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Previous issue date: 2012-01-16 / Cellular therapy represent a new frontier for the treatment of several diseases, including diseases related to central nervous system. Human umbilical cord blood is an attractive source of stem cells; however, it has a heterogeneous population with a small amount of mesenchymal stem cells. Currently, cell reprogramming induced by different methodologies can confer pluripotency to differentiated adult cells. The objective of the study was to evaluate the reprogramming potential of fibroblasts and neural differentiation after co-culture with umbilical cord blood mononuclear cells. This study was approved by the Ethics Committee of PUCRS (CEP 11/05504). Cells were obtained from four human umbilical cord blood. Cell suspensions were fractionated at gradient of density 1077. The mononuclear cells of each sample were cultured for 7 days and later, the culture was infected with 105 cells of mouse fibroblast cell line NIH 3T3 and co-cultured for 6 days. To evaluate the pluripotency it was performed RNA extraction and later, amplification using primers specific for pluripotency genes. The differentiation was also confirmed by the adipogenic and osteogenic differentiation. Neural differentiation of the reprogrammed cells was obtained by the method described by Song et. al (2008) and evaluated by immunofluorescence. Population growth was quantified by hemocytometer. All co-cultured cells showed adipogenic and osteogenic differentiation capacity. After co-cultivation cells began transcribing the pluripotency gene KLF4. Statistically significant differences in the parameters area, diameter, optical density and fractal dimension were observed by confocal microscopy in reprogrammed and neural differentiated cells. The population of reprogrammed cells doubled every three days until the addition of the last medium of neural differentiation, when cultures became quiescent until the end of the neural differentiation. The contact in form of co-cultivation of fibroblasts with umbilical cord blood mononuclear fraction for six days promoted the reprogramming of these cells to a indifferentiation stage, allowing the induction of neural differentiation later. / As terapias celulares representam uma nova fronteira para o tratamento de diversas doen?as, inclusive patologias relacionadas ao sistema nervoso central. O sangue de cord?o umbilical humano ? uma atrativa fonte de c?lulas-tronco, por?m, apresenta uma popula??o heterog?nea com pouca quantidade de c?lulas-tronco mesenquimais. Atualmente, a reprograma??o celular induzida atrav?s de metodologias distintas, pode conferir pluripot?ncia ?s c?lulas adultas diferenciadas. O objetivo deste trabalho foi avaliar o potencial de reprograma??o de fibroblastos e a diferencia??o neural ap?s co-cultivo com fra??o mononuclear de sangue de cord?o umbilical. Foram obtidas c?lulas da fra??o mononuclear de quatro cord?es umbilicais humanos de pacientes atendidas no Centro Obst?trico do Hospital S?o Lucas da PUCRS. As suspens?es celulares foram fracionadas em gradiente de densidade 1077. A fra??o mononuclear de cada amostra foi cultivada por sete dias e ap?s, contaminada com 105 c?lulas da linhagem de fibroblasto de camundongos NIH 3T3 e co-cultivadas por 6 dias. Para avalia??o da pluripot?ncia, foi realizada extra??o de RNA e posterior amplifica??o utilizando oligonulceot?deos espec?ficos para os genes de pluripot?ncia Oct3/4, Sox2 e KLF4. O fen?tipo mesenquimal foi confirmado pela diferencia??o adipog?nica e osteog?nica. A neurodiferencia??o das c?lulas reprogramadas seguiu o m?todo descrito por Song et. al (2008) e foi avaliada por imunofluoresc?ncia. O crescimento populacional foi quantificado em hemocit?metro. Todas as c?lulas co-cultivadas mostraram capacidade de diferencia??o adipog?nica e osteog?nica. Ap?s o co-cultivo, as c?lulas passaram a transcrever o gene de pluripot?ncia KLF4. Diferen?as estatisticamente significativas nos par?metros ?rea, di?metro, densidade ?ptica e dimens?o fractal foram observadas por microscopia confocal nas c?lulas reprogramadas e neurodiferenciadas. A popula??o de c?lulas reprogramadas duplicou a cada tr?s dias at? o momento da adi??o do ultimo meio de neurodiferencia??o, quando as culturas tornaram-se quiescentes at? o final do per?odo de neurodiferecia??o. O contato em forma de co-cultivo dos fibroblastos com a fra??o mononuclear de cord?o umbilical por seis dias promoveu a reprograma??o dessas c?lulas a um est?gio com certa plasticidade, permitindo a indu??o posterior de neurodiferencia??o.
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/1664 |
| Date | 16 January 2012 |
| Creators | Marinowic, Daniel Rodrigo |
| Contributors | Costa, Jaderson Costa da |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Medicina e Ci?ncias da Sa?de, PUCRS, BR, Faculdade de Medicina |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 7620745074616285884, 500, 600, -8624664729441623247 |
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