人iPSC衍生心血管祖细胞（iPSC-CVPCs）的体外分化

Agnieszka D’Antonio-Chronowska; Matteo D’Antonio; Kelly A. Frazer

doi:10.21769/BioProtoc.3755

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Peer-reviewed

In vitro Differentiation of Human iPSC-derived Cardiovascular Progenitor Cells (iPSC-CVPCs)

人iPSC衍生心血管祖细胞（iPSC-CVPCs）的体外分化

Agnieszka D’Antonio-Chronowska email

MD Matteo D’Antonio

Kelly A. Frazer

发布: 2020年09月20日第10卷第18期 DOI: 10.21769/BioProtoc.3755 浏览次数: 8723

评审: Vivien Jane Coulson-ThomasAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Stem Cell Reports, featuring study using the protocol.

Nov 2019

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Abstract

Induced pluripotent stem cell derived cardiovascular progenitor cells (iPSC-CVPCs) provide an unprecedented platform for examining the molecular underpinnings of cardiac development and disease etiology, but also have great potential to play pivotal roles in the future of regenerative medicine and pharmacogenomic studies. Biobanks like iPSCORE ( Stacey et al., 2013; Panopoulos et al., 2017), which contain iPSCs generated from hundreds of genetically and ethnically diverse individuals, are an invaluable resource for conducting these studies. Here, we present an optimized, cost-effective and highly standardized protocol for large-scale derivation of human iPSC-CVPCs using small molecules and purification using metabolic selection. We have successfully applied this protocol to derive iPSC-CVPCs from 154 different iPSCORE iPSC lines obtaining large quantities of highly pure cardiac cells. An important component of our protocol is Cell confluency estimates (ccEstimate), an automated methodology for estimating the time when an iPSC monolayer will reach 80% confluency, which is optimal for initiating iPSC-CVPC derivation, and enables the protocol to be readily used across iPSC lines with different growth rates. Moreover, we showed that cellular heterogeneity across iPSC-CVPCs is due to varying proportions of two distinct cardiac cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs), both of which have been shown to have a critical function in heart regeneration. This protocol eliminates the need of iPSC line-to-line optimization and can be easily adapted and scaled to high-throughput studies or to generate large quantities of cells suitable for regenerative medicine applications.

Keywords: Human induced pluripotent stem (iPSC) (人诱导多能干细胞（iPSC）)

Cardiovascular progenitor cells (CVPCs) (心血管祖细胞(CVPCs))

cardiomyocytes (CMs) (心肌细胞(CMs))

Epicardium cells (EPDCs) (心外膜细胞（EPDCs）)

Human induced pluripotent stem cell-derived cardiovascular progenitor cells (iPSC-CVPCs) (人诱导多能干细胞衍生心血管祖细胞（iPSC-CVPCs）)

Human induced pluripotent stem cell-derived cardiomyocytes cells (iPSC-CMs) (人诱导多能干细胞衍生心肌细胞（iPSC CMs）)

Human induced pluripotent stem cell-derived epicardium derived cells (iPSC-EPDCs) (人诱导多能干细胞衍生心外膜源性细胞（iPSC-epdc）)

Cardiovascular disease (心血管疾病)

Heart (心脏)

Differentiation (分化)

Genetic studies (遗传研究)

Small molecules (小分子)

Pharmacogenomics (基因测试)

Regenerative medicine (再生医学)

Background

Cardiovascular diseases (CVDs) remain the leading cause of death worldwide and account for about 30% of all mortality causes globally. Coronary artery disease (CAD) and myocardial infarction (MI) are among the most common CVDs, and in the USA alone, every 40 s someone suffers a heart attack (Association, 2016; Heron, 2017; WHO, 2018; Benjamin et al., 2019; D'Antonio-Chronowska et al., 2019a). Heart failure results in the death of cardiac muscle cells and is the consequence of morphological and functional changes (cardiac remodeling: necrosis, scar formation, inflammation, fibrosis, dilation and reshaping) that occur in response to pre-existing cardiac conditions, including CAD, MI, hypertension, cardiomyopathy, myocarditis and abnormal cardiac valve function (Cohn et al., 2000; Reed et al., 2017). Heart failure is commonly treated with beta blockers, ACE inhibitors and aldosterone antagonists which partially reverse cardiac remodeling and thereby improve prognosis (Reis Filho et al., 2015), but does not result in the regeneration of cardiac tissue. There are currently several ongoing clinical trials, including ESCORT and DREAM-HF (Menasche et al., 2015 and 2018; Borow et al., 2019), which are aimed at evaluating the effectiveness of transplanting iPSC-derived Cardiovascular Progenitor Cells (iPSC-CVPCs) or embryonic stem cell derived CVPCs (ESC-CVPCs) as a therapeutic treatment for heart failure. The ability to generate iPSC-CVPCs in large quantities, as is required for regenerative medicine, using biological material obtained directly from the patient would enable autologous transplantations and thereby eliminate the need of immunosuppression. Thus, the development of a robust and cost-effective protocol for generating large amounts of high-quality iPSC-CVPCs without requiring individualized optimization for each iPSC line is imperative for the advancement of future therapeutic treatments of heart failure.

