Femur bone marrow from brain death deceased donors as source of human mesenchymal stromal cells for cell therapy.

Background: The use of a deceased donor (DD) as an alternative source of human mesenchymal stromal cells (hMSC) is promising, but has been little explored. This study evaluated the potential of femur bone marrow (FBM) from brain-death donors as a source of hMSC and compared this with hMSC from matched iliac crest bone marrow (ICBM). Methods: Sixteen donor-matched FBM and ICBM samples were processed from brain-death donors. We analyzed the starting material and compared cell yield, phenotypic profile and differentiation capacity of hMSC. Results: Neither the amount of nucleated cells per gram (14.6×106±10.3×106 from FBM vs. 38.8×106±34.6×106 from ICBM, P≥0.09) nor the frequency of CFU-F (0.0042%±0.0036% in FBM vs. 0.0057%±0.0042% in ICBM, P≥0.73) differ significantly from FBM or ICBM. Cell cultures from both sources were obtained and hMSC yields showed that there were no significant differences in hMSC obtained per gram of bone marrow (BM) when comparing femur with iliac crest samples. At passage 2, 12.5×106±12.9×106 and 5.0×106±4.4×106 hMSC per gram of BM were obtained from FBM and ICBM, respectively. FBM and ICBM hMSC express CD73, CD90, CD105, but not hematopoietic lineage markers [CD45, CD34, CD11, CD19 and isotype of HLA clase II (HLA-DR)]. HLA-A expression from both sources was clearly detected, while HLA-B was weakly expressed or undetectable and HLA-DR was undetectable. Cells from both sources were differentiated in vitro into osteoblasts, adipocytes and chondroblasts. Conclusions: To our knowledge, there are no previous studies evaluating BM from femur dead donors as a source of hMSC. Our findings confirm that it is feasible to expand cells from FBM from brain-death donors meeting in vitro characteristics of hMSC, making them a promising source for clinical translation.

5-Azacytidine restores interleukin 6-increased production in mesenchymal stromal cells from myelodysplastic patients.

INTRODUCTION: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematological diseases. In addition to defects in hematologic progenitor and stem cells, dysfunctions in the bone marrow microenvironment (BMM) participate in the MDS pathogenesis. Furthermore, the immune response is deregulated by the pro-inflammatory response prevailing in low-risk MDS, while immunosuppression predominates in high-risk MDS. Mesenchymal stromal cells (MSC), part of the BMM, are characterized by plastic adherent growth and multipotentiality. They exhibit immunomodulatory properties and sustain hematopoiesis. There is conflicting evidence regarding their status in MDS. The aim of this study was to characterize MDS-MSC and evaluate the effect of 5-Azacytidine. METHODS: The MSC from MDS patients and controls were cultured and characterized according to the International Society of Cell Therapy recommendations. Immunomodulatory properties were assessed by studying the MSD cytokine production, using the cytometric bead array. We evaluated the effect of 5-Azacytidine on the MSC cytokine production. RESULTS: We included 35 MDS patients and 22 controls. The MSC from patients and controls were cultured and characterized. The MSC from patients showed morphological differences, but there were no differences in immunophenotype or multipotentiality. The interleukin 6 (IL-6) was the main MSC secreted cytokine. The MDS-MSC produced higher levels of IL-6, IL-17, interferon gamma, or interferon γ (INF-γ), and tumor necrosis factor alpha (TNF-α). The in vitro 5-Azacytidine treatment induced a significant decrease in the IL-6 production by MDS-MSC. CONCLUSIONS: The MDS-MSC show an increased production of pro-inflammatory cytokines. The in vitro treatment with 5-Azacytidine lead to a significant reduction in the IL-6 production by the MDS-MSC, restoring the IL-6 levels to those found in controls. The MSC produced inflammatory cytokines involved in the MDS pathogenesis, representing a potential future therapeutic target. Moreover, 5-Azacytidine may have a stromal effect, modulating the immune response in MDS.

Establecimiento e implementación de un protocolo simplificado de expansión y cultivo de Células Madre de Pulpa Dental Humana (DPSCh) / Estabelecimento e implementação de um protocolo simplificado para expansão e cultura de células-tronco da polpa dentária humana (DPSCh) / Establishing and implementing a simplified protocol for the expansion and culture of human dental pulp stem cells (hDPSCs)

Resumen Objetivos: Establecer e implementar un protocolo simplificado de extracción, aislamiento primario y cultivo de células madre derivadas de la pulpa dental humana (DPSCh). Analizar cuantitativamente y cualitativamente las células aisladas. Metodología: 10 terceros molares sanos donados por pacientes que concurrieron a la Facultad de Odontología, UdelaR y otorgaron su consentimiento escrito fueron procesados antes de las 48 hs. Se realizó la fractura de la pieza para la obtención del tejido pulpar y se procesó por el método explante. Se analizó viabilidad celular y expresión de marcadores por citometría de flujo en pasajes 4 y 12 y se corroboró mediante inmunocitoquímica. Resultados: Las células obtenidas presentaron una vitalidad mayor al 90% en todos los pasajes, observándose una morfología característica y expresión de marcadores de células madre mesenquimales CD90, C105, CD73, CD29 y 166 mediante citometría de flujo en ambos pasajes. Conclusiones: Se logró establecer un protocolo de aislamiento y expansión celular, con alta tasa de éxito de una población de DPSCh.

