Methods in Molecular Biology DOI 10.1007/7651_2014_119 © Springer Science+Business Media New York 2014
Generation, Expansion, and Differentiation of Cardiovascular Progenitor Cells from Human Pluripotent Stem Cells Nan Cao, He Liang, and Huang-Tian Yang Abstract Cardiovascular progenitor cells (CVPCs) derived from human embryonic stem cells and human induced pluripotent stem cells represent an invaluable potential source for the study of early embryonic cardiovascular development and stem cell-based therapies for congenital and acquired heart diseases. To fully realize their values, it is essential to establish an efficient and stable differentiation system for the induction of these pluripotent stem cells (PSCs) into the CVPCs and robustly expand them in culture, and then further differentiate these CVPCs into multiple cardiovascular cell types. Here we describe the protocols for efficient derivation, expansion, and differentiation of CVPCs from hPSCs in a chemically defined medium under feeder- and serum-free culture conditions. Keywords: Human pluripotent stem cells, Human embryonic stem cells, Human induced pluripotent stem cells, Cardiovascular progenitor cells, Directed differentiation, Progenitor maintenance, Chemically defined medium, Cardiomyocytes, Smooth muscle cells, Endothelial cells
1
Introduction Pluripotent stem cells (PSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) possess unlimited proliferation capacity and can differentiate into derivatives of the three primary germ layers in vitro, including major cell types that form the heart (1–4). Thus they hold tremendous promise for the study of cardiac development and regenerative medicine (5, 6). Heart development is a well-organized process that involves the sequential induction of mesoderm, multipotent cardiovascular progenitor cells (CVPCs), and functional derivatives (7). The process of hPSC differentiation into cardiomyocytes (CMs) is thought to proceed through a similar hierarchy of CVPCs (4, 8). The CVPCs derived from hPSCs are definitively committed cells at earlier developmental stage than CMs and are capable of populating multiple cardiovascular lineages including CMs, smooth muscle cells (SMCs), and endothelial cells (ECs) (9–14). The in vitro
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differentiation of PSCs into CVPCs allows investigators (1) to study human early embryonic developmental processes during the differentiation of PSCs into specialized CVPCs; (2) to identify factors, reagents, and drugs that promote or suppress the formation and maintenance of CVPCs; (3) to understand the characteristics of cardiovascular progenitors and the mechanisms for their selfrenewal; and (4) to evaluate the therapeutic value of hCVPCs for myocardial regeneration. To realize these full values, methodologies for efficient generation, expansion, and differentiation of the CVPCs must be developed. In this chapter, we describe methods to efficiently induce and expand CVPCs from hPSCs. Multipotent CVPCs are generated from monolayer-cultured hESCs and hiPSCs in a chemically defined medium within 3 days by a combined treatment of bone morphogenetic protein 4 (BMP4), glycogen synthase kinase-3 (GSK3) inhibitor CHIR99021, and ascorbic acid. These CVPCs stably self-renew and expand >107 fold in feeder- and serum-free conditions when the differentiation-inducing signals from BMP, GSK3, and Activin/Nodal pathways are synchronizedly inhibited. Furthermore, the protocols are presented for the differentiation of these CVPCs into major cardiovascular lineages including CMs, SMCs, and ECs in vitro when guided by appropriate extrinsic influences.
2 2.1
Materials Cells
1. Human ESC lines H1 and H9 (WiCell). 2. hiPSC line hAFDC-iPS-36 generated from human amniotic fluid-derived cells via ectopic expression of human OCT4, SOX2, KLF4, and C-MYC (15). The use of human cell lines is subject to regulatory guidelines in related country.
2.2
Reagents
1. DMEM/F12 (Life Technologies, cat. no. 11330-057). 2. mTeSR1 (STEMCELL Technologies, cat. no. 05850). 3. RPMI1640 (Life Technologies, cat. no. 11875-119). 4. B27 supplement (Life Technologies, cat. no. 17504-044). 5. B27 supplement without vitamin A (Life Technologies, cat. no. 12587-010). 6. B27 supplement without insulin (Life Technologies, cat. no. 0050129SA). 7. Dulbecco’s PBS (D-PBS) Ca- and Mg-free (Life Technologies, cat. no. 14200067). 8. N2 supplement (Life Technologies, cat. no.17502048).
hPSC-Derived Cardiovascular Precursor Cells
9. A83-01 (Stemgent, cat. no. 04-0014). 10. Accutase (Stem Cell Technologies, cat. no. 07920). 11. Ascorbic acid (AA) (Sigma, cat. no. A4544-25G). 12. bFGF (Life Technologies, cat. no. 13256-029). 13. β-mercaptoethanol (Sigma, cat. no. M3148). 14. BMP4 (R&D Systems, cat. no. 314-BP-010). 15. CHIR99021 (Stemgent, cat. no. 04-0004). 16. Dispase (Life Technologies, cat. no. 17105041). 17. Dorsomorphin (Sigma, cat. no. p5499). 18. Growth factor-reduced matrigel (BD Biosciences, cat. no. 354277). 19. L-glutamine (Life Technologies, cat. no. 25030-081). 20. IWR1 (Calbiochem, cat. no. 681669-10MG). 21. Nonessential amino acids (Life Technologies, cat. no. 11140-050). 22. PDGF-BB (R&D Systems, cat. no. 220-BB-010). 23. Penicillin/streptomycin (Life Technologies, cat. no. 15140-122). 24. TGF-β1 (R&D Systems, cat. no. 240-B-010/CF). 25. 1-Thioglycerol (Sigma, cat. no. M6145). 26. VEGF (R&D Systems, cat. no. 493-MV-005/CF). 27. Y-27632 (Calbiochem, cat. no. 688000). 28. Anti-α-Actinin (sarcomeric) clone EA-53 (Sigma, cat. no. A7811). 29. Anti-α-SMA (Sigma, cat. no. A2547). 30. Anti-GATA4 (Santa Cruz Biotechnology, cat. no. sc-25310). 31. Anti-ISL1 (Developmental Studies Hybridoma Bank, clone 39.4D5). 32. Anti-MEF2C (Cell Signaling, cat. no. 5030). 33. Anti-MESP1/2 (Aviva ARP39374_P050).
Systems
Biology,
cat.
no.
