Methods in Molecular Biology DOI 10.1007/7651_2014_162 © Springer Science+Business Media New York 2014
Mesoderm Differentiation from hiPS Cells Hiroyuki Miwa and Takumi Era Abstract Human induced pluripotent stem (hiPS) cells are very attractive tools for modeling diseases and regenerative medicine. However, to achieve them, the efficient differentiation methods of hiPS cells into aimed cell type in vitro are necessary. Because mesoderm cells are useful in particular, we have developed the differentiation of mouse embryonic stem (mES) cells into mesoderm cells previously. In this time, these methods were improved for hiPS cells and now human mesoderm cells are able to be obtained efficiently. It is certain that the new methods are applicable to various studies and therapies. Keywords: Human induced pluripotent stem (hiPS) cells, Mesoderm, Vascular endothelial growth factor receptor 2 (VEGFR2), Platelet-derived growth factor receptor α (PDGFRα), Bone, Cartilage
1
Introduction Human induced pluripotent stem (hiPS) cells with pluripotency and self-renewal capability can be induced from fibroblasts or hematopoietic cells by overexpression of four factors, Oct 3/4, Sox2, Klf4, and c-Myc (1). Such hiPS cells are thus very attractive tools in terms of both basic and clinical medicine. For instance, targeted differentiation of hiPS cells into specific cells could be used in cell therapy to regenerate tissue and organ defects. In addition, inducing hiPS cells from patients and differentiating them into cells of lesional tissue could provide important cellular models of disease for basic and clinical experimentation. Furthermore, drug screening for diseases could also utilize hiPS cells differentiated from patients. However, increasing the efficiency of differentiation is paramount for realizing any or all of the proposed applications for hiPS cells. In this context, we have been investigating the differentiation of mouse embryonic stem (mES) cells and hiPS cells into mesoderm and their descendant cells, bone and cartilage, which are important in the formation of various tissues and involved in many injury and disease processes (2, 3). For the mES cells, we are using two main methods of differentiation. One is formation of the commonly used embryoid body (EB) and involves aggregating the mES cells on low-attachment dishes and then culturing with specific factors (4). The other is
Hiroyuki Miwa and Takumi Era
culturing on OP9 cells or collagen IV-coated dishes, without any factors used during the differentiation (5). In this method, dissociated mES cells are seeded and cultured with αMEM containing fetal bovine serum (FBS). After a few days, FACS analysis showed differentiated mES cells to be positive for vascular endothelial growth factor receptor 2 (VEGFR2, FLK1) and platelet-derived growth factor receptor alpha (PDGFRα), i.e., PDGFRα+VEGFR2+ (DP) cells. After another few days, the DP cells differentiated into PDGFRα+VEGFR2 (PSP) or PDGFRα VEGFR2+ (VSP) cells. The PSP is likely to be paraxial mesoderm and the VSP lateral mesoderm, because PSP cells can differentiate into cartilage and bone, while VSP differentiates into endothelial cells (6–12). In regenerative medicine, many researchers have tried to generate mesoderm and their descendant cells from hES cells (13–16). Xu et al. (15) differentiated hESCs into fibroblast-like cells, and after EB formation followed by culture on gelatin-coated plates, these cells overexpressed human telomerase reverse transcriptase (hTERT). They also expressed CD29, CD44, CD71, and CD90 and could be differentiated into an osteocytic lineage, but not chondrocytic or adipocytic lineage. Other groups also induced hESCs into fibroblastic cells that express CD90 and CD44 and can give rise to mesoderm-descendant cells (16). Such mesodermal multipotent stem cells were also purified from in vitro ES/iPS cell culture by FACS using surface markers such as CD73 and CD105 (14). In that study, gene expression analysis isolated cells expressing surface markers such as CD44 and STRO-1, as well as other markers including DSC54, neuropilin 1, hepatocyte growth factor, forkhead box D1, and notch homolog 2 (14). The culture conditions for selectively inducing descendants of mesoderm cells from ES/iPS cells were also reported, involving the culturing of hESCs with bFGF and PDGF-AB without feeder cells and then isolating the CD105+CD24 population by FACS (13). This population could then be differentiated into adipocytic, chondrocytic, and osteocytic lineages. Mouse iPS cell-derived EBs have also been cultured with TGF-β1 and retinoic acid (RA) to generate cells that are able to differentiate into osteoblasts (17). Similarly, hiPS cells were cultured with bFGF, PDGF-AB, and epidermal growth factor (EGF), followed by FACS isolation of the CD105+CD24 population to generate multipotent stem cells (18). Herein, we describe the latest method of mesoderm differentiation to generate bone and cartilage cells from hiPS cells. This was developed by combining and modifying previous methods and is expected to efficiently differentiate into mesoderm lineages (4, 5, 12, 19–21). In brief, EBs are transferred onto collagen IV-coated dishes for culturing together with various factors, including bFGF, BMP-4, and Activin A and then sorted for VEGFR2 and PDGFRα expression by FACS. The subsequent PSP contains colony-forming unit-fibroblasts (CFU-Fs) and can differentiate into bone and
Mesoderm Differentiation from hiPS Cells
Fig. 1 Differentiation of hiPS cell into mesoderm. hiPS cells formed EBs differentiate into the three types of mesoderm cells, PDGFRα+VEGFR2+ population (PDGFRα and VEGFR2 double-positive population, DP), PDGFRα+VEGFR2 population (PDGFRα single-positive population, PSP), and PDGFRα VEGFR2+ population (VEGFR2 single-positive population, VSP). The DP is the most immature and can give rise to both the PSP and the VSP. The PSP can differentiate into bone and cartilage
cartilage cells (Fig. 1), as confirmed by CFU-F assay and staining using Alizarin Red and Alcian Blue. This method for efficiently differentiating hiPS cells into mesoderm cells should be useful for many studies and therapies in this field.