Resumo Objetivos: Estabelecer e implementar um protocolo simplificado para a extração, isolamento primário e cultura de células-tronco da polpa dentária humana (DPSCh). Analise as células isoladas quantitativa e qualitativamente. Metodologia: 10 terceiros molares saudáveis ​​doados por pacientes que frequentaram a Faculdade de Odontologia UdelaR e deram consentimento por escrito foram processados ​​antes de 48 horas. A fratura da peça foi realizada para obtenção do tecido pulpar e processada pelo método do explante. A viabilidade celular e a expressão do marcador foram analisadas por citometría de fluxo nas passagens 4 e 12 e confirmadas por inmunocitoquímica. Resultados: As células obtidas apresentaram viabilidade superior a 90% em todas as passagens, observando uma morfologia característica e expressão dos marcadores de células-tronco mesenquimais CD90, C105, CD73, CD29 e 166 por citometría de fluxo em ambas as passagens. Conclusões: Foi possível estabelecer um protocolo de isolamento celular, com alta taxa de sucesso e segurança para isolar o DPSCh.

Abstract Objectives: To establish and implement a simplified protocol for the extraction, primary isolation, and culture of human dental pulp stem cells (hDPSCs). To analyze the isolated cells quantitatively and qualitatively. Methodology: Ten healthy third molars were donated by patients who attended the School of Dentistry, UdelaR, and gave their written consent. The teeth were processed within 48 hours. The teeth were sectioned to obtain the pulp tissue and processed with the explant method. Cell viability and marker expression were analyzed by flow cytometry at passages 4 and 12 and verified by immunocytochemistry. Results: The cells obtained had a vitality greater than 90% in all passages. We found the characteristic morphology and the expression of CD90, C105, CD73, CD29 and 166 mesenchymal stem cell markers by flow cytometry in both passages. Conclusion: It was possible to establish a cell isolation protocol that is highly successful and safe to isolate hDPSC.

Platelet-rich plasma for male genital lichen sclerosus resistant to conventional therapy: First prospective study.

Ultrapotent topical corticosteroids and circumcision are usually effective for male genital lichen sclerosus (MGLSc); however, refractory cases are often referred to our Male Genital Dermatology Unit. Treatment with autologous platelet-rich plasma (TPRP) has recently been advocated as a safe and effective treatment option, but there have been no prospective studies in men to date. The objective of this study is to assess the safety and efficacy of TPRP for MGLSc resistant to conventional therapy. A prospective, open-label, single-arm, therapeutic study was carried out in this study. Inclusion criteria: resistant to conventional therapy for at least 6 months. Procedure: infiltration of 0.1 mL/cm2 PRP every 8 weeks. Monthly data recording: visual appearance with photographs and external scoring by an expert using Investigator's Global Assessment Scale (IGA scale 0-5), symptoms (scale 0-5), quality of life (QoL; Dermatology Life Quality Index [DLQI]), and complications. No. of patients included was n = 5. No. of patients excluded during treatment was n = 1. Mean initial IGA: 3.6. Mean initial DLQI: 6. TPRP n = 34 (range: 2-9; average: 6.8 per patient). Mean IGA at 18 months: 3.25. Mean DLQI at 18 months: 1.25. All patients reported being completely asymptomatic at 10 months. No. of patients with complications is n = 1 (balanitis). TPRP seems to be safe and effective, regarding symptom control and improvement in QoL; however, visual changes were minimal.

Autologous bone marrow-derived cells for venous leg ulcers treatment: a pilot study.