34. Anti-PECAM1 clone 9G11 (R&D System, cat. no. BBA7) PE-conjugated Anti-SSEA1 (eBioscience, cat. no. 12-8813-42). 2.3 Cell Culture Media (See Note 1)
1. Basal CVPC induction medium (CIM) (500 mL): in a sterile environment, mix 480 mL of DMEM/F12, 10 mL of B27 supplement without vitamin A, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin, 400 μM 1-thioglycerol. 2. CIM (500 mL): in a sterile environment, mix 480 mL of DMEM/F12, 10 mL of B27 supplement without vitamin A, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin,
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400 μM 1-thioglycerol, 50 μg/mL AA, 25 ng/mL BMP4, and 3 μM GSK3 inhibitor CHIR99021. 3. Basal CVPC propagation medium (CPM) (500 mL): in a sterile environment, mix 470 mL of DMEM/F12, 10 mL of B27 supplement without vitamin A, 5 mL of N2 supplement, 5 mL of nonessential amino acids, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin, 400 μM 1-thioglycerol, 0.1 mM β-mercaptoethanol. 4. CPM (500 mL): in a sterile environment, mix 470 mL of DMEM/F12, 10 mL of B27 supplement without vitamin A, 5 mL of N2 supplement, 5 mL of nonessential amino acids, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin, 400 μM 1-thioglycerol, 0.1 mM β-mercaptoethanol, 3 μM CHIR99021, 2 μM BMP inhibitor dorsomorphin, and 0.5 μM Activin/Nodal inhibitor A83-01. 5. Cardiac differentiation medium 1 (CDM1) (500 mL): in a sterile environment, mix 480 mL of RPMI1640, 10 mL of B27 supplement without insulin, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin, 10 ng/mL BMP4, and 5 μM Wnt antagonist IWR1. 6. Cardiac differentiation medium 2 (CDM2) (500 mL): in a sterile environment, mix 480 mL of RPMI1640, 10 mL of B27 supplement, 5 mL of 200 mM L-glutamine, and 5 mL penicillin/streptomycin. 7. SMC differentiation medium (SDM) (500 mL): in a sterile environment, mix 480 mL of DMEM/F12, 10 mL of B27 supplement without vitamin A, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin, 400 μM 1-thioglycerol, 10 ng/ mL PDGF-BB, and 2 ng/mL TGF-β1. 8. EC differentiation medium (EDM) (500 mL): in a sterile environment, mix 480 mL of DMEM/F12, 10 mL of B27 supplement without vitamin A, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin, 400 μM 1-thioglycerol, 50 ng/ mL VEGF, and 10 ng/mL bFGF. 2.4
Equipment
1. Flow cytometry FACS Aria (Becton Dickinson). 2. Humidified tissue culture incubator (Thermo, 37 C, 5 % CO2). 3. Inverted TS100).
phase-contrast
microscope
(Nikon,
ECLIPSE
4. Conical tubes (15 and 50 mL; Corning Biosciences, cat. nos. 430791 and 430829). 5. Flow round-bottom tube (5 mL; BD Biosciences, cat. no. 352052).
hPSC-Derived Cardiovascular Precursor Cells
6. Microcentrifuge tube (1.5 mL; Axygen, cat. no. MCT-150-C). 7. Plates (6-, 12-, 24-, and 96-well; Nunc, cat. nos. 140675, 150628, 142475, and 165306). 8. Serological pipettes (1, 5, 10, and 25 mL; Corning, cat. nos. 4011, 4050, 4100, and 4250). 9. Stericup filtration system (Millipore, cat. no. SCGPU02RE, 250 mL). 10. Stericup filtration system (Millipore, cat. no. SCGPU05RE, 500 mL).
3
Methods
3.1 Preparation of Matrigel-Coated Plates
1. In a sterile hood, add 23 mL of cold (4 C) DMEM/F12 to a 50-mL conical tube and keep it cold by placing it on ice. 2. Remove one matrigel aliquot (2 mg) from the 80 C freezer, and add 1 mL of cold DMEM/F12 to it. Gently pipette the Matrigel solution with a P1000 tip to thaw and dissolve the Matrigel. 3. After that, immediately transfer the Matrigel solution to the 50-mL conical tube that contains 23 mL of cold DMEM/F12, mix them well with pipette, and then add Matrigel in DMEM/ F12 at a volume of 1 mL/well for 6-well plates, 0.5 mL/well for 12-well plates, 250 μL/well for 24-well plates, or 100 μL/ well for 96-well plates. 4. The Matrigel-coated plates are stored in a freezer at 4 C overnight before use and they can be stored up to 1 week (see Note 2).
3.2 Thawing and Recovering hPSCs in Feeder-Free Culture
1. Pre-warm the required volume of mTeSR1 medium at room temperature until it is no longer cool to the touch. 2. Take a Matrigel-coated 6-well plate from 4 C and put it at 37 C for at least 1 h. 3. Remove the vial of hPSCs from liquid nitrogen storage using metal forceps and immerse the vial in a 37 C water bath without submerging the cap. Swirl the vial gently. When only an ice crystal remains, remove the vial from the water bath. 4. Spray the vial with 70 % ethanol and place it in tissue culture hood. Pipet cells gently into a sterile 15-mL conical tube, add 4 mL of pre-warmed mTeSR1 medium drop-wise using a 5-mL sterile pipette. While adding the medium, gently shake the tube to mix the PSCs. This reduces osmotic shock to the cells. 5. Centrifuge the cells at 200 g for 5 min. Aspirate and discard the supernatant with a sterilized Pasteur pipette. Aspirate the liquid from the wells of the Matrigel-coated plate.
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6. Resuspend the cell pellet in 2 mL mTeSR1 medium. Slowly add 2 mL of cell suspensions into each well of the Matrigel-coated 6-well plate. Place the plate gently into the incubator. Move the plate in several quick, short, back-and-forth, and side-to-side motions to further disperse cells across the surface of the wells. 7. The next day, replace the spent medium with fresh mTeSR1 medium. 8. Replace the medium daily thereafter until the cells are approximately 80–90 % confluent (see Note 3). 3.3 Passaging hPSCs (See Note 4)
1. Pre-warm the required volume of mTeSR1 medium at room temperature until it is no longer cool to the touch. 2. Take a Matrigel-coated 6-well plate from 4 C and put it at 37 C for at least 1 h. 3. Aspirate the old medium from the vessel containing PSCs with a Pasteur pipette, and rinse the vessel with D-PBS. 4. Add 1 mL of 1 mg/mL Dispase solution (room temperature) to each well and incubate the plate at 37 C for 7 min. 5. Aspirate the Dispase solution with a Pasteur pipet. Remove the Dispase carefully without disturbing the attached cell layer. 6. Gently wash the cells with 2 mL of DMEM/F12, aspirate the medium, and repeat twice. 7. Add 2 mL pre-warmed mTeSR1 medium to the well and remove the cells from the well(s) by gently scraping them into small clusters. 8. After the cells are removed from the surface of the well, pool the contents of the well into a sterile conical tube containing 12 mL mTeSR1 medium. Gently mix 2–4 times using a 5 mL pipette and seed 2 mL of the cell suspension into each well of a Matrigel-coated 6-well plate (split ratio of 1:6 is performed here). 9. Put the plate back into the incubator. Move the plate in several quick, short, back-and-forth, and side-to-side motions to further disperse cells across the surface of the wells. 10. The next day, replace the spent medium with fresh mTeSR1 medium; change medium daily until cells are 80–90 % confluent.
