Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Summary

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Abstract

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

Introduction

Ischemic heart disease, including myocardial infarction (MI), is a major cause of death around the world¹. Stem cell therapy for regenerating dead myocardium remains a major approach to improve the cardiac function of an MI heart^2,3,4,5. Different types of stem cells have been used to replenish dead myocardium and to improve the cardiac function of an MI heart. They can be broadly categorized into embryonic stem cells⁶ and adult stem cells. In adult stem cells, various types of stem cells have been used, such as bone marrow-derived mononuclear cells^7,8, mesenchymal stem cells derived from bone marrow^9,10, adipose tissue^11,12, and umbilical cord¹³, and CSCs^14,15. Stem cells can promote cardiac regeneration through endocrine and/or paracrine actions^{16,17,18,19,20}. However, a major limitation of stem cell therapy is obtaining an adequate number of stem cells that can proliferate and/or differentiate toward a specific cardiac lineage^21,22. Autologous and allogenic transplantation of stem cells is an important challenge in stem cell therapy⁹. CSCs could be a better approach for cardiac regeneration because they are derived from the heart and they can be more easily differentiated into cardiac lineages than non-cardiac stem cells. Thus, it reduces the risk of teratoma. In addition, the endocrine and paracrine effects of CSCs, such as exosomes and miRNAs derived from the CSCs, could be more effective than other types of stem cells. Thus, CSCs remains a better option for cardiac regeneration^23,24.

Although CSCs are a better candidate for cardiac regeneration in an MI heart due to their cardiac origin, a major limitation with CSCs is less yield due to the lack of an efficient isolation method. Another limitation could be the impaired differentiation of CSCs toward cardiomyocytes lineage^2,25,26,27. To circumvent these limitations, it is important to develop an efficient protocol for CSC isolation, characterization, and differentiation towards cardiac lineage. There is no single acceptable marker for CSCs and a specific cell-surface marker-based isolation of CSCs yields less CSCs. Here, we standardize a simple gradient centrifugation approach to isolate CSCs from the mouse heart that is cost-effective and results in an increased yield of CSCs. These isolated CSCs can be selected for specific cell-surface markers by fluorescence-activated cell shorting. In addition to CSCs isolation, we provided a protocol for CSC culture, characterization, and differentiation towards cardiomyocyte lineage. Thus, we present an elegant method to isolate, characterize, culture, and differentiate CSCs from adult mouse hearts (Figure 5).

Protocol

The housing, anesthesia, and sacrifice of mice were performed following the approved IACUC protocol of the University of Nebraska Medical Center.

1. Materials

1. Use 10- to 12-week-old C57BL/6J black male mice, kept in-house at the institutional animal facility, for the isolation of CSCs. CSCs can also be isolated from non-pregnant female mice.
2. Sterilize all the necessary surgical instruments, including surgical scissors, fine surgical scissors, curved shank forceps, and a surgical blade, by autoclaving them before the euthanasia of the mouse.
3. Prepare CSC isolation buffers in a sterilized condition and store them at 4 °C on ice. These buffers include polysucrose and a sodium diatrizoate solution adjusted to a density of 1.077 g/mL, incomplete Dulbecco’s modified Eagle’s Medium, 1x Ham’s balanced salt solution (HBSS), and cell culture-grade 1x phosphate-buffered saline (PBS). Prepare a 1% collagenase II stock solution (filtered and sterilized) and a 0.2% working solution in HBSS.
4. Keep the tissue culture materials in the sterilized condition in the biosafety cabinet, including a 10-mm Petri dish, a 6-well plate, a 24-well plate, T-25 and T-75 culture flasks, 15-mL and 50-mL conical tubes, and disposable serological pipettes with 40-µm and 100-µm cell strainers with 0.22-µm filters.
5. Sterilize the 10-µL, 200-µL, and 1,000-µL pipette tips by autoclave.
6. Use powder-free nitrile gloves and 70% ethanol to maintain a sterile condition throughout the experiment.
7. Use 10% bleach as a disinfectant.
8. Use commercially available CSC maintenance medium and cardiomyocytes differentiation medium for the culture and differentiation of CSCs, respectively.

