Exosomal-miRNas expression and growth factors released by mononuclear cells of CLAD patients in response to extracorporeal photopheresis.

BACKGROUND: CLAD (Chronic Lung Allograft Dysfunction) remains a serious complication following lung transplantation. Some evidence shows that portions of Extracorporeal Photopheresis (ECP)-treated patients improve/stabilize their graft function. In spite of that, data concerning molecular mechanisms are still lacking. Aims of our study were to assess whether ECP effects are mediated by Mononuclear Cells (MNCs) modulation in term of microRNAs (miRNAs) expression and growth factors release. METHODS: Cells from leukapheresis of 16 CLAD patients, at time 0 and 6-months (10 cycles), were cultured for 48h ± PHA (10 ug/ml) or LPS (2 ug/ml). Expression levels of miR-146a-5p, miR-155-5p, miR-31-5p, miR181a-5p, miR-142-3p, miR-16-5p and miR-23b-5p in MNCs-exosomes were evaluated by qRT-PCR, while ELISA assessed different growth factors levels on culture supernatants. RESULTS: Our result showed miR-142-3p down-regulation (p = 0.02) in MNCs of ECP-patients after the 10 cycles and after LPS stimulation (p = 0.005). We also find miR-146a-5p up-regulation in cells after the 10 cycles stimulated with LPS (p = 0.03). Connective tissue growth factor (CTGF) levels significantly decreased in MNCs supernatant (p = 0.04). The effect of ECP is translated into frequency changes of Dendritic Cell (DC) subpopulations and a slight increase in T regulatory cells (Treg) number and a significant decrease in CTGF release. CONCLUSIONS: ECP might affect regulatory T cell functions, since both miR-142 and miR-146a have been shown to be involved in the regulation of suppressor regulatory T cell functions and DCs. On the other side ECP, possibly by regulating macrophage activation, is able to significantly down modulate CTGF release.

Peripheral blood stem and progenitor cell collection in pediatric candidates for ex vivo gene therapy: a 10-year series.

Hematopoietic stem and progenitor cell (HSPC)-based gene therapy (GT) requires the collection of a large number of cells. While bone marrow (BM) is the most common source of HSPCs in pediatric donors, the collection of autologous peripheral blood stem cells (PBSCs) is an attractive alternative for GT. We present safety and efficacy data of a 10-year cohort of 45 pediatric patients who underwent PBSC collection for backup and/or purification of CD34+ cells for ex vivo gene transfer. Median age was 3.7 years and median weight 15.8 kg. After mobilization with lenograstim/plerixafor (n = 41) or lenograstim alone (n = 4) and 1-3 cycles of leukapheresis, median collection was 37 × 106 CD34+ cells/kg. The procedures were well tolerated. Patients who collected ≥7 and ≥13 × 106 CD34+ cells/kg in the first cycle had pre-apheresis circulating counts of at ≥42 and ≥86 CD34+ cells/µL, respectively. Weight-adjusted CD34+ cell yield was positively correlated with peripheral CD34+ cell counts and influenced by female gender, disease, and drug dosage. All patients received a GT product above the minimum target, ranging from 4 to 30.9 × 106 CD34+ cells/kg. Pediatric PBSC collection compares well to BM harvest in terms of CD34+ cell yields for the purpose of GT, with a favorable safety profile.

Photopheresis Abates the Anti-HLA Antibody Titer and Renal Failure Progression in Chronic Antibody-Mediated Rejection.

OBJECTIVE: Chronic renal antibody-mediated rejection (ABMR) is a common cause of allograft failure, but an effective therapy is not available. Extracorporeal photopheresis (ECP) has been proven successful in chronic lung and heart rejection, and graft versus host disease. The aim of this study was to evaluate the effectiveness of ECP in chronic ABMR patients. PATIENTS AND METHODS: We investigated ECP treatment in 14 patients with biopsy-proven chronic ABMR and stage 2-3 chronic renal failure. The primary aim was to e valuate the eGFR lowering after 1 year of ECP therapy. The ECP responders (R) showed eGFR reduction greater than 20% vs the basal levels. We also evaluated the effectiveness of ECP on proteinuria, anti-HLA antibodies (HLAab), interleukin 6 (IL-6) serum levels, and CD3, CD4, CD8, CD19, NK, Treg and T helper 17 (Th17) circulating cells. RESULTS: Three patients dropped out of the study. The R patients were eight (72.7%) out of the 11 remaining patients. Because ECP was not associated with any adverse reaction, the R patients continued such treatment for up to 3 years, showing a persisting eGFR stabilization. Twenty four hour proteinuria did not increase in the R patients over the follow-up when compared to the non-responder patients (NR). In the R patients, the HLAab levels were reduced and completely cleared in six out of eight patients when compared with the NR patients. The NR HLAab levels also increased after the discontinuation of the ECP. The ECP in the R patients showed a decrease in CD3, CD4, CD8, CD19, and NK circulating cells. The ECP treatment in the R patients also induced Tregs and Th17 cell increases, and a decrease of the IL-6 serum levels. CONCLUSIONS: ECP abates the HLAab titer and renal failure progression in patients with chronic renal ABMR, modulating the immune cellular and humoral responses.