3.4 Induction of CVPCs from hPSCs
1. Pre-warm the required volume of CIM at room temperature until it is no longer cool to the touch. 2. Take a Matrigel-coated plate from 4 C and put it at 37 C for at least 1 h. 3. Take hPSCs cultured on Matrigel-coated 6-well plates in mTeSR1 medium at 80–90 % confluence (see Note 5).
hPSC-Derived Cardiovascular Precursor Cells
Aspirate the medium and rinse the vessel twice with D-PBS. Add 1 mL of room-temperature Accutase to each well. Put the plate in an incubator and wait for exactly 5 min (see Note 6). 4. Add 2 mL of mTeSR1 medium into each well and pool all of the cells in a 15-mL conical tube. Count the total cell number with a hemocytometer. Centrifuge the cells at 200 g for 5 min at room temperature. 5. Aspirate the supernatant, resuspend the cells in CIM + 5 μM Y27632 at a cell density of 0.25 million cells per mL, and plate them onto Matrigel-coated culture plates at a density of 5 104 cells/cm2 (see Note 7). 6. Put the plate back into the incubator. Move the plate in several quick figure eight motions to disperse cells across the surface of the wells. This time point corresponds to day 0 of differentiation. 7. Twenty-four hours later, aspirate the old medium and then replace it with room-temperature CIM. Place the plate in a 37 C, 5 % CO2 incubator for 2 days without changing the medium. 8. The monolayer-cultivated cells were harvested at differentiation day 3 for further examination or passaging. 9. The CVPCs exhibit homogenous morphology and express several CVPC markers including SSEA1, MESP1/2, ISL1, GATA4, and MEF2C but not pluripotent markers, which can be monitored by fluorescence-activated cell sorter (FACS), immunostaining analyses, and quantitative reverse transcription PCR (Fig. 1) as previously described (12). 3.5 Propagation of CVPCs from hPSCs
1. Pre-warm the required volume of CPM at room temperature until it is no longer cool to the touch. 2. Take a Matrigel-coated 6-well plate from 4 C and put it at 37 C for at least 1 h (see Note 8). 3. When CVPCs are induced on day 3 (typically 80–90 % confluence) (see Note 9), aspirate the medium and rinse the vessel twice with D-PBS. Add 1 mL of room-temperature Accutase to each well of a 6-well plate. Place the plate in an incubator and wait for exactly 5 min. 4. Add 2 mL of CPM into each well and pool all of the cells in a 15-mL conical tube. Centrifuge the cells at 200 g for 5 min at room temperature. 5. Remove the supernatant and resuspend in 6 mL CPM + 5 μM Y27632 (see Note 10). Gently mix 5–10 times using 5 mL pipette and seed 2 mL of the cell suspension into each well of a Matrigel-coated 6-well plate (split ratio of 1:3 is performed here) (see Note 11).
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a
b CIM
CIM+Y
MESP1/2+ (%)
Cell
N
SSEA1+ (%)
Cell
N
H1
3
89.7 ± 1.9
H1
3
94.0 ± 2.3
H9
15
86.8 ± 2.2
H9
7
94.4 ± 2.4
hiPSC
3
86.5 ± 2.2
hiPSC
3
92.4 ± 4.2
0 1 3 Differentiation time (days)
c
Relative expression
d
Nuclei
MESP1/2
Merge
Nuclei
MEF2C
Merge
Nuclei
GATA4
Merge
Nuclei
ISL1
Merge
1.5 1.2 0.9 0.6 0.3 0 50 40 30 20 10 0
OCT4
SOX17
3 2 1
0
1
2
3
MESP2
0
0
1
40 20 0
1
2
3
0
2
PDGFRA
80 60
3
2.5 2.0 1.5 1.0 0.5 0 40 30
PAX6
0
1
2
3
NKX2-5
20 10 0
1
0 2 3 0 1 2 Differentiation time (days)
3
100 80 60 40 20 0 300 240 180 120 60 0
T
0
1
2
3
ISL1
0
1
2
3
Fig. 1 Induction of cardiovascular progenitor cells (CVPCs) from human PSCs. (a) An outline of the differentiation protocol. CIM, CVPC induction medium; Y, Y27632. (b) Intracellular flow cytometry analysis of induction efficiency of CVPCs in various hPSC lines. (c) Immunofluorescence analysis showing the expression of CVPC markers in cells at differentiation day 3 following CIM treatment. Scale Bars ¼ 25 μm. (d) qPCR analysis showing the downregulation of pluripotency markers and upregulation of CVPC markers during cardiovascular induction (n ¼ 3) (reproduced from (12) with permission from Cell Res)
6. Place the plate back into the CO2 incubator. Move the plate in several quick, short, back-and-forth, and side-to-side motions to further disperse cells across the surface of the wells. 7. The medium is renewed daily until cells reach 80–90 % confluence (typically 3–4 days) that is ready for passage. 8. The expanded CVPCs can actively proliferate and form roundshaped colonies with clear edge during expansion. They uniformly express primitive CVPC markers including SSEA1, MESP1/2, and ISL1 (Fig. 2) as previously described (12).
hPSC-Derived Cardiovascular Precursor Cells
b
Basal CPM
# Cells
a
H1-CVPCs 97.6
93.7
6.8
H9-CVPCs
# Cells
CPM
94.0
92.8
90.1
hiPSC-CVPCs MESP1/2
93.8
91.8
SSEA1
c
H1-CVPCs
MESP1 hiPSC-CVPCs
H9-CVPCs
Nuclei
ISL1
Nuclei
ISL1
Nuclei
ISL1
MESP1/2
Merge
MESP1/2
Merge
MESP1/2
Merge
Fig. 2 Maintenance of hPSC-derived CVPCs. (a) Phase contrast images (left panels) and percentage of MESP1/ 2+ cells analyzed by intracellular flow cytometry (right panels. CPM, CVPC propagation medium; Basal CPM, CPM without CHIR99021, dorsomorphin and A83-01). (b) Representative images showing the typical morphology (left panels), and the SSEA1 and MESP1 expression analyzed by intracellular flow cytometry (middle and right panels). The control sample used in this assay was a 1:1:1 mixture from all three types of cells examined. (c) Representative images of the ISL1 and MESP1 expression analyzed by immunostaining of CVPC colonies at passage 15. Scale bar ¼ 100 μm (reproduced from (12) with permission from Cell Res) 3.6 Differentiation of CVPCs into CMs
1. Pre-warm the required volume of CDM1 at room temperature until it is no longer cool to the touch. 2. Take a Matrigel-coated 6-well plate from 4 C and put it at 37 C for at least 1 h. 3. Aspirate the medium containing CVPCs with a Pasteur pipette and rinse the vessel twice with D-PBS. Add 1 mL of roomtemperature Accutase to each well of a 6-well plate. Place the plate in an incubator and wait for exactly 5 min. 4. Add 2 mL of CDM1 into each well and pool all of the cells in a 15-mL conical tube. Count the total cell number with a
Nan Cao et al.
