DAPI diffusion after intravitreal injection of mesenchymal stem cells in the injured retina of rats.

To evaluate DAPI (4',6-diamidino-2-phenylindole) as a nuclear tracer of stem cell migration and incorporation it was observed the pattern of retinal integration and differentiation of mesenchymal stem cells (MSCs) injected into the vitreous cavity of rat eyes with retinal injury. For this purpose adult rat retinas were submitted to laser damage followed by transplantation of DAPI-labeled BM-MSCs grafts and double-labeled DAPI and quantum dot-labeled BM-MSCs. To assess a possible DAPI diffusion as well as the integration and differentiation of DAPI-labeled BM-MSCs in laser-injured retina, host retinas were evaluated 8 weeks after injury/transplantation. It was demonstrated that, 8 weeks after the transplant, most of the retinal cells in all neural retinal presented nuclear DAPI labeling, specifically in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). Meanwhile, at this point, most of the double-labeled BM-MSCs (DAPI and quantum dot) remained in the vitreous cavity and no retinal cells presented the quantum dot marker. Based on these evidences we concluded that DAPI diffused to adjacent retinal cells while the nanocrystals remained labeling only the transplanted BM-MSCs. Therefore, DAPI is not a useful marker for stem cells in vivo tracing experiments because the DAPI released from dying cells in moment of the transplant are taken up by host cells in the tissue.

Stem cells: potential source for retinal repair and regeneration.

Stem cells have been studied in several fields of Medicine, and their applications are not too far from the clinical practice. Retinal impairment by neuronal death has been considered incurable due to the limited regenerative capacity of the central nervous system. The capacity of stem cells to regenerate tissues, as well as their plasticity makes them a potential source for retinal repair. The stem cells are a great promise for the therapy of inherited retinal disorders and retinal-neuronal degenerative diseases, such as retinitis pigmentosa and allied retinal dystrophies, which can result in blindness. Because of the accessibility, expansibility, and multipotentiality mesenchymal stem cells are expected to be useful for clinical applications, especially in regenerative medicine and tissue engineering. Mesenchymal stem cells are clonogenic, nonhematopoietic stem cells present in the bone marrow. Given the appropriate microenvironment, they could differentiate into cardiomyocytes or even into cells of nonmesodermal derivation including hepatocytes and neurons. So far, the results of a few studies are consistent with the belief that cell-based therapies using mesenchymal stem cells may be effective when it comes to retinal damaged tissue repair.

Protection against dermonecrotic and lethal activities of Loxosceles intermedia spider venom by immunization with a fused recombinant protein.

We report the use of a recombinant Loxosceles intermedia spider protein in the form of a fusion protein as an antigen for immunization in rabbits and mice. The aim is to produce model protective antisera in these animals against dermonecrotic and lethal activities of the venom from the Brazilian spider responsible for 3000 cases, reported annually, of spider bites in South Brazil. A protein homologous to the dermonecrotic toxin was cloned from a cDNA expression library made with L. intermedia venom glands, expressed in E. coli cells as a fusion protein with beta-galactosidase and the recombinant protein (Li-rec protein) was purified by molecular filtration and affinity chromatography [Kalapothakis et al., Toxicon (2002) in press]. The Li-rec protein was characterized and used as an antigen to generate antibodies in rabbits and mice. These specifically raised antibodies recognized the native venom. In vitro neutralization assay of lethal effects indicated that 1 ml of rabbit serum raised against Li-rec protein was able to neutralize 25 LD(50) of the whole venom. In vivo protection experiments, the fusion proteins induced a long-term protection in rabbits against the dermonecrotic activity of the native venom. Immunized mice were challenged with various doses of the Loxosceles venom. Mice were fully protected against 2.5 LD(50) of venom. This result provides basic data for the use of such recombinant spider proteins as immunogens in the development of anti-venoms for clinical use or can be used as a vaccine providing efficient immune protection against L. intermedia venom.

Time-dependent migration of systemically delivered bone marrow mesenchymal stem cells to the infarcted heart.