BACKGROUND: Chronic venous leg ulcers (VLUs) are a common problem in clinical practice and available treatments are not satisfactory. The use of adjuvant therapies in combination with lower limb compression may lead to improved healing rates. Chronic wounds are candidates for new strategies in the emergent field of regenerative medicine. Bone marrow-derived cells (BMDCs) contain cells and secrete cytokines known to participate in wound healing. Thus, BMDC therapy seems a logical strategy for the treatment of chronic wounds. Our objective was to evaluate feasibility, safety and initial clinical outcome of autologous BMDC therapy associated with standard treatment in patients with VLUs. METHODS: We conducted an open-label, single-arm, prospective pilot clinical trial in four patients with six chronic VLUs. The study protocol was approved by the institutional and national review boards and ethics committees. Bone marrow was harvest, processed and then administered by multiple injections into the ulcers. All patients received standard treatment and non-healing characteristics of the VLUs were confirmed at study entry. RESULTS: Ulcer size and wound pain evaluated 12 months after BMDC treatment were significantly reduced (P < 0.05). BMDC treatment was safe and well tolerated in long-term follow-up. DISCUSSION: Despite the low number of patients studied, our results showed that autologous BMDC treatment could be a useful, feasible and safe procedure to enhance ulcer healing. However, randomized controlled trials with more patients are needed to address this question and translate this approach into clinical practice.

Tratamiento complementario de heridas crónicas con factor de crecimiento estimulante de colonias de granulocitos. A propósito de dos casos clínicos / Granulocyte Colony-Stimulating Growth Factor complementary treatment in chronic wounds. Report of two cases / Tratamento complementar de feridas crônicas com colônias de granulócitos estimulantes do fator de crescimento. Cerca de dois casos clínicos

RESUMEN: Introducción: Independientemente de su etiología, las heridas crónicas, representan un desafío terapéutico debido a que muchas veces son refractarias a tratamientos convencionales. Es por este motivo que en los últimos años se han desarrollado de forma creciente estrategias complementarias en el área de la medicina regenerativa, como: terapias con células madre, ingeniería de tejidos, plasma rico en plaquetas y factores de crecimiento aplicados en las heridas crónicas. Dichas terapias complementarias han mostrado ciertos beneficios en la cicatrización de heridas complejas. Materiales y métodos: Se presentan dos casos clínicos de pacientes con heridas crónicas de diferente etiología, refractarias al tratamiento con cura avanzada de heridas, las cuales recibieron de forma complementaria factor estimulante de colonias de granulocitos (G-CSF; Filgen®). Se realizaron inyecciones locales de G-CSF a una dosis de 300 mcg/ml en piel peri úlcera en forma semanal completando dos series de cuatro inyecciones cada una. Resultados. Ambos casos presentaron una reducción en el área de las mismas alcanzándose la cicatrización total en un paciente y una reducción del 37% en el otro luego de dos series. El procedimiento fue bien tolerado y no se reportaron efectos adversos relacionados al mismo. Conclusiones: Los resultados obtenidos en estos pacientes mostraron beneficios en la cicatrización con la aplicación del G-CSF. Se requieren de ensayos clínicos controlados que permitan establecer el rol de dicho factor en el tratamiento de las mismas. Por este motivo nuestro grupo de trabajo se encuentra desarrollando un protocolo que permita evaluar este aspecto.

ABSTRACT: Introduction: Regardless of their etiology, chronic wounds, represent a therapeutic challenge because they are often refractory to conventional treatments. Due to this observation, in the last few years, new complementary strategies have emerged in the area of regenerative medicine, including stem cell therapeutics, tissue engineering, platelet rich plasma, and growth factors applied to chronic wounds. These complementary therapies have shown certain benefits in healing of complex wounds. Materials and methods: We present two clinical cases of patients with chronic wounds of different etiology, refractory to advanced and conventional wound treatments, which received complementary granulocyte colony-stimulating growth factor (G-CSF; Filgen®). Local injections with G-CSF were administered weekly in periulcer skin at a dose of 300 mcg/ml, completing two series of four injections each. Results: Both cases showeda reduction in their areas, reaching to total healing inone patient, and a reduction of 37% in the other one after two series of treatment. The procedure was well tolerated and no adverse effects were detected. Conclusions: The results obtained with these patients showed a benefit in cicatrization with the administration of G-CSF. Controlled clinical trials are needed to establish the role of G-CSF in these wounds. Thus, our group is developing a protocol to evaluate this aspect.

RESUMO: Introdução: Independentemente da sua etiologia, as feridas crônicas representam um desafio terapêutico porque muitas vezes são refratárias aos tratamentos convencionais. É por esta razão que nos últimos anos foram desenvolvidas estratégias complementares na área da medicina regenerativa, tais como: terapias de células estaminais, engenharia de tecidos, plasma rico em plaquetas e fatores de crescimento aplicados a feridas crônicas. Tais terapias complementares mostraram certos benefícios na cicatrização de feridas complexas. Materiais e métodos: Dois casos de pacientes com feridas crónicas de diferentes etiologias, refractárias ao tratamento com a cura da ferida avançado, que receberam complementaria factor estimulante forma de colónias de granulócitos (Filgen® FEC-G) presente. As injeções locais de G-CSF foram feitas a uma dose de 300 mcg / ml na pele peri úlcera, semanalmente, completando duas séries de quatro injeções cada. Resultados: Ambos os casos mostraram uma redução na área do mesmo atingindo a cicatrização total em um paciente e uma redução de 37% no outro após duas séries. O procedimento foi bem tolerado e nenhum efeito adverso relacionado a ele foi relatado. Conclusões: Os resultados obtidos nestes pacientes mostraram benefícios na cura com a aplicação de G-CSF. Ensaios clínicos controlados são necessários para estabelecer o papel desse fator no tratamento deles. Por esse motivo, nosso grupo de trabalho está desenvolvendo um protocolo que nos permite avaliar esse aspecto.

