In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Podsumowanie

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

Streszczenie

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) — are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

Wprowadzenie

The use of pluripotent stem cells (PSCs) has revolutionized the field of cardiac regeneration and personalized medicine with patient-specific myocytes for disease modeling and drug therapies^1,2,3,4. More recently, in vitro protocols for the generation of atrial vs ventricular as well as pacemaker-like PSC-derived cardiomyocytes have been developed^5,6. However, whether cardiogenesis can be recreated in vitro to study cardiac development and subsequently generate ventricular chamber-specific cardiac cells is still unclear.

During early embryonic development, mesodermal cells under the influence of secreted morphogens such as BMP4, Wnts and Activin A form the primitive streak⁷. Cardiac mesodermal cells marked by the expression of Mesp1, migrate anteriorly and latterly to form the cardiac crescent and then the primitive heart tube^7,8. This migratory group of cells includes two very distinct populations of cardiac progenitor cells (CPCs), namely the first and the second heart field (FHF and SHF)^9,10. Cells from the SHF are highly proliferative and migratory and are primarily responsible for the elongation and looping of the heart tube. Additionally, SHF cells differentiate to cardiomyocytes, fibroblasts, smooth muscle and endothelial cells as they enter the heart tube to form the right ventricle, right ventricular outflow tract and large part of both atria^7,10. In contrast, FHF cells are less proliferative and migratory and differentiate mainly to cardiomyocytes as they give rise to the left ventricle and a smaller part of the atria¹¹. Moreover, SHF progenitors are marked by the expression of Tbx1, FGF8, FGF10 and Six2 while FHF cells express Hcn4 and Tbx5^{11,12,13,14,15}.

PSCs can differentiate to all three germ layers and subsequently to any cell type in the body^4,16. Therefore, they offer tremendous potential for understanding heart development and for modelling specific developmental defects resulting in congenital heart disease, the most frequent cause of birth defects¹⁷. A large subgroup of congenital heart disease includes chamber-specific cardiac abnormalities^18,19. However, it still unclear whether these originate from anomalous heart field development. In addition, given the inability of cardiomyocytes to proliferate after birth, there have been extensive efforts to create cardiac tissue for heart regeneration^1,7,20. Considering the physiological and morphological differences between cardiac chambers, generation of chamber-specific cardiac tissue using PSCs is of significant importance. While recent advances in developmental cardiology have led to robust generation of cardiac cells from PSCs, it is still unclear if the two heart fields can be induced in PSC systems.

To recapitulate cardiogenesis in vitro and study the specification and properties of CPCs, we previously used a system based on differentiating PSC-derived cardiac spheroids^21,22,23,24. Recently, we generated mouse embryonic stem cells (mESCs) with GFP and RFP reporters under the control of the FHF gene Hcn4 and the SHF gene Tbx1, respectively (mESCs^{Tbx1-Cre; Rosa-RFP; HCN4-GFP}) ²⁵. In vitro differentiated mESCs formed cardiac spheroids in which GFP+ and RFP+ cells appeared from two distinct areas of mesodermal cells and patterned in a complementary manner. The resulting GFP+ and RFP+ cells exhibited FHF and SHF characteristics, respectively, determined by RNA-sequencing and clonal analyses. Importantly, using mESCs carrying the Isl1-RFP reporter (mESC^Isl1-RFP), we discovered that SHF cells were faithfully marked by the cell-surface protein CXCR4, and this can enable isolation of heart field-specific cells without transgenes. The present protocol will describe the generation and isolation of heart field-specific CPCs from mESCs, which may serve as a valuable tool for studying chamber-specific heart disease.

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Protokół

NOTE: In vitro generation of heart field-specific mouse cardiac progenitor cells (Figure 1).

1. Maintenance of Mouse ESCs

1. Grow mESCs (mESCs^{Tbx1-Cre; Rosa-RFP; HCN4-GFP}, mESC^Isl1-RFP)²⁵ on 0.1% (w/v) gelatin coated T25 flasks in 2i medium (870 mL of glascow minimum essential medium (GMEM), 100 mL of fetal bovine serum (FBS), 10 mL of GlutaMAX, 10 mL of non-essential amino acids, 10 mL of sodium pyruvate, 3 μL of beta-mercaptoethanol, 20 μL of Lif (200 U/mL), 0.3 μM CHIR99021 and 0.1 μM PD0325901).
2. When the cells reach 70-80% confluence, rinse the cells once with phosphate buffer solution (PBS) and then dissociate into single cells by adding 1 mL of Trypsin and incubating at 37 °C for 3 min.
3. Neutralize Trypsin by adding 4 mL of 10% FBS in Dulbecco’s Modified Eagle Medium (DMEM). Count the cells using an automated cell counter.
4. Centrifuge ~3 x 10⁵ cells for 3 min at 270 x g and room temperature.
5. Aspirate the supernatant, resuspend the cells in 5 mL of 2i medium and replate on 0.1% (w/v) gelatin coated T25 flasks for maintenance.

