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.
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.
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 heart2,3,4,5. 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 cells7,8, mesenchymal stem cells derived from bone marrow9,10, adipose tissue11,12, and umbilical cord13, and CSCs14,15. Stem cells can promote cardiac regeneration through endocrine and/or paracrine actions16,17,18,19,20. 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 lineage21,22. 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 regeneration23,24.
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 lineage2,25,26,27. 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).
The housing, anesthesia, and sacrifice of mice were performed following the approved IACUC protocol of the University of Nebraska Medical Center.
1. Materials
2. Isolation Method of Cardiac Stem Cells
3. Culture Maintenance and Passage of Cardiac Stem Cells
4. Characterization of Cardiac Stem Cells
5. Differentiation of Cardiac Stem Cells into Cardiomyocyte
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 1B). We found a change in the morphology of CSCs at 7 days of culture in maintenance medium, during which time the stem cells become elongated (Figure 1C). We characterized CSCs for markers of pluripotency and found that they express OCT4, SOX2, and Nanog (Figure 2A – 2C). We also found that CSCs proliferate in culture medium and express the proliferation marker Ki-67 (Figure 2D). To characterize the cardiac origin of CSCs, we determined the expression of cardiac markers Sca-1, NKX2-5, and GATA4 in CSCs. We found an expression of cardiac markers in the cultured CSCs (Figure 3A – 3C). To determine the differentiation of CSCs toward cardiomyocyte lineage, we cultured CSCs in cardiomyocyte differentiation medium for 12 days. After 12 days, we imaged the differentiated CSCs for the cardiomyocyte markers actinin and troponin I. We observed that cardiomyocyte markers were expressed in differentiated CSCs (Figure 4A – 4B). Overall, these results demonstrate that we successfully isolated CSCs from mouse heart and that these CSCs can proliferate and differentiate toward cardiomyocytes lineage.
Figure 1: Morphology of cultured CSCs. These panels show phase-contrast imaging of cultured CSCs, namely representative CSCs after 2 days of culturing in maintenance medium at (A1) 20X and (A2) 40X objectives, representative CSCs after 3 days of culturing in maintenance medium at (B1) 20X and (B2) 40X objectives, and representative CSCs after 7 days of culturing in maintenance medium at (C1) 20X and (C2) 40X objectives. The scale bars are 100 µm for the 40X magnifications and 200 µm for the 20X magnifications.
Figure 2: Characterization of CSCs for pluripotency markers. These panels show representative fluorescence imaging of cultured CSCs for markers of pluripotency (OCT4, SOX2, and Nanog) and proliferation (Ki-67). Panels A show expressions of OCT4 (green) and DAPI (blue) in CSCs at (A1) 20X and (A2) 40X magnifications. Panels B show expressions of SOX2 (green) and DAPI (blue) in CSCs at (B1) 20X and (B2) 40X magnifications. Panels C show expressions of Nanog (green) and DAPI (blue) in CSCs at (C1) 20X and (C2) 40X magnifications. Panels D show expressions of Ki-67 (red) and DAPI (blue) in CSCs at (D1) 20X and (D2) 40X magnifications. The scale bars are 100 µm for the 40X magnifications and 200 µm for the 20X magnifications. Please click here to view a larger version of this figure.
Figure 3: Characterization of CSCs for cardiac markers. These panels show representative fluorescence imaging of cultured CSCs for cardiac markers. Panels A show expressions of Sca-1 (red) and DAPI (blue) in CSCs at (A1) 20X and (A2) 40X magnifications. Panels B show expressions of NKX2-5 (green) and DAPI (blue) in CSCs at (B1) 20X and (B2) 40X magnifications. Panels C show expressions of GATA4 (red) and DAPI (blue) in CSCs at (C1) 20X and (C2) 40X magnifications. The scale bars are 100 µm for the 40X magnifications and 200 µm for the 20X magnifications.
Figure 4: Characterization of CSC differentiation toward cardiomyocyte lineage. These panels show representative phase-contrast and fluorescence imaging of 12-days differentiated CSCs. (A) This panel shows the expression of cardiomyocyte marker actinin in differentiated CSCs, with (a) a phase-contract image, (b) a fluorescence image of DAPI (staining nucleus), (c) a fluorescence image of actinin (green), and (d) a merged image of panels a – c. The magnifications are 40X. (B) This panel shows the expression of the cardiomyocyte marker troponin I in differentiated CSCs, with (a) a phase-contract image, (b) a fluorescence image of DAPI (staining nucleus), (c) a fluorescence image of troponin I (green), and (d) a merged image of panels a – c. The magnifications are 40X. The scale bars are 100 µm for the 40X magnifications and 200 µm for the 20X magnifications. Please click here to view a larger version of this figure.
Figure 5: Schematic representation of the different steps of CSC isolation, culture, and differentiation. We used surgically removed hearts from four to five adult mice for the isolation of CSCs. The stepwise process of CSC isolation, culture, and differentiation toward cardiomyocytes lineages are presented. Please click here to view a larger version of this figure.
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 sodium diatrizoate gradient solution for the separation of the cells must be prewarmed. 5) The medium for the CSC culture must be prewarmed. 6) The confluence of the CSCs during the seeding in a culture dish should be high because, in low confluency, CSCs may change their morphology. 7) The CSC number should be scored after the isolation from the heart and before the plating into a culture dish. The number of CSCs indicates the efficiency of the isolation from the heart. The counting of the CSCs is important for the seeding of the CSCs in the culture plate.
We used adult mice to isolate CSCs, which has been used by other investigators in rodent models23,28. CSCs can be isolated from neonatal rodent hearts29,30 and even from the biopsy of a human heart31. The previously reported CSC isolation protocols have some limitations, such as less yield, a complex isolation procedure, a long duration for differentiation, and less cardiomyocyte differentiation potential32,33,34. The CSC isolation protocol presented here is simple: it takes less time and is cost-effective and more efficient in CSC yield. Moreover, the isolated CSCs are Sca-1+ve (a well-accepted marker for mouse CSCs) (Figures 1A1 – 3C2). For the differentiation of CSCs, other protocols need three to four weeks35,36. However, this protocol requires 12 days (Figures 4A – 4B). The gradient centrifugation-based CSC isolation presented here yields ~1.8 million CSCs from four male mice hearts.
Although the yield of CSCs is high, a limitation of this protocol is the uniformity and purity of the isolated CSCs. Because there is no single acceptable marker for CSCs, it is difficult to use a single-cell-surface marker to select CSCs. However, CSCs isolated by this protocol can be used to obtain a uniform population of CSCs based on a specific marker or several markers of CSCs, using fluorescence-activated cell sorting. Thus, this method of CSC isolation is cost-effective with a high yield and has the option to get a uniform CSC population based on specific cell-surface marker(s).
The authors have nothing to disclose.
This work is supported, in parts, by the National Institutes of Health grants HL-113281 and HL116205 to Paras Kumar Mishra.
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 |