Circulating endothelial progenitor cells during pregnancy in multiple sclerosis.

BACKGROUND: Endothelial progenitor cells (EPCs) have been shown to increase during physiological pregnancy and are believed to play a fundamental role in the process of placentation. Reduced levels of EPCs during pregnancy have been associated with preeclampsia and miscarriage. Women with multiple sclerosis (MS) are not at increased risk of preeclampsia nor of general adverse obstetric outcome, in contrast with some other autoimmune diseases. OBJECTIVE: The aim of this study was to evaluate circulating EPCs levels in pregnant patients with MS. METHODS: CD34+ and CD133+ were longitudinally detected by flow cytometry in the maternal plasma of 29 healthy controls and 9 MS patients and in the cord blood of their newborns. RESULTS: EPCs were affected by pregnancy with the same trend in both groups (CD34+ p = 0.0342; CD133+ p = 0.0347). EPCs during pregnancy were increased in MS (mean ± SD: CD34+ cells 0.038 ± 0.010; CD133+ 0.024 ± 0.009) with respect to healthy controls (mean ± SD: CD34+ cells 0.022 ± 0.006; CD133+ 0.016 ± 0.004), CD34+ p = 0.0004; CD133+ p = 0.0109. EPCs levels of the cord blood of MS patients' newborns mild correlated with maternal EPC levels at delivery (CD34+: spearman's Rho 0.658, p = 0.054; CD133+: spearman's Rho 0.758, p = 0.018). CONCLUSIONS: This work identified increased circulating EPC levels during pregnancy, following the same trend both in MS patients and healthy controls. Despite the similar trend, the levels of circulating EPCs were significantly higher in MS patients with respect to the control population. A correlation was also found in MS patients between cord blood EPCs and circulating EPCs at delivery.

A retrospective study assessing the characteristics of COVID-19 convalescent plasma donors and donations.

BACKGROUND: Although many trials are currently investigating the safety and efficacy of convalescent plasma (CP) in critically ill COVID-19 patients, there is a paucity of ongoing and published studies evaluating the CP donors' side. This retrospective study reports the first Italian experience on CP donors' selection and donations. METHODS: Patients aged 18-68 years who had recovered from COVID-19 at least 2 weeks previously were recruited between March 18 and June 30, 2020 in a study protocol at the Italian hospitals of Pavia and Mantova. RESULTS: During the study period, 494 of 512 donors recruited were judged eligible and underwent 504 plasmapheresis procedures. Eighty-five percent (437/512) of the CP donors were males. The average time between symptom recovery and CP donation was 36.6 (±20.0) days. Four hundred and eighty-eight plasmapheresis procedures (96.8%) were concluded and each unit was divided into two subunits (total 976) with an average volume of 316.2 (±22.7) mL. Ninety-three percent (460/494) of CP donors at the time of plasma donation had a neutralizing IgG titer ≥1:80. Plasmapheresis-related adverse reactions occurred in 2.6% (13/504) of cases; all the reactions were mild and none required therapeutic intervention. Donors' age and COVID-19 severity were positively associated with greater antibody responses. CONCLUSION: This study demonstrates the feasibility and safety of a pilot CP program conducted in Italy. The identification of factors (ie, age and severity of COVID-19) positively associated with higher neutralizing antibody titers at the time of donation may help to optimize the selection of CP donors.

Endothelial Progenitor Cell CD34+ and CD133+ Concentrations and Soluble HLA-G Concentrations During Pregnancy and in Cord Blood in Undifferentiated Connective Tissue Diseases Compared to Controls.