2. Generation of Cardiac Progenitor Cells Using Cardiac Spheroids

1. Centrifuge 2.5 x 10⁶ cells from step 1.3 for 3 min at 270 x g and room temperature.
2. Aspirate the supernatant and resuspend the cells in 25 mL of SFD medium (10⁵ cells/mL). Depending on the scale of the experiment, mESC number can be adjusted accordingly.
   NOTE: SFD medium contains 715 mL of Iscove’s Modified Dulbecco’s Medium (IMDM), 250 mL of Ham’s F12, 5 mL of N2-supplement, 10 mL of B27 minus Vitamin A, 5 mL of 10% (w/v) BSA (in PBS), 7.5 mL of GlutaMAX and 7.5 mL of Penicillin-Streptomycin. Add ascorbic acid (50 μg/mL) and 3.9 x 10^-3% (v/v) of monothioglycerol prior to using.
3. Plate the cell suspension into one 150 mm x 25 mm sterile plate and incubate at 37 °C in the 5% CO₂ incubator for 48 h. Cardiac spheroids should be formed within 24 h.
4. Collect all the formed cardiac spheroids and centrifuge for 3 min at 145 x g and room temperature to selectively isolate spheroids and avoid single cells.
5. Aspirate the supernatant and resuspend the spheroids in 25 mL of SFD medium with 1 ng/mL of Activin A and 1.5 ng/mL of BMP4 for differentiation induction. Plate the spheroids in the same 150 mm x 25mm sterile plate and incubate them at 37 °C in the 5% CO₂ incubator for 40 h.
   NOTE: Different concentrations of Activin A (0-3 ng/mL) and BMP4 (0.5-2 ng/mL) can be used for differentiation optimization depending on the mESC line.
6. Collect all the cardiac spheroids and centrifuge for 3 min at 145 x g and room temperature.
7. Aspirate the supernatant and resuspend the spheroids in 25 mL of SFD medium. Transfer the resuspended EBs in an ultra-low attachment 75 cm² flask and incubate them at 37 °C in the 5% CO₂ incubator for 48 h. 

3. Isolation of Heart Field Specific Cardiac Progenitor Cells Using Fluorescent Reporters

1. Centrifuge cardiac spheroids at 145 x g and room temperature for 3min and aspirate the supernatant. Add 1 mL of Trypsin and incubate at 37 °C for 3 min. Mix well by pipetting to dissociate the cells.
2. Add 4 mL of 10% FBS in DMEM to inactivate Trypsin and mix well by pipetting. To remove the non-dissociated EBs, filter the mix using a 70 μm strainer and centrifuge the filtrated cells for 3 min at 270 x g and room temperature.
3. To sort CPCs carrying fluorescent reporters (CPCs derived from mESCs^{Tbx1-Cre; Rosa-RFP; HCN4-GFP}), aspirate the supernatant and add 500 μL of FACS sorting solution (1% (v/v) FBS, 200 mM HEPES and 10 mM of EDTA in PBS) to resuspend.
4. To remove all cell clusters prior to sorting, filter the cells again using a 5 mL polystyrene round-bottom tube with a 40 μm cell strainer. Keep the cells on ice until sorting.
5. Sort the cells to isolate Tbx1-Cre; Rosa-RFP and HCN4-GFP positive CPCs using a fluorescent activated cell sorter (FACS). Collect the sorted cells in 1 mL of FBS. Keep the cell sample and sorted cells at 4 °C.

4. Isolation of Heart Field Specific Cardiac Progenitor Cells Using Cxcr4 as a Cell Surface Protein Marker

1. To isolate first vs second heart field CPCs based on the expression of the surface protein receptor Cxcr4, use the mESC^Isl1-RFP line. Aspirate the supernatant from step 3.3 and resuspend the single CPCs in 300 μL of 10% FBS in PBS containing 1:200 (vol/vol) PerCP-efluor 710 conjugated anti-Cxcr4 antibody.
2. Incubate at room temperature for 5min and wash by adding 1-2 mL of cold PBS. Centrifuge the single CPCs for 3 min at 270 x g and room temperature and wash two more times followed by centrifugation.
3. Aspirate the supernatant and add 500 μL of FACS sorting solution to resuspend the single CPCs and filter as in step 3.4.
4. Isolate Cxcr4+ and Cxcr4- cells using FACS. Collect the sorted cells in 1 mL of FBS. Keep the cell sample and sorted cells at 4 °C.