Activity of the Calcineurin Pathway in Patients on the Liver Transplantation Waiting List: Factors of Variability and Response to Tacrolimus Inhibition.

BACKGROUND: We sought to evaluate, in patients on a liver transplantation waiting list, potential biomarkers of the base calcineurin pathway activity with use of a new model of nonstimulated peripheral blood mononuclear cells (PBMC) and ex vivo response to tacrolimus (TAC). METHODS: The calcineurin pathway activity was explored ex vivo in stimulated and nonstimulated PBMC from 19 patients. The inhibition of NFAT1 translocation to PBMC nuclei, expression of intracellular IL-2, and membrane CD25 in different T-cell subsets were measured by multiparametric flow cytometry before and after exposure to TAC. We also studied the influence on the individual response of polymorphisms in 3 key genes of the calcineurin pathway: PPIA, PPP3CA, and IL2RA. RESULTS: All pharmacodynamics profiles closely fitted an I/Imax sigmoid model. Interindividual variability was higher in nonstimulated than in stimulated conditions, as well as in the presence of TAC. IL-2+CD8+ cells at TAC Imax showed the highest interindividual variability, suggesting its usefulness as a biomarker of individual TAC effects integrating many different sources of regulation and variability. Moreover, in the absence of TAC, patients with end-stage liver disease exhibited lower NFAT1 translocation and T-cell activation than healthy volunteers from a previous study under similar conditions. Multivariate statistical analysis showed strong and significant associations between TAC pharmacodynamic parameters and 2 polymorphisms in the gene-coding cyclophilin A (rs8177826 and rs6850). CONCLUSIONS: We show the feasibility of using nonstimulated PBMCs to explore the calcineurin pathway under more physiologic conditions and point toward potential biomarkers for TAC pharmacodynamic monitoring. ClinicalTrials.gov Identifier: NCT01760356.

N-acetylcysteine improves the quality of red blood cells stored for transfusion.

Storage inflicts a series of changes on red blood cells (RBC) that compromise the cell survival and functionality; largely these alterations (storage lesions) are due to oxidative modifications. The possibility of improving the quality of packed RBC stored for transfusion including N-acetylcysteine (NAC) in the preservation solution was explored. Relatively high concentrations of NAC (20-25 mM) were necessary to prevent the progressive leakage of hemoglobin, while lower concentrations (≥2.5 mM) were enough to prevent the loss of reduced glutathione during the first 21 days of storage. Peroxiredoxin-2 was also affected during storage, with a progressive accumulation of disulfide-linked dimers and hetero-protein complexes in the cytosol and also in the membrane of stored RBC. Although the presence of NAC in the storage solution was unable to avoid the formation of thiol-mediated protein complexes, it partially restored the capacity of the cell to metabolize H2O2, indicating the potential use of NAC as an additive in the preservation solution to improve RBC performance after transfusion.

Human Mesenchymal Stromal Cells Improve Cardiac Perfusion in an Ovine Immunocompetent Animal Model.

BACKGROUND: Mesenchymal stromal cells (MSCs) hold considerable promise in the treatment of ischemic heart disease. Most preclinical studies of MSCs for acute myocardial infarction (AMI) have been performed either in syngeneic animal models or with human cells in xenogeneic immunodeficient animals. A preferable pre-clinical model, however, would involve human MSCs in an immunocompetent animal. METHODS: AMI was generated in adult sheep by inducing ischemia reperfusion of the second diagonal branch. Sheep (n = 10) were randomized to receive an intravenous injection of human MSCs (1 × 10(6) cells/kg) or phosphate buffered saline. Cardiac function and remodeling were evaluated with echocardiography. Perfusion scintigraphy was used to identify sustained myocardial ischemia. Interaction between human MSCs and ovine lymphocytes was assessed by a mixed lymphocyte response (MLR). RESULTS: Sheep receiving human MSCs showed significant improvement in myocardial perfusion at 1 month compared with baseline measurements. There was no change in ventricular dimensions in either group after 1 month of AMI. No adverse events or symptoms were observed in the sheep receiving human MSCs. The MLR was negative. CONCLUSION: The immunocompetent ovine AMI model demonstrates the clinical safety and efficacy of human MSCs. The human cells do not appear to be immunogenic, further suggesting that immunocompetent sheep may serve as a suitable pre-clinical large animal model for testing human MSCs.

