Effects of EPC capture stent and CD34+ mobilization in acute myocardial infarction.

AIM: Percutaneous coronary intervention (PCI) is the gold standard for the treatment of acute myocardial infarction (AMI), with the main limitation of in-stent restenosis for BMS and late stent thrombosis (ST) for both BMS and DES. Endothelial progenitor cells (EPC) CD34+ capture stents, promoting vascular healing, may be advantageous in preventing ST. Aim of the study is to evaluate the outcomes of AMI patients treated with EPC CD34+ capture stent and describe the mobilization kinetics of CD34+ and their clinical correlation. METHODS: Fifty AMI patients underwent primary PCI with EPC CD34+ capture stent. Serial assays of CD34+ were performed by flow-cytometric analysis. RESULTS: Procedural success rate was 100%. At six-months follow-up cardiac death, myocardial infarction, target lesion revascularization (TLR) and target vessel revascularization (TVR) occurred respectively in 2%, 4%, 10% and 12% of patients. No case of ST was observed. The MACE-free survival was 81,2%. The mean peak value of plasmatic CD34+ was 4.69±3.76 cells/µL. A positive correlation was found between CD34+ concentration, age and infarct area. No correlation was detected between CD34+ concentration and occurrence of TVR, TLR and MACE. CONCLUSION: EPC capture stent implantation seems to be safe and effective in the clinical setting of AMI, representing a possible alternative to BMS and DES. CD34+ cells plasmatic concentration seems not to correlate to coronary restenosis and atheromasic disease progression.

Mesenchymal stem cell transplantation in amyotrophic lateral sclerosis: A Phase I clinical trial.

Amyotrophic Lateral Sclerosis (ALS) is a devastating incurable disease. Stem-cell-based therapies represent a new possible strategy for ALS clinical research. The objectives of this Phase 1 clinical study were to assess the feasibility and toxicity of mesenchymal stem cell transplantation and to test the impact of a cell therapy in ALS patients. The trial was approved and monitored by the National Institute of Health and by the Ethics Committees of all participating Institutions. Autologous MSCs were isolated from bone marrow, expanded in vitro and analyzed according to GMP conditions. Expanded MSCs were suspended in the autologous cerebrospinal fluid (CSF) and directly transplanted into the spinal cord at a high thoracic level with a surgical procedure. Ten ALS patients were enrolled and regularly monitored before and after transplantation by clinical, psychological, neuroradiological and neurophysiological assessments. There was no immediate or delayed transplant-related toxicity. Clinical, laboratory, and radiographic evaluations of the patients showed no serious transplant-related adverse events. Magnetic resonance images (MRI) showed no structural changes (including tumor formation) in either the brain or the spinal cord. However the lack of post mortem material prevents any definitive conclusion about the vitality of the MSCs after transplantation. In conclusion, this study confirms that MSC transplantation into the spinal cord of ALS patients is safe and that MSCs might have a clinical use for future ALS cell based clinical trials.

Role of different medium and growth factors on placental blood stem cell expansion: an in vitro and in vivo study.

Expansion of haemopoietic stem cells from placental blood has been obtained with a combination of flt3 ligand (FL), thrombopoietin (TPO), kit-ligand (KL) with or without interleukin-6 (IL6) in serum-replete medium. For clinical use, cell expansion in the absence of serum is a clear advantage. Therefore, stem cell expansion in serum-free (SF) medium with a combination of three (FL, TPO, KL) or four (FL, TPO, KL, IL6) growth factors was compared with the results obtained using fetal calf serum (FCS) or human serum (HS). Human CD34(+) placental blood cells were cultured in the presence of FL, TPO, KL +/- IL6 with SF medium, HS and FCS for up to 8 weeks. CD34(+), CFC, LTC-IC content was measured at intervals. To determine the in vivo repopulating capacity of expanded cells, CD34(+) expanded cells were transplanted in sublethally irradiated NOD/SCID mice. With the three growth factor combination the CD34(+) cell number increased steadily up to the 8 weeks of culture. CD34(+) cells were expanded 67.5-fold with SF, 11.7 with HS and 49.2 with FCS. However, when CFCs and LTC-ICs were considered, a continuous expansion was observed only with HS and FCS, whereas in SF medium after 6 weeks their number started to decline. The addition of IL-6 did not change the expansion significantly. Cells grown ex vivo for 14 days were transplanted into NOD/SCID mice. The engraftment of human cells in mice was higher for serum-replete than for SF expanded cells. Nevertheless, SF cultured cells were also able to engraft both marrow and spleen in all animals. In addition, engrafted human cells still maintained clonogenic ability. With KL, FL, TPO +/- IL6 it is possible to expand haemopoietic progenitor cells in a SF medium. Compared with serum-replete cultures, the absolute number of clonogenic cells and in vivo repopulating cells is lower. Although the degree of expansion remains significant, a clinical trial still needs to be carried out to address the question of whether this expansion might be useful in reducing post-transplant aplasia.