Large collections of iPSC-CVPCs (D'Antonio-Chronowska et al., 2019b) generated from genetically and ethnically diverse individuals could also be used for cost-effective large-scale testing of drugs for cardiotoxicity or proarrhythmic effects. Previous studies (Burridge et al., 2016) and initiatives like CiPA Project (Blinova et al., 2017) have shown the utility of iPSC-CVPCs for testing drugs for cardiotoxicity, which, if scaled to examine large collections of iPSC-CVPCs derived from both healthy or disease bearing individuals, could greatly improve the efficiency of testing new drugs for safety, and in turn decrease the cost of drug development.

We have previously demonstrated the feasibility of using a highly standardized protocol for successfully deriving high quality iPSC-CVPCs from hundreds of iPSC lines reprogrammed from ethnically diverse individuals (D'Antonio-Chronowska et al., 2019b). In this study, we performed 193 differentiations to derive iPSC-CVPCs from 154 iPSCORE iPSC lines (Panopoulos et al., 2017) from 144 individuals. We obtained large numbers of high quality cells, specifically, on average we derived 1.5 x 10⁸ (and up to 6 x 10⁸) cells from a 450 cm² culture with median cardiac troponin T (cTnT; TNNT2) positive cells of 89.2%. Importantly, while previous differentiation studies acknowledged cellular heterogeneity and the presence of beating cardiomyocytes and non-contractile cell types, the origin and cellular identity of the non-contractile cells had not been addressed. We characterized the 154 iPSC-CVPCs lines using single cell RNA-seq and bulk RNAs-seq and determined that across all the iPSC-CVPC samples there were two distinct fetal-like cardiac cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs), which were present in varying proportions. Of note, both CMs and EPDCs have been show to contribute to the post-infarction heart regeneration (Bargehr et al., 2019). Moreover, our previous studies have shown how molecular characterization of iPSC-CVPCs can result in the identification of genetic variants that contribute to heart development and cardiac pathologies (Benaglio et al., 2019).