2
Materials
2.1 hiPS Cell Maintenance
1. Phosphate-buffered saline without calcium and magnesium chloride (PBS). 2. Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12, Sigma-Aldrich). Store at 4 C. 3. KnockOut SR (KSR, Gibco). Store at
20 C.
4. 2-Mercaptoethanol (0.1 M, 1,000) (2-ME). Add 70 μL 2-ME to 10 mL PBS and sterilized by 0.22 μm filter. Store at 4 C. 5. L-Glutamine 200 mM (100) (Gibco). Store at 20 C. 6. Penicillin-streptomycin (100) (P/S). Store at
20 C.
7. MEM Non-Essential Amino Acid Solution (100) (NEAA, Sigma-Aldrich). Store at 4 C. 8. hiPS cell culture medium: DMEM/F12, 20 % KSR, 0.1 mM 2-ME, 2 mM L-glutamine, P/S, 0.1 mM NEAA. Store at 4 C (see Note 1). 9. Fibroblast Growth Factor (basic), Human, recombinant (rhbFGF) (Wako). Store at 20 C. 10. Trypsin 250 (Difco). Store at 4 C. 11. Collagenase Type IV (Gibco). Store at 4 C. 12. CTK solution: PBS, 20 % KSR, 0.25 % Trypsin, 1 mg/mL Collagenase, 1 mM CaCl2. Store at 20 C.
Hiroyuki Miwa and Takumi Era
2.2
Formation of EB
1. hiPS cell culture medium. 2. Recombinant Human BMP-4 (R&D Systems). Store at 20 C. 3. 100-mm Petri dish (BIO-BIK) (see Note 2).
2.3 Induction of Mesoderm Cells
1. Minimum Essential Medium Alpha Medium (αMEM, Gibco). Store at 4 C. 2. Fetal bovine serum (FBS) (see Note 3). Store at
20 C.
3. BMP-4. 4. Recombinant Human/Mouse/Rat Activin A (R&D Systems). Store at 20 C. 5. bFGF. 6. Collagen Type IV Cellware 100-mm dish (BD Biosciences). Store at 4 C. 2.4 Purification of Mesoderm Cells by FACS
1. TrypLE Express (Gibco). 2. HBSS (10) (Gibco). 3. HBSS/BSA: HBSS, 1 % bovine serum albumin (BSA). Store at 4 C. 4. Clear Back (human Fc receptor blocking reagent) (MBL). Store at 4 C. 5. Biotin antihuman CD140a (PDGFRα) (BioLegend). Store at 4 C. 6. Antihuman VEGF R2/KDR Phycoerythrin (R&D Systems). Store at 4 C. 7. Streptavidin APC (eBioscience). Store at 4 C. 8. 7-AAD Viability Dye (Beckman Coulter). Store at 4 C.
2.5 Culture of Mesoderm Cells
1. αMEM. 2. FBS. 3. 2-ME. 4. P/S 5. rhPDGF-BB (R&D Systems). Store at
20 C.
6. Mesoderm cell culture medium: αMEM, 10 % FBS, 0.1 mM 2ME, P/S, 10 ng/mL PDGF-BB. Store at 4 C. 7. 0.25 % Trypsin-EDTA (Gibco). Store at 2.6
CFU-F Assay
20 C.
1. Mesoderm cell culture medium. 2. Collagen Type IV Cellware six-well plate (BD Biosciences). Store at 4 C. 3. Leishman’s eosin methylene blue solution (Merck).
Mesoderm Differentiation from hiPS Cells
2.7 Bone Cell Differentiation from Mesoderm Cells
1. Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco). Store at 4 C. 2. FBS. 3. P/S. 4. Dexamethasone. 5. Ascorbic acid 2-phosphate. 6. β-Glycerophosphate. 7. BMP-4. 8. 24-well culture plate (BD Biosciences). 9. Bone cell differentiation medium: DMEM, 10 % FBS, P/S, 0.1 mM dexamethasone, 50 mM ascorbic acid 2-phosphate, 10 mM β-glycerophosphate, 10 ng/mL BMP-4. 10. 4 % PFA: PBS, 4 % paraformaldehyde (PFA). 11. Alizarin Red staining solution. Add 0.1 g Alizarin Red S into 10 mL water.
2.8 Cartilage Cell Differentiation from Mesoderm Cells
1. αMEM. 2. FBS. 3. P/S. 4. Dexamethasone. 5. Ascorbic acid 2-phosphate. 6. Recombinant Human TGF-β3 (R&D Systems). Store at 20 C. 7. Recombinant Human BMP-2 (R&D Systems). Store at 20 C. 8. 24-well culture plate. 9. Cartilage cell differentiation medium: αMEM, 10 % FBS, P/S, 0.1 mM dexamethasone, 170 mM ascorbic acid 2-phosphate, 10 ng/mL TGF-β3. 10. 4 % PFA. 11. Alcian Blue staining solution. Add 50 mg Alcian Blue into 10 mL 0.1 M HCl.
3
Methods
3.1 In Vitro hiPS Cell Differentiation 3.1.1 Formation of EB
In this method, hiPS cells are maintained on mitomycin C-treated mouse embryonic fibroblasts (MEFs) as feeder cells. 1. Expand hiPS cells with hiPS cell culture medium and 5 ng/mL bFGF at 37 C, 3 % CO2 (see Note 4).