hemocytometer. Centrifuge the cells at 200 g for 5 min at room temperature. 5. Aspirate the supernatant, resuspend the cells in CDM1 at a cell density of four million cells/mL and plate them onto Matrigelcoated culture plates at a density of 4 105 cells/cm2. 6. Put the plate back into the incubator. Move the plate in several quick, short, back-and-forth, and side-to-side motions to further disperse cells across the surface of the wells. 7. Cultivate the cells for 3 days without changing the medium. 8. Three days later, aspirate the CDM1 medium and add 3 mL of room-temperature CDM2 medium to each well of a 6-well plate. 9. Cultivate the cells in CDM2 for another 9 days. Medium is renewed every 2–3 days. 10. After 12 days of differentiation, majority of the cells should exhibit hallmarks of CMs including spontaneous beating and cardiac-specific marker expression (Fig. 3) as previously described (12). 3.7 Differentiation of CVPCs into SMCs or ECs
1. Pre-warm the required volume of SDM or EDM at room temperature until they are no longer cool to the touch. 2. Take Matrigel-coated 6-well plates from 4 C and put them at 37 C for at least 1 h. 3. Aspirate the medium containing CVPCs with a Pasteur pipette and rinse the vessel twice with D-PBS. Add 1 mL of roomtemperature Accutase to each well of a 6-well plate. Put the plate in the incubator and wait for exactly 5 min. 4. Add 2 mL of SDM or EDM into each well and pool all of the cells in a 15-mL conical tube. Count the total cell number with a hemocytometer. Centrifuge the cells at 200 g for 5 min at room temperature. 5. Aspirate the supernatant, resuspend the cells in SDM or EDM at a cell density of 0.1 million cells/mL, and plate them onto Matrigel-coated culture plates at a density of 104 cells/cm2 in SDM or EDM respectively. 6. Put the plate back into the incubator. Move the dish in several quick figure eight motions to disperse cells across the surface of the wells. 7. Cultivate the cells for 12 days. Medium is renewed every 2–3 days. 8. After 12 days of differentiation, majority of the cells should express SMC marker α-SMA or EC marker PECAM1 respectively (Fig. 3) as previously described (12).
hPSC-Derived Cardiovascular Precursor Cells
PDGF-BB+TGF β 1 in Basal CIM
Smooth muscles
VEGF+FGF2 in Basal CIM
Endothelial cells 0
b
CDM2
BMP4+IWR1 in CDM1
Cardiomyocytes
3 Differentiation time (days)
12
cTNT
Nuclei/a-Actinin
NKX2-5
Merge
Nuclei/cTNT
Smooth muscles
Cardiomyocytes
Nuclei
Endothelial cells
a
Nuclei/a-SMA
Nuclei/SM-MHC
Nuclei/PECAM1
Nuclei/CDH5/CD34
Fig. 3 In vitro differentiation potential of CVPCs. (a) An outline of the conditions for inducing CVPC differentiation. (b) Differentiation potential of P15 CVPCs into cardiomyocytes, smooth muscle cells, and endothelial cells determined by immunostaining analyses for Nkx2-5 (green), cTnT (red or green), α-actinin (red) and merged Nkx-2.5 with cTnT in cardiomyocytes, α-SMA (red) and SM-MHC (green) in smooth muscle cells, and PECAM1 (red), CDH5 (red), and CD34 (green) in endothelial cells derived from CVPCs at differentiation day 12. Scale Bars ¼ 50 μm (reproduced from (12) with permission from Cell Res)
4
Notes 1. All the medium used in this protocol can be stored at 70 C for more than 3 months, but they are not stable in 4 C and should be used immediately. 2. Different batch of Matrigel may affect the induction and maintenance of CVPCs and should be tested before use. 3. Never let the hPSC colonies reach confluence and they may start to differentiate. 4. Pluripotent stem cell culture can be maintained by repeating passage before moving to the next step for CVPC induction. The induction efficiency of CVPCs is not significantly changed by using hPSCs with passages range from 20 to 60. 5. HPSCs are cultured on Madrigal in mTeSR1 for at least two passages. The homogeneous maintenance of hESCs in undifferentiated state is critical for the efficient induction of CVPCs.
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6. Incubation should be carried out for exactly 5 min under the hood. This is important to digest the hPSCs into single cells that is critical for the efficient CVPC without decreasing cell variability. 7. ROCK inhibitor Y27632 at 5 μM is helpful for increasing cell viability after thawing but it is better not over 24 h. 8. Different batch of Madrigal may affect the propagation efficiency of CVPCs and should be tested before use. 9. The high efficiency in CVPC induction is critical for their selfrenewal and propagation. Make sure the expression of CVPC markers in majority of the cells (>90 %) before long-term expansion (12). If not, the CVPCs can be sorted out using anti-SSEA-1 antibody-conjugated magnetic beads as previously described (11, 16). 10. During the initial five passages, overnight treatment of 5 μM Y27632 is used to improve cell survival, but it is not required in the following passages. 11. Split ratio for the CVPCs can be variable (1:2–1:5) between lines and passage times. A general rule is to observe the last split ratio and adjust the ratio according to the appearance of the CVPC colonies. If the cells are actively proliferating, increase the split ratio.
Acknowledgments The work was supported by grants of the Strategic Priority Research Program of CAS (XDA01020204), National Natural Science Foundation of China (31030050), National Basic Research Program of China (2011CB965300), National Science and Technology Project of China (2012ZX09501-001-001), and CAS (GJHZ1225). We are grateful to WiCell Research Institute for providing us H1 and H9 hESC lines and to Dr. Ying Jin (Institute of Health Sciences, China) for kindly providing the hiPSC line hAFDC-iPS-36. We also thank the members from our laboratory for valuable discussions. References 1. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147 2. Takahashi K, Tanabe K, Ohnuki M et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872
3. Park IH, Lerou PH, Zhao R et al (2008) Generation of human-induced pluripotent stem cells. Nat Protoc 3:1180–1186 4. Burridge PW, Keller G, Gold JD et al (2012) Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell 10:16–28
hPSC-Derived Cardiovascular Precursor Cells 5. Murry CE, Keller G (2008) Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132:661–680 6. Blin G, Neri T, Stefanovic S et al (2010) Human embryonic and induced pluripotent stem cells in basic and clinical research in cardiology. Curr Stem Cell Res Ther 5:215–226 7. Noseda M, Peterkin T, Simoes FC et al (2011) Cardiopoietic factors: extracellular signals for cardiac lineage commitment. Circ Res 108:129–152 8. Mummery CL, Zhang J, Ng ES et al (2012) Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circ Res 111:344–358 9. Bu L, Jiang X, Martin-Puig S et al (2009) Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature 460:113–117 10. Yang L, Soonpaa MH, Adler ED et al (2008) Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453:524–528 11. Blin G, Nury D, Stefanovic S et al (2010) A purified population of multipotent
cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates. J Clin Invest 120:1125–1139 12. Cao N, Liang H, Huang J et al (2013) Highly efficient induction and long-term maintenance of multipotent cardiovascular progenitors from human pluripotent stem cells under defined conditions. Cell Res 23:1119–1132 13. Elliott DA, Braam SR, Koutsis K et al (2011) NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat Methods 8:1037–1040 14. Kattman SJ, Witty AD, Gagliardi M et al (2011) Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8:228–240 15. Li C, Zhou J, Shi G et al (2009) Pluripotency can be rapidly and efficiently induced in human amniotic fluid-derived cells. Hum Mol Genet 18:4340–4349 16. Leschik J, Stefanovic S, Brinon B et al (2008) Cardiac commitment of primate embryonic stem cells. Nat Protoc 3:1381–1387
Generation, Expansion, and Differentiation of Cardiovascular Progenitor Cells from Human Pluripotent Stem Cells Nan Cao, He Liang, and Huang-Tian Yang Abstract Cardiovascular progenitor cells (CVPCs) derived from human embryonic stem cells and human induced pluripotent stem cells represent an invaluable potential source for the study of early embryonic cardiovascular development and stem cell-based therapies for congenital and acquired heart diseases. To fully realize their values, it is essential to establish an efficient and stable differentiation system for the induction of these pluripotent stem cells (PSCs) into the CVPCs and robustly expand them in culture, and then further differentiate these CVPCs into multiple cardiovascular cell types. Here we describe the protocols for efficient derivation, expansion, and differentiation of CVPCs from hPSCs in a chemically defined medium under feeder- and serum-free culture conditions. Keywords: Human pluripotent stem cells, Human embryonic stem cells, Human induced pluripotent stem cells, Cardiovascular progenitor cells, Directed differentiation, Progenitor maintenance, Chemically defined medium, Cardiomyocytes, Smooth muscle cells, Endothelial cells
1
Introduction Pluripotent stem cells (PSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) possess unlimited proliferation capacity and can differentiate into derivatives of the three primary germ layers in vitro, including major cell types that form the heart (1–4). Thus they hold tremendous promise for the study of cardiac development and regenerative medicine (5, 6). Heart development is a well-organized process that involves the sequential induction of mesoderm, multipotent cardiovascular progenitor cells (CVPCs), and functional derivatives (7). The process of hPSC differentiation into cardiomyocytes (CMs) is thought to proceed through a similar hierarchy of CVPCs (4, 8). The CVPCs derived from hPSCs are definitively committed cells at earlier developmental stage than CMs and are capable of populating multiple cardiovascular lineages including CMs, smooth muscle cells (SMCs), and endothelial cells (ECs) (9–14). The in vitro
Nan Cao et al.