Protocols to derive cardiac cells from ESCs or iPSCs have been developed to mimic the processes naturally occurring during cardiogenesis. Initially, cardiac cells were derived as embryoid bodies cultures, first as spontaneous differentiations by culturing ESCs in medium containing 20% fetal calf serum or by stimulation with several reagents known to enhance cardiac differentiation like dimethyl sulfoxide, retinoic acid, or 5-aza-2’-deoxycytidine, after which beating cardiac cells were manually or mechanically purified (Maltsev et al., 1993; Burridge et al., 2014). Differentiation efficiency was greatly improved by the development of directed differentiation protocols that incorporated recombinant proteins including fibroblast growth factor 2, transforming growth factor β, superfamily growth factors activin A and BMP4, vascular endothelial growth factor and the WNT inhibitor DKK-1 proteins (Schneider and Mercola, 2001; Marvin et al., 2001; Beqqali et al., 2006; Laflamme et al., 2007), and by modification of the format of cell differentiation from embryoid bodies to monolayer culture (Paige et al., 2010; Lian et al., 2012). Further advancements were made by the introduction of small molecule protocols (Lian et al., 2012 and 2013) and chemically defined differentiation media (Burridge et al., 2014). Finally, by taking advantage of the adaptation of the developing heart to metabolize lactate, we and others were able to eliminate all non-cardiac cells (Burridge et al., 2014; D'Antonio-Chronowska et al., 2019b; Tohyama et al., 2013). Importantly, previous studies have optimized differentiation protocols to derive cardiac cells from a limited number of iPSC or ESC lines, and in most cases utilized small format culture vessels. Here, we present an optimized, cost-effective and highly standardized protocol which we applied to derive iPSC-CVPCs from 154 genetically and ethnically diverse human iPSC lines in large-sized culture flasks. We optimized the concentration of IWP-2 used to drive the cardiac cell differentiation, which resulted in improved formation of a thick cardiac syncytium and strong wave-like beating (Video 1) (D'Antonio-Chronowska et al., 2019b). We also demonstrated that simple mechanical disruption of the cardiac syncytium prior to metabolic purification of iPSC-CVPCs using lactate results in improved selection and virtually pure cardiac cells (CMs and EPDCs). Additionally, we developed Cell confluency estimates (ccEstimate), an automated method for estimation of cell confluency during the monolayer stage. ccEstimate estimates the point in time for each iPSC line when the monolayer will reach 80% of confluency, which is optimal condition at which to initiate iPSC-CVPC differentiation. Thereby ccEstimate overcomes some of the technical issues in standardizing a differentiation protocol across iPSC lines which have widely varying growth rates. The derived iPSC-CVPCs beat synchronously, are positive for multiple cardiac markers and can be used directly for molecular or electrophysiological assays like multielectrode array (MEA), or they can be cryopreserved for future analysis. Our optimized protocol allowed us to derive high quality iPSC-CVPC samples from 154 iPSC lines generated from ethnically diverse individuals under identical culturing conditions without the requirement of any individualized optimization steps.