Stem cell research in Latin America: update, challenges and opportunities in a priority research area.

Stem cell research is attracting wide attention as a promising and fast-growing field in Latin America, as it is worldwide. Many countries in the region have defined Regenerative Medicine as a research priority and a focus of investment. This field generates not only opportunities but also regulatory, technical and operative challenges. In this review, scientists from Uruguay, Mexico, Chile, Brazil and Argentina provide their view on stem cell research in each of their countries. Despite country-specific characteristics, all countries share several issues such as regulatory challenges. Key initiatives of each country to promote stem cell research are also discussed. As a conclusion, it is clear that regional integration should be more emphasized and international collaboration, promoted.

Ischemia-induced model of diastolic dysfunction in sheep.

BACKGROUND: Ischemic heart failure with normal ejection fraction (HFNEF) is an increasingly recognized entity that carries similar morbidity and mortality than low ejection fraction heart failure. Animal models of diastolic dysfunction mimicking this condition are lacking and are essential for the development of therapeutic strategies. METHODS AND RESULTS: Eight Corriedale sheep, 18 ± 5 months old, were included in the study. Basal echocardiography and myocardial perfusion evaluation (SPECT) was performed. Acute myocardial infarction (AMI) was made by occlusion (90 min) and reperfusion of the second diagonal branch of the anterior descending coronary artery. Two months after AMI, echocardiography and SPECT evaluation were performed prior to sacrifice. Basal and 2 months echocardiography showed similar fractional shortening and ventricular dimensions in each animal except for an increase in left atrial diameter. No mitral regurgitation was evidenced. SPECT imaging and pathology confirmed infarction in the apical, apico-anterior, and apico-septal segments. CONCLUSION: A novel model of ischemia-induced diastolic dysfunction with preserved ventricular thickness and ejection fraction is described. This model opens the possibility of testing therapeutic options for patients with this condition.

Tacrolimus pharmacodynamics and pharmacogenetics along the calcineurin pathway in human lymphocytes.

BACKGROUND: Although therapeutic drug monitoring has improved the clinical use of immunosuppressive drugs, there is still interpatient variability in efficacy and toxicity that pharmacodynamic monitoring may help to reduce. To select the best biomarkers of tacrolimus pharmacodynamics, we explored the strength and variability of signal transduction and the influence of polymorphisms along the calcineurin pathway. METHODS: Peripheral blood mononuclear cells from 35 healthy volunteers were incubated with tacrolimus (0.1-50 ng/mL) and stimulated ex vivo. Inhibition of NFAT1 (nuclear factor of activated T cells 1) translocation to the nucleus and intracellular expression of interleukin-2 in CD4(+) and CD8(+) T cells and the surface activation marker CD25 in CD3(+) cells were measured by flow cytometry. We sequenced the promoter regions of immunophilins and calcineurin subunits and characterized selected single nucleotide polymorphisms in the genes of the calcineurin pathway with allelic discrimination assays. RESULTS: All responses closely fitted an I/Imax sigmoid model. Large interindividual variability (n = 30) in I0 and IC50 was found for all biomarkers. Moreover, strong and statistically significant associations were found between tacrolimus pharmacodynamic parameters and polymorphisms in the genes coding cyclophilin A, the calcineurin catalytic subunit α isoenzyme, and CD25. CONCLUSIONS: This study demonstrates the consistency and large interindividual variability of signal transduction along the calcineurin pathway, as well as the strong influence of pharmacogenetic polymorphisms in the calcineurin cascade on both the physiological activity of this route and tacrolimus pharmacodynamics.

Stem cell research and clinical development in tendon repair.

Stem cells are one of the most fascinating areas in regenerative medicine today. They play a crucial role in development and regeneration and are defined as cells that continuously reproduce themselves while maintaining the ability to differentiate into various cell types. Stem cells are found at all developmental stages, from embryonic stem cells (ESCs) which differentiate into all cell types, to adult stem cells (ASCs) which are responsible for tissue regeneration. Studies using animal models have shown promising results following cell therapy for induced injury in musculoskeletal system, including tendon healing, but the results can be variable. Alternative sources for cell therapy in tendon pathology may include ESCs, ASCs (bone marrow, adipose tissue or tendon derived stem cells) or induced pluripotent stem cells (iPSCs). While ethical and safety concerns currently forbid clinical application of ESCs and iPSCs, initial clinical trials with ASCs are promising.