Different growth factor requirements for the ex vivo amplification of transplantable human cord blood cells in a NOD/SCID mouse model.

The growth factor combination containing early acting cytokines FLT-3 ligand (FL), Stem Cell Factor (SCF) and thrombopoietin (TPO) is able to maintain, for an extended culture period, early stem cells, defined as long-term repopulating NOD/SCID mice (Scid Repopulating Cell-SRC) contained in cord blood (CB). In this culture system, the role of IL-6 and IL-3 has not been clearly established. Using a combination of FL+TPO+SCF with or without IL-6, we were able to form CB CD34+ cells for 30 weeks. The CB CD34+ cells cultured in this system engrafted NOD/SCID mice after 6 weeks of culture; the cells from primary recipients were also able to engraft secondary NOD/SCID mice. When CB CD34+ cells were cultured in the presence of IL-3 in the place of IL-6 we observed an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of culture. However, more primitive LTC-IC output increased up to week 6 with the growth factor combination containing IL-3 and then decreased and disappeared, while with the growth factor combination with or without IL-6 increased up to week 23. Cells cultured for 4 weeks with the 4-factor combination containing IL-3 engrafted NOD/SCID mice less efficiently. Repopulation of NOD/SCID mice was no longer observed when ex vivo expansion was performed for 6 weeks. This study provides some evidence that no differences could be detected in long-term maintenance and even expansion of human primitive cord blood cells cultured with FL+TPO+SCF in the presence or absence of IL-6. Under the culture conditions employed in this study, the presence of IL-3 reduced the repopulating potential of expanded CB CD34+ cells.

Negative influence of IL3 on the expansion of human cord blood in vivo long-term repopulating stem cells.

Identification of culture conditions that support expansion or even long-term maintenance of in vivo repopulating human hematopoietic stem cells is still a major challenge. Using a combination of FLT3 ligand (FL), Stem Cell Factor (SCF), Thrombopoietin (TPO) and Interleukin 6 (IL6), we cultured cord blood (CB) CD34+ cells for up to 12 weeks and transplanted their progeny into sublethally irradiated NOD/SCID mice. Bone marrow engraftment was considered successful when recipients contained measurable numbers of human CD45+, CD71+ and Glycophorin A+(GpA) cells 8 weeks after transplantation. Twelve-week expanded cells with FL+SCF+TPO+IL6 successfully engrafted all of the recipients and human CD45(+)+CD71(+)+GpA(+) cells represented 4.3 to 22.4% of bone marrow. Substitution of IL6 with IL3 led to an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of culture; however, LTC-IC output increased up to week 6 and then decreased and disappeared. By contrast, with FL+SCF+TPO+IL6, LTC-IC kept increasing up to week 12. Four-week cultured cells with FL+SCF+TPO+IL3 less efficiently engrafted NOD/SCID mice, both as measured by frequency of positive recipients (4 out of 10) and percentage of engrafted human cells (< or =2%). Six-week expanded cells failed to engraft. This study provides evidence that many, but not all, of the so-called "early acting" cytokines, can sustain long-term maintenance and even expansion of human primitive in vivo repopulating stem cells. In particular, in the culture conditions used in this study, the presence of IL3 greatly reduces the repopulating potential of expanded CD34+ CB cells.