Materials and Reagents

iPSC Cell Culture as described also in detail in D’Antonio-Chronowska et al. (2019)
1. 6-well plates (Corning, catalog number: 3506 )
2. Syringe filter 0.2 μm (VWR, catalog number: 28145-501 )
3. Soft-Ject^® 3-Part Disposable Syringe, Air-Tite-3 ml (VWR, catalog number: 89215-234 )
4. 5 ml Borosilicate serological pipettes (Fisher Scientific, catalog number: 1367827E )
5. 5 ml Serological pipettes (Bio Pioneer, catalog number: GEX0050-S01 )
6. 10 ml Serological pipettes (Bio Pioneer, catalog number: GEX0100-S01 )
7. P20 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-00 ) or other equivalent sterile tips with filter compatible with pipettes
8. P1000 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-02 ) or other equivalent P1000 sterile tips with filter compatible with pipettes
9. 15 ml conical tubes (Bio Pioneer, catalog number: CNT-15R )
10. iPSC cells
11. UltraPure^TM DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, catalog number: 10977023 )
12. Corning^® Matrigel^®Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, catalog number: 354230 )
13. mTeSR^TM 1 (Stem Cell Technologies, catalog number: 85850 )
14. DMEM/F-12 medium (Thermo Fisher Scientific, catalog number: 11330057 )
15. Dispase II, powder (Thermo Fisher Scientific, catalog number: 17105041 )
16. 200 proof Ethanol denatured (i.e., VWR, catalog number: 71002-398 )–to prepare 70% ethanol
17. 70% ethanol (see Recipes: Table 20)
18. Matrigel solution (Matrigel) (see Recipes: Table 1)
19. 10 mM ROCK inhibitor, Y-27632 dihydrochloride solution (ROCK Inhibitor) (see Recipes: Table 3)
20. 10x Dispase (see Recipes: Table 4)
21. mTeSR^TM 1 complete medium (mTeSR) (see Recipes: Table 5)
PSC passaging using Versene–Versene I passage
1. 6-well plates (Corning, catalog number: 3506 )
2. Automated cell counter slides (Bio-Rad Laboratories, catalog number: 1450019 ) or a hemocytometer (Hausser Scientific, catalog number: 1483 ) or equivalent
3. 5 ml Serological pipettes (Bio Pioneer, catalog number: GEX0050-S01 )
4. P20 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-00 ) or other equivalent P200 sterile tips with filter compatible with pipettes
5. P1000 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-02 ) or other equivalent P1000 sterile tips with filter compatible with pipettes
6. 15 ml conical tubes (Bio Pioneer, catalog number: CNT-15R )
7. iPSC cells
8. UltraPure^TM DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, catalog number: 10977023 )
9. Corning^® Matrigel^® Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, catalog number: 354230 )
10. mTeSR^TM 1 (Stem Cell Technologies, catalog number: 85850 )
11. DMEM/F-12 medium (Thermo Fisher Scientific, catalog number: 11330057 )
12. Versene^® (EDTA) 0.02% (Lonza, catalog number: 17-711E )
13. 200 proof Ethanol denatured (i.e., VWR, catalog number: 71002-398 )–to prepare 70% ethanol
14. 70% ethanol (see Recipes: Table 20)
15. Matrigel solution (Matrigel) (see Recipes: Table 1)
16. 10 mM ROCK inhibitor, Y-27632 dihydrochloride solution (ROCK Inhibitor) (see Recipes: Table 3)
17. mTeSR^TM 1 complete medium (mTeSR) (see Recipes: Table 5)
iPSC passaging using Versene - Versene II passage
1. 100 mm tissue culture dishes (Corning, catalog number: 430167 )
2. Automated cell counter slides (Bio-Rad Laboratories, catalog number: 1450019 ) or a hemocytometer (Hausser Scientific, catalog number: 1483 ) or equivalent
3. 5 ml Serological pipettes (Bio Pioneer, catalog number: GEX0050-S01 )
4. P20 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-00 ) or other equivalent sterile tips with filter compatible with pipettes