2. Isolation Method of Cardiac Stem Cells

1. Euthanize four to five adult male (or non-pregnant female) mice using a CO₂ chamber. Fix each mouse’s arms on a separate dissection cardboard with pins or sticky tapes.
2. Spray 70% ethanol on the mouse to get rid of outside germs and maintain the sterilized condition during the harvesting of the heart. Make an incision with scissors in the middle of the abdomen and cut the skin from the abdomen up to the thorax in a straight line. Remove the skin from both sides of the middle cut to clear the abdominal area of skin and hairs. Using scissors and tweezers, cut the diaphragm below the ribcage.
3. Open the thoracic cavity of the mouse by cutting the ribcage inside the hood. Expose the heart and remove the blood near the heart. Wash the surrounding area of the heart with ice-cold PBS to get rid of any blood in the vicinity of the heart.
4. Dissect the heart using surgical scissors and tweezers. Place the heart in a 100-mm Petri dish containing 10 mL of ice-cold PBS.
5. Palpitate the heart using curved shank forceps and remove the residual blood inside the heart.
6. Use the whole heart for the isolation of CSCs.
7. Transfer all four or five whole hearts to a 50-mL conical tube containing 10 mL of ice-cold HBSS. Keep the tube on ice until further processing.
8. Wipe inside and outside of a biosafety cabinet with 70% ethanol to create a sterilized environment. Sterilize the heart-containing tube and other required materials with 70% ethanol. Place the heart-containing tube into the biosafety cabinet for further processing.
9. Transfer the hearts to a 100-mm Petri dish containing 10 mL of ice-cold HBSS. Wash the hearts again by palpating to remove any residual blood inside the heart. Change the HBSS solution after each wash to ensure the complete removal of the blood.
10. Cut the hearts into small pieces using fine surgical scissors or a surgical blade in a 100-mm Petri dish containing 5 - 7 mL of HBSS solution. Hold each heart with a pair of forceps and use a pair of surgical fine scissors or a surgical blade to cut the heart into 2- to 4-mm pieces. Change the HBSS solution frequently to ensure blood-free HBSS. Mince the hearts in the HBSS solution.
11. Centrifuge the minced tissue at 500 x g at 4 °C for 5 min. Discard the supernatant and keep the pellet.
12. Resuspend the pellet in 5 - 6 mL of 0.2% collagenase II solution in a 50-mL conical tube. The volume of collagenase solution should be almost 1.5- to 2-fold to the volume of the pellet.
    Note: For the digestion of tissue, 0.2% collagenase I and 2.5 U/mL dispase solution can replace 0.2% collagenase II. Use an almost equal volume of dispase solution to the collagenase I solution.
13. Thoroughly mix the pellet with 0.2% collagenase II solution by agitating the tube or by rocking the tube on a shaker at 75 x g for 45 - 60 min at 37 °C. Shake the tube vigorously by hand after every 20 - 30 min to enhance the lysis.
14. Triturate the lysed tissue mix using a 5 mL serological pipette and a 1-mL pipette tip to dissociate the tissue lump into the single-cell suspension.