Hiroyuki Miwa and Takumi Era
2. When confluent on a 100-mm dish, aspirate medium and wash cells twice with PBS. 3. Add 500 μL CTK solution to dissociate cells from dish. 4. Incubate at 37 C for 5 min. 5. Remove CTK solution and add 10 mL hiPS cell culture medium. 6. Transfer cells into a 15-mL centrifuge tube and spin them down at 20 g for 5 min. 7. Resuspend cells in 10 mL hiPS cell culture medium and transfer them on a gelatin-coated 100-mm dish (see Note 5). 8. Incubate at 37 C for 30 min to remove feeder cells (see Note 6). 9. Transfer supernatant on a 100-mm Petri dish and add BMP-4 (10 ng/mL final conc.). 10. Incubate at 37 C, 3 % CO2 overnight. 3.1.2 Induction of Mesoderm Cells
1. Transfer EBs into a 15-mL centrifuge tube and spin them down at 20 g for 5 min. 2. Resuspend EBs in 10 mL αMEM with 10 % FBS, 3 ng/mL Activin A, 10 ng/mL BMP-4, and 5 ng/mL bFGF and seed them on Collagen IV-coated dish. 3. Change medium every 2 days. 4. Culture for 5 days (see Note 7).
3.1.3 Purification of Mesoderm Cells by FACS
1. Aspirate medium and wash cells twice with PBS. 2. Add 2 mL TrypLE Express and incubate at 37 C for 5 min. 3. Add 8 mL PBS with 10 % FBS and repeat pipetting 10–15 times. 4. Transfer cells into a 15-mL centrifuge tube and spin down them at 4 C, 270 g for 5 min. 5. Resuspend 1 107 cells in 100 μL Fc receptor blocking reagent and put them at room temperature for 5 min. 6. Add 300 μL HBSS/BSA and antibody to cell suspension and put them on ice for 20 min (see Note 8). 7. Add 400 μL HBSS/BSA and spin them down at 4 C, 270 g for 5 min. 8. Resuspend 400 μL HBSS/BSA and add 1 μL SA-APC. Put them on ice for 20 min. 9. Add 400 μL HBSS/BSA and spin them down at 4 C, 270 g for 5 min.
Mesoderm Differentiation from hiPS Cells
Fig. 2 Analysis of hiPS-derived mesoderm cells by FACS. hiPS cells formed EBs were cultured on Collagen IV-coated dish with αMEM containing FBS, Activin A, BMP-4, and bFGF. After 5 days of culture, cells were dissociated and analyzed with anti-PDGFRα and anti-VEGFR2 antibodies by FACS. Four populations (PDGFRα+VEGFR2+, DP; PDGFRα+VEGFR2 , PSP; PDGFRα VEGFR2+, VSP; PDGFRα VEGFR2 , DN) were observed in differentiated hiPS cells
10. Resuspend 800 μL HBSS/BSA and spin them down at 4 C, 270 g for 5 min. 11. Resuspend 1 mL HBSS/BSA and add 5 μL 7-AAD. Filter them into a 5-mL PS tube. 12. Sort PSP cells by FACS (Fig. 2). 1. Resuspend 1 105 hiPS-derived mesoderm cells in 10 μL mesoderm cell culture medium and put this solution on a well of Collagen IV-coated 24-well plate.
3.2 Maintenance, Expansion, and Validation of Mesoderm Cells
2. Incubate at 37 C until cells attach to plate (1–3 h).
3.2.1 Culture of Mesoderm Cells
3. Add slowly 500 μL mesoderm cell culture medium into the well of plate and incubate at 37 C, 5 % CO2. 4. Change medium every 3 days. 5. When confluent, aspirate medium and wash cells twice with PBS. 6. Add 500 μL 0.25 % Trypsin-EDTA to dissociate cells from dish. 7. Incubate at 37 C for 5 min.
Hiroyuki Miwa and Takumi Era
8. Add 4.5 mL medium. 9. Transfer cells into a 15-mL centrifuge tube and spin them down at 270 g for 5 min. 10. Resuspend and seed cells on new dishes (see Note 9). 3.2.2 CFU-F Assay
1. Seed 3 102–3 hiPS-derived mesoderm cells into 1 well of Collagen IV-coated six-well plate. 2. Change medium every 3 days. 3. On day 14, wash cells twice with PBS. 4. Add 1 mL Leishman’s eosin methylene blue solution. Keep at 4 C overnight. 5. Discard Leishman’s eosin methylene blue solution and wash with water. 6. Dry up and count the number of colonies (see Note 10).
3.3 Differentiation into Descendants of Mesoderm Cells
Differentiation and confirmation can be performed in the same manner as described previously (14).
3.3.1 Induction of Bone Cells
1. Seed 1 105 hiPS-derived mesoderm cells into a well of gelatinized 24-well plate (see Note 5). 2. In the next day, change medium to 500 μL bone cell differentiation medium. 3. Change medium every 3 days. 4. The calcium deposit can be observed around on day 28.
3.3.2 Alizarin Red Staining
1. Wash cells twice with PBS. 2. Add 500 μL 4 % PFA into the well for fixture. Keep at room temperature for 10 min. 3. Wash twice with PBS. 4. Add 1 mL of Alizarin Red staining solution and keep at room temperature for 5 min. 5. Quickly wash three times with PBS. 6. Observation: calcium deposit is stained to red color.
