drop culture. (A) Three-day EB collected from hanging drop. (B) Three-day EB under UV light. (C) Six-day EB under UV light. GFP expression in some parts of the EB has decreased indicating the cells in these parts of EB had differentiated. Magnification 100.
3.3. Osteogenic, chondrogenic and adipogenic differentiation assays of MPC
decreased, indicating that the cells in these parts of EB had differentiated (Fig. 8).
After being cultured with osteo-inductive medium for 2 weeks, substantial calcium deposition could be identified by von Kossa staining in the cultured cells. When cells were cultured in adipo-inductive medium, intercellular lipid vacuoles could be seen from day 10 by Oil Red-O staining. By culture with chondrogenic differentiation medium for 2 weeks, sulfated proteoglycans produced by cell plaque were detected by Alcian Blue staining (Fig. 3).
3.7. Germ-like cell differentiation
3.4. Characteristic of ES cells cultured on an MPC monolayer After being cultured for 15 passages with MPCs as the feeder cells, the ES cell clones were similar to those formed by MEF supported ES cells, and expressed GFP (Fig. 4) and ALP (Fig. 5). In this cell line, GFP expression was controlled by the Oct4 promoter; when ES cells start differentiation, GFP expression is diminished. Immunofluorescence examination showed that these cells were positive for Sox2 and SSEA-1 (Fig. 6). The results indicate that the cultured ES cells kept their undifferentiated characteristics.
SSEA-1þ cells isolated from 6-day EBs expressed GFP, indicating that the Oct4 promoter was functioning (Fig. 9). After being treated with RA for 7 days, some of the treated cells expressed Mvh, Stra-8, Hsp90-a, integrinb1 and a6 (Fig. 10). The expression of these markers indicated that some RA-treated SSEA-1þ cells had started differentiating towards spermatogonial stem cells. 4. Discussion Recently, two groups have reported that embryonic stemlike cells could be isolated from bone marrow (Kucia et al., 2007), and bone marrow derived adherent cells could be induced to form male germ cells (Nayernia et al., 2006). To a certain degree, these results demonstrate that bone marrow
3.5. FACS analysis of GFP-expressing ES cells After being cultured for 2 days, fluorescence of GFPexpressing ES cells cultured on an MPC feeder layer was compared with that on MEF. Fluorescence was measured in arbitrary units on a log scale. Results showed the average percent of GFP-expressing cells in the two different culture systems was very similar in three experiments: MPC culture system 85.5 5.1% vs MEF culture system 84.1 6.2%. Values are mean SEM. p > 0.05 (Fig. 7). This result indicates that an MPC feeder layer is as effective as an MEF feeder layer in supporting ES cell growth. 3.6. EB differentiation ES cells cultured in hanging drops formed EBs. By the end of the sixth day, GFP expression in some parts of the EB had
Fig. 9. SSEA-1þ cells isolated form 6-day EBs expressed GFP indicating the working of Oct4 promoter. Magnification 100.
440
G. Cui et al. / Cell Biology International 33 (2009) 434e441
Fig. 10. After being treated by RA for 7 days, some cells isolated from EB expressed spermatogonial stem cell marker. (A) Expression of Mvh. (B) Expression of Stra-8. (C) Expression of Hsp90-a. (D) Expression of integrin b1. (E) Expression of integrin a6. (F) Negative control. Magnification 600.