differentiation of PSCs into CVPCs allows investigators (1) to study human early embryonic developmental processes during the differentiation of PSCs into specialized CVPCs; (2) to identify factors, reagents, and drugs that promote or suppress the formation and maintenance of CVPCs; (3) to understand the characteristics of cardiovascular progenitors and the mechanisms for their selfrenewal; and (4) to evaluate the therapeutic value of hCVPCs for myocardial regeneration. To realize these full values, methodologies for efficient generation, expansion, and differentiation of the CVPCs must be developed. In this chapter, we describe methods to efficiently induce and expand CVPCs from hPSCs. Multipotent CVPCs are generated from monolayer-cultured hESCs and hiPSCs in a chemically defined medium within 3 days by a combined treatment of bone morphogenetic protein 4 (BMP4), glycogen synthase kinase-3 (GSK3) inhibitor CHIR99021, and ascorbic acid. These CVPCs stably self-renew and expand >107 fold in feeder- and serum-free conditions when the differentiation-inducing signals from BMP, GSK3, and Activin/Nodal pathways are synchronizedly inhibited. Furthermore, the protocols are presented for the differentiation of these CVPCs into major cardiovascular lineages including CMs, SMCs, and ECs in vitro when guided by appropriate extrinsic influences.
2 2.1
Materials Cells
1. Human ESC lines H1 and H9 (WiCell). 2. hiPSC line hAFDC-iPS-36 generated from human amniotic fluid-derived cells via ectopic expression of human OCT4, SOX2, KLF4, and C-MYC (15). The use of human cell lines is subject to regulatory guidelines in related country.
2.2
Reagents
1. DMEM/F12 (Life Technologies, cat. no. 11330-057). 2. mTeSR1 (STEMCELL Technologies, cat. no. 05850). 3. RPMI1640 (Life Technologies, cat. no. 11875-119). 4. B27 supplement (Life Technologies, cat. no. 17504-044). 5. B27 supplement without vitamin A (Life Technologies, cat. no. 12587-010). 6. B27 supplement without insulin (Life Technologies, cat. no. 0050129SA). 7. Dulbecco’s PBS (D-PBS) Ca- and Mg-free (Life Technologies, cat. no. 14200067). 8. N2 supplement (Life Technologies, cat. no.17502048).
hPSC-Derived Cardiovascular Precursor Cells
9. A83-01 (Stemgent, cat. no. 04-0014). 10. Accutase (Stem Cell Technologies, cat. no. 07920). 11. Ascorbic acid (AA) (Sigma, cat. no. A4544-25G). 12. bFGF (Life Technologies, cat. no. 13256-029). 13. β-mercaptoethanol (Sigma, cat. no. M3148). 14. BMP4 (R&D Systems, cat. no. 314-BP-010). 15. CHIR99021 (Stemgent, cat. no. 04-0004). 16. Dispase (Life Technologies, cat. no. 17105041). 17. Dorsomorphin (Sigma, cat. no. p5499). 18. Growth factor-reduced matrigel (BD Biosciences, cat. no. 354277). 19. L-glutamine (Life Technologies, cat. no. 25030-081). 20. IWR1 (Calbiochem, cat. no. 681669-10MG). 21. Nonessential amino acids (Life Technologies, cat. no. 11140-050). 22. PDGF-BB (R&D Systems, cat. no. 220-BB-010). 23. Penicillin/streptomycin (Life Technologies, cat. no. 15140-122). 24. TGF-β1 (R&D Systems, cat. no. 240-B-010/CF). 25. 1-Thioglycerol (Sigma, cat. no. M6145). 26. VEGF (R&D Systems, cat. no. 493-MV-005/CF). 27. Y-27632 (Calbiochem, cat. no. 688000). 28. Anti-α-Actinin (sarcomeric) clone EA-53 (Sigma, cat. no. A7811). 29. Anti-α-SMA (Sigma, cat. no. A2547). 30. Anti-GATA4 (Santa Cruz Biotechnology, cat. no. sc-25310). 31. Anti-ISL1 (Developmental Studies Hybridoma Bank, clone 39.4D5). 32. Anti-MEF2C (Cell Signaling, cat. no. 5030). 33. Anti-MESP1/2 (Aviva ARP39374_P050).
Systems
Biology,
cat.
no.
34. Anti-PECAM1 clone 9G11 (R&D System, cat. no. BBA7) PE-conjugated Anti-SSEA1 (eBioscience, cat. no. 12-8813-42). 2.3 Cell Culture Media (See Note 1)
1. Basal CVPC induction medium (CIM) (500 mL): in a sterile environment, mix 480 mL of DMEM/F12, 10 mL of B27 supplement without vitamin A, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin, 400 μM 1-thioglycerol. 2. CIM (500 mL): in a sterile environment, mix 480 mL of DMEM/F12, 10 mL of B27 supplement without vitamin A, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin,
Nan Cao et al.