3.3.3 Induction of Cartilage Cells
1. Resuspend 1 105 hiPS-derived mesoderm cells in 10 μL medium and put this solution on a well of 24-well plate. 2. Incubate at 37 C until cells attach to plate (1–3 h). 3. Add slowly 500 μL cartilage cell differentiation medium into the well of plate. 4. One week later, change medium to new cartilage cell differentiation medium with 10 ng/mL BMP2.
Mesoderm Differentiation from hiPS Cells
5. Change medium every 3 days. 6. Analyze the cartilage generation on day 21. 3.3.4 Alcian Blue Staining
1. Wash cells twice with PBS. 2. Add 500 μL 4 % PFA into the well for fixture. Keep at room temperature for 10 min. 3. Wash twice with PBS. 4. Add 1 mL of Alcian Blue staining solution. Keep at room temperature for 30 min. 5. Quickly wash three times with PBS. 6. Observation: mucoglycoprotein is stained to blue color.
4
Notes 1. The medium is used less than 2 week as old medium affects growth rate and differentiation potency of cells. 2. Ultralow-attachment multiwell plates (Corning) can also be used for formation of EB. 3. FBS is a critical factor to induce a high rate of PDGFRα+ cell induction. Lot no. checks of sera are highly recommended for finding appropriate serum lot. They are usually examined by the induction rate of hiPS cell-derived mesoderm cells. In general, using 20 different sera lots, the frequency of PSP cells is generated after 5 days from 10 to 20 %. 4. Avoid long-term culture. High passages (>30 passages) easily induce hiPS cells to be transformed and may lose their ability to support the in vitro hiPS cell differentiation. 5. To coat wells or dishes with gelatin before seeding cells, add 0.1 % gelatin solution into wells or dishes. Put them at room temperature for more than 5 min and aspirate gelatin solution. 6. If feeder cells remain, repeat the same progression. Or transfer supernatant into a 15-mL centrifuge tube and then put it at room temperature for 5 min. Aspirate medium and resuspend cells. 7. The proper number of days may differ from one cell line to another. At least, hiPS cells need longer differentiation than mES cells (12, 15). It is recommended to examine a rate of PSP from day 4 to 8 during induction of mesoderm cells. 8. It is recommended to check the titer of antibodies before using. The ratio in which differentiated cells can be divide into positive and negative should be decided between 1:10 and 1:1,000. 9. Differentiated cells can be maintained for more than 1 month and stored by freezing with FBS containing 10 % dimethyl sulfoxide (DMSO). When passage, split cells at 1:2–1:4.
Hiroyuki Miwa and Takumi Era
10. A colony is defined as a cluster of more than 50 cells. It is appropriate that about 50 colonies appear per well of six-well plate.
Acknowledgment This work was supported in part by grants from the Ministry of Health, Labour, and Welfare of Japan and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency. References 1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872 2. Kitagawa M, Takebe A, Ono Y, Imai T, Nakao K, Nishikawa S, Era T (2012) Phf14, a novel regulator of mesenchyme growth via plateletderived growth factor (PDGF) receptor-α. J Biol Chem 287:27983–27996 3. Hamasaki M, Hashizume Y, Yamada Y, Katayama T, Hohjoh H, Fusaki N, Nakashima Y, Furuya H, Haga N, Takami Y, Era T (2012) Pathogenic mutation of ALK2 inhibits induced pluripotent stem cell reprogramming and maintenance: mechanisms of reprogramming and strategy for drug identification. Stem Cells 30:2437–2449 4. Keller G (2005) Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev 19:1129–1155 5. Tada S, Era T, Furusawa C, Sakurai H, Nishikawa S, Kinoshita M, Nakao K, Chiba T, Nishikawa S-I (2005) Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 132:4363–4374 6. Kabrun N, Buhring HJ, Choi K, Ullrich A, Risau W, Keller G (1997) Flk-1 expression defines a population of early embryonic hematopoietic precursors. Development 124:2039–2048 7. Nishikawa SI, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama H (1998) Progressive lineage analysis by cell sorting and culture identifies FLK1 + VE-cadherin + cells at a diverging point of endothelial and hemopoietic lineages. Development 125:1747–1757
8. Yamashita J, Itoh H, Hirashima M, Ogawa M, Nishikawa S, Yurugi T, Naito M, Nakao K, Nishikawa S (2000) Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408:92–96 9. Soriano P (1997) The PDGF alpha receptor is required for neural crest cell development and for normal patterning of the somites. Development 124:2691–2700 10. Kataoka H, Takakura N, Nishikawa S, Tsuchida K, Kodama H, Kunisada T, Risau W, Kita T, Nishikawa SI (1997) Expressions of PDGF receptor alpha, c-Kit and Flk1 genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells. Dev Growth Differ 39:729–740 11. Takebe A, Era T, Okada M, Jakt LM, Kuroda Y, Nishikawa S-I (2006) Microarray analysis of PDGFR alpha + populations in ES cell differentiation culture identifies genes involved in differentiation of mesoderm and mesenchyme including ARID3b that is essential for development of embryonic mesenchymal cells. Dev Biol 293:25–37 12. Sakurai H, Era T, Jakt LM, Okada M, Nakai S, Nishikawa S, Nishikawa S-I (2006) In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility. Stem Cells 24:575–586 13. Lian Q, Lye E, Suan Yeo K, Khia Way Tan E, Salto-Tellez M, Liu TM, Palanisamy N, El Oakley RM, Lee EH, Lim B, Lim SK (2007) Derivation of clinically compliant MSCs from CD105+, CD24 differentiated human ESCs. Stem Cells 25:425–436 14. Barberi T, Willis LM, Socci ND, Studer L (2005) Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Med 2:e161