Angiogênese coronariana como resposta endógena da isquemia miocárdica no adulto / Coronary angiogenesis as an endogenous response to myocardial ischemia in adults / Angiogénesis coronaria como respuesta endógena de la isquemia miocárdica en el adulto

O processo de angiogênese envolve uma sequência complexa de estímulos e respostas integradas, como estimulação das células endoteliais (CE) para sua proliferação e migração, estimulação da matriz extracelular, para atração de pericitos e macrófagos, estimulação das células musculares lisas, para sua proliferação e migração, e formação de novas estruturas vasculares. Angiogênese é principalmente uma resposta adaptativa à hipóxia tecidual e depende do acúmulo do fator de crescimento induzido pela hipóxia (FIH-1 α) na zona do miocárdio isquêmico, que serve para aumentar a transcrição do fator de crescimento endotelial vascular (VEGF) e seus receptores VEGF-R, pelas CE em sofrimento isquêmico. Esses passos envolvem mecanismos enzimáticos e proteases ativadoras do plasminogênio, metaloproteinases (MMP) da matriz extracelular (MEC) e cinases que provocam a degradação molecular proteolítica da MEC, bem como pela ativação e a liberação de fatores de crescimento, tais como: fator básico de crescimento dos fibroblastos (FCFb), VEGF e fator de crescimento insulínico-1 (FCI-1). Posteriormente, vem a fase intermediária de estabilização do novo broto neovascular imaturo e a fase final de maturação vascular da angiogênese fisiológica. Como conclusões generalizáveis, é possível afirmar que a angiogênese coronária em adultos é, fundamentalmente, uma resposta parácrina da rede capilar preexistente em condições fisiopatológicas de isquemia e inflamação.

The process of angiogenesis involves a complex sequence of stimuli and integrated responses, such as stimulation of endothelial cells (ECs) for their proliferation and migration, stimulation of the extracellular matrix (ECM) for the attraction of pericytes and macrophages, stimulation of smooth muscle cells for their proliferation and migration, and formation of new vascular structures. Angiogenesis is mainly an adaptive response to tissue hypoxia and depends on the accumulation of the hypoxia-inducible factor (HIF-1α) in the ischemic myocardial area, which increases the transcription of the vascular endothelial growth factor (VEGF) and its receptors VEGF-R by the ECs undergoing ischemia. Those steps involve enzymatic mechanisms and plasminogen activator proteases, metalloproteinases (MMP) of the ECM, and kinases that cause proteolytic molecular degradation of the ECM and activation and release of growth factors, such as: basic fibroblast growth factor (bFGF), VEGF, and insulin growth factor-1 (IGF-1). In the intermediate phase, stabilization of the immature neovascular sprout occurs. The final phase is characterized by vascular maturation of the physiological angiogenesis. In conclusion, coronary angiogenesis in adults is fundamentally a paracrine response of the preexisting capillary network under pathophysiological condition of ischemia and inflammation.

El proceso de angiogénesis involucra una serie compleja de estímulos y de respuestas integradas, como la estimulación de las células endoteliales (CE), para su proliferación y migración, estimulación de la matriz extracelular, para la atracción de pericitos y macrófagos, estimulación de las células musculares lisas, para su proliferación y migración, y formación de nuevas estructuras vasculares. La angiogénesis es principalmente una respuesta adaptativa a la hipoxia tisular y depende de la acumulación del factor de crecimiento inducido por la hipoxia (FIH-1 α) en la zona del miocardio isquémico, que sirve para aumentar la transcripción del factor de crecimiento endotelial vascular (con sus siglas en inglés: VEGF vascular endotelial growth factor), y sus receptores VEGF-R, por las CE en el sufrimiento isquémico. Esos pasos aglutinan mecanismos enzimáticos y proteasas activadoras del plasminógeno, metaloproteinasas (MMP) de la matriz extracelular (MEC), y cinasas que provocan la degradación molecular proteolítica de la MEC, como también la activación y la liberación de factores de crecimiento, tales como: factor básico de crecimiento de los fibroblastos (FCFb), VEGF y factor de crecimiento insulínico-1 (FCI-1). Posteriormente, viene la fase intermedia de estabilización del nuevo brote neovascular inmaduro y la fase final de maduración vascular de la angiogénesis fisiológica. Como conclusiones generales, podemos afirmar que la angiogénesis coronaria en adultos es fundamentalmente, una respuesta paracrina de la red capilar preexistente en condiciones fisiopatológicas de isquemia e inflamación.

Coronary angiogenesis as an endogenous response to myocardial ischemia in adults.

