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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

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

Published: January 7th, 2019

DOI:

10.3791/58448

1Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 2Department of Anesthesiology, University of Nebraska Medical Center

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 ....

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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 s.......

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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 .......

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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.......

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This work is supported, in parts, by the National Institutes of Health grants HL-113281 and HL116205 to Paras Kumar Mishra.

....

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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

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