Mesoderm Differentiation from hiPS Cells 15. Xu C, Jiang J, Sottile V, McWhir J, Lebkowski J, Carpenter MK (2004) Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells 22:972–980 16. Stojkovic P, Lako M, Stewart R, Przyborski S, Armstrong L, Evans J, Murdoch A, Strachan T, Stojkovic M (2005) An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 23:306–314 17. Li F, Bronson S, Niyibizi C (2010) Derivation of murine induced pluripotent stem cells (iPS) and assessment of their differentiation toward osteogenic lineage. J Cell Biochem 109:643–652 18. Lian Q, Zhang Y, Zhang J, Zhang HK, Wu X, Zhang Y, Lam FF, Kang S, Xia JC, Lai WH, Au KW, Chow YY, Siu CW, Lee CN, Tse HF
(2010) Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice. Circulation 121:1113–1123 19. Takashima Y, Era T, Nakao K, Kondo S, Kasuga M, Smith AG, Nishikawa S (2007) Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 129:1377–1388 20. Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM, Field LJ, Keller GM (2008) Human cardiovascular progenitor cells develop from a KDR+embryonicstem-cell-derived population. Nature 453:524–528 21. Era T (2010) Mesoderm cell development from ES cells. Methods Mol Biol 636:87–103
Mesoderm Differentiation from hiPS Cells Hiroyuki Miwa and Takumi Era Abstract Human induced pluripotent stem (hiPS) cells are very attractive tools for modeling diseases and regenerative medicine. However, to achieve them, the efficient differentiation methods of hiPS cells into aimed cell type in vitro are necessary. Because mesoderm cells are useful in particular, we have developed the differentiation of mouse embryonic stem (mES) cells into mesoderm cells previously. In this time, these methods were improved for hiPS cells and now human mesoderm cells are able to be obtained efficiently. It is certain that the new methods are applicable to various studies and therapies. Keywords: Human induced pluripotent stem (hiPS) cells, Mesoderm, Vascular endothelial growth factor receptor 2 (VEGFR2), Platelet-derived growth factor receptor α (PDGFRα), Bone, Cartilage
1
Introduction Human induced pluripotent stem (hiPS) cells with pluripotency and self-renewal capability can be induced from fibroblasts or hematopoietic cells by overexpression of four factors, Oct 3/4, Sox2, Klf4, and c-Myc (1). Such hiPS cells are thus very attractive tools in terms of both basic and clinical medicine. For instance, targeted differentiation of hiPS cells into specific cells could be used in cell therapy to regenerate tissue and organ defects. In addition, inducing hiPS cells from patients and differentiating them into cells of lesional tissue could provide important cellular models of disease for basic and clinical experimentation. Furthermore, drug screening for diseases could also utilize hiPS cells differentiated from patients. However, increasing the efficiency of differentiation is paramount for realizing any or all of the proposed applications for hiPS cells. In this context, we have been investigating the differentiation of mouse embryonic stem (mES) cells and hiPS cells into mesoderm and their descendant cells, bone and cartilage, which are important in the formation of various tissues and involved in many injury and disease processes (2, 3). For the mES cells, we are using two main methods of differentiation. One is formation of the commonly used embryoid body (EB) and involves aggregating the mES cells on low-attachment dishes and then culturing with specific factors (4). The other is
Hiroyuki Miwa and Takumi Era
culturing on OP9 cells or collagen IV-coated dishes, without any factors used during the differentiation (5). In this method, dissociated mES cells are seeded and cultured with αMEM containing fetal bovine serum (FBS). After a few days, FACS analysis showed differentiated mES cells to be positive for vascular endothelial growth factor receptor 2 (VEGFR2, FLK1) and platelet-derived growth factor receptor alpha (PDGFRα), i.e., PDGFRα+VEGFR2+ (DP) cells. After another few days, the DP cells differentiated into PDGFRα+VEGFR2 (PSP) or PDGFRα VEGFR2+ (VSP) cells. The PSP is likely to be paraxial mesoderm and the VSP lateral mesoderm, because PSP cells can differentiate into cartilage and bone, while VSP differentiates into endothelial cells (6–12). In regenerative medicine, many researchers have tried to generate mesoderm and their descendant cells from hES cells (13–16). Xu et al. (15) differentiated hESCs into fibroblast-like cells, and after EB formation followed by culture on gelatin-coated plates, these cells