Paulo S. Brofman

Expression of cardiac function genes in adult stem cells is increased by treatment with nitric oxide agents.

Mesenchymal stem cells (MSCs) have received special attention for cardiomyoplasty because several studies have shown that they differentiate into cardiomyocytes both in vitro and in vivo. Nitric oxide (NO) is a free radical signaling molecule that regulates several differentiation processes including cardiomyogenesis. Here, we report an investigation of the effects of two NO agents (SNAP and DEA/NO), able to activate both cGMP-dependent and -independent pathways, on the cardiomyogenic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs). The cells were isolated, cultured and treated with NO agents. Cardiac- and muscle-specific gene expression was analyzed by indirect immunofluorescence, flow cytometry, RT-PCR and real-time PCR. We found that untreated (control) ADSCs and BM-MSCs expressed some muscle markers and NO-derived intermediates induce an increased expression of some cardiac function genes in BM-MSCs and ADSCs. Moreover, NO agents considerably increased the pro-angiogenic potential mostly of BM-MSCs as determined by VEGF mRNA levels.

Transplantation of SNAP-treated adipose tissue-derived stem cells improves cardiac function and induces neovascularization after myocardium infarct in rats

Stem cell therapy has been considered a promise for damaged myocardial tissue. We have previously shown that S-nitroso-N-acetyl-D,L-penicillamine (SNAP) increases the expression of several muscular markers and VEGF in mesenchymal stem cells, indicating that transplantation of SNAP-treated cells could provide better functional outcomes. Here, we transplanted SNAP-treated adipose tissue-derived stem cells (ADSCs) in rat infarcted myocardium. After 30days, we observed a significant improvement of the ejection fraction in rats that received SNAP-treated ADSCs, compared with those that received untreated cells (p=0.008). Immunohistochemical reactions showed an increased expression of troponin T-C and von Willebrand factor, and organized vascular units in the infarcted area of tissue transplanted with treated ADSCs. SNAP exposure induced intracellular S-nitrosation, a decreased GSH/GSSG ratio, but did not increase cGMP levels. Collectively, these results indicate that SNAP alters the redox environment of ADSCs, possibly associated with a pre-differentiation state, which may improve cardiac function after transplantation.

PUMILIO-2 Is Involved in the Positive Regulation of Cellular Proliferation in Human Adipose-Derived Stem Cells

Stem cells can either differentiate into more specialized cells or undergo self-renewal. Several lines of evidence from different organisms suggest that these processes depend on the post-transcriptional regulation of gene expression. The presence of the PUF [Pumilio/FBF (fem-3 binding factor)] domain defines a conserved family of RNA binding proteins involved in repressing gene expression. It has been suggested that a conserved function of PUF proteins is to repress differentiation and sustain the mitotic proliferation of stem cells. In humans, Pumilio-2 (PUM2) is expressed in embryonic stem cells and adult germ cells. Here we show that PUM2 is expressed in a subpopulation of adipose-derived stem cell (ASC) cultures, with a granular pattern of staining in the cytoplasm. Protein levels of PUM2 showed no changes during the differentiation of ASCs into adipocytes. Moreover, RNAi knockdown of pum2 did not alter the rate of adipogenic differentiation compared with wild-type control cells. A ribonomic approach was used to identify PUM2-associated mRNAs. Microarray analysis showed that PUM2-bound mRNAs are part of gene networks involved in cell proliferation and gene expression control. We studied pum2 expression in cell cultures with low or very high levels of proliferation and found that changes in pum2 production were dependent on the proliferation status of the cell. Transient knockdown of pum2 expression by RNAi impaired proliferation of ASCs in vitro. Our results suggest that PUM2 does not repress differentiation of ASCs but rather is involved in the positive control of ASCs division and proliferation.

Emergence of clonal chromosomal alterations during the mesenchymal stromal cell cultivation