400 μM 1-thioglycerol, 50 μg/mL AA, 25 ng/mL BMP4, and 3 μM GSK3 inhibitor CHIR99021. 3. Basal CVPC propagation medium (CPM) (500 mL): in a sterile environment, mix 470 mL of DMEM/F12, 10 mL of B27 supplement without vitamin A, 5 mL of N2 supplement, 5 mL of nonessential amino acids, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin, 400 μM 1-thioglycerol, 0.1 mM β-mercaptoethanol. 4. CPM (500 mL): in a sterile environment, mix 470 mL of DMEM/F12, 10 mL of B27 supplement without vitamin A, 5 mL of N2 supplement, 5 mL of nonessential amino acids, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin, 400 μM 1-thioglycerol, 0.1 mM β-mercaptoethanol, 3 μM CHIR99021, 2 μM BMP inhibitor dorsomorphin, and 0.5 μM Activin/Nodal inhibitor A83-01. 5. Cardiac differentiation medium 1 (CDM1) (500 mL): in a sterile environment, mix 480 mL of RPMI1640, 10 mL of B27 supplement without insulin, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin, 10 ng/mL BMP4, and 5 μM Wnt antagonist IWR1. 6. Cardiac differentiation medium 2 (CDM2) (500 mL): in a sterile environment, mix 480 mL of RPMI1640, 10 mL of B27 supplement, 5 mL of 200 mM L-glutamine, and 5 mL penicillin/streptomycin. 7. SMC differentiation medium (SDM) (500 mL): in a sterile environment, mix 480 mL of DMEM/F12, 10 mL of B27 supplement without vitamin A, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin, 400 μM 1-thioglycerol, 10 ng/ mL PDGF-BB, and 2 ng/mL TGF-β1. 8. EC differentiation medium (EDM) (500 mL): in a sterile environment, mix 480 mL of DMEM/F12, 10 mL of B27 supplement without vitamin A, 5 mL of 200 mM L-glutamine, 5 mL penicillin/streptomycin, 400 μM 1-thioglycerol, 50 ng/ mL VEGF, and 10 ng/mL bFGF. 2.4
Equipment
1. Flow cytometry FACS Aria (Becton Dickinson). 2. Humidified tissue culture incubator (Thermo, 37 C, 5 % CO2). 3. Inverted TS100).
phase-contrast
microscope
(Nikon,
ECLIPSE
4. Conical tubes (15 and 50 mL; Corning Biosciences, cat. nos. 430791 and 430829). 5. Flow round-bottom tube (5 mL; BD Biosciences, cat. no. 352052).
hPSC-Derived Cardiovascular Precursor Cells
6. Microcentrifuge tube (1.5 mL; Axygen, cat. no. MCT-150-C). 7. Plates (6-, 12-, 24-, and 96-well; Nunc, cat. nos. 140675, 150628, 142475, and 165306). 8. Serological pipettes (1, 5, 10, and 25 mL; Corning, cat. nos. 4011, 4050, 4100, and 4250). 9. Stericup filtration system (Millipore, cat. no. SCGPU02RE, 250 mL). 10. Stericup filtration system (Millipore, cat. no. SCGPU05RE, 500 mL).
3
Methods
3.1 Preparation of Matrigel-Coated Plates
1. In a sterile hood, add 23 mL of cold (4 C) DMEM/F12 to a 50-mL conical tube and keep it cold by placing it on ice. 2. Remove one matrigel aliquot (2 mg) from the 80 C freezer, and add 1 mL of cold DMEM/F12 to it. Gently pipette the Matrigel solution with a P1000 tip to thaw and dissolve the Matrigel. 3. After that, immediately transfer the Matrigel solution to the 50-mL conical tube that contains 23 mL of cold DMEM/F12, mix them well with pipette, and then add Matrigel in DMEM/ F12 at a volume of 1 mL/well for 6-well plates, 0.5 mL/well for 12-well plates, 250 μL/well for 24-well plates, or 100 μL/ well for 96-well plates. 4. The Matrigel-coated plates are stored in a freezer at 4 C overnight before use and they can be stored up to 1 week (see Note 2).
3.2 Thawing and Recovering hPSCs in Feeder-Free Culture
1. Pre-warm the required volume of mTeSR1 medium at room temperature until it is no longer cool to the touch. 2. Take a Matrigel-coated 6-well plate from 4 C and put it at 37 C for at least 1 h. 3. Remove the vial of hPSCs from liquid nitrogen storage using metal forceps and immerse the vial in a 37 C water bath without submerging the cap. Swirl the vial gently. When only an ice crystal remains, remove the vial from the water bath. 4. Spray the vial with 70 % ethanol and place it in tissue culture hood. Pipet cells gently into a sterile 15-mL conical tube, add 4 mL of pre-warmed mTeSR1 medium drop-wise using a 5-mL sterile pipette. While adding the medium, gently shake the tube to mix the PSCs. This reduces osmotic shock to the cells. 5. Centrifuge the cells at 200 g for 5 min. Aspirate and discard the supernatant with a sterilized Pasteur pipette. Aspirate the liquid from the wells of the Matrigel-coated plate.
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6. Resuspend the cell pellet in 2 mL mTeSR1 medium. Slowly add 2 mL of cell suspensions into each well of the Matrigel-coated 6-well plate. Place the plate gently into the incubator. Move the plate in several quick, short, back-and-forth, and side-to-side motions to further disperse cells across the surface of the wells. 7. The next day, replace the spent medium with fresh mTeSR1 medium. 8. Replace the medium daily thereafter until the cells are approximately 80–90 % confluent (see Note 3). 3.3 Passaging hPSCs (See Note 4)
1. Pre-warm the required volume of mTeSR1 medium at room temperature until it is no longer cool to the touch. 2. Take a Matrigel-coated 6-well plate from 4 C and put it at 37 C for at least 1 h. 3. Aspirate the old medium from the vessel containing PSCs with a Pasteur pipette, and rinse the vessel with D-PBS. 4. Add 1 mL of 1 mg/mL Dispase solution (room temperature) to each well and incubate the plate at 37 C for 7 min. 5. Aspirate the Dispase solution with a Pasteur pipet. Remove the Dispase carefully without disturbing the attached cell layer. 6. Gently wash the cells with 2 mL of DMEM/F12, aspirate the medium, and repeat twice. 7. Add 2 mL pre-warmed mTeSR1 medium to the well and remove the cells from the well(s) by gently scraping them into small clusters. 8. After the cells are removed from the surface of the well, pool the contents of the well into a sterile conical tube containing 12 mL mTeSR1 medium. Gently mix 2–4 times using a 5 mL pipette and seed 2 mL of the cell suspension into each well of a Matrigel-coated 6-well plate (split ratio of 1:6 is performed here). 9. Put the plate back into the incubator. Move the plate in several quick, short, back-and-forth, and side-to-side motions to further disperse cells across the surface of the wells. 10. The next day, replace the spent medium with fresh mTeSR1 medium; change medium daily until cells are 80–90 % confluent.
