Homing of cardiac progenitor cells to ischemic heart via hypoxic preconditioning 
Lijuan Chen 1, 2, Mi Zhou1, Gengshan Ma2, John Zhang3, Neal Weintraub1, Yaoliang Tang1
•	Cardiovascular disease, Internal Medicine, University of Cincinnati, 231 Albert Sabin way, Cincinnati, OH 45243
•	 Department of Cardiology, Zhongda Hospital, Southeast University, Nanjing 210009, China
•	Keck Graduate Institute, 535 Watson Dr. Claremont, CA 91711
Corresponding authors:
Yaoliang Tang MD, Ph.D
Associate Professor of Internal Medicine, Division of Cardiovascular Disease
College of Medicine, University of Cincinnati
231 Albert Sabin Way, ML0542
Cincinnati, OH 45267
Tel: (513)558-3091
Email: tangyg@ucmail.uc.edu
Keywords:
Cardiac progenitor cells, Hypoxia preconditioning, Homing, Ischemic heart disease
Short Abstract

We demonstrate in the video a method for isolating, culturing and preconditioning cardiac progenitor cells from mouse hearts. We also show how to transplant CPCs intravenously via jugular vein injection. 
Long Abstract
Ischemic heart attacks remain the leading cause of death worldwide. Resident cardiac progenitor cells (CPCs) are particularly suitable for cardiomyocyte replacement because they are endogenous components of the adult heart and responsible for the physiological and pathological heart repair1-4. Although CPCs seem to be the obvious choice for cell-based cardiac repair, the success of isolation of CPCs and homing capacity of CPCs in vivo are quite limited because of extremely low frequency of CPCs in adult heart, and lack of activation of main cell homing signaling pathway. To overcome these limitations, we developed a two-step protocol for CPC isolation5, and used hypoxia treatment to activate cell homing signaling pathway6. Our protocol can help us generate a sufficient number of cells for transplantation purpose, and increase cell retention in the ischemic myocardium to maximize CPC regenerative potential.

Protocol Text
Two step protocol to isolate and culture of cardiac progenitor cells
•	Before we start the experiment, we will coat cell culture dishes with Matrigel: Slowly thaw Matrigel (354230 BD, Bioscience) overnight at 4oC on ice, dilute Matrigel (1:30) using cold serum-free DMEM(1:30 final), and add diluted Matrigel Basement Membrane Matrix to dishes being coated. Quantity should be sufficient to cover entire growth surface easily. Coated dish should be incubated at 4°C overnight before use
•	Adult mouse heart  Tissues were minced into small pieces(1x1mm2), and then subjected to enzymatic dissociation with a mixture of 0.125% trypsin and 0.1% collagenase IV three times for 5 minutes at 38 °C. 
•	After digestion, the remaining tissue fragments are cultured as explants in explant medium (DMEM with 10% fetal calf serum (FBS), 100U/mL penicillin G, 100ug/ml streptomycin, 2 mmol/L L-glutamine, and 0.1mmoL/L 2-mercaptoethanol) at 37°C and 5% CO2. 
•	After 1 week, a fibroblast lay will formed from the adherent explants, and at about 2 weeks, the heart explants will de-differentiate, and many small, phase-bright cells will migrate above the fibroblast layer formed from adherent explants. 
•	To avoid damaging the integrity of cell surface antigen, we collected these phase-bright cells simply by washing with D-Hanks.
•	Newly isolated cardiac progenitor cells will be seeded at multiwell plates precoated with Matrigel in media designated for cell growth medium (CGM) (DMEM/F12, 10% FBS, 200 mmol/L L-glutamine, 0.1 mmol/L β-mercaptoethanol, 1% nonessential amino acids, 1000 units/ml LIF, 0.1 unit/ml thrombin and 5 ng/ml basic fibroblast growth factor (bFGF)). Some CPCs will form spheres, which show positive for c-kit staining.

Hypoxic preconditioning of cardiac progenitor cells

CPC cells will be incubated under hypoxia chamber(37°C; 0.1%Oxygen, 5%Carbon Dioxide, 94.9%Nitrogen, billups-rothenberg, http://www.brincubator.com) for 8hrs before in vivo transplantation.
1. Open both inlet and outlet ports (tygon tubing with white plastic clamps).
2. Attach inlet port tygon tubing to a source containing the gas mixture.
3. Purge chamber at 20 liters/min for 5 minutes . After chamber has been purged disconnect gas source.
4. Seal chamber by closing plastic clamps, and put Chamber in a conventional continuous flow CO2 incubator.