The objective of this study is to evaluate endothelial progenitor cells (EPCs) CD34+ CD133- and CD34+ CD133+ and soluble HLA-G (sHLA-G) concentrations among undifferentiated connective tissue disease (UCTD) subjects, compared to controls, during pregnancy and in cord blood. This is a case-control study including 29 controls and 29 UCTDs. CD34+ CD133-, CD34+ CD133+, and sHLA-G concentrations were detected in maternal plasma and in cord blood. This study was approved by the Medical-Ethical Committee of our Institution (Current Research Project N. 901-rcr2017i-23 of IRCCS Foundation Policlinico San Matteo of Pavia). Circulating CD34+ CD133- and CD34+ CD133+ counts and sHLA-G (soluble human leucocyte antigen G) concentrations in maternal peripherical blood were higher in UCTD compared to those in controls in first and third trimester of pregnancy and at delivery (p < 0.001). Maternal CD34+ CD133- and CD34+ CD133+ counts were strongly and significantly correlated in UCTD (Spearman's rho = 0.79, p < 0.0001) but not in controls (Spearman's rho = 0.10, p = 0.35). Cord blood CD34+ CD133- and CD34+ CD133+ median counts and median sHLA-G concentrations were higher among UCTD subjects than in controls (p < 0.001). Cord blood CD34+ and CD133+ counts were inversely and significantly correlated with sHLA-G concentrations among UCTDs, but not in controls. Early UCTD is characterized by increased EPC levels in maternal plasma and in cord blood and higher levels of sHLA-G, compared to controls. Data suggest that fetoplacental unit plays an independent role in the EPC response to a systemic autoimmune disease.

Automated mononuclear cell collection: a feasibility study employing a new software for extracorporeal photopheresis.

BACKGROUND AND OBJECTIVES: Very recently, Fresenius Kabi, improved the software (autoMNC lymphocytes, SW 04.03.08) for mononuclear cells (MNCs) collection with the aim to ameliorate the quality of harvest, employing the automated autoMNC lymphocytes software SW 04.03.09. Herein, we report the results of an observational study evaluating the feasibility of MNCs collection in patients undergoing extracorporeal photopheresis (ECP) at our centre, using the new COM.TEC software 04.03.08c for MNC collection, afterwards integrated in the software 04.03.09, available on the market since November 2018. MATERIALS AND METHODS: Thirty adult patients (21 males and 9 females) with GvHD, Chronic Lung Allograft Dysfunction or renal rejection, were consecutively enrolled to undergo 1 ECP procedure by the offline technique, according to our internal protocol, processing 1·5 blood volumes. Feasibility of collection was defined as: Hct in collection bag ≤5%, MNCs purity (percentage of MNCs/bag) ≥80%, MNCs collection efficiency (CE2) ≥60%, patient's platelet depletion ≤50%. RESULTS: Thirty ECP procedures were evaluated. Feasibility (defined by the four parameters previously described) of MNCs collection was observed in 1 out of the 30 harvests analysed. Median Hct in the product was 3·45% (IQR: 2·6-5·0), and median MNCs purity was 97·2% (IQR 89·1-98·6). Median CE2 for MNCs was 21·4% (IQR: 11·9-41·2), and median patient's platelet depletion was 36·2% (IQR 21·9-51·4). CONCLUSION: The autoMNC lymphocytes software SW 04.03.08c for MNCs collection in ECP setting demonstrated to collect a good quality product in terms of purity and RBC contamination even if the collection efficiency and platelet contamination must be improved.

Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent ß-thalassemia.

ß-thalassemia is caused by ß-globin gene mutations resulting in reduced (ß+) or absent (ß0) hemoglobin production. Patient life expectancy has recently increased, but the need for chronic transfusions in transfusion-dependent thalassemia (TDT) and iron chelation impairs quality of life1. Allogeneic hematopoietic stem cell (HSC) transplantation represents the curative treatment, with thalassemia-free survival exceeding 80%. However, it is available to a minority of patients and is associated with morbidity, rejection and graft-versus-host disease2. Gene therapy with autologous HSCs modified to express ß-globin represents a potential therapeutic option. We treated three adults and six children with ß0 or severe ß+ mutations in a phase 1/2 trial ( NCT02453477 ) with an intrabone administration of HSCs transduced with the lentiviral vector GLOBE. Rapid hematopoietic recovery with polyclonal multilineage engraftment of vector-marked cells was achieved, with a median of 37.5% (range 12.6-76.4%) in hematopoietic progenitors and a vector copy number per cell (VCN) of 0.58 (range 0.10-1.97) in erythroid precursors at 1 year, in absence of clonal dominance. Transfusion requirement was reduced in the adults. Three out of four evaluable pediatric participants discontinued transfusions after gene therapy and were transfusion independent at the last follow-up. Younger age and persistence of higher VCN in the repopulating hematopoietic cells are associated with better outcome.