    Note: To get the maximum recovery of the CSCs, triturate the lysis mix for at least 5 min. If the tissue lumps are relatively big, cut the tip of a 1-mL pipette tip with scissors or a surgical blade and use it for trituration.
15. Stop the enzymatic lysis of the tissue by adding commercially available CSC maintenance medium. Add almost 2 - 3x as much maintenance medium as the volume of the lysed tissue in step 2.14.
16. Pass the digested cell suspension through a 100-µm cell strainer to remove any undigested tissue pieces.
17. Repeat step 2.16 using a 40-µm cell strainer.
18. Separate the cells through density gradient centrifugation. For the separation of cells, gently overlay an equal volume of the filtrate over polysucrose and sodium diatrizoate solution. For example, first, add 20 mL of polysucrose and sodium diatrizoate solution in a 50-mL conical tube and then, add 20 mL of filtrate from step 2.17 over the polysucrose and sodium diatrizoate solution.
    NOTE: Prewarm the polysucrose and sodium diatrizoate solution at 37 °C before use with the cells. Add the filtrate from step 2.17 over the polysucrose and sodium diatrizoate solution very carefully and slowly to avoid any mixing of the two solutions. This is considered to be a crucial step.
19. Centrifuge the tube containing both solutions at 500 x g for 20 min at room temperature using a swing bucket centrifuge. Set the centrifuge at a lower acceleration and deceleration speed to avoid mixing the two solutions and for proper separation of the CSCs. This is an important step in CSC isolation. Put the tube containing the gradient mixture very slowly without disturbance inside the centrifuge and set it for centrifugation.
20. Take out the buffy coat along with extra solution from the upper layer using either a 1-mL pipette or a plastic Pasteur pipette in a 15-mL sterilized tube. This buffy layer will contain the CSCs.
    Note: Take extra precaution during the pipetting of the buffy coat not to take out the polysucrose and sodium diatrizoate solution layer because it is toxic to CSCs.
21. Add an equal volume of CSC maintenance medium to the cell suspension from step 2.20 and mix it properly to neutralize the residual polysucrose and sodium diatrizoate solution, if present.
22. Centrifuge the above cell suspension at 500 x g for 5 min at 4 °C. Discard the supernatant.
23. Resuspend the pellet in 7 - 10 mL of incomplete DMEM medium and centrifuge it at 500 x g for 5 min at 4 °C to get rid of any residual polysucrose and sodium diatrizoate solution.
24. Collect the pellet, which contains the purified CSCs.
25. Resuspend the pellet in 1 mL of CSC maintenance medium, count the CSC number, and use the pellet for culture. To count the CSC number, use Trypan blue staining and a hemocytometer. With four male mice hearts, the yield of CSCs is ~1.8 million.
26. Seed the CSCs in a 6-well culture plate coated with a 0.02% gelatin solution containing 0.5% fibronectin. Use 2 mL of complete maintenance medium for seeding. Incubate the culture plate in an incubator at 37 °C with 5% CO₂.
27. Replace the medium of culture CSCs with complete CSC maintenance medium every day until the third day. After that, change the medium on alternate days.
28. Determine the growth of the CSCs by observing them under a microscope.

