Stem cells for transplantation and manufacturing method therefor
a technology of stem cells and stem cells, applied in the field of stem cells for transplantation, can solve the problems of inability to stably express a therapeutic gene, difficult to maintain, etc., and achieve the effects of convenient production of high post-transplantation engraftment rate, high post-transplantation cell survival rate and engraftment rate, and high safety
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example 1
[0149](Objective)
[0150]The objective is to verify the effect of improving the survival rate of MSCs after the intracellular expression level of IL-10 in MSCs was increased by use of an IL-10 expression plasmid, and an IL-10 concentration in serum.
[0151](Method)
[0152]1. Gene Transfection of MSCs with IL-10 Expression Plasmid DNA
[0153]2 μg of a mouse IL-10 expression plasmid DNA (pW-CAG-mIL-10-WPRE) or a control GFP expression plasmid DNA (pW-CAG-EGFP-WPRE) was mixed with 100 μL of a Nucleofection solution (Human MSC Nucleofector kit, Lonza Group Ltd.) / 5×105 MSCs, and each mixture was used in gene transfection using an Amaxa Nucleofector system (C-17 mode). The pW-CAG-mIL-10-WPRE was prepared by introducing the recombinant mouse IL-10 gene prepared in Comparative Example 2 to the BamHI site of pW-CAG-WPRE. The pW-CAG-EGFP-WPRE was prepared by introducing an EGFP gene to the BamHI site of pW-CAG-WPRE (Okada, T., et al., 2005, Hum. Gene Ther., 16: 1212-1218). The detailed procedures fol...
example 2
[0163](Objective)
[0164]The objective is to verify the effect of improving the survival rate and engraftment rate of MSCs after the intracellular expression level of IL-10 in the MSCs was increased by use of an IL-10 expression AAV vector.
[0165](Method)
[0166]1. Gene Transfection of MSCs with AAV Vector and Confirmation of Expression
[0167]The recombinant AAV was prepared according to the method described in Comparative Example 2. SD-rat MSCs-Luc was inoculated at 1×105 cells / well to a 24 well-plate (IWAKI / AGC Techno Glass Co., Ltd.) containing a DMED / F-12 (1:1) medium containing 10% FBS. After cell adhesion, an unpurified culture supernatant containing the mouse IL-10 expression AAV vector AAV1-IL-10 or a GFP expression AAV vector AAV1-CAG-EGFP-WPRE(EW) (hereinafter, referred to as “AAV1-GFP”) at 5.0×1010 g.c. was added thereto for the gene transfection of the SD-rat MSCs. After overnight culture at 37° C. in the presence of 5% CO2, the medium was replaced with a fresh one every 2 day...
example 3
[0179]
[0180](Objective)
[0181]The objective is to verify, in dogs, the effect of improving the survival and engraftment rates of AAV1-IL-10-introduced MSCs, which was demonstrated in mice.
[0182](Method)
[0183]1. Preparation of Dog Bone Marrow-Derived MSCs
[0184]Donor and recipient dogs were selected from DLA (dog leukocyte antigen)-matched male and female pairs of beagles. As illustrated in FIG. 8, 2 mL of a bone marrow fluid was collected from the fore-leg right and left upper arms of the donor normal dog. The bone marrow fluid was cultured in 2 mL of RPMI-1640 (Life Technologies Corporation) containing 20 U / mL heparin. Monocytes were isolated (1.3×108 cells) under a density gradient using Histopaque-1077 (Sigma-Aldrich Corp.). Then, a CD271-positive fraction having the high ability to grow (MSC Research Tool Box-CD271 (LNGFR) containing CD271 (LNGFR)-PE & Anti-PE Micro Beads (Miltenyi Biotec) was concentrated using immuno-magnetic beads (MACS® Columns and MACS Separators, Miltenyi Bi...
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