Stem cells for transplantation and manufacturing method therefor
a technology of stem cells and stem cells, applied in the direction of cell culture active agents, viruses, peptide/protein ingredients, etc., can solve the problems of inability to stably express a therapeutic gene, difficult to maintain, etc., and achieve the effect of high post-transplantation cell survival rate and engraftment rate, high post-transplantation engraftment rate, and high safety
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example 1
Verification of Effect of Improving Survival Rate of MSCs by MSCs Intracellularly Overexpressing IL-10
[0143](Objective)
[0144]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.
[0145](Method)
[0146]1. Gene Transfection of MSCs with IL-10 Expression Plasmid DNA
[0147]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...
example 2
[0157]Verification of Effect of Improving Survival Rate and Engraftment Rate by MSCs Overexpressing IL-10
[0158](Objective)
[0159]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.
[0160](Method)
[0161]1. Gene Transfection of MSCs with AAV Vector and Confirmation of Expression
[0162]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 ov...
example 3
[0174]Verification of Improvement in Survival Rate and Engraftment Rate in Experiment of Transplantation of MSCs Transduced with IL-10 Expression AAV Vector into Dog
[0175](Objective)
[0176]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.
[0177](Method)
[0178]1. Preparation of Dog Bone Marrow-Derived MSCs
[0179