overexpressed human telomerase reverse transcriptase (hTERT). They also expressed CD29, CD44, CD71, and CD90 and could be differentiated into an osteocytic lineage, but not chondrocytic or adipocytic lineage. Other groups also induced hESCs into fibroblastic cells that express CD90 and CD44 and can give rise to mesoderm-descendant cells (16). Such mesodermal multipotent stem cells were also purified from in vitro ES/iPS cell culture by FACS using surface markers such as CD73 and CD105 (14). In that study, gene expression analysis isolated cells expressing surface markers such as CD44 and STRO-1, as well as other markers including DSC54, neuropilin 1, hepatocyte growth factor, forkhead box D1, and notch homolog 2 (14). The culture conditions for selectively inducing descendants of mesoderm cells from ES/iPS cells were also reported, involving the culturing of hESCs with bFGF and PDGF-AB without feeder cells and then isolating the CD105+CD24 population by FACS (13). This population could then be differentiated into adipocytic, chondrocytic, and osteocytic lineages. Mouse iPS cell-derived EBs have also been cultured with TGF-β1 and retinoic acid (RA) to generate cells that are able to differentiate into osteoblasts (17). Similarly, hiPS cells were cultured with bFGF, PDGF-AB, and epidermal growth factor (EGF), followed by FACS isolation of the CD105+CD24 population to generate multipotent stem cells (18). Herein, we describe the latest method of mesoderm differentiation to generate bone and cartilage cells from hiPS cells. This was developed by combining and modifying previous methods and is expected to efficiently differentiate into mesoderm lineages (4, 5, 12, 19–21). In brief, EBs are transferred onto collagen IV-coated dishes for culturing together with various factors, including bFGF, BMP-4, and Activin A and then sorted for VEGFR2 and PDGFRα expression by FACS. The subsequent PSP contains colony-forming unit-fibroblasts (CFU-Fs) and can differentiate into bone and
Mesoderm Differentiation from hiPS Cells
Fig. 1 Differentiation of hiPS cell into mesoderm. hiPS cells formed EBs differentiate into the three types of mesoderm cells, PDGFRα+VEGFR2+ population (PDGFRα and VEGFR2 double-positive population, DP), PDGFRα+VEGFR2 population (PDGFRα single-positive population, PSP), and PDGFRα VEGFR2+ population (VEGFR2 single-positive population, VSP). The DP is the most immature and can give rise to both the PSP and the VSP. The PSP can differentiate into bone and cartilage
cartilage cells (Fig. 1), as confirmed by CFU-F assay and staining using Alizarin Red and Alcian Blue. This method for efficiently differentiating hiPS cells into mesoderm cells should be useful for many studies and therapies in this field.
2
Materials
2.1 hiPS Cell Maintenance
1. Phosphate-buffered saline without calcium and magnesium chloride (PBS). 2. Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12, Sigma-Aldrich). Store at 4 C. 3. KnockOut SR (KSR, Gibco). Store at
20 C.
4. 2-Mercaptoethanol (0.1 M, 1,000) (2-ME). Add 70 μL 2-ME to 10 mL PBS and sterilized by 0.22 μm filter. Store at 4 C. 5. L-Glutamine 200 mM (100) (Gibco). Store at 20 C. 6. Penicillin-streptomycin (100) (P/S). Store at
20 C.
7. MEM Non-Essential Amino Acid Solution (100) (NEAA, Sigma-Aldrich). Store at 4 C. 8. hiPS cell culture medium: DMEM/F12, 20 % KSR, 0.1 mM 2-ME, 2 mM L-glutamine, P/S, 0.1 mM NEAA. Store at 4 C (see Note 1). 9. Fibroblast Growth Factor (basic), Human, recombinant (rhbFGF) (Wako). Store at 20 C. 10. Trypsin 250 (Difco). Store at 4 C. 11. Collagenase Type IV (Gibco). Store at 4 C. 12. CTK solution: PBS, 20 % KSR, 0.25 % Trypsin, 1 mg/mL Collagenase, 1 mM CaCl2. Store at 20 C.
Hiroyuki Miwa and Takumi Era
2.2
Formation of EB
1. hiPS cell culture medium. 2. Recombinant Human BMP-4 (R&D Systems). Store at 20 C. 3. 100-mm Petri dish (BIO-BIK) (see Note 2).
2.3 Induction of Mesoderm Cells
1. Minimum Essential Medium Alpha Medium (αMEM, Gibco). Store at 4 C. 2. Fetal bovine serum (FBS) (see Note 3). Store at
20 C.
3. BMP-4. 4. Recombinant Human/Mouse/Rat Activin A (R&D Systems). Store at 20 C. 5. bFGF. 6. Collagen Type IV Cellware 100-mm dish (BD Biosciences). Store at 4 C. 2.4 Purification of Mesoderm Cells by FACS
1. TrypLE Express (Gibco). 2. HBSS (10) (Gibco). 3. HBSS/BSA: HBSS, 1 % bovine serum albumin (BSA). Store at 4 C. 4. Clear Back (human Fc receptor blocking reagent) (MBL). Store at 4 C. 5. Biotin antihuman CD140a (PDGFRα) (BioLegend). Store at 4 C. 6. Antihuman VEGF R2/KDR Phycoerythrin (R&D Systems). Store at 4 C. 7. Streptavidin APC (eBioscience). Store at 4 C. 8. 7-AAD Viability Dye (Beckman Coulter). Store at 4 C.
2.5 Culture of Mesoderm Cells
1. αMEM. 2. FBS. 3. 2-ME. 4. P/S 5. rhPDGF-BB (R&D Systems). Store at
20 C.
6. Mesoderm cell culture medium: αMEM, 10 % FBS, 0.1 mM 2ME, P/S, 10 ng/mL PDGF-BB. Store at 4 C. 7. 0.25 % Trypsin-EDTA (Gibco). Store at 2.6
CFU-F Assay
20 C.
1. Mesoderm cell culture medium. 2. Collagen Type IV Cellware six-well plate (BD Biosciences). Store at 4 C. 3. Leishman’s eosin methylene blue solution (Merck).