5. Analysis of Isolated Heart Field Specific Cardiac Progenitor Cells

1. Centrifuge sorted CPCs for 3 min at 270 x g and room temperature. Sorted cells can be used for gene and protein expression analyses or they can be recultured for analyses at later time points. 
2. To re-culture isolated CPCs, aspirate the supernatant, resuspend the cells in SFD medium and replate ~3 x 10⁴ cells per well of a 384-well plate coated with 0.1% (w/v) gelatin.  If increased cell death is noted after sorting, add 10 μM of Y-27632 (ROCK inhibitor) to the sample. Two days after reculture, spontaneous beating should be noted.
3. To analyze the ability of plated CPCs to differentiate to cardiomyocytes, collect the cells at day 12 of differentiation. Use Trypsin as described in steps 1.2-1.5 to isolate single CMs. Resuspend the cells in 4% (w/v) paraformaldehyde (PFA) and incubate for 30 min at room temperature to fix the cells.
4. Centrifuge the cells for 3 min at 895 x g, and room temperature. Aspirate the supernatant and resuspend the cells in PBS to wash the PFA. Repeat this step once more.
5. Aspirate the supernatant and resuspend the cells in 10% FBS in PBS. Incubate half of the cell sample with mouse anti-Troponin T antibody (1:500) and use the rest of the sample as a negative control. Incubate for 30 min at room temperature.
6. Wash the cells twice as described in step 5.4 using PBS. Aspirate the supernatant and resuspend both cell samples in 10% FBS in PBS with 1:500 donkey anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 647 conjugate. Incubate for 30 min at room temperature.
7. Wash twice with PBS as in step 5.6. Aspirate the supernatant and resuspend the cells in 200 μL of PBS. Use a flow cytometer to analyze the cells.

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Wyniki

After approximately 132 h of differentiation, Tbx1-RFP and Hcn4-GFP CPCs can be detected using a fluorescent microscope (Figure 2). Generally, GFP and RFP cells appear approximately around the same time. The two populations of CPCs continue to expand in close proximity and commonly in a complementary pattern. Adjusting the concentrations of Activin A and BMP4 will alter the percentages of FHF vs SHF CPCs (Figure 3). CPC specification in vitro was primarily deter...

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Dyskusje

In our protocol, we describe a methodology to generate cardiac spheroids and isolated heart field-specific CPCs. Those can be used to study mechanisms of CPC specification and their properties, as well as for cardiac chamber-specific disease modelling. One previously published work used a mESC line with two fluorescent reporters (Mef2c/Nkx2.5) to study cardiogenesis in vitro, however, both those markers are expressed at embryonic day 9.5-10 when cardiomyocytes are already formed²⁶. To our knowledg...

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Materiały

Name	Company	Catalog Number	Comments
β-mercaptoethanol	Sigma	M6250
0.1% (w/v) Gelatin	EMD Millipore	ES-006-B
100 mM Sodium Pyruvate	Gibco	11360
100x Pen/Strep	Gibco	15070-063
1x PBS w/o Calcium and Magnesium	Thermo Fisher Scientific	21-040-CV
20% Paraformaldehyde	Thermo Fisher Scientific	50-980-493
5 mL Polystyrene round-bottom tube with a 40μm cell strainer	BD Falcon	35223
Activin A	R & D Systems	338-AC-010
Ascorbic Acid	Sigma	A-4544
B27 minus vitamin A (50x)	Thermo Fisher Scientific	12587010
BMP4	R & D Systems	314-BP
Bovine Serum Albumin	Sigma	A2153
Cell sorter	Sony	SH800	Sony or any other fluorescence-activated cell sorter
Cell strainer 70μm	Thermo Fisher Scientific	08-771-2
Centrifuge Sorvall Legend XT	Thermo Fisher Scientific	75004508
CHIR99021	Selleck chemicals	S2924
CO2 Incubator	Thermo Fisher Scientific	51030285
Corning Ultra Low Attachment T75 flask	Corning	07-200-875
Countless II FL automated cell counter	Thermo Fisher Scientific
Donkey anti-mouse IgG secondary antibody, Alexa Fluor 647 conjugate	Thermo Fisher Scientific	A-31571, Lot #1757130
Dulbecco's Modified Eagle's Medium high glucose (DMEM)	Gibco	11965-092
EDTA	Sigma	E6758
ESGRO (LIF)	Millipore	ESG1106
EVOS FL microscope	Thermo Fisher Scientific	AMF4300
Fetal Bovine Serum	Invitrogen	SH30071.03
Glasgow’s MEM (GMEM)	Gibco	11710035
GlutaMAX (100x)	Gibco	35050-061
Ham’s F12	Gibco	10-080-CV
HEPES	Sigma	H3375
IMDM	Gibco	12440053
Monothioglycero (MTG)	Sigma	M-6145
Mouse anti-Troponin T antibody	Thermo Fisher Scientific	MS-295-P1
N2-SUPPLEMENT	Gibco	17502-048
Non-essential amino acid solution (NEAA	Invitrogen	11140-050
PD0325901	Selleckchem	S1036
PerCP-efluor 710 conjugated anti-Cxcr4 antibody	Thermo Fisher Scientific	46-9991-82
Suspension culture dish 150 mm x 25 mm	Corning	430597
T25 flasks	Corning	353109
TrypLE (Trypsin)	Gibco	12604
Y-27632 (ROCK inhibitor)	Stem cell technologies	72304