Mild-to-moderate foeto-maternal haemorrhage in the third trimester and at term of pregnancy: quantitative determination and clinical-diagnostic evaluation.

BACKGROUND: Foeto-maternal haemorrhage (FMH), a gestational event that occurs before or during delivery, consists of a loss of foetal blood into the maternal circulation. FMH occurs more frequently during the third trimester or labour both in normal and complicated pregnancies. In the case of alloimmunisation, the maternal immunological response and the severity of the resulting foetal or neonatal disease depend on the amount of foetal blood that passes into the maternal circulation. The aim of this study was to determine FMH in the third trimester and at term of pregnancy and to evaluate the role of clinical and ultrasound markers in the prediction of FMH. MATERIALS AND METHODS: FMH was quantified by cytofluorimetric testing at 28 to 35 weeks of gestation in 223 women and at term in 465 women, all with risk factors. Foetal evaluation included foetal movement profile, middle cerebral artery peak velocity of systolic blood flow (MCA-PSV) and cardiotocographic monitoring. RESULTS: All women tested negative for FMH in the third trimester. Four patients (0.9%) tested positive at term, with estimated volumes of bleeding of 2.2, 8.1, 12.3 and 39.8 mL. Three FMH cases (75%) had a non-reassuring cardiotocography compared to 8.9% (42/461) of women without FMH (p=0.003) and two FMH cases reported a reduction in foetal movements reduction compared to four of those without FMH (p=0.001). Mean MCA-PSV was normal in both the groups with and without FMH (p=0.22). DISCUSSION: FMH is rare in pregnancy and at term. Cytofluorimetric testing is a specific method to detect mild-to-moderate FMH even when the MCA-PSV is not informative. Mild-to-moderate FMH is significantly associated with reduced foetal movements and non-reassuring cardiotocographic monitoring.

Automated red blood cell depletion in ABO incompatible grafts in the pediatric setting.

Bone marrow ABO incompatible transplantations require graft manipulation prior to infusion to avoid potentially lethal side effects. We analyzed the influence of pre-manipulation factors (temperature at arrival, transit time, time of storage at 4°C until processing and total time from collection to red blood cell depletion) on the graft quality of 21 red blood cell depletion procedures in ABO incompatible pediatric transplants. Bone marrow collections were processed using the Spectra Optia® (Terumo BCT) automated device. Temperature at arrival ranged between 4°C and 6°C, median transit time was 9.75h (range 0.33-28), median time of storage at 4°-6°C until processing was 1.8h (range 0.41-18.41) and median time from collection to RBC depletion was 21h (range1-39.4). Median percentage of red blood cell depletion was 97.7 (range 95.4-98.5), median mononuclear cells recovery was 92.2% (range 40-121.2), median CD34+ cell recovery was 93% (range 69.9-161.2), median cell viability was 97.7% (range 94-99.3) and median volume reduction was 83.9% (range 82-92). Graft quality was not significantly different between BM units median age. Our preliminary data show that when all good manifacturing practices are respected the post-manipulation graft quality is excellent also for those units processed after 24h.

Primary myelofibrosis: Older age and high JAK2V617F allele burden are associated with elevated plasma high-sensitivity C-reactive protein levels and a phenotype of progressive disease.