stromal cells provide a suitable environment for maintenance of ES-like cell and primitive male germ cells. MPCs isolated from mouse bone marrow is difficult to amplify in the long term. As a result, we isolated MPCs from rat bone marrow, and amplified the cells by at least 10 doublings. The isolated cells expressed CD44, CD105, CD90 and Sca-1, and were able to differentiate into osteogenic, chondrogenic and adipogenic cells. With these properties, we have the tendency to define these cells as bone marrow derived mesenchymal stem cells. After being cultured on MPC monolayer for 15 generations without the addition of LIF, the cultured ES cells kept expressing Oct4 controlled GFP, ALP, Sox2 and SSEA-1. Oct4, ALP, Sox2 and SSEA-1 expression have long been regarded as the markers of undifferentiated ES cells. EB formation is a widely accepted way to induce ES cell differentiation to various kinds of somatic cells and PGCs (Geijsen et al., 2004; Shiota et al., 2007). Without interaction with feeder cells and their secreted cytokines, the isolated PGCs quickly enter apoptosis (Farini et al., 2005). We isolated SSEA1þ cells from EB, and cultured these cells on MPC monolayer in the presence of 2 mM RA for 4 days. RA acts rapidly to aid differentiation into ES cells while stimulating proliferation of PGCs (Geijsen et al., 2004). After being treated by RA for 4 days, large colonies of cells positive for ALP could still be found in EB derived SSEA-1þ culture, indicating the existence of RA-resistant PGCs, which express ALP. However, in RAtreated ES cell culture, no ALP-positive cell colonies were found (data not shown). This result indicates that this culture system successfully protected the PGCs existing in the isolated SSEA-1þ cells from becoming apoptotic. At present, the commonly used feeder cells for ES and PGC culture are MEFs, which are widely accepted as being
complicated and time-consuming to prepare, as explained in Section 1. Additionally, there are other inconveniences in using MEF as feeder cells; (i) MEF is liable to undergo senescence (only 4e6 passages can be achieved, after which cells become senescent). (ii) MEF needs mitomycin treatment before being used as a feeder layer. (iii) When MEF serves as the feeder layer for culturing Oct4-GFP ES cells, LIF cannot be omitted from the medium. As a result, different media for the culture of MEF and ES cells have to be prepared. Compared with the preparation of MEF, obtaining MPC from rat bone marrow is easier, and time saving, the process only involving bone marrow cell collection and adherent cell amplification. MPC can be amplified to at least 10 generations without obvious senescence. MPC grows at a very low rate, and mitomycin treatment is not necessary when it is made a feeder cell layer. Because of Oct4-GFP, ES cells grow well in MPC culture medium, and therefore no special ES culture medium containing LIF is needed. Acknowledgements This work was supported by grants from Shen Zhen and Hong Kong Creative Circle Plan [2008] 121 and China Post doctor fund 20080430012. We thank Professor Bing Huang at the Ophthalmology Center of Zhongshan University for kindly providing the Oct4-GFP ES cell line. References Farini D, Scaldaferri ML, Iona S, Sala GL, Felici MD. Growth factors sustain primordial germ cell survival, proliferation and entering into
G. Cui et al. / Cell Biology International 33 (2009) 434e441 meiosis in the absence of somatic cells. Developmental Biology 2005; 285:49e56. Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 2004;427:148e154. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, OrtizGonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41e49. Kassem M. Mesenchymal stem cells: biological characteristics and potential clinical applications. Cloning and Stem Cells 2004;6:369e374. Kucia M, Zuba-Surma EK, Wysoczynski M, Wu W, Ratajczak J, Machalinski B, et al. Adult marrow-derived very small embryonic-like stem cells and tissue engineering. Expert Opinion on Biological Therapy 2007;7(10):1499e 1514. Lawson KA, Hage WJ. Clonal analysis of the origin of primordial germ cells in the mouse. Ciba Foundation Symposium 1994;182:68e91. Nayernia K, Lee JH, Drusenheimer N, Nolte J, Wulf G, Dressel R, et al. Derivation of male germ cells from bone marrow stem cells. Laboratory Investigation 2006;86:654e663.
441
Ratajczak MZ, Kucia M, Majka M, Reca R, Ratajcak J. Heterogeneous populations of bone marrow stem cells: are we spotting on the same cells from the different angles? Folia Histochemica et Cytobiologica 2004;42:139e 146. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. Journal of Clinical Investigation 2002;109:337e346. Schrepfer S, Deuse T, Lange C, Katzenberg R, Reichenspurner H, Robbins R, et al. Simplified protocol to isolate, purify, and culture expand mesenchymal stem cells. Stem Cells and Development 2007;16:105e107. Shiota M, Heike T, Haruyama M, Baba S, Tsuchiya A, Fujino H, et al. Isolation and characterization of bone marrow-derived mesenchymal progenitor cells with myogenic and neuronal properties. Experimental Cell Research 2007;313(5):1008e1023. Sun S, Guo ZK, Xiao XR, Liu B, Liu X, Tang PH, et al. Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method. Stem Cells 2003;21:527e535. Zhao GQ, Garbers DL. Male germ cell specification and differentiation. Developmental Cell 2002;5:537e547.