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 ....

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 s.......

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

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