BackgroundUse of human mesenchymal stromal cells (MSCs) is a promising strategy for cell therapy in injured tissues recovery. However, MSCs acquire genetic changes when cultivated in vitro that make them more susceptible to undergo neoplastic transformation. Therefore, genomic integrity of stem cells should be monitored carefully for the use in basic research and clinical trials, including karyotype analysis to confirm the absence of genetic instability. Here, we report a case of a male 67-year-old patient selected to join the study: “Autologous transplantation of mesenchymal cells for treatment of severe and refractory ischemic cardiomyopathy”. He underwent nephrectomy for malignant tumor on the right kidney. Cytogenetic analysis on a bone marrow sample showed a normal karyotype: 46,XY[20]. However, the MSC at second passage showed a hyperdiploid clone, with clonal trisomies of chromosomes 4, 5, 10 and X. In order to investigate more, another sample from the same patient was used for a second cultivation and, at third passage, these cells showed a clonal translocation t(9;18)(p24;q11). The recurrent aberrations in MSC may indicate the beginning of a spontaneous transformation in culture, so, these cells were not used for cell therapy. Several analyses were performed at the Center for Cell Technology (152 samples), however this was the only case to show clonal cytogenetic abnormalities. Interestingly, two distinct clonal alterations were seen in two parallel cell cultivations from the same patient, suggesting a propensity for genetic instability. This highlights the need to evaluate these cells on a case-by-case basis, especially in patients with a history of cancer. Although there is controversy about the use of cells with cytogenetic abnormality for therapy, because their tumorigenic doubtful potential, we decided against the use of these cells for regenerative medicine.Case presentationHere, we report a case of a male 67-year-old patient selected to join the study: “Autologous transplantation of mesenchymal cells for treatment of severe and refractory ischemic cardiomyopathy”. He underwent nephrectomy for malignant tumor on the right kidney. Cytogenetic analysis on a bone marrow sample showed a normal karyotype: 46,XY[20]. However, the MSC at second passage showed a hyperdiploid clone, with clonal trisomies of chromosomes 4, 5, 10 and X. In order to investigate more, another sample from the same patient was used for a second cultivation and, at third passage, these cells showed a clonal translocation t(9;18)(p24;q11). The recurrent aberrations in MSC may indicate the beginning of a spontaneous transformation in culture, so, these cells were not used for cell therapy. Several analyses were performed at the Center for Cell Technology(152 samples), however this was the only case to show clonal cytogenetic abnormalities. Interestingly, two distinct clonal alterations were seen in two parallel cell cultivations from the same patient, suggesting a propensity for genetic instability. This highlights the need to evaluate these cells on a case-by-case basis, especially in patients with a history of cancer.ConclusionsAlthough there is controversy about the use of cells with cytogenetic abnormality for therapy, because their tumorigenic doubtful potential, we decided against the use of these cells forregenerative medicine.

The transplant of cardiac cells and myoblast skeletal cells in myocardial infarction

OBJETIVO:Comparar o resultado funcional e anatomo-patologico entre o transplante de celulas mioblasticas e cardiacas no infarto do miocardio. METODO: Realizado infarto da parede ântero-lateral do ventriculo esquerdo em 26 ratos Wistar, com ligadura da arteria coronaria esquerda. Apos cinco dias, os animais foram submetidos a ecocardiografia transtoracica para calculo dos volumes sistolico (VSFVE) e diastolico (VDFVE) finais e da fracao de ejecao do ventriculo esquerdo (FEVE). Os animais foram divididos em tres grupos: 1) controle (n=10), 2) celulas cardiacas adultas (n=8) e 3) celulas musculares esqueleticas adultas (n=8). Sete dias apos o infarto do miocardio, os animais foram reoperados por esternotomia mediana, sendo identificada a regiao de fibrose no ventriculo esquerdo e nela, injetado 0.15ml de meio de cultura no grupo I, 8.5x106/0.15ml de celulas cardiacas heterologas no grupo II e 8.5x106/0.15ml de celulas musculares esqueleticas heterologas no grupo III. Todos os animais receberam ciclosporina (15mg/kg/dia). Apos dois meses do transplante, realizou-se nova ecocardiografia avaliando os mesmos parâmetros. RESULTADOS: Apos dois meses do transplante celular, o grupo I apresentou um decrescimo da FEVE (48.18% vs. 33.25% p=0.0003), sendo que houve um acrescimo dos VSFVE e VDFVE (0.308ml vs. 0.536ml p=0.026 e 0.597ml vs. 0.776ml p=0.054, respectivamente). No grupo II houve uma estabilizacao da FEVE (42.48% vs. 41.31% p=0.4968, respectivamente) e um discreto aumento do VDFVE (0.602ml vs. 0.771ml p=0.0711). O VSFVE variou de 0.358ml a 0.450ml p=0.0400. O grupo III apresentou um acrescimo da FEVE, VDFVE e VSFVE (40% vs. 47.35% p=0.0142, 0.643ml vs. 0.931ml p=0.0026 e 0.388ml vs. 0.491ml p=0.0557 (sem significância), respectivamente. O GIII apresentou um maior valor, considerado estatisticamente significativo, da fracao de ejecao do ventriculo esquerdo, em comparacao ao GI e ao GII (47.35% + 6.89% vs. 41.31% + 8.46% vs. 33.25% + 12.41% p=0.0200, respectivamente). Identificou-se uma diferenca estatisticamente significativa da fracao de ejecao do ventriculo esquerdo entre o GIII e o GI, apos dois meses do transplante (47.35% + 6.891% vs. 33.25% + 12.41% p=0.0213, respectivamente). Identificou-se uma diferenca da fracao de ejecao, apos dois meses, entre o GIII e o GII, todavia nao foi considerada estatisticamente significativa (47.35% + 6.891% vs. 41.31% + 8.46% p= 0.481, respectivamente). Tambem se identificou uma diferenca deste mesmo parâmetro entre o GII e o GI, mas sem ser significativa (41.31% + 8.461% vs. 33.25% + 12.41% p=0.245, respectivamente). CONCLUSAO: As celulas mioblasticas mantiveram suas caracteristicas morfologicas apos o transplante no infarto do miocardio. Os fibroblastos foram as celulas encontradas em maior quantidade, durante o processo de cultura celular no grupo das celulas cardiacas. Apos dois meses do transplante das celulas, houve uma melhora significativa da FEVE do grupo III em comparacao ao grupo I, uma preservacao da contratilidade ventricular no grupo III e uma estabilizacao da fracao de ejecao do grupo II.