The process of angiogenesis involves a complex sequence of stimuli and integrated responses, such as stimulation of endothelial cells (ECs) for their proliferation and migration, stimulation of the extracellular matrix (ECM) for the attraction of pericytes and macrophages, stimulation of smooth muscle cells for their proliferation and migration, and formation of new vascular structures. Angiogenesis is mainly an adaptive response to tissue hypoxia and depends on the accumulation of the hypoxia-inducible factor (HIF-1α) in the ischemic myocardial area, which increases the transcription of the vascular endothelial growth factor (VEGF) and its receptors VEGF-R by the ECs undergoing ischemia. Those steps involve enzymatic mechanisms and plasminogen activator proteases, metalloproteinases (MMP) of the ECM, and kinases that cause proteolytic molecular degradation of the ECM and activation and release of growth factors, such as: basic fibroblast growth factor (bFGF), VEGF, and insulin growth factor-1 (IGF-1). In the intermediate phase, stabilization of the immature neovascular sprout occurs. The final phase is characterized by vascular maturation of the physiological angiogenesis. In conclusion, coronary angiogenesis in adults is fundamentally a paracrine response of the preexisting capillary network under pathophysiological condition of ischemia and inflammation.

Revascularización miocárdica biológica complementaria a la revascularización miocárdica quirúrgica convencional. Comunicación preliminar de estudio prospectivo de casos y controles, randomizado y doble ciego / Biological myocardial revascularization complementary to conventional surgical myocardial revascularization Preliminary communication of a prospective, randomized, double-blind case-control study

Introducción: la revascularización miocárdica quirúrgica (RVMQ) incompleta es un factor determinante en la reaparición precoz de angina y determina una menor sobrevida para los pacientes. Objetivo: el objetivo del presente trabajo es evaluar la eficacia perfusional de la terapia angiogénica celular con células derivadas de la médula ósea (CMO) autólogas por inyección directa intramiocárdica complementaria a la RVMQ convencional. Material y método: la evaluación perfusional luego de la cirugía se realiza a los tres, seis y 12 meses mediante SPECT y ecocardiograma Doppler Tisular-Strain (DTI-Strain). Resultados: en esta comunicación preliminar (precedida por ocho casos pilotos) se reportan resultados de cuatro pacientes (dos casos y dos controles) que completaron el seguimiento al tercer mes, teniendo todos oclusión crónica de coronaria derecha (CD) sin recanalización, fracción de eyección del ventrículo izquierdo (FEVI) media de 50%. En los territorios de la descendente posterior (DP) y posterolateral (PL) de CD se inyectó un promedio de 5,7 x 106 células CD34+ autólogas en un volumen promedio de 5 ml por territorio. En el control perfusional con SPECT al tercer mes de los casos se registró un incremento perfusional en los territorios inyectados de aproximadamente 10%. En los controles, los sectores miocárdicos no revascularizados tuvieron un decremento perfusional en promedio de 3%. En el DTI-Strain se observó mejoría de los valores en todas las muestras estudiadas en el caso con FEVI conservada, y peoría en el caso con FEVI en rango inferior. La terapia celular angiogénica con CMO autólogas por vía intramiocárdica tiene beneficios prefusionales, es una técnica factible y segura complementaria al tratamiento quirúrgico.

Summary Introduction: incomplete surgical myocardial revascularization is a determining factor for early flare of subsequent angina which results in lower patient survival. Objective: the present study aims to evaluate the perfusional efficacy of cell angiogenic therapy, using autologic bone-marrow derived stem cells, by means of direct intramyocardial injections, as a complementary therapy when combined with myocardial surgical revascularization. Method: perfusional assessment subsequent to surgery is performed after three, six and twelve months with Single Photon Emission Computed Tomography (SPECT) and DTI-Strain. Results: the present preliminary communication/notice (preceded by eight pilot cases) reports results obtained in four patients (two cases and two controls), completed follow-up in the third month, all of which evidenced chronic right coronary (RC) occlusion with no reversal, 50% average left ventricle ejection fraction. An average of 5.7 x 106 CD34+ autologic cells was injected into the descending posterior and posterolateral territory. The average injection volume was 5 ml per territory. Upon SPECT perfusional control in the third month, a 10% perfusional increase was detected in the injected territories. As to the control cases, nonrevascularized myocardial sectors evidenced a perfusional decrease of 3%, on average. DTI-Strain showed values improvement for all samples studied in the case of the preserved left ventricle ejection fraction (LVEF), and worsening in the LVEF lower range. Angiogenic cell therapy by intramyocardial autologic bone-marrow stem cells implies perfusional benefits. It is a feasible and safe technique to complement surgical treatment.

