stem cell transplantation in patients with peripheral arterial disease (pad)

regenerative therapy for cardiovascular diseases. Several celltypes from different spaces have been used for this purpose.However, MSCs from porcine bone marrow have not been verywell characterized.Methods and results: For our future study in porcine model ofmyocardial ischemia, adult pig bone marrow was aspirated fromthe iliac crest. The mononuclear cells were then isolated bygradient centrifugation and cultured in DMEM supplementedwith 10% FBS and 1X pen/strep in a density of 106/cm2 at37 °C with 5% CO2 in T-75 culture flasks. The phenotype of thecells at passage 0 was heterogeneous (round shaped, irregularshaped and spindle shaped cells) forming different cell colonies.After the attached cells were confluent, they were split andexpanded for 2–4 passages. The growth speed of the culturedbone marrow cells was much slower at passage 0 to passage 2.However, the growth speed dramatically increased starting frompassage 3, and showing uniformed fibroblast-like morphology.The bone marrow derived cells were further analyzed usingFACS for cell surface marker. FACS analysis at passage twoshowed strong positive for CD44, CD90, and negative forCXCR4, CD34, C-kit, CD45, CD31 and CD54, suggesting thatthey are Mesenchymal stem cells. The differentiation studies ofthe cells showed multi-mesenchymal lineages, such as adipo-cytes and osteocytes. Under hypoxic condition, these cellsproduce more VEGF and other growth factors compared withcells under normal growth condition. Bothmale and female bonemarrow-derived MSCs were performed karyotyping analysis atpassage 4 and showed normal chromosome number of 38.Conclusions: Pig bone-marrow derived MSCs can be culturedand expanded without losing their multi-mesenchymal lineagepotency and maintained a normal ploidy of chromosomenumber. The experiments provide us with an opportunity toperform the novel regenerative therapy in porcine model ofmyocardial ischemia.

doi:10.1016/j.vph.2006.08.129

A4.18

Stem cell transplantation in patients with peripheralarterial disease (PAD)

Nathalie Hernandez1, Gloria Franco1, Juan Duque2, JulietaHenao3

1Clinica Cardiovascular, Medellin, Colombia2Hospital Pablo Tobon Uribe, Medellin, Colombia3Universidad de Antioquia, Medellin, Colombia

The present preliminary report aims to demonstrate if bonemarrow stem cell transplantation helps to develop collateralvessels in patients with PAD, and fontaine IIb, III and IVavoiding progression. We describe sociodemography and cliniccharacteristics.Materials and methods: pseudoexperimental clinical essay,6 months following, the intervention is defined as local

implantation of autologous stem cells in patients with PADand no other option to treatment. The controls were patients withPAD without option of intervention and only medical treatment.All patients have during the study time PVR and angiographypre intervention and 6 months post-intervention. We describechanges in the pain scale and long distance walking.Results: Cases were 12 extremities, since January to November2005 and controls: 24 extremities.

Variable Cases ControlsSex: female 8 (67%) 10 (41%)Age (years) 69.33±10.6 72.1±8.7Side:R 6 (50%) 13 (54.2%)Initial stage IIa 0% 4.2%

IIb 58.3% 37.5%III 33.3% 41.7%IV 16.7% 16.7%

End stage IIb 100% 12.5%III 0% 25%IV 0% 62.5%

6 m following amputation 0% 41.7%Walking distance 224±100 70.4±59.2Meters worsing 0% 75%

During the following PVR didn't show significant changes,but angiography revelated significant increase in colateralvessels and mild positive changes in the infrapopliteal nativearteries. The ARR respecting negative change in fontaineclassification was 75% and the NNT was 1.3.Conclusion: this clinical assay results show that localimplantation of stem cells in patients with PAD change thenatural course of disease, improving local blood flow byangiography and clinical characteristics as scale of ischemicpain, healing ulcer and long distance walking.

doi:10.1016/j.vph.2006.08.130

A4.19

Differentiation of embryonic stem cells into cardiacmyocytes

Kristina Shevchenko, Yevgen Zadorin, Olga Zadorina

Clinical Research Institute, Kiev, Ukraine

Background: Embryonic stem (ES) cells are capable of self-renewal and differentiation into cellular derivatives of all 3 germlayers. In appropriate culture conditions, ES cells candifferentiate into specialized cells, including cardiac myocytes,but the efficiency is typically low and the process isincompletely understood.Methods and results: We evaluated a chemical library for itspotential to induce cardiac differentiation of ES cells in theabsence of embryoid body formation. Using ES cells stablytransfected with cardiac-specific-cardiac myosin heavy chain(MHC) promoter-driven enhanced green fluorescent protein(EGFP), 880 compounds approved for human use were

e34 14th IVBM Abstracts