3. Culture Maintenance and Passage of Cardiac Stem Cells

1. Culture the isolated CSCs in a commercially available maintenance medium. Replace the culture medium with fresh maintenance medium on alternate days. When the CSC culture has reached confluency, transfer the CSCs to a new plate coated with gelatin and fibronectin, as mentioned in step 2.26. Detach the cultured CSCs by enzymatic cell dissociation buffer. Follow the standard protocol of cell dissociation from the culture plate. At this stage, CSCs are considered at passage 0 (P0) stage.
2. Culture P0 CSCs in a new gelatin and fibronectin-coated plate in complete CSC maintenance medium at 37 °C in a 5% CO₂ incubator, allow them to be confluent, and then follow step 3.1 to get passage 1 (P1) CSCs. Store P1 CSCs in liquid nitrogen or use them for experiments.
3. Seed 0.5 million cells in a 6-well plate or 1 million cells in T-25 to maintain the CSC culture at its initial stage.
4. Passage the CSCs when they become 85% - 90% confluent. CSCs can be cultured in the maintenance medium up to four to six weeks.
   Note: Keep the initial seeding density of the CSCs relatively high (40% - 50%) because, at a lower seeding density, the CSCs may change their morphology to flat and elongated.

4. Characterization of Cardiac Stem Cells

1. To characterize the cultured CSCs, observe them under a phage-contrast or a bright-field microscope. Place the CSC-containing plate on the objective of a fluorescent microscope. Set the magnification fairly low (10X) to focus the cells. Use phase-contrast aperture to observe the cells. Increase the magnification to 20X by changing the objective lens and image the CSCs. Increase the objective to 40X and image the CSCs. In two days, the CSCs appear almost round in shape, but the size of the cell increases with time, and in seven days, the CSCs appear almost cylindrical in shape (Figure 1A - 1C).
2. Perform immunostaining of the CSCs with markers of pluripotency/stemness (OCT4, SOX2, or Nanog), proliferation (Ki-67) (Figure 2A - 2D), and cardiac origin/progenitor cells (Sca-1, NKX2-5, or GATA4) (Figure 3A - 3C).
   1. For the immunostaining of the CSCs, seed 10,000 CSCs in a 24-well culture plate.
   2. The next day (after 18 - 24 h), fix the CSCs in 300 µL of 4% paraformaldehyde for 10 min.
   3. Wash the CSCs with 500 µL of ice-cold PBS, 3x with 5-min intervals.
   4. Permeabilize the CSCs with 300 µL of 0.2% Triton X-100 diluted in PBS for 10 min.
   5. Wash the CSCs with 500 µL of ice-cold PBS, 3x with 5-min intervals.
   6. Perform the blocking of the CSCs with 300 µL of 1% BSA diluted in PBST (PBS + 0.1% Tween 20) or 300 µL of 10% normal chicken serum diluted in PBST for 1 h.
   7. Incubate the CSCs in 200 µL of primary antibody diluted in blocking solution overnight at 4 °C. Dilute the primary antibody as recommended by the datasheet of antibodies or as per standardization.
   8. Wash the CSCs with 500 µL of ice-cold PBS, 3x with 5-min intervals.
   9. Incubate the CSCs in 200 µL of secondary antibody diluted in blocking solution (see step 4.2.6) for 1 h at room temperature. Dilute the secondary antibody as recommended by the datasheet of antibodies or as per standardization.
   10. Wash the CSCs with 500 µL of ice-cold PBS, 3x with 5-min intervals.
   11. Counterstain the CSCs with 200 µL of DAPI (1 µg/mL) diluted in PBS for 5 min.
   12. Wash the CSCs 1x with 500 µL of ice-cold PBS. Then, add 300 µL of ice-cold PBS in each well.
   13. Perform imaging using a fluorescence microscope. Follow the instructions of fluorescent imaging, such as to avoid direct light on the plate. Keep the culture plate under the microscope. Focus the cells at different magnifications. Observe the cells under phase-contrast and/or different excitation filters and save the images.
   14. Store the plate in the dark at 4 °C.

5. Differentiation of Cardiac Stem Cells into Cardiomyocyte

1. For the differentiation of CSCs, seed 500,000 CSCs in a 6-well plate with 2 mL of prewarm (37 °C) commercially available CSC maintenance medium.
2. Incubate the plate containing the CSCs in a CO₂ incubator at 37 °C. Allow the CSCs to attach and proliferate until sub-confluent growth. If the medium turns yellow, replace the culture medium with fresh maintenance medium.
3. At 85% - 90% confluency, replace the maintenance medium with 2 mL of prewarm (37 °C) cardiomyocytes differentiation medium. Incubate the plate in a CO₂ incubator at 37 °C for up to 2 - 3 weeks.
4. Change the differentiation medium every 2 - 3 d. Observe the CSCs’ morphology under a microscope every time during the medium change to ensure the good culture conditions.
5. After 12 d of CSC culture in differentiation medium, image the CSCs for differentiation markers following a standard immunostaining protocol (see steps 4.2.1 - 4.2.14). Save the fluorescent images for the expression of cardiomyocytes markers (Figure 4A - 4B).

Results

In the present study, we isolated CSCs from 10- to 12-week-old C57BL/6J male mice hearts. Here, we have presented a simple method for CSC isolation and characterization using markers of pluripotency. We also presented an elegant method for CSC differentiation and the characterization of CSCs that differentiated toward cardiomyocytes lineage. We observed a spindle shape morphology of 2- to 3-days-cultured CSCs under a phase-contrast microscope (Figure 1A and