5. P1000 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-02 ) or other equivalent P1000 sterile tips with filter compatible with pipettes
6. 15 ml conical tubes (Bio Pioneer, catalog number: CNT-15R )
7. iPSC cells
8. UltraPure^TM DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, catalog number: 10977023 )
9. Corning^® Matrigel^® Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, catalog number: 354230 )
10. mTeSR^TM 1 (Stem Cell Technologies, catalog number: 85850 )
11. DMEM/F-12 medium (Thermo Fisher Scientific, catalog number: 11330057 )
12. Versene^® (EDTA) 0.02% (Lonza, catalog number: 17-711E )
13. 200 proof Ethanol denatured (i.e., VWR, catalog number: 71002-398 )–to prepare 70% ethanol
14. 70% ethanol (see Recipes: Table 20)
15. Matrigel solution (Matrigel) (see Recipes: Table 1)
16. 10 mM ROCK inhibitor, Y-27632 dihydrochloride solution (ROCK Inhibitor) (see Recipes: Table 3)
17. mTeSR^TM 1 complete medium (mTeSR) (see Recipes: Table 5)
Monolayer plating is also described in detail in D’Antonio-Chronowska et al. (2019a)
1. 100 mm tissue culture dishes (Corning, catalog number: 430167 )
2. Automated cell counter slides (Bio-Rad Laboratories, catalog number: 1450019 ) or a hemocytometer (Hausser Scientific, catalog number: 1483 ) or equivalent
3. 5 ml Serological pipettes (Bio Pioneer, catalog number: GEX0050-S01 )
4. 10 ml Serological pipettes (Bio Pioneer, catalog number: GEX0100-S01 )
5. P20 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-00 ) or other equivalent P20 sterile tips with filter compatible with pipettes
6. P200 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-01 ) or other equivalent P200 sterile tips with filter compatible with pipettes
7. P1000 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-02 ) or other equivalent P1000 sterile tips with filter compatible with pipettes
8. 15 ml conical tubes (Bio Pioneer, catalog number: CNT-15R )
9. 50 ml conical tubes (Bio Pioneer catalog number: CNT-50R )
10. Corning^® Matrigel^® Growth Factor Reduced (GFR) Basement Membrane Matrix (Matrigel) (Corning, catalog number: 354230 )
11. mTeSR^TM 1 (Stem Cell Technologies, catalog number: 85850 )
12. DMEM/F-12 medium (Thermo Fisher Scientific, catalog number: 11330-057 )
13. Accutase (Innovative Cell Technologies, Inc., catalog number: AT 104 )
14. Trypan Blue Solution, 0.4% (Thermo Fisher Scientific, catalog number: 15250061 )
15. ROCK inhibitor, Y-27632 dihydrochloride (Selleck hem, catalog number: S1049 )
16. iPSC cell culture
17. 200 proof Ethanol denatured (i.e., VWR, catalog number: 71002-398 )–to prepare 70% ethanol
18. 70% ethanol (see Recipes: Table 20)
19. Matrigel solution (see Recipes: Table 1)
20. 10 mM ROCK inhibitor, Y-27632 dihydrochloride solution (see Recipes: Table 3)
21. mTeSR^TM 1 complete medium (see Recipes: Table 5)
Estimation of optimal time for initiation of iPSC-CVPCs differentiation using ccEstimate
1. iPSCs monolayer
2. Marker pen resistant to 70% ethanol
iPSC-CVPCs differentiation
1. T150 tissue culture flasks, vented (Sigma, catalog number: Z707929 )
  Note: At the time of preparation of this manuscript Z707929 was no longer available. The same flasks are available under the catalog number Z707511-36EA (Sigma, catalog number: Z707511-36EA ).
2. Automated cell counter slides (Bio-Rad Laboratories, catalog number: 1450019 ) or a hemocytometer (Hausser Scientific, catalog number: 1483 ) or equivalent
3. 10 ml Serological pipettes (Bio Pioneer, catalog number: GEX0100-S01 )
4. 25 ml Serological pipettes (Bio Pioneer catalog number: GEX250-S01 )