Cell transplantation through jugular vein in mouse with myocardial ischemic/reperfusion
1. Mouse myocardial ischemia/reperfusion model, the procedure is carried out using a stereo microscope at 2 times magnification.:
1) Anesthetize adult(25-30g) C57BL/6 mice with ketamine(80mg/kg) /xylazin (25mg/kg), and underwent oral intubation, and connected to ventilator.
2) Make an incision on the left side of the throax and the muscles were retracted to expose the fourth and fifth intercostals space.
3) We open the chest and remove the pericardial sac to expose the heart.
4) Place 8-0 silk suture around the left coronary artery and tied around the PE tubing, the heart was returned to the thoracic cavity and the chest wall and skin were closed by 6-0 silk suture. The tubing will be removed 45min after ischemia to make myocardial reperfusion. ECG was carried out before and after surgery to confirm MI.
2. Intravenous injection of CPCs through right jugular vein 
Mice will receive intravenous injections of CPCs within 1 day after MIR surgery.
1) Shave the right side of neck of mouse, and disinfect the skin using an iodine solution.
2) Make a longitudinal incision of about 10mm in the neck, Jugular vein is on the right lateral of right thyroid gland, after careful removal of the connective tissue surrounding the jugular vein, Gently lift the right thyroid gland to expose the whole jugular vein.
3) Load 200 μL (5x105 cells labeled with lentiviral GFP vector) into a 1mL insulin syringe with a 30G needle. 
4) Inject 200ul of cells into the jugular vein of recipient mice slowly. 
5) 24 hrs after injection, harvest the heart, and make 5µm cryo-tissue section, cell recruitment will be quantified by analyzing GFP positive cells in the heart section.

Representative Results

Fig. 1. Isolation and expansion of cardiac progenitor cells.  Phase-contrast microscopy of cells migrated from primary culture of mouse ventricular explants. Migrated cells aggregated and proliferated above the coating of fibroblasts.

Fig. 2.  (A) Culture of migrated round, bright progenitor cells, which can form cardiosphere. (B)  cardiosphere showes positive for c-kit markers . 

Discussion
Cardiac progenitor cells are promising for repairing heart tissue, however, it is still very challenge to isolate intact CPCs from small heart tissue because the CPCs are extremely rare in adult heart, and these cells are very sensitive to regular one-step enzyme digestion protocol, which will make CPCs lose their cell surface stem cell markers, and lead to spontaneous differentiation. The CPCs isolated with our novel two stage protocol show their capacity for self-renewal and clonogenic in vitro with fibroblast-free conditional medium, and can differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells after being transplanted into ischemia-induced heart of mice5. Moreover, we found that hypoxic preconditioning increases CPC homing to injured hearts6. When administered systemically, the hypoxia-preconditioned CPC cells homed to the ischemic myocardium more efficiently than normoxia-cultured cells, which led to significantly improved heart repair. Our presented protocol is easily translatable to clinical applications. 

Disclosures

No conflicts of interest declared.

Acknowledgements
We would like to acknowledge our funding sources: NIH grants HL086555 (to
Y.T.)
Materials
Materials
Material name	type	company	Catalogue Number	commet	
Collagenase type4		MP Biomedicals LLC	195110		
Trypson0.25%(1X)solution		Hyclone	SH30042.01		
Matrigel Basement Membrane (growth factor reduced)		BD Bioscience	354230		
DMEM		Hyclone	SH30243.01		
DMEM/F12		Hyclone	SH30261.01		
Fetal Bovine Serum		Biowest	S01520HI		
Antibiotic/antimycotic solution 100x		Hyclone	SV30079.01		
MEM Non-Essential Amino Acids		invitrogen	11140		
2-Mercaptoethanol 1000x		invitrogen	21985-023		
Leukemia inhibitory factor		Santa Cruz	SC4988		
basic fibroblast growth factor(bFGF)		Millipore	GF003AF		
PGK-GFP plasmid		Addgene	12252		
Modular incubator chamber	MIC101	Billups-rothenberg inc			
8-0 prolene(0.4metric)		Ethicon	BV175-6		
6-0 (0.7metric)Nonabsorbable		Surgik LC	ESILRC10387560		

References

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