We measured plasma levels of high-sensitivity C-reactive protein (hs-CRP) in 526 subjects with primary myelofibrosis (PMF). Thirty-eight percent had an elevated hs-CRP level (≥0.3mg/dL). Elevated hs-CRP levels were associated with a progressive disease phenotype, including anemia, high white blood cell count, low platelet count, increased splenomegaly, increased risk of blast transformation, and worse survival. Age≥52years, but no other demographic characteristics, was associated with an elevated hs-CRP level in multivariable logistic regression (odds ratio [OR], 4.29; 95% CI, 2.73-6.77; P <0.001). Subjects with JAK2V617F mutation and an allele burden≥50% had an age-independent higher incidence of elevated hs-CRP level (OR=1.97; 95% CI,1.21-3.22; P=0.006) compared with a combined cohort of subjects with JAK2V617F <50% allele burden, CALR, MPL mutations, or no detectable driver mutations. Neither ASXL1 or EZH2 sub-clonal mutations, nor JAK2 46/1 haplotype or the A3669G polymorphism of glucocorticoid receptor were significantly associated with increased hs-CRP levels. Subjects with age≥52years and JAK2V617F with≥50% allele burden had a phenotype of progressive disease. Our data indicate that older age and high JAK2V617 allele burden are major determinants of inflammation in PMF, and are associated with disease progression.

Comparison of two automated mononuclear cell collection systems in patients undergoing extracorporeal photopheresis: a prospective crossover equivalence study.

BACKGROUND: Extracorporeal photopheresis (ECP) is an effective cell therapy employed in several diseases, including graft versus host disease (GVHD) and organ rejection. When ECP is performed using an off-line technique, mononuclear cell (MNC) collection by leukapheresis is necessary for further manipulation (addition of 8-methoxypsoralen and ultraviolet A irradiation before reinfusion to the patient). We report the results of the first crossover equivalence study on yield and purity of MNCs collected from patients undergoing ECP with two different automated systems: MNC and CMNC (working with intermittent and continuous-flow collection, respectively), released by Terumo BCT. STUDY DESIGN AND METHODS: Fifty-one patients (15 males and 36 females) with GVHD or chronic lung allograft dysfunction were consecutively enrolled and randomly assigned to MNC collection alternatively by the CMNC or MNC system within each ECP cycle (two procedures) in two consecutive cycles. ECP procedures were performed using the off-line technique, according to our internal protocol, processing 1.5 blood volumes. RESULTS: A total of 204 ECP procedures were evaluated. The MNC system showed a higher MNC concentration capacity than the CMNC (mean difference, -13.46; 95% confidence interval, -21.05 to -5.47; p < 0.001). Collection efficiency was higher with the CMNC system as well as total MNC bag content, while MNC bag purity was equivalent for both systems. Platelet loss was higher with the CMNC. Equivalence was shown for MNC purity and anticoagulant infused to the patient. CONCLUSION: CMNC and MNC systems offer different advantages in different clinical conditions, and both are safe and efficient in collecting MNCs for ECP.

Increased plasma nicotinamide phosphoribosyltransferase is associated with a hyperproliferative phenotype and restrains disease progression in MPN-associated myelofibrosis.

Myeloproliferative neoplasm (MPN)-associated myelofibrosis is a clonal, neoplastic disorder of the hematopoietic stem cells, in which inflammation and immune dysregulation play an important role. Extracellular nicotinamide phosphoribosyltransferase (eNAMPT), also known as visfatin, is a cytokine implicated in a number of inflammatory and neoplastic diseases. Here plasma levels of eNAMPT in patients with MPN-associated myelofibrosis and their effects on disease phenotype and outcomes were examined. The concordance of eNAMPT levels with the marker of general inflammation high-sensitivity C-reactive protein (hs-CRP) was also studied. A total of 333 MPN-associated myelofibrosis patients (187 males and 146 females) and 31 age- and gender-matched normal-weight healthy subjects were enrolled in the study main body. Levels of eNAMPT and hs-CRP were simultaneously assayed in 209 MPN-associated myelofibrosis patients. Twenty-four polycythemia vera or essential thrombocythemia patients were used as controls. eNAMPT was over expressed in MPN-associated myelofibrosis, and eNAMPT expression was correlated with higher white blood cell count, higher hemoglobin, and higher platelet count, suggesting that eNAMPT is an indispensable permissive agent for myeloproliferation of MPN-associated myelofibrosis. The lack of correlation between eNAMPT and hs-CRP revealed that eNAMPT in MPN-associated myelofibrosis does not behave as a canonical inflammatory cytokine. In addition, higher levels of eNAMPT predicted longer time to blast transformation, and protected against progression toward thrombocytopenia and large splenomegaly. In conclusion, in MPN-associated myelofibrosis high levels of eNAMPT mark the myeloproliferative potential and, at variance with a high number of cancers, are protective against disease progression. Am. J. Hematol. 91:709-713, 2016. © 2016 Wiley Periodicals, Inc.

