This is a retrospective study conducted in a tertiary medical center. Data were retrieved from our phase I clinical trial entitled “intra-coronary transfusion of circulation-derived CD34+ cells improves left ventricular function in patients with end-stage diffuse coronary artery disease unsuitable for coronary intervention” (registration number: ISRCTN72853206)  and phase II clinical trial entitled “Intracoronary injection of autologous CD34+ cells improves one-year left ventricular systolic function in patients with diffuse coronary artery disease and partially preserved cardiac performance unsuitable for coronary intervention - a randomized, open-label, controlled phase II clinical trial (registration number: ISRCTN26002902) .
A total of 38 patients with EnD-CAD receiving intracoronary CD34+ cell therapy in the phase I trial between December 2011 and March 2014, and 30 patients undergoing CD34+ cell therapy in the phase II trial between December 2013 and November 2017, were selected in the present study. The inclusion and exclusion criteria have been thoroughly described in our clinical trials [11, 12, 21]. The written informed consents were obtained from all participants before enrollment. All variables in the phase I/II trials were collected and subjected to detailed analysis.
The data acquisitions during study period, including the clinical and laboratory parameters and imaging studies such as echocardiographic and coronary angiographic findings, have been approved by Taiwan Food and Drug Administration (TFDA) (IRB No: 99-3985A [phase I], 1066062944 [phase II]) and the Institutional Review Committee on Human Research at Chang Gung Memorial Hospital (IRB No: 96-1381A [phase I], 201003985A0 [phase II]). Both phases I and II clinical trials were conducted at Kaohsiung Chang Gung Memorial Hospital, a tertiary referral center. Additionally, a long-term 5-year follow-up for the phase I study was also permitted to perform in the same institute .
Definition of “good responder” after cell therapy for EnD-CAD
The good responder was defined as 1-year improvement of LVEF ≥7.0% after CD34+ cell therapy for EnD-CAD. A cutoff value of LVEF ≥7.0% or <7% was calculated by the average change of LVEF from baseline to 1-year measurement among all participants in our previous phase I [11, 12] and phase II trials . This cutoff value was also considered reasonable after taking several previous reports [13, 22, 23] as references in which the great responder was defined as the mean improvement of at least 5% in LVEF after cell therapy.
Definition of end-stage diffuse CAD (EnD-CAD)
The definition of EnD-CAD has been described in detail in our previous phase I clinical trial . Briefly, the EnD-CAD was confirmed by coronary angiographic findings which showed more than or equal to one obstructive CAD with severe diffuse morphological feature (defined as the diffuse lesion ≥ 50.0 mm in lengths, especially in relatively distal portion) or total occlusion of the vessel with unclarified length of the obstruction and non-candidates for PCI or CABG (i.e., since vessel involvement was too diffuse and the diameter was too small for intervention).
Retrospective collection of the variables and one-year clinical follow-up
Enrolled patients who successfully underwent stem cell therapy were followed-up for one year in the previous phase I/II studies [11, 21]. The baseline characteristics, laboratory data, bench-work results and imaging findings of coronary angiography (CAG), transthoracic echocardiography and cardiac magnetic resonance imaging were retrospectively retrieved from our stem cell research database that entered in computers during the previous phase I and II trials. The clinical and preclinical measures at baseline, after granulocyte-colony stimulating factor (G-CSF) administration but prior to CD34+ cell therapy, as well as 1, 3, 6, 9 and 12 months after cell delivery were collected to ensure a thorough analysis. Each patient was regularly followed up at our outpatient clinic, and the relevant clinical information including presentation of symptoms, presence or absence of adverse clinical events, and drug prescription was recorded by research nurses with case report forms, as well as telephone interviews on an irregular basis.
Procedure and protocol for CD34+ cell isolation
The procedure and protocol were based on our previous report . In detail, the number of CD34+ cells in the mononuclear cell preparation isolated during leukapheresis was enriched by utilizing a commercially available device [COBE Spetra 6.1 (Terumo BCT, INC.)] at 8:00 a.m. through a double lumen catheter inserted into the right femoral vein.