5. 50 ml Serological pipettes (Bio Pioneer, catalog number: GEX500-S01 )
6. P20 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-00 ) or other equivalent P20 sterile tips with filter compatible with pipettes
7. P200 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-01 ) or other equivalent P200 sterile tips with filter compatible with pipettes
8. P1000 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-02 ) or other equivalent P1000 sterile tips with filter compatible with pipettes
9. Cell scraper (VWR International, catalog number: 179707 )
10. 15 ml conical tubes (Bio Pioneer, catalog number: CNT-15R )
11. 50 ml conical tubes (Bio Pioneer, catalog number: CNT-50R )
12. 125 ml Nalgene^® PET sterile bottle (Fisher Scientific, catalog number: 342040-0125 )
13. Nalgene^® Cryogenic vials (Thermo Fisher Scientific, catalog number: 5000-1020 )
14. iPSCs monolayer
15. Corning^® Matrigel^® Growth Factor Reduced (GFR) Basement Membrane Matrix (Matrigel) (Corning^®, catalog number: 354230 )
16. RPMI 1640 medium (Thermo Fisher Scientific, catalog number: 11875119 )
17. RPMI 1640 medium, no glucose (Thermo Fisher Scientific, catalog number: 11879020 )
18. 1x Dulbecco’s phosphate buffered saline (DPBS; PBS) without calcium and magnesium (Thermo Fisher Scientific, catalog number: 14190250 )
19. B-27^TM Supplement, minus insulin (Thermo Fisher Scientific, catalog number: A1895601 )
20. B-27^TM Supplement (50x), serum free (Thermo Fisher Scientific, catalog number: 17504044 )
21. FBS (Omega Scientific, catalog number: FB-02 ) or equivalent, or KnockOut^TM Serum Replacement (KOSR) (Thermo Fisher Scientific, catalog number: 10828028 )
22. MEM Non-Essential Amino Acids Solution 100x (Thermo Fisher Scientific, catalog number: 11140050 )
23. Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, catalog number: 15140122 )
24. CHIR-99021 (CT99021) (CHIR99021) HCl (Selleckchem, catalog number: S2924 )
25. IWP-2 (Tocris, catalog number: 3533 )
26. Sodium L-lactate (Sigma, catalog number: 71718-10G )
27. HEPES sodium salt solution 1M, BioReagent, suitable for cell culture (Sigma, catalog number: H3662-100ML )
28. Accutase (Innovative Cell Technologies, Inc., catalog number: AT 104 )
29. Trypan Blue Solution, 0.4% (Thermo Fisher Scientific, catalog number: 15250061 )
30. Dimethyl Sulfoxide (DMSO) (Sigma, catalog number: D2650-100ML )
31. Liquid nitrogen
32. 200 proof Ethanol denatured (i.e., VWR, catalog number: 71002-398 ) – to prepare 70% ethanol
33. 70% ethanol (see Recipes: Table 20)
34. RPMI Minus (-) medium (see Recipes: Table 6)
35. RPMI Plus (+) medium (see Recipes: Table 7)
36. RPMI Lactate medium (see Recipes: Table 8)
37. iPSC-CVPCs Harvest Medium (see Recipes: Table 9)
38. iPSC-CVPCs 2x freezing medium (see Recipes: Table 10)
39. 10 mM CHIR-992021 solution (see Recipes: Table 11)
40. 5 mM IWP-2 solution (see Recipes: Table 12)
41. 1M Sodium L-lactate solution (see Recipes: Table 13)
Flow cytometry
1. 96-well round bottom assay plates (Genesee Scientific, catalog number: 25-224 )
2. Corning^TM Falcon^TM Test Tube with Cell Strainer Snap Cap (Fisher Scientific, catalog number: 352235 )
3. Corning^TM Costar^TM Sterile Disposable Reagent Reservoirs (Fisher Scientific, catalog number: 4870 )
4. 5 ml Serological pipettes (Bio Pioneer, catalog number: GEX0050-S01 )
5. 10 ml Serological pipettes (Bio Pioneer, catalog number: GEX0100-S01 )
6. P20 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-00 ) or other equivalent P20 sterile tips with filter compatible with pipettes
7. P200 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-01 ) or other equivalent P200 sterile tips with filter compatible with pipettes