Autologous immuno magnetically selected CD133+ stem cells in the treatment of no-option critical limb ischemia: clinical and contrast enhanced ultrasound assessed results in eight patients.

OBJECTIVES: Demonstrate the safety and effectiveness of highly purified CD133+ autologous stem cells in critical limb ischemia (CLI). DESIGN: Prospective single-center not randomized. Clinicaltrials.gov identifier: NCT01595776 METHODS: Eight patients with a history of stable CLI were enrolled in a period of 2 years. After bone marrow stimulation and single leukapheresis collection, CD133+ immunomagnetic cell selection was performed. CD133+ cells in buffer phosphate suspension was administered intramuscularly. Muscular and arterial contrast enhanced ultra sound (CEUS), lesion evolution and pain management were assessed preoperatively and 3, 6 and 12 months after the implant. RESULTS: No patient had early or late complications related to the procedure. Two patients (25 %) didn't get any relief from the treatment and underwent major amputation. Six patients (75 %) had a complete healing of the wounds, rest pain cessation and walking recovery. An increase in CEUS values was shown in all eight patients at 6 months and in the six clinical healed patients at 12 months and had statistical relevance. CONCLUSIONS: Highly purified autologous CD133+ cells can stimulate neo-angiogenesis, as based on clinical and CEUS data.

Long-Term Off-Line Extracorporeal Photochemotherapy in Patients with Chronic Lung Allograft Rejection Not Responsive to Conventional Treatment: A 10-Year Single-Centre Analysis.

BACKGROUND: Extracorporeal photochemotherapy (ECP) for chronic lung allograft dysfunction (CLAD) has been reported as beneficial in a few short-term studies. OBJECTIVES: In this retrospective cohort study on 48 CLAD patients treated by ECP (off-line technique) for a period of >8 years (compared to 58 controls), we explored potential predictors of survival and response. METHODS: Failures were defined as a decrease in forced expiratory volume in 1 s (FEV1) of >10% from ECP initiation. RESULTS: ECP patients were enrolled between February 2003 and December 2013; 14 (29.2%) with restrictive allograft syndrome (RAS) and 34 with bronchiolitis obliterans syndrome. Grade 1 severity was indicated in 58.3%, grade 2 in 20.8%, and grade 3 in 20.8% of patients. The median follow-up was 65 months (cumulative 2,284.4 person-months). Twenty (41.7%) patients died, including 17 (85%) CLAD-related deaths. Among the controls, there were 42 deaths (72.4%), of which 32 (76.2%) were CLAD related, over a median of 51 months (cumulative 3,066.5 person-months; p = 0.09). Among ECP patients, the FEV1 slope flattened out after a decline in the initial months (slope -19 ml/month in months 0-6, +4 in months 36-48 and later; p = 0.001). RAS was associated with poorer survival, whereas a 'rapid decline in the previous 6 months' was not. No ECP side effects or complications were observed. CONCLUSION: Long-term ECP for CLAD is safe and reduces FEV1 decline over time; the RAS phenotype might show a poorer response. ECP deserves to be evaluated in a randomized controlled trial.

Conditioned medium from human amniotic mesenchymal stromal cells limits infarct size and enhances angiogenesis.

The paracrine properties of human amniotic membrane-derived mesenchymal stromal cells (hAMCs) have not been fully elucidated. The goal of the present study was to elucidate whether hAMCs can exert beneficial paracrine effects on infarcted rat hearts, in particular through cardioprotection and angiogenesis. Moreover, we aimed to identify the putative active paracrine mediators. hAMCs were isolated, expanded, and characterized. In vitro, conditioned medium from hAMC (hAMC-CM) exhibited cytoprotective and proangiogenic properties. In vivo, injection of hAMC-CM into infarcted rat hearts limited the infarct size, reduced cardiomyocyte apoptosis and ventricular remodeling, and strongly promoted capillary formation at the infarct border zone. Gene array analysis led to the identification of 32 genes encoding for the secreted factors overexpressed by hAMCs. Among these, midkine and secreted protein acidic and rich in cysteine were also upregulated at the protein level. Furthermore, high amounts of several proangiogenic factors were detected in hAMC-CM by cytokine array. Our results strongly support the concept that the administration of hAMC-CM favors the repair process after acute myocardial infarction.