After a time-interval about four hours, an adequate amount of circulatory-derived CD34+ cells was collected and well-prepared for intra-coronary infusion. According to the International Society of Hematotherapy and Grafting Engineering (ISHAGE) Guidelines for CD34+ cell determination with flow cytometric measurement of circulating CD34+ cells, hematological stem cells are characterized by the presence of surface markers CD34high/CD45dim/SSClow that were used to quantify the number of isolated CD34+ cells. The formula for the number of circulation-derived CD34+ cells was: Number of CD34+ cells = (percentage of CD34+ cells) x WBC count x 103 x peripheral-blood stem cell (PBSC) volume (mL). The flow cytometric analysis followed current guideline of the College of American Pathology with a performance coefficient of variation (CV) <4.0% (3.4 ± 2.5) (by definition, CV <10.0% is acceptable).
After finishing the CD34+ collection without any cell culture for the differentiation of CD34, the patients were immediately sent to cardiac catheterization room for receiving the intra-coronary CD34+ cell injection.
Laboratory assessment of circulating levels of soluble angiogenesis factors
Circulating levels of vascular endothelial growth factor (VEGF), angiopoietin, epithelial growth factor (EGF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-β, and stromal cell-derived growth factor (SDF)-1α, six indicators of soluble angiogenesis biomarkers, were measured by duplicated determination with a commercially available ELISA method (R&D Systems, Minneapolis, MN, USA). Intra-observer variability of the measurements was also assessed, and the mean intra-assay coefficients of variance were all <4.5%.
Cardiac magnetic resonance image (MRI) was performed (i.e., prior to and at six months after cell therapy) by a radiologist blinded to the treatment allocation of the patients using current standard evaluation method. In addition, 2D and 3D transthoracic echocardiography were performed by an experienced cardiologist blinded to the patient grouping. The procedure and protocol of 3D transthoracic echocardiography were previously described .
Circulatory derived mononuclear cells for EPC culture and Matrigel assay for evaluating angiogenesis
The protocol and procedure of EPC culture and the assessment of angiogenesis were based on our previous report . In brief, mononuclear cells (MNCs) were isolated cells and cultivated in differential endothelial cell culture medium (endothelial cell basal medium-2, Cambrex) with 10% fetal bovine serum (FBS), 50 U/mL penicillin, 50 g/mL streptomycin and 2 mmol/L L-glutamine (Invitrogen) with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (10 ng/mL) plated on gelatin-coated tissue culture flasks and incubated at 37°C with 5% CO2 for 21 days. The culture medium was changed every 48 hours. By day 21, cells with spindle-shaped and cobblestone-like phenotype typical of endothelial cells were found attached on the plate.
The cells with endothelial cell phenotype were then plated in 96-well plates at 1.0×104 cells/well in 150 µL serum-free M199 culture medium mixed with 50 µL cold Matrigel (Chemicon international, Inc. Temecula, CA, USA) for 24 hours using passages 3 to 4 EPCs incubated at 37°C in 5% CO2. Three random microscopic images (200 ×) were taken from each well to count cluster, tube, and network formations and the mean values were derived. Both cumulative and mean tube lengths were calculated by Image-Pro Plus software (Media Cybernetics, Bethesda, MD, USA).
Cardiopulmonary exercise testing
Cardiopulmonary exercise testing (CPET) was used to objectively assess the patients’ functional capacity. The result of peak oxygen update (peak VO2) at maximal exercise was recorded as metabolic equivalents, i.e., the best index of aerobic capacity and cardiorespiratory function.
All variables are expressed as mean ± standard deviation or number with percentage. Independent t and Mann-Whitney U tests were used to compare the difference in continuous variables between two groups as appropriate. For categorical variables between groups, the variables were compared with chi-square analysis with Fisher exact test. Logistic regression models with univariate and multivariate analyses were performed to identify potential independent predictors of good responder to cell therapy, followed by Hosmer–Lemeshow (H-L) test for goodness of fit in the logistic regression model. In addition, those variables with p-value <0.08 in univariate analysis were chosen into multivariate analysis for adjustment. Finally, a nomogram was drawn based on the identified predictors to facilitate the calculation of probability rate of good response to cell therapy. Statistical analysis was performed using SPSS statistical software for Windows version 22 (SPSS for Windows, version 22; SPSS, IL, USA). A p-value <0.05 was considered statistically significant.