In this study the time course of homing and the body distribution of systemically delivered bone marrow mesenchymal stem cells (BM-MSCs) after myocardial infarction (MI) were evaluated. BM-MSCs were isolated from Wistar rats, expanded in vitro, and their phenotypical characterization was performed by flow cytometer. Rats were randomly divided into three groups: control, sham MI, and MI. BM-MSCs (5 x 10(6)) were labeled with (99m)Tc-HMPAO and injected through the tail vein 7 days after MI. Gamma camera imaging was performed at 5, 15, 30, and 60 min after cell inoculation. Due to the (99m)Tc short half-life, cell migration and location were also evaluated in heart sections using DAPI-labeled cells 7 days after transplantation. Phenotypical characterization showed that BM-MSCs were CD90(+), CD73(+), CD54(+), and CD45(-). Five minutes after (99m)Tc-HMPAO-labeled cell injection, they were detected in various tissues. The cells migrated mainly to the lungs (approximately 70%) and, in small amounts, to the heart, kidneys, spleen, and bladder. The number of cells in the heart and lungs decreased after 60 min. MI markedly increased the amount of cells in the heart, but not in the lungs, during the period of observation (4.55 +/- 0.32 vs. 6.34 +/- 0.67% of uptake in infarcted hearts). No significant differences were observed between control and sham groups. Additionally, 7 days after DAPI-labeled cells injection, they were still detected in the heart but only in infarcted areas. These results suggest that the migration of systemically delivered BM-MSCs to the heart is time dependent and MI specifically increases BM-MSCs homing to injured hearts. However, the systemic delivery is limited by cell entrapment in the lungs.

Retinal incorporation and differentiation of mesenchymal stem cells intravitreally injected in the injured retina of rats.

PURPOSE: To evaluate the pattern of retinal integration and differentiation of mesenchymal stem cells (MSCs) injected into the vitreous cavity of rat eyes with retinal injury. METHODS: Adult rat retinas were submitted to laser damage followed by transplantation of DAPI-labeled BM-MSCs grafts. To assess the integration and differentiation of BM-MSCs in laser-injured retina, host retinas were evaluated 2.4 and 8 weeks after injury/transplantation. RESULTS: Our results demonstrated that the grafted cells survived in the retina for at least 8 weeks and almost all BM-MSCs migrated and incorporated into the neural retina, specifically in the outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL) while a subset of grafted cells were found in the subretinal space posttransplantation. At 8 weeks immunohistochemical analysis with several retinal specific markers revealed that the majority of the grafted cells expressed rhodopsin, a rod photoreceptor marker, followed by parvalbumin, a marker for bipolar and amacrine cells. A few subsets of cells were able to express a glial marker, glial fibrillary acidic protein. However, grafted cells failed to express pan-cytokeratin, a retinal pigment epithelium marker. CONCLUSIONS: These results suggest the potential of BM-MSCs to differentiate into retinal neurons. Taken together, these findings might be clinically relevant for future mesenchymal stem cell therapy studies concerning retinal degeneration repair.

Retinal incorporation and differentiation of mesenchymal stem cells intravitreally injected in the injured retina of rats / Incorporação e diferenciação retiniana de células tronco mesenquimais intravítreas em ratos

PURPOSE: To evaluate the pattern of retinal integration and differentiation of mesenchymal stem cells (MSCs) injected into the vitreous cavity of rat eyes with retinal injury. METHODS: Adult rat retinas were submitted to laser damage followed by transplantation of DAPI-labeled BM-MSCs grafts. To assess the integration and differentiation of BM-MSCs in laser-injured retina, host retinas were evaluated 2.4 and 8 weeks after injury/transplantation. RESULTS: Our results demonstrated that the grafted cells survived in the retina for at least 8 weeks and almost all BM-MSCs migrated and incorporated into the neural retina, specifically in the outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL) while a subset of grafted cells were found in the subretinal space posttransplantation. At 8 weeks immunohistochemical analysis with several retinal specific markers revealed that the majority of the grafted cells expressed rhodopsin, a rod photoreceptor marker, followed by parvalbumin, a marker for bipolar and amacrine cells. A few subsets of cells were able to express a glial marker, glial fibrillary acidic protein. However, grafted cells failed to express pan-cytokeratin, a retinal pigment epithelium marker. CONCLUSIONS: These results suggest the potential of BM-MSCs to differentiate into retinal neurons. Taken together, these findings might be clinically relevant for future mesenchymal stem cell therapy studies concerning retinal degeneration repair.