3.4 Induction of CVPCs from hPSCs
1. Pre-warm the required volume of CIM at room temperature until it is no longer cool to the touch. 2. Take a Matrigel-coated plate from 4 C and put it at 37 C for at least 1 h. 3. Take hPSCs cultured on Matrigel-coated 6-well plates in mTeSR1 medium at 80–90 % confluence (see Note 5).
hPSC-Derived Cardiovascular Precursor Cells
Aspirate the medium and rinse the vessel twice with D-PBS. Add 1 mL of room-temperature Accutase to each well. Put the plate in an incubator and wait for exactly 5 min (see Note 6). 4. Add 2 mL of mTeSR1 medium into each well and pool all of the cells in a 15-mL conical tube. Count the total cell number with a hemocytometer. Centrifuge the cells at 200 g for 5 min at room temperature. 5. Aspirate the supernatant, resuspend the cells in CIM + 5 μM Y27632 at a cell density of 0.25 million cells per mL, and plate them onto Matrigel-coated culture plates at a density of 5 104 cells/cm2 (see Note 7). 6. Put the plate back into the incubator. Move the plate in several quick figure eight motions to disperse cells across the surface of the wells. This time point corresponds to day 0 of differentiation. 7. Twenty-four hours later, aspirate the old medium and then replace it with room-temperature CIM. Place the plate in a 37 C, 5 % CO2 incubator for 2 days without changing the medium. 8. The monolayer-cultivated cells were harvested at differentiation day 3 for further examination or passaging. 9. The CVPCs exhibit homogenous morphology and express several CVPC markers including SSEA1, MESP1/2, ISL1, GATA4, and MEF2C but not pluripotent markers, which can be monitored by fluorescence-activated cell sorter (FACS), immunostaining analyses, and quantitative reverse transcription PCR (Fig. 1) as previously described (12). 3.5 Propagation of CVPCs from hPSCs
1. Pre-warm the required volume of CPM at room temperature until it is no longer cool to the touch. 2. Take a Matrigel-coated 6-well plate from 4 C and put it at 37 C for at least 1 h (see Note 8). 3. When CVPCs are induced on day 3 (typically 80–90 % confluence) (see Note 9), aspirate the medium and rinse the vessel twice with D-PBS. Add 1 mL of room-temperature Accutase to each well of a 6-well plate. Place the plate in an incubator and wait for exactly 5 min. 4. Add 2 mL of CPM into each well and pool all of the cells in a 15-mL conical tube. Centrifuge the cells at 200 g for 5 min at room temperature. 5. Remove the supernatant and resuspend in 6 mL CPM + 5 μM Y27632 (see Note 10). Gently mix 5–10 times using 5 mL pipette and seed 2 mL of the cell suspension into each well of a Matrigel-coated 6-well plate (split ratio of 1:3 is performed here) (see Note 11).
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a
b CIM
CIM+Y
MESP1/2+ (%)
Cell
N
SSEA1+ (%)
Cell
N
H1
3
89.7 ± 1.9
H1
3
94.0 ± 2.3
H9
15
86.8 ± 2.2
H9
7
94.4 ± 2.4
hiPSC
3
86.5 ± 2.2
hiPSC
3
92.4 ± 4.2
0 1 3 Differentiation time (days)
c
Relative expression
d
Nuclei
MESP1/2
Merge
Nuclei
MEF2C
Merge
Nuclei
GATA4
Merge
Nuclei
ISL1
Merge
1.5 1.2 0.9 0.6 0.3 0 50 40 30 20 10 0
OCT4
SOX17
3 2 1
0
1
2
3
MESP2
0
0
1
40 20 0
1
2
3
0
2
PDGFRA
80 60
3
2.5 2.0 1.5 1.0 0.5 0 40 30
PAX6
0
1
2
3
NKX2-5
20 10 0
1
0 2 3 0 1 2 Differentiation time (days)
3
100 80 60 40 20 0 300 240 180 120 60 0
T
0
1
2
3
ISL1
0
1
2
3
Fig. 1 Induction of cardiovascular progenitor cells (CVPCs) from human PSCs. (a) An outline of the differentiation protocol. CIM, CVPC induction medium; Y, Y27632. (b) Intracellular flow cytometry analysis of induction efficiency of CVPCs in various hPSC lines. (c) Immunofluorescence analysis showing the expression of CVPC markers in cells at differentiation day 3 following CIM treatment. Scale Bars ¼ 25 μm. (d) qPCR analysis showing the downregulation of pluripotency markers and upregulation of CVPC markers during cardiovascular induction (n ¼ 3) (reproduced from (12) with permission from Cell Res)
6. Place the plate back into the CO2 incubator. Move the plate in several quick, short, back-and-forth, and side-to-side motions to further disperse cells across the surface of the wells. 7. The medium is renewed daily until cells reach 80–90 % confluence (typically 3–4 days) that is ready for passage. 8. The expanded CVPCs can actively proliferate and form roundshaped colonies with clear edge during expansion. They uniformly express primitive CVPC markers including SSEA1, MESP1/2, and ISL1 (Fig. 2) as previously described (12).
hPSC-Derived Cardiovascular Precursor Cells
b
Basal CPM
# Cells
a
H1-CVPCs 97.6
93.7
6.8
H9-CVPCs
# Cells
CPM
94.0
92.8
90.1
hiPSC-CVPCs MESP1/2
93.8
91.8
SSEA1
c
H1-CVPCs
MESP1 hiPSC-CVPCs
H9-CVPCs
Nuclei
ISL1
Nuclei
ISL1
Nuclei
ISL1
MESP1/2
Merge
MESP1/2
Merge
MESP1/2
Merge
Fig. 2 Maintenance of hPSC-derived CVPCs. (a) Phase contrast images (left panels) and percentage of MESP1/ 2+ cells analyzed by intracellular flow cytometry (right panels. CPM, CVPC propagation medium; Basal CPM, CPM without CHIR99021, dorsomorphin and A83-01). (b) Representative images showing the typical morphology (left panels), and the SSEA1 and MESP1 expression analyzed by intracellular flow cytometry (middle and right panels). The control sample used in this assay was a 1:1:1 mixture from all three types of cells examined. (c) Representative images of the ISL1 and MESP1 expression analyzed by immunostaining of CVPC colonies at passage 15. Scale bar ¼ 100 μm (reproduced from (12) with permission from Cell Res) 3.6 Differentiation of CVPCs into CMs
1. Pre-warm the required volume of CDM1 at room temperature until it is no longer cool to the touch. 2. Take a Matrigel-coated 6-well plate from 4 C and put it at 37 C for at least 1 h. 3. Aspirate the medium containing CVPCs with a Pasteur pipette and rinse the vessel twice with D-PBS. Add 1 mL of roomtemperature Accutase to each well of a 6-well plate. Place the plate in an incubator and wait for exactly 5 min. 4. Add 2 mL of CDM1 into each well and pool all of the cells in a 15-mL conical tube. Count the total cell number with a
Nan Cao et al.
