Highly efficient transfer and stable expression of two genes upon lentivirus transduction of mesenchymal stem cells from human bone marrow

Abstract

The efficiency of human bone marrow (BM) mesenchymal stem cell (MSC) transduction with a bicistronic lentivirus vector was estimated, and the stability of transgene expression in genetically modified MSCs was determined. First passage BM MSCs were capable of efficient transduction with the bicistronic lentivirus vector. The transduction efficiency depended on the multiplicity of infection (MOI), being 64.64 ± 6.5 and 88.6 ± 2.9% at MOI 10 and 20, respectively. The lentivirus transduction efficiency proved indepen dent on the number of passages of a BM MSC culture, and expression of the egfp and dsRed1 transgenes in genetically modified MSCs remained stable for one month of culturing. A comparison showed that the level of egfp and dsRed1 transgene expression was preserved upon hepatogenic differentiation in vitro. The results provide a basis for further development of multigenic modification of human BM MSCs for research and/or therapeutic purposes.

A simple and highly efficient method for transduction of human adipose‐derived mesenchymal stem cells

“…Optimization of transduction can be achieved by several conditions including types of viral vectors, viral concentration, initial target cells number, transduction time points, and positively charged components to reduce the electrostatic repulsion forces between viruses and target cells. Currently, several optimized methods for transduction of MSCs have been described . However, most of those approaches contain tedious steps such as concentrating a large amount of virus‐containing supernatant and measuring viral titers.…”

“…Currently, several optimized methods for transduction of MSCs have been described. [27][28][29][30][31] However, most of those approaches contain tedious steps such as concentrating a large amount of virus-containing supernatant and measuring viral titers. While time-consuming because of several demanding steps, these methods are not efficient enough.…”

Estudo da função de CD90 na proliferação e diferenciação de células-tronco mesenquimais humanas

“…Outra vantagem é que os vetores lentivirais são integrados no genoma das células hospedeiras, apresentando resistência à supressão epigenética da transcrição, com uma expressão por longo tempo dos transgenes. A atividade promotora interna estabelecida é mantida e a atividade promotora do lentivírus é bloqueada (117) Uma forma de modificação genética das células pode ser a inativação de genes, com knock-out do gene-alvo, mas demanda tempo maior para a técnica e possui custo elevado.…”

O papel da quimiocina CXCL12 na diferenciação e autorrenovação de células-tronco hematopoéticas

“…Inicialmente tentamos isolar através a técnica de sorting subpopulações de CTMs que a literatura descreve como importantes para a secreção no nicho medular de quimiocina CXCL12, como por exemplo, o marcador NG2. (75,77) Entretanto, após inúmeras tentativas não obtivemos sucesso, após a separação celular, A literatura relata muitos desafios e diversos trabalhos mostrando a dificuldade de otimização das técnicas de transfecção e transdução das CTMs, (137,139,140,(171)(172)(173)(174) apesar do efeito de silenciamento muito transitório do siRNA, o agente de transfecção siPORT ® NeoFx™ e a técnica de transfecção reversa mostrou eficiência na entrega do siRNA e não causou danos as células.…”