Mesoderm Differentiation from hiPS Cells
2.7 Bone Cell Differentiation from Mesoderm Cells
1. Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco). Store at 4 C. 2. FBS. 3. P/S. 4. Dexamethasone. 5. Ascorbic acid 2-phosphate. 6. β-Glycerophosphate. 7. BMP-4. 8. 24-well culture plate (BD Biosciences). 9. Bone cell differentiation medium: DMEM, 10 % FBS, P/S, 0.1 mM dexamethasone, 50 mM ascorbic acid 2-phosphate, 10 mM β-glycerophosphate, 10 ng/mL BMP-4. 10. 4 % PFA: PBS, 4 % paraformaldehyde (PFA). 11. Alizarin Red staining solution. Add 0.1 g Alizarin Red S into 10 mL water.
2.8 Cartilage Cell Differentiation from Mesoderm Cells
1. αMEM. 2. FBS. 3. P/S. 4. Dexamethasone. 5. Ascorbic acid 2-phosphate. 6. Recombinant Human TGF-β3 (R&D Systems). Store at 20 C. 7. Recombinant Human BMP-2 (R&D Systems). Store at 20 C. 8. 24-well culture plate. 9. Cartilage cell differentiation medium: αMEM, 10 % FBS, P/S, 0.1 mM dexamethasone, 170 mM ascorbic acid 2-phosphate, 10 ng/mL TGF-β3. 10. 4 % PFA. 11. Alcian Blue staining solution. Add 50 mg Alcian Blue into 10 mL 0.1 M HCl.
3
Methods
3.1 In Vitro hiPS Cell Differentiation 3.1.1 Formation of EB
In this method, hiPS cells are maintained on mitomycin C-treated mouse embryonic fibroblasts (MEFs) as feeder cells. 1. Expand hiPS cells with hiPS cell culture medium and 5 ng/mL bFGF at 37 C, 3 % CO2 (see Note 4).
Hiroyuki Miwa and Takumi Era
2. When confluent on a 100-mm dish, aspirate medium and wash cells twice with PBS. 3. Add 500 μL CTK solution to dissociate cells from dish. 4. Incubate at 37 C for 5 min. 5. Remove CTK solution and add 10 mL hiPS cell culture medium. 6. Transfer cells into a 15-mL centrifuge tube and spin them down at 20 g for 5 min. 7. Resuspend cells in 10 mL hiPS cell culture medium and transfer them on a gelatin-coated 100-mm dish (see Note 5). 8. Incubate at 37 C for 30 min to remove feeder cells (see Note 6). 9. Transfer supernatant on a 100-mm Petri dish and add BMP-4 (10 ng/mL final conc.). 10. Incubate at 37 C, 3 % CO2 overnight. 3.1.2 Induction of Mesoderm Cells
1. Transfer EBs into a 15-mL centrifuge tube and spin them down at 20 g for 5 min. 2. Resuspend EBs in 10 mL αMEM with 10 % FBS, 3 ng/mL Activin A, 10 ng/mL BMP-4, and 5 ng/mL bFGF and seed them on Collagen IV-coated dish. 3. Change medium every 2 days. 4. Culture for 5 days (see Note 7).
3.1.3 Purification of Mesoderm Cells by FACS
1. Aspirate medium and wash cells twice with PBS. 2. Add 2 mL TrypLE Express and incubate at 37 C for 5 min. 3. Add 8 mL PBS with 10 % FBS and repeat pipetting 10–15 times. 4. Transfer cells into a 15-mL centrifuge tube and spin down them at 4 C, 270 g for 5 min. 5. Resuspend 1 107 cells in 100 μL Fc receptor blocking reagent and put them at room temperature for 5 min. 6. Add 300 μL HBSS/BSA and antibody to cell suspension and put them on ice for 20 min (see Note 8). 7. Add 400 μL HBSS/BSA and spin them down at 4 C, 270 g for 5 min. 8. Resuspend 400 μL HBSS/BSA and add 1 μL SA-APC. Put them on ice for 20 min. 9. Add 400 μL HBSS/BSA and spin them down at 4 C, 270 g for 5 min.
Mesoderm Differentiation from hiPS Cells
Fig. 2 Analysis of hiPS-derived mesoderm cells by FACS. hiPS cells formed EBs were cultured on Collagen IV-coated dish with αMEM containing FBS, Activin A, BMP-4, and bFGF. After 5 days of culture, cells were dissociated and analyzed with anti-PDGFRα and anti-VEGFR2 antibodies by FACS. Four populations (PDGFRα+VEGFR2+, DP; PDGFRα+VEGFR2 , PSP; PDGFRα VEGFR2+, VSP; PDGFRα VEGFR2 , DN) were observed in differentiated hiPS cells
10. Resuspend 800 μL HBSS/BSA and spin them down at 4 C, 270 g for 5 min. 11. Resuspend 1 mL HBSS/BSA and add 5 μL 7-AAD. Filter them into a 5-mL PS tube. 12. Sort PSP cells by FACS (Fig. 2). 1. Resuspend 1 105 hiPS-derived mesoderm cells in 10 μL mesoderm cell culture medium and put this solution on a well of Collagen IV-coated 24-well plate.
3.2 Maintenance, Expansion, and Validation of Mesoderm Cells
2. Incubate at 37 C until cells attach to plate (1–3 h).
3.2.1 Culture of Mesoderm Cells
3. Add slowly 500 μL mesoderm cell culture medium into the well of plate and incubate at 37 C, 5 % CO2. 4. Change medium every 3 days. 5. When confluent, aspirate medium and wash cells twice with PBS. 6. Add 500 μL 0.25 % Trypsin-EDTA to dissociate cells from dish. 7. Incubate at 37 C for 5 min.