8. P1000 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-02 ) or other equivalent P1000 sterile tips with filter compatible with pipettes
9. P20 sterile pipette tips without filter (VWR, catalog number: 83009-694 ) or other equivalent P20 sterile tips without filter compatible with pipettes
10. (Optional) P200 sterile pipette tips without filter (VWR, catalog number: 89495-378 ) or other equivalent P20 sterile tips without filter compatible with pipettes
11. 1x Dulbecco’s phosphate buffered saline (PBS) without calcium and magnesium (Thermo Fisher Scientific, catalog number: 14190250 )
12. Methanol, ACS reagent, ≥ 99.8% (Sigma, catalog number: 179337-4L-PB )
13. Bovine Serum Albumin (BSA) (Sigma, catalog number: A2153-100G )
14. (Optional) NaN₃ (Sigma, catalog number: S2002-5G )
15. 37% Formaldehyde (Sigma, catalog number: F-1635-500ML )
16. Triton^TM X-100 (Sigma, catalog number: X-100-500ML )
17. Goat serum, New Z
18. Mouse monoclonal anti-Troponin T, Cardiac Isoform (cTNT; TNNT2) Ab-1antibody, clone 13-11 (Thermo Fisher Scientific, catalog number: MS-295-P0 )
19. Mouse IgG1 antibody (Thermo Fisher Scientific, catalog number: MG100 )
20. Goat-anti-Mouse Alexa Fluor^TM 488 conjugated antibody (Thermo Scientific, catalog number: A-11001 )
21. FACS Buffer (see Recipes: Table 14)
22. FACS-FIX Buffer (see Recipes: Table 16)
Note: For antibody working concentration, see Recipes: Table 21.
Immunofluorescence
1. Millicell EZ SLIDE 8-well glass slides (Millipore, catalog number: PEZGS0816 )
2. Coverslip glass slides (Fisherbrand, catalog number: 12-545-F [coverslip thickness #1]). Different coverslip thickness may be used if required
3. 5 ml Serological pipettes (Bio Pioneer, catalog number: GEX0050-S01 )
4. 10 ml Serological pipettes (Bio Pioneer, catalog number: GEX0100-S01 )
5. P20 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-00 ) or other equivalent P20 sterile tips with filter compatible with pipettes
6. P200 pipette tips sterile with filter (Fisher Scientific, catalog number: 21-403-01 ) or other equivalent P200 sterile tips with filter compatible with pipettes
7. 1x Dulbecco’s phosphate buffered saline (PBS) without calcium and magnesium (Thermo Fisher Scientific, catalog number: 14190250 )
8. Bovine Serum Albumin (BSA) (Sigma, catalog number: A2153-100G )
9. Paraformaldehyde (PFA) (Sigma, catalog number: 158127-100G )
10. Tween^® 20 (Sigma, catalog number: P9416-100ML )
11. Triton^TM X-100 (Sigma, catalog number: X100-500ML )
12. Gelatin from porcine skin (Sigma, catalog number: G1890-100G )
13. Mouse monoclonal anti-α-Actinin (Sarcomeric) antibody, clone EA-53 (Sigma, catalog number: A7811 )
14. Rabbit polyclonal anti-Connexin 43 (CX43/GJA1) antibody (Invitrogen, catalog number: 71-0700 )
15. Rabbit polyclonal anti-Myosin Light Chain 2 (MYL2; MLC2v) antibody (Proteintech, catalog number: 10906-1-AP )
16. Mouse monoclonal anti-Myosin Atrial Light Chain 2 (MYL7; MLC2a) antibody clone 56F5 (Synaptic Systems, catalog number: 311 011 )
17. Donkey anti-Rabbit Alexa Fluor^TM 488 conjugated antibody (Invitrogen, catalog number: A21206 )
18. Goat anti-Mouse Alexa Fluor^TM 568 conjugated antibody (Invitrogen, catalog number: A-11004 )
19. ProLong^® Gold Antifade Reagent with DAPI (Cell Signaling Technologies, catalog number: 8961 )
20. 0.1% Gelatin solution (see Recipes: Table 2)
21. 4% PFA solution in PBS
22. IF Perm Buffer II (see Recipes: Table 17)
23. IF Blocking Buffer II (see Recipes: Table 18)
24. IF Staining Buffer (see Recipes: Table 19)
Note: For antibody working concentration, see Recipes: Table 21.

