RESUMEN
BACKGROUND AIMS: The number of hematopoietic stem cells (HSCs) is critical for transplantation. The ex vivo expansion of mobilized peripheral blood (MPB) HSCs is of clinical value for reconstitution to meet clinical need. METHODS: This study proposed a simple, defined, stromal-free and serum-free culture system (SF-HSC medium) for clinical use, which is composed of Iscove's modified Dulbecco's medium, cytokine cocktails and serum substitutes. This study also characterized the cellular properties of expanded MPB CD133(+) HSCs from patients with hematologic malignancies and healthy donors by surface antigen, colony-forming cell, long-term culture-initiating cell, gene expression and in vivo engraftment assays. RESULTS: The expanded fold values of CD45(+) white blood cells and CD34(+), CD133(+), CD34(+)CD38(-), CD133(+)CD38(-), CD34(+)CD133(+), colony-forming and long-term culture-initiating cells at the end of 7-day culture from CD133(+) MPB of hematologic malignancies were 9.4-fold, 5.9-fold, 4.0-fold, 35.8-fold, 21.9-fold, 3.8-fold, 11.8-fold and 6.7-fold, and values from healthy donor CD133(+) MPB were 20.7-fold, 14.5-fold, 8.5-fold, 83.8-fold, 37.3-fold, 6.2-fold, 19.1-fold and 14.6-fold. The high enrichment of CD38(-) cells, which were either CD34(+) or CD133(+), sustained the proliferation of early uncommitted HSCs. The expanded cells showed high levels of messenger RNA expression of HOBX4, ABCG2 and HTERT and had the in vivo ability to re-populate NOD/SCID mice. CONCLUSIONS: Our results demonstrated that an initial, limited number of MPB CD133(+) HSCs could be expanded functionally in SF-HSC medium. We believe that this serum-free expansion technique can be employed in both basic research and clinical transplantation.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Medio de Cultivo Libre de Suero/metabolismo , Neoplasias Hematológicas/fisiopatología , Células Madre Hematopoyéticas/fisiología , Células del Estroma/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/fisiología , Células Cultivadas , Neoplasias Hematológicas/metabolismo , Movilización de Célula Madre Hematopoyética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , ARN Mensajero/genética , Células Madre/metabolismo , Células Madre/fisiología , Células del Estroma/metabolismo , Donantes de TejidosRESUMEN
BACKGROUND: Hyaluronic acid (HA) and chondroitin sulfate (CD) are endogenous polysaccharides. In recent years, they have aroused the interest of scientists because of specific binding to CD44 receptors, which are overexpressed in several types of tumors. METHODS: In this study, HA- and CD-modified poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) copolymers were synthesized and applied to encapsulate 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP)/pDNA (D/P) lipoplex as CD44 receptor targeting gene delivery nanoparticles (NPs). RESULTS: The particle size of CD-PEG-PLGA-D/P (186.8 ± 21.7 nm) was smaller than that of HA-PEG-PLGA-D/P (270.2 ± 13.8 nm), with narrow size distribution, and both HA-PEG-PLGA-D/P NPs and CD-PEG-PLGA NPs possessed negative zeta potentials (-39.63 ± 5.44 mV and -38.9 ± 2.0 mV, respectively), which prevent erythrocytes from agglutination. Both NPs exhibited pH-dependent release and had faster release in pH 4.0 than in pH 7.4. Generally, the CD-PEG-PLGA-D/P NPs possessed less cytotoxicity than HA-PEG-PLGA-D/P NPs. The D/P-loaded HA-PEG-PLGA and CD-PEG-PLGA NPs expressed significantly higher transfection in CD44 high-expressed U87 (30.1% ± 2.1% and 40.7% ± 4.3%, respectively) than in CD44-negative HepG2 (3.3% ± 1.5% and 1.4% ± 1.0%, respectively) (p < 0.001). It was revealed that the endocytosis of HA-PEG-PLGA-D/P NPs was majorly dominated by macropinocytosis and the endocytosis of CD-PEG-PLGA-D/P NPs was dominated by clathrin-mediated endocytosis pathway (p < 0.001). CONCLUSION: The high selectivity to CD44-positive U87 cancer cells and low cytotoxicity in L929 normal cells assured the promising potential of CD-PEG-PLGA NPs as gene delivery nano-carriers.
Asunto(s)
Técnicas de Transferencia de Gen , Receptores de Hialuranos/metabolismo , Nanopartículas/química , Polisacáridos/química , Animales , Supervivencia Celular , Sulfatos de Condroitina/química , ADN/metabolismo , Liberación de Fármacos , Endocitosis , Eritrocitos/metabolismo , Ácidos Grasos Monoinsaturados/química , Células Hep G2 , Humanos , Ácido Hialurónico/química , Ratones , Tamaño de la Partícula , Plásmidos/metabolismo , Poliésteres/química , Polietilenglicoles/química , Espectroscopía de Protones por Resonancia Magnética , Compuestos de Amonio Cuaternario/química , Electricidad EstáticaRESUMEN
The high- and low-molecular weight hyaluronic acid (HHA and LHA) were used to conjugate with PLGA-PEG copolymers which were applied to encapsulate DOTAP/pDNA (D/P) lipoplex as a CD44-targeted micelle delivery system. The size and zeta potential of DNA loaded micelles were measured. The cytotoxicity and cellular transfection of DNA loaded micelles were performed in CD44-positive MDA-MB-231 and MCF-7 cancer cells and CD44-negative HepG2 cells. The endocytosis mechanism of micelles was investigated further. The DNA loaded HA-conjugated micelles possessed negative-charged character which prevented erythrocytes from agglutination. Both LHA-PEG-PLGA and HHA-PEG-PLGA micelles had comparable cellular viability in L929 normal cells. The cellular transfection of HHA-PEG-PLGA micelles was much higher than of LHA-PEG-PLGA micelles in CD44-positive cells. The specific and strong binding of HHA to CD44-positive cells resulted in the cellular transfection of HHA-PEG-PLGA micelles in CD44-positive cells significantly higher than in CD44-negative cells.