Hiroyuki Miwa and Takumi Era
8. Add 4.5 mL medium. 9. Transfer cells into a 15-mL centrifuge tube and spin them down at 270 g for 5 min. 10. Resuspend and seed cells on new dishes (see Note 9). 3.2.2 CFU-F Assay
1. Seed 3 102–3 hiPS-derived mesoderm cells into 1 well of Collagen IV-coated six-well plate. 2. Change medium every 3 days. 3. On day 14, wash cells twice with PBS. 4. Add 1 mL Leishman’s eosin methylene blue solution. Keep at 4 C overnight. 5. Discard Leishman’s eosin methylene blue solution and wash with water. 6. Dry up and count the number of colonies (see Note 10).
3.3 Differentiation into Descendants of Mesoderm Cells
Differentiation and confirmation can be performed in the same manner as described previously (14).
3.3.1 Induction of Bone Cells
1. Seed 1 105 hiPS-derived mesoderm cells into a well of gelatinized 24-well plate (see Note 5). 2. In the next day, change medium to 500 μL bone cell differentiation medium. 3. Change medium every 3 days. 4. The calcium deposit can be observed around on day 28.
3.3.2 Alizarin Red Staining
1. Wash cells twice with PBS. 2. Add 500 μL 4 % PFA into the well for fixture. Keep at room temperature for 10 min. 3. Wash twice with PBS. 4. Add 1 mL of Alizarin Red staining solution and keep at room temperature for 5 min. 5. Quickly wash three times with PBS. 6. Observation: calcium deposit is stained to red color.
3.3.3 Induction of Cartilage Cells
1. Resuspend 1 105 hiPS-derived mesoderm cells in 10 μL medium and put this solution on a well of 24-well plate. 2. Incubate at 37 C until cells attach to plate (1–3 h). 3. Add slowly 500 μL cartilage cell differentiation medium into the well of plate. 4. One week later, change medium to new cartilage cell differentiation medium with 10 ng/mL BMP2.
Mesoderm Differentiation from hiPS Cells
5. Change medium every 3 days. 6. Analyze the cartilage generation on day 21. 3.3.4 Alcian Blue Staining
1. Wash cells twice with PBS. 2. Add 500 μL 4 % PFA into the well for fixture. Keep at room temperature for 10 min. 3. Wash twice with PBS. 4. Add 1 mL of Alcian Blue staining solution. Keep at room temperature for 30 min. 5. Quickly wash three times with PBS. 6. Observation: mucoglycoprotein is stained to blue color.
4
Notes 1. The medium is used less than 2 week as old medium affects growth rate and differentiation potency of cells. 2. Ultralow-attachment multiwell plates (Corning) can also be used for formation of EB. 3. FBS is a critical factor to induce a high rate of PDGFRα+ cell induction. Lot no. checks of sera are highly recommended for finding appropriate serum lot. They are usually examined by the induction rate of hiPS cell-derived mesoderm cells. In general, using 20 different sera lots, the frequency of PSP cells is generated after 5 days from 10 to 20 %. 4. Avoid long-term culture. High passages (>30 passages) easily induce hiPS cells to be transformed and may lose their ability to support the in vitro hiPS cell differentiation. 5. To coat wells or dishes with gelatin before seeding cells, add 0.1 % gelatin solution into wells or dishes. Put them at room temperature for more than 5 min and aspirate gelatin solution. 6. If feeder cells remain, repeat the same progression. Or transfer supernatant into a 15-mL centrifuge tube and then put it at room temperature for 5 min. Aspirate medium and resuspend cells. 7. The proper number of days may differ from one cell line to another. At least, hiPS cells need longer differentiation than mES cells (12, 15). It is recommended to examine a rate of PSP from day 4 to 8 during induction of mesoderm cells. 8. It is recommended to check the titer of antibodies before using. The ratio in which differentiated cells can be divide into positive and negative should be decided between 1:10 and 1:1,000. 9. Differentiated cells can be maintained for more than 1 month and stored by freezing with FBS containing 10 % dimethyl sulfoxide (DMSO). When passage, split cells at 1:2–1:4.
Hiroyuki Miwa and Takumi Era
10. A colony is defined as a cluster of more than 50 cells. It is appropriate that about 50 colonies appear per well of six-well plate.
Acknowledgment This work was supported in part by grants from the Ministry of Health, Labour, and Welfare of Japan and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency. References 1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872 2. Kitagawa M, Takebe A, Ono Y, Imai T, Nakao K, Nishikawa S, Era T (2012) Phf14, a novel regulator of mesenchyme growth via plateletderived growth factor (PDGF) receptor-α. J Biol Chem 287:27983–27996 3. Hamasaki M, Hashizume Y, Yamada Y, Katayama T, Hohjoh H, Fusaki N, Nakashima Y, Furuya H, Haga N, Takami Y, Era T (2012) Pathogenic mutation of ALK2 inhibits induced pluripotent stem cell reprogramming and maintenance: mechanisms of reprogramming and strategy for drug identification. Stem Cells 30:2437–2449 4. Keller G (2005) Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev 19:1129–1155 5. Tada S, Era T, Furusawa C, Sakurai H, Nishikawa S, Kinoshita M, Nakao K, Chiba T, Nishikawa S-I (2005) Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 132:4363–4374 6. Kabrun N, Buhring HJ, Choi K, Ullrich A, Risau W, Keller G (1997) Flk-1 expression defines a population of early embryonic hematopoietic precursors. Development 124:2039–2048 7. Nishikawa SI, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama H (1998) Progressive lineage analysis by cell sorting and culture identifies FLK1 + VE-cadherin + cells at a diverging point of endothelial and hemopoietic lineages. Development 125:1747–1757
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