The protocol of the present study was approved by the Institutional Review Board and the Ethics Committee of Siping Hospital of China Medical University. The study was conducted in compliance with current Good Clinical Practice standards and in accordance with the principles set forth under the Declaration of Helsinki (1989).

The HUC-MSC doses used in this study were derived from two donated UCs obtained from healthy mothers during routine term elective caesarean section birth. Fully informed consent was obtained several weeks prior to delivery. HUC-MSC were isolated and propagated as previously described (10–13). UCs were filled with 0.1% collagenase (Sigma-Aldrich, St. Louis, MO, USA) in PBS and incubated at 37°C for 20 min. Each UC was washed with proliferation medium [a-minimal essential medium (MEM), 10% human AB serum; Gibco, Grand Island, NY, USA], and the detached cells were harvested after gentle massage of the UC. The cells were centrifuged at 300 × g for 10 min, resuspended in proliferation medium to seed in 75-cm 2 flasks at the density of 5×10⁷ cells/ml. After 24 h of incubation, non-adherent cells were removed and the culture medium was replaced every 3 days. The adherent cells were cultured until they reached 80–90% confluence.

Flow cytometry was performed to analyze the cell-surface expression of typical protein markers. The adherent cells were incubated with the following anti-human primary antibodies CD31-phycoerythrin (PE), CD45-fluorescein isothiocyanate (FITC), CD90-R-PE, HLA-DR-R-PE (Becton-Dickinson, Franklin Lakes, NJ, USA). The total of 10,000 labeled cells were analyzed using a Guava easyCyte flow cytometer running Guava Express Plus software (Guava Technologies, Inc., Hayward, CA, USA).

The inclusion criteria were stable symptomatic patients of ischemic cardiomyopathy [New York Heart Association (NYHA) functional class II/III], older than 18 years, left ventricular ejection fraction (LVEF) <40%. The exclusion criteria were non-cardiac serious diseases expected to reduce the patients's short-time survival, recent (<6 months) myocardial infarction or an implanted pacemaker. The patients provided written informed consent stating agreement to the treatment according to the Siping Hospital of China Medical University. The general characteristics of the patients are shown in Table I.

HUC-MSCs (10 ml) with a cell density of 5×10⁶-1×10⁷/ml was given intravenously at a rate of no more than 12.5×10⁶/min and flushed with 20 ml saline to ensure full cell dose delivery. Once the needle was fully withdrawn, the puncture site was wrapped with sterilized dressing. The patients remained in the supine decubitus on the operation bed for another 30 min before off-bed activities. The patient was monitored [temperature, blood pressure, pulse and electrocardiograph (ECG)] at 0, 15, 30, 45 and 60 min, and then hourly for a minimum of 4 h.

Patients underwent exercise testing using six-minute walk test a modified Bruce treadmill test. The patients were monitored throughout with tests being terminated by physiological markers (ST changes, arrhythmias, or chest pain) or by patient request.

i) Primary safety assessments included monitoring and recording of all adverse and serious adverse events. All patient were monitored (temperature, blood pressure, pulse and oxygen saturation) at 15, 30, 45 and 60 min, and then hourly for a minimum of 4 h. They were discharged 24-h post-transplantation given that the patient was afebrile and hemodynamically stable with no signs of infection or any type of allergic reaction. Mortality and major adverse cardiovascular events (MACE) defined as all-cause death, myocardial infarction, hospitalization for HF, or major arrhythmias were assessed at 3 months and 1 year.

ii) As exploratory secondary endpoints we investigated the efficacy of HUC-MSC infusion as follows: The change in global LVEF at 3, 6 and 12 months compared with baseline as assessed by advanced cardiac imaging and changes in left ventricular (LV) volumes; exercise capacity (six-minute walk test), and NYHA classification at 3, 6 and 12 months compared with baseline.

The patient's pharmacological therapy consisted of: i) Digoxin, 0.125 mg, once daily, p.o.; ii) β-acceptor blockers: Metoprolol, 6.25 mg, twice daily, p.o., or bisoprolol 2.5 mg once daily, p.o.; iii) diuretic: Furosemide, 20 mg once daily, i.v.; and/or spironolactone: 20 mg once daily, p.o.; and iv) angiotensin-converting enzyme inhibitors: Enalapril, 5 mg orally twice daily; irbesartan: 150 mg orally once daily.

Statistical analysis was performed using SPSS 16.0 software (Chicago, IL, USA). Safety and exploratory efficacy secondary endpoints were observed for each patient against the baseline values. P<0.05 was considered to indicate a statistically significant difference.

The cells derived from UC were observed 24 h after seeding (Fig. 1A), when part of the round mononuclear cells was adherent. Three days after inoculation, small colonies of the adherent cells with typical fibroblast-shaped morphology were obtained (Fig. 1B). These primary cells reached monolayer confluence, after planting for 5–6 days, when passaged for the first time. The fifth passage cells were analyzed by flow cytometry, and were strongly positive for CD105 and CD90, but negative for CD45 and HLA-DR (Fig. 1C-F).

The general characteristics of the patients are shown in Table I. The patients included 2 males and 1 female with a mean age of 51.7 years (range, 37–65 years) at HUC-MSCs infusion. The patients were enrolled between January 2010 and January 2012. The etiology of the HF was ischemic cardiomyopathy. All the patients reached the 3, 6 and 12 months primary endpoint (Table I).

Two patients demonstrated a 65.1% increase in LVEF at the end of 3 months, which was maintained increasing to 47.8% at the end of 12 months post-HUC-MSC intravenous infusion. LVEF of patient 1 decreased slowly in the observation period (Table II).

All the patients underwent a six-minute walk test at baseline, 3, 6 and 12 months. Patient 1 got a transient decrease at the end of 3 months, patient 2 got a transient increase at the end of 3 months, and then decreased slowly. After 12 months, there was significant improvement in six-minute walk test in two patients post-transplantation (Table II).

Each patient who showed improvement in the NYHA classification improved within 3 months of post-transplantation. After 12 months, this pattern continued with the three (100%) patients improving (Table II).

There were no complications or adverse events associated with HUC-MSC transplantation. No cases of distal coronary artery occlusion, acute cardiac dysfunction, and ventricular arrhythmia occurred.