OBJETIVO: Avaliar o padrão de integração e diferenciação retiniana de células tronco mesenquimais (CTM) injetadas na cavidade vítrea de ratos portadores de lesões retinianas. MÉTODOS: Ratos Wistar adultos foram submetidos a múltiplas lesões retinianas utilizando-se YAG laser e injeção intravítrea de células tronco mesenquimais. A fim de se avaliar a integração e diferenciação retiniana, o tecido retiniano lesado pelo YAG laser / tratado pelas células tronco, foi avaliado 2, 4 e 8 semanas após a lesão. RESULTADOS: As células injetadas na cavidade vítrea sobreviveram na retina por pelo menos 8 semanas e quase todas células tronco mesenquimais migraram e incorporaram-se na retina neural, especificamente nas camadas nucleares externa e interna e camada de células ganglionares. Uma pequena quantidade de células foi encontrada no espaço sub-retiniano. A análise imuno-histoquímica de 8 semanas mostrou que a maioria das células injetadas expressou rodopsina (marcador para fotorreceptores), parvalbumina (marcador para células bipolares e amácrinas), GFAP (marcador de células gliais). As células injetadas não expressaram a pancitoqueratina, que é a marcadora de células do epitélio pigmentar da retina. CONCLUSÕES: Ocorre aparente diferenciação e incorporação de células tronco mesenquimais na retina de ratos após injeção intravitrea destas células.

Stem cells: potential source for retinal repair and regeneration

Stem cells have been studied in several fields of Medicine, and their applications are not too far from the clinical practice. Retinal impairment by neuronal death has been considered incurable due to the limited regenerative capacity of the central nervous system. The capacity of stem cells to regenerate tissues, as well as their plasticity makes them a potential source for retinal repair. The stem cells are a great promise for the therapy of inherited retinal disorders and retinal-neuronal degenerative diseases, such as retinitis pigmentosa and allied retinal dystrophies, which can result in blindness. Because of the accessibility, expansibility, and multipotentiality mesenchymal stem cells are expected to be useful for clinical applications, especially in regenerative medicine and tissue engineering. Mesenchymal stem cells are clonogenic, nonhematopoietic stem cells present in the bone marrow. Given the appropriate microenvironment, they could differentiate into cardiomyocytes or even into cells of nonmesodermal derivation including hepatocytes and neurons. So far, the results of a few studies are consistent with the belief that cell-based therapies using mesenchymal stem cells may be effective when it comes to retinal damaged tissue repair.

Células-tronco têm sido estudadas em várias áreas da Medicina e suas aplicações brevemente deverão estar incorporadas à prática clínica. O dano retiniano pela morte neuronal é considerado incurável devido a pobre capacidade regenerativa do sistema nervoso central. A capacidade das células-tronco em regenerar tecidos, assim como sua plasticidade, faz que estas sejam uma fonte potencial de células para a regeneração retiniana. Células-tronco são muito promissoras para o tratamento das distrofias retinianas, como a retinose pigmentar e outras doenças neurodegenerativas, que podem evoluir para cegueira. As células-tronco mesenquimais são o tipo mais provável de células-tronco a serem utilizadas na prática clínica devido a sua fácil acessibilidade e multipotencialidade de diferenciação em vários tecidos. As células-tronco mesenquimais são células clonogênicas, não-hematopoiéticas, localizadas na medula óssea. Desde que seja proporcionado um microambiente apropriado, estas células podem se diferenciar em cardiomiócitos e até mesmo em células de origem não-mesodérmica, como hepatócitos e neurônios. Até o presente momento, os resultados dos estudos iniciais são animadores em relação ao uso de células-tronco mesenquimais e uso eficaz destas no reparo de tecidos retinianos lesados.