Discussion

The critical steps of this CSC isolation protocol are as follows. 1) A sterilized condition must be maintained for extraction of the hearts from the mice. Any contamination during the heart extraction may compromise the quality of the CSCs. 2) The blood must be completely removed before mincing the heart, which is done by several washes of the whole heart and the heart pieces with HBSS solution. 3) The heart pieces must be completely lysed into a single-cell suspension with collagenase solution. 4) The polysucrose and so...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mice	The Jackson laboratory, USA	Stock no. 000664
Antibodies:
OCT4-	Abcam	ab18976 (rabbit polyclonal)	OCT4-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution
SOX2	Abcam	ab97959 (rabbit polyclonal)	SOX2-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution
Nanog	Abcam	ab80892 (rabbit polyclonal)	Nanog-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution
Ki67	Abcam	ab16667 (rabbit polyclonal)	Ki67-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution
Sca I	Millipore	AB4336 (rabbit polyclonal)	Sca I Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution
NKX2-5	Santa Cruz	sc-8697 (goat polyclonal)	NKX2-5-Primary antibody- 1:50 dilution, Secondar antibody- 1:200 dilution, in blocking solution
GATA4	Abcam	ab84593 (rabbit polyclonal)	GATA4-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution
MEF2C	Santa Cruz	sc-13268 (goat polyclonal)	MEF2C-Primary antibody- 1:50 dilution, Secondar antibody- 1:200 dilution, in blocking solution
Troponin I	Millipore	MAB1691 (mouse monoclonal)	Troponin I-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution
Actinin	Millipore	MAB1682 (mouse monoclonal)	Actinin-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution
ANP	Millipore	AB5490 (mouse polyclonal)	ANP-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution
Alex Fluor-488 checken anti-rabbit	Life technology	Ref no. A21441
Alex Fluor-594 goat anti-rabbit	Life technology	Ref no. A11012
Alex Fluor-594 rabbit anti-goat	Life technology	Ref no. A11078
Alex Fluor-488 checken anti-mouse	Life technology	Ref no. A21200
Alex Fluor-594 checken anti-goat	Life technology	Ref no. A21468
Name	Company	Catalog Number	Comments
Culture medium:
CSC maintenance medium	Millipore	SCM101	Note: For CSC culture, PBS or incomplete DMEM medium was used for washing the cells
cardiomyocytes differentiation medium	Millipore	SCM102
DMEM	Sigma-Aldrich	D5546
Name	Company	Catalog Number	Comments
Cell Isolation buffer:
polysucrose and sodium diatrizoate solution (Histopaque1077)	Sigma	10771
HBSS	Gibco	2018-03
Collagenase I	Sigma	C0130
Dispase solution	STEMCELL Technologies	7913
PBS	LONZA	S1226
StemPro Accutase Cell Dissociation Reagent	Thermoscientific	A1110501
Other reagents:
BSA	Sigma	A7030
Normal checken serum	Vector laboratory	S3000
DAPI solution	Applichem	A100,0010	Dapi, working concentration-1 µg/mL
Trypan blue	Biorad	145-0013
Trypsin	Sigma	T4049
StemPro Accutase Cell Dissociation Reagent	Thermo Fisher Scientific	A1110501
Formaldehyde	Sigma	158127
Triton X-100	ACROS	Cas No. 900-293-1
Tween 20	Fisher Sceintific	Lot No. 160170
Ethanol	Thermo Scientific
Name	Company	Catalog Number	Comments
Tissue culture materials:
100 mm petri dish	Thermo Scientific
6-well plate	Thermo Scientific
24-well plate	Thermo Scientific
T-25 flask	Thermo Scientific
T-75 flask	Thermo Scientific
15 ml conical tube	Thermo Scientific
50 mL conical tube	Thermo Scientific
40 µm cell stainer	Fisher Scientific	22363547
100 µm cell stainer	Fisher Scientific	22363549
0.22 µm filter	Fisher Scientific	09-719C
10 mL syring	BD	Ref no. 309604
10 µL, 200 µL, 1000 µL pipette tips	Fisher Scientific
5 mL, 10mL, 25 mL disposible plastic pipette	Thermo Scientific
Name	Company	Catalog Number	Comments
Instruments
Centrufuge machine	Thermo Scientific	LEGEND X1R centrifuge
EVOS microscope	Life technology
Automated cell counter	Biorad
Cell counting slide	Biorad	145-0011
Pippte aid	Thermo Scientific	S1 pipet filler
Name	Company	Catalog Number	Comments
Surgical Instruments:
Surgical scissors	Fine Scientific Tool
Fine surgical scissors	Fine Scientific Tool
Curve shank forceps	Fine Scientific Tool
Surgical blade	Fine Scientific Tool