Résumé Introduction: la revascularisation myocardique chirurgicale (RVMQ) incomplète est un facteur primordial à la réapparition précoce d’angine et détermine une survie raccourcie des patients. Objectif: le but de ce travail est d’évaluer l’efficacité de perfusion de la thérapie angiogénique cellulaire avec des cellules extraites de la moelle osseuse (CMO) autologues par injection directe intra-myocardique complémentaire à la RVMQ conventionnelle. Matériel et méthode: l’évaluation de la perfusion post-chirurgicale est faite trois, six et douze mois après avec SPECT et échocardiogramme Doppler Tisular-Strain (DTI-Strain). Résultats: dans cette communication préalable, précédée de huit cas pilotes, on reporte les résultats de quatre patients (deux cas et deux contrôles) ayant complété le suivi au bout de trois mois et présentant tous occlusion chronique de coronaire droite (CD) sans recanalisation, fraction d’éjection du ventricule gauche (FEVG) moyenne de 50%. Dans les territoires de la descendante postérieure (DP) et postéro latérale (PL) de CD on a injecté une moyenne de 5,7 x 106 cellules CD34+autologues dans un volume moyen de 5 ml par territoire. Lors du contrôle de perfusion avec SPECT des cas au troisième mois, on a repéré une hausse de perfusion dans les territoires injectés de 10% environ, les secteurs myocardiques non revascularisés ayant subi une décroissance perfusionnelle de 3% environ. Au DTI-Strain, on a observé une amélioration des valeurs dans tous les échantillons prélevés au cas de FEVG conservée et aggravation au cas avec FEVG à rang inférieur. La thérapie cellulaire angiogénique avec CMO autologues par voie intra-myocardique a des bénéfices de perfusion, est une technique faisable et sûre complémentaire au traitement chirurgical.

Resumo Introdução: a revascularização cirúrgica incompleta do miocárdio (RVCM) é um fator determinante no reaparecimento precoce da angina que implica uma sobrevida menor para os pacientes. Objetivo: o objetivo deste trabalho é avaliar a eficácia perfusional da terapia angiogênica celular com células autólogas obtidas da medula óssea por injeção direta intra-miocárdica como complemento a RVCM tradicional. Material e método: a avaliação perfusional pós-cirurgia é feita aos três, seis e doze meses usando SPECT e ecocardiograma Doppler Tisular-Strain (DTI-Strain). Resultados: nesta comunicação preliminar, (depois de oito casos piloto) relatamos os resultados de quatro pacientes (dois casos e dois controles) que terminaram o seguimento no terceiro mês, todos com oclusão crônica de coronária direita (CD) sem recanalizaçao com fração de ejeção do ventrículo esquerdo (FEVE) média de 50%. Na região do ramo descendente posterior (DP) e póstero-lateral (PL) da CD, injetaram-se uma média de 5,7 x 106 células CD34+ autólogas em um volume médio de 5 ml por região. No controle perfusional dos casos, realizado no terceiro mês, foi registrado um aumento da perfusão nas regiões injetadas de aproximadamente 10%. Nos controles, os setores do miocárdio não revascularizados apresentaram uma redução média da perfusão de 3%. No DTI-Strain foi observada uma melhora dos valores em todas as amostras estudadas no caso com FEVE conservada, e uma piora no caso com FEVE com valor inferior. A terapia celular angiogênica com CMO autólogas por via intra-miocárdica apresenta melhoras da perfusão, é uma técnica viável e segura, complementar ao tratamento cirúrgico.

Mesenchymal cells generated from patients with myelodysplastic syndromes are devoid of chromosomal clonal markers and support short- and long-term hematopoiesis in vitro.

Myelodysplastic syndromes (MDS) are clonal malignant stem cell disorders characterized by inefficient hematopoiesis. The role of the marrow microenvironment in the pathogenesis of the disease has been controversial and no study has been performed so far to characterize mesenchymal cells (MC) from MDS patients and to analyse their ability to support hematopoiesis. To this end, we have isolated and characterized MC at diagnostic marrow samples (n=12) and have purified their CD34+CD38- and CD34+CD38+ counterparts (n=7) before using MC as a short- and long-term hematopoietic support. We show that MC can be readily isolated from MDS marrow and exhibit a major expansion potential as well as an intact osteoblastic differentiation ability. They do not harbor the abnormal marker identified by FISH in the hematopoietic cells and they stimulate the growth of autologous clonogenic cells. Conversely, highly purified stem cells and their cytokine-expanded progeny harbor the clonal marker with variable frequencies, and both normal and abnormal long-term culture-initiating cell-derived progeny can be effectively supported by autologous MC. Thus, we demonstrate that MDS marrow is an abundant source of MC appearing both cytogenetically and functionally noninvolved by the malignant process and able to support hematopoiesis, suggesting their possible usefulness in future cell therapy approaches.