hemocytometer. Centrifuge the cells at 200 g for 5 min at room temperature. 5. Aspirate the supernatant, resuspend the cells in CDM1 at a cell density of four million cells/mL and plate them onto Matrigelcoated culture plates at a density of 4 105 cells/cm2. 6. Put the plate back into the incubator. Move the plate in several quick, short, back-and-forth, and side-to-side motions to further disperse cells across the surface of the wells. 7. Cultivate the cells for 3 days without changing the medium. 8. Three days later, aspirate the CDM1 medium and add 3 mL of room-temperature CDM2 medium to each well of a 6-well plate. 9. Cultivate the cells in CDM2 for another 9 days. Medium is renewed every 2–3 days. 10. After 12 days of differentiation, majority of the cells should exhibit hallmarks of CMs including spontaneous beating and cardiac-specific marker expression (Fig. 3) as previously described (12). 3.7 Differentiation of CVPCs into SMCs or ECs
1. Pre-warm the required volume of SDM or EDM at room temperature until they are no longer cool to the touch. 2. Take Matrigel-coated 6-well plates from 4 C and put them at 37 C for at least 1 h. 3. Aspirate the medium containing CVPCs with a Pasteur pipette and rinse the vessel twice with D-PBS. Add 1 mL of roomtemperature Accutase to each well of a 6-well plate. Put the plate in the incubator and wait for exactly 5 min. 4. Add 2 mL of SDM or EDM into each well and pool all of the cells in a 15-mL conical tube. Count the total cell number with a hemocytometer. Centrifuge the cells at 200 g for 5 min at room temperature. 5. Aspirate the supernatant, resuspend the cells in SDM or EDM at a cell density of 0.1 million cells/mL, and plate them onto Matrigel-coated culture plates at a density of 104 cells/cm2 in SDM or EDM respectively. 6. Put the plate back into the incubator. Move the dish in several quick figure eight motions to disperse cells across the surface of the wells. 7. Cultivate the cells for 12 days. Medium is renewed every 2–3 days. 8. After 12 days of differentiation, majority of the cells should express SMC marker α-SMA or EC marker PECAM1 respectively (Fig. 3) as previously described (12).
hPSC-Derived Cardiovascular Precursor Cells
PDGF-BB+TGF β 1 in Basal CIM
Smooth muscles
VEGF+FGF2 in Basal CIM
Endothelial cells 0
b
CDM2
BMP4+IWR1 in CDM1
Cardiomyocytes
3 Differentiation time (days)
12
cTNT
Nuclei/a-Actinin
NKX2-5
Merge
Nuclei/cTNT
Smooth muscles
Cardiomyocytes
Nuclei
Endothelial cells
a
Nuclei/a-SMA
Nuclei/SM-MHC
Nuclei/PECAM1
Nuclei/CDH5/CD34
Fig. 3 In vitro differentiation potential of CVPCs. (a) An outline of the conditions for inducing CVPC differentiation. (b) Differentiation potential of P15 CVPCs into cardiomyocytes, smooth muscle cells, and endothelial cells determined by immunostaining analyses for Nkx2-5 (green), cTnT (red or green), α-actinin (red) and merged Nkx-2.5 with cTnT in cardiomyocytes, α-SMA (red) and SM-MHC (green) in smooth muscle cells, and PECAM1 (red), CDH5 (red), and CD34 (green) in endothelial cells derived from CVPCs at differentiation day 12. Scale Bars ¼ 50 μm (reproduced from (12) with permission from Cell Res)
4
Notes 1. All the medium used in this protocol can be stored at 70 C for more than 3 months, but they are not stable in 4 C and should be used immediately. 2. Different batch of Matrigel may affect the induction and maintenance of CVPCs and should be tested before use. 3. Never let the hPSC colonies reach confluence and they may start to differentiate. 4. Pluripotent stem cell culture can be maintained by repeating passage before moving to the next step for CVPC induction. The induction efficiency of CVPCs is not significantly changed by using hPSCs with passages range from 20 to 60. 5. HPSCs are cultured on Madrigal in mTeSR1 for at least two passages. The homogeneous maintenance of hESCs in undifferentiated state is critical for the efficient induction of CVPCs.
Nan Cao et al.
6. Incubation should be carried out for exactly 5 min under the hood. This is important to digest the hPSCs into single cells that is critical for the efficient CVPC without decreasing cell variability. 7. ROCK inhibitor Y27632 at 5 μM is helpful for increasing cell viability after thawing but it is better not over 24 h. 8. Different batch of Madrigal may affect the propagation efficiency of CVPCs and should be tested before use. 9. The high efficiency in CVPC induction is critical for their selfrenewal and propagation. Make sure the expression of CVPC markers in majority of the cells (>90 %) before long-term expansion (12). If not, the CVPCs can be sorted out using anti-SSEA-1 antibody-conjugated magnetic beads as previously described (11, 16). 10. During the initial five passages, overnight treatment of 5 μM Y27632 is used to improve cell survival, but it is not required in the following passages. 11. Split ratio for the CVPCs can be variable (1:2–1:5) between lines and passage times. A general rule is to observe the last split ratio and adjust the ratio according to the appearance of the CVPC colonies. If the cells are actively proliferating, increase the split ratio.
Acknowledgments The work was supported by grants of the Strategic Priority Research Program of CAS (XDA01020204), National Natural Science Foundation of China (31030050), National Basic Research Program of China (2011CB965300), National Science and Technology Project of China (2012ZX09501-001-001), and CAS (GJHZ1225). We are grateful to WiCell Research Institute for providing us H1 and H9 hESC lines and to Dr. Ying Jin (Institute of Health Sciences, China) for kindly providing the hiPSC line hAFDC-iPS-36. We also thank the members from our laboratory for valuable discussions. References 1. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147 2. Takahashi K, Tanabe K, Ohnuki M et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872
3. Park IH, Lerou PH, Zhao R et al (2008) Generation of human-induced pluripotent stem cells. Nat Protoc 3:1180–1186 4. Burridge PW, Keller G, Gold JD et al (2012) Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell 10:16–28
hPSC-Derived Cardiovascular Precursor Cells 5. Murry CE, Keller G (2008) Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132:661–680 6. Blin G, Neri T, Stefanovic S et al (2010) Human embryonic and induced pluripotent stem cells in basic and clinical research in cardiology. Curr Stem Cell Res Ther 5:215–226 7. Noseda M, Peterkin T, Simoes FC et al (2011) Cardiopoietic factors: extracellular signals for cardiac lineage commitment. Circ Res 108:129–152 8. Mummery CL, Zhang J, Ng ES et al (2012) Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circ Res 111:344–358 9. Bu L, Jiang X, Martin-Puig S et al (2009) Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature 460:113–117 10. Yang L, Soonpaa MH, Adler ED et al (2008) Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453:524–528 11. Blin G, Nury D, Stefanovic S et al (2010) A purified population of multipotent
cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates. J Clin Invest 120:1125–1139 12. Cao N, Liang H, Huang J et al (2013) Highly efficient induction and long-term maintenance of multipotent cardiovascular progenitors from human pluripotent stem cells under defined conditions. Cell Res 23:1119–1132 13. Elliott DA, Braam SR, Koutsis K et al (2011) NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat Methods 8:1037–1040 14. Kattman SJ, Witty AD, Gagliardi M et al (2011) Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8:228–240 15. Li C, Zhou J, Shi G et al (2009) Pluripotency can be rapidly and efficiently induced in human amniotic fluid-derived cells. Hum Mol Genet 18:4340–4349 16. Leschik J, Stefanovic S, Brinon B et al (2008) Cardiac commitment of primate embryonic stem cells. Nat Protoc 3:1381–1387