Equipment

iPSC Cell Culture
1. Biosafety cabinet (Labconco, model: Logic+ )
2. Incubator with humidity and gas control set to maintain 37 °C and 95% humidity in an atmosphere of 5% CO₂ in air (Panasonic, model: MCO-170AICUVH-PA )
3. Water bath (Thermo Scientific, model: Precision )
4. Tissue culture centrifuge with rotors for 15 ml conical tubes and 50 ml conical tubes (Thermo Scientific, model: Legend RT+ )
5. Phase contrast inverted microscope (objectives: x4, x10, x20) (Olympus, model: CKX41SF )
6. (Optional) Phase contrast inverted microscope with camera (objectives: x4, x10, x20) (Thermo Scientific, model: EVOS XL Core )
7. Microscope Object marker (Nikon, model: MBW10020 )
8. Pipette aid (Labnet, catalog number: FastPette^TM V2 P2000) or other equivalent available pipette aid
9. P20 Micropipette (Rainin, catalog number: 17014392 ) or other available P20 pipette
10. Non-frost-free freezer -20 °C
11. Refrigerator 2-8 °C
iPSC passaging using Versene (Versene I and Versene II passage)
1. Biosafety cabinet (Labconco, model: Logic+ )
2. Incubator with humidity and gas control set to maintain 37 °C and 95% humidity in an atmosphere of 5% CO₂ in air (Panasonic, model: MCO-170AICUVH-PA )
3. Tissue culture centrifuge with rotors for 15 ml conical tubes and 50 ml conical tubes (Thermo Scientific, model: Legend RT+ )
4. Phase contrast inverted microscope (objectives: x4, x10, x20) (Olympus, model: CKX41SF )
5. (Optional) Phase contrast inverted microscope with camera (objectives: x4, x10, x20) (Thermo Scientific, model: EVOS XL Core )
6. Pipette aid (Labnet, catalog number: FastPette^TM V2 P2000) or other equivalent available pipette aid
7. P20 Micropipette (Rainin, catalog number: 17014392 ) or other available P20 pipette
8. P200 Micropipette (Rainin, catalog number: 17014391 ) or other available P200 pipette
9. P1000 Micropipette (Rainin, catalog number: 17014382 ) or other available P1000 pipette
10. Automated cell counter (Bio-Rad, model: TC20 ) or a hemocytometer (Hausser Scientific, catalog number: 1483 ) or equivalent
11. Non-frost-free freezer -20 °C
12. Refrigerator 2-8 °C
Monolayer plating
1. Biosafety cabinet (Labconco, model: Logic+ )
2. Incubator with humidity and gas control set to maintain 37 °C and 95% humidity in an atmosphere of 5% CO₂ in air (Panasonic, model: MCO-170AICUVH-PA )
3. Tissue culture centrifuge with rotors for 15 ml conical tubes and 50 ml conical tubes (Thermo Scientific, model: Legend RT+ )
4. Phase contrast inverted microscope (objectives: x4, x10, x20) (Olympus, model: CKX41SF )
5. (Optional) Phase contrast inverted microscope with camera (objectives: x4, x10, x20) (Thermo Scientific, model: EVOS XL Core )
6. Pipette aid (Labnet, catalog number: FastPette^TM V2 P2000 or other equivalent available pipette aid
7. P20 Micropipette (Rainin, catalog number: 17014392 ) or other available P20 pipette
8. P200 Micropipette (Rainin, catalog number: 17014391 ) or other available P200 pipette
9. P1000 Micropipette (Rainin, catalog number: 17014382 ) or other available P1000 pipette
10. Automated cell counter (Bio-Rad, model: TC20 ) or a hemocytometer (Hausser Scientific, catalog number: 1483 ) or equivalent
11. Non-frost-free freezer -20 °C
12. Refrigerator 2-8 °C
iPSC-CVPCs differentiation and cryopreservation
1. Biosafety cabinet (Labconco, model: Logic+ )
2. Incubator with humidity and gas control set to maintain 37 °C and 95% humidity in an atmosphere of 5% CO₂ in air (Panasonic, model: MCO-170AICUVH-PA )
3. Tissue culture centrifuge with rotors for 15 ml conical tubes and 50 ml conical tubes (Thermo Scientific, model: Legend RT+ )
4. Phase contrast inverted microscope (objectives: x4, x10, x20) (Olympus, model: CKX41SF )
5. (Optional) Phase contrast inverted microscope with camera (objectives: x4, x10, x20) (Thermo Scientific, model: EVOS XL Core )
6. Pipette aid (Labnet, catalog number: FastPette^TM V2 P2000) or other equivalent available pipette aid
7. P20 Micropipette (Rainin, catalog number: 17014392 ) or other available P20 pipette
8. P200 Micropipette (Rainin, catalog number: 17014391 ) or other available P200 pipette
9. P1000 Micropipette (Rainin, catalog number: 17014382 ) or other available P1000 pipette
10. Automated cell counter (Bio-Rad, model: TC20 ) or a hemocytometer (Hausser Scientific, catalog number: 1483 ) or equivalent.
11. Mr. Frosty freezing container (Corning, model: CoolCell^® FTS30)
12. Refrigerator 2-8 °C
13. Non-frost-free freezer -20 °C
14. Freezer -80 °C
15. Liquid nitrogen vapor tank
Estimation of optimal time for initiation of iPSC-CVPCs differentiation using ccEstimate
1. Phase contrast inverted microscope with camera (objective: x4) (Thermo Scientific, model: EVOS XL Core ) or equivalent or automatic imaging system
2. Any computer with R 3.5.1 and the R package EBImage (Pau et al., 2010) installed and 4GB RAM
Flow cytometry
1. Pipette aid (Labnet, catalog number: FastPette^TM V2 P2000) or other equivalent available pipette aid
2. P20 Micropipette (Rainin, catalog number: 17014392 ) or other available P20 pipette
3. P200 Micropipette (Rainin, catalog number: 17014391 ) or other available P200 pipette
4. P1000 Micropipette (Rainin, catalog number: 17014382 ) or other available P1000 pipette
5. P200 Multichannel micropipette (Rainin, catalog number: 17013805 ) or other available multichannel P200 pipette
6. Tissue culture centrifuge with rotors suitable to centrifuge 96-well plates (Thermo Scientific, model: Legend RT+ )
7. Refrigerator 2-8 °C
8. Non-frost-free freezer -20 °C
9. Flow cytometer (BD Biosciences, model: FACSCanto II ) or equivalent.
Immunofluorescence
1. Pipette aid (Labnet, catalog number: FastPette^TM V2 P2000) or other equivalent available pipette aid
2. P20 Micropipette (Rainin, catalog number: 17014392 ) or other available P20 pipette
3. P200 Micropipette (Rainin, catalog number: 17014391 ) or other available P200 pipette
4. P1000 Micropipette (Rainin, catalog number: 17014382 ) or other available P1000 pipette
5. Refrigerator 2-8 °C
6. Non-frost-free freezer -20 °C
7. Confocal laser scanning fluorescence microscope (Olympus, FluoView1000)

Software

FlowJo (Version 10) (FlowJo, LLC, https://www.flowjo.com/)
FlowView ASW V03.01.03.03 or V4.2a (Olympus Life Science, https://www.olympus-lifescience.com/en/support/downloads/)
ccEstimate available on request
R 3.5.1
R package EBImage (Pau et al., 2010)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

D’Antonio-Chronowska, A., D’Antonio, M. and Frazer, K. A. (2020). In vitro Differentiation of Human iPSC-derived Cardiovascular Progenitor Cells (iPSC-CVPCs). Bio-protocol 10(18): e3755. DOI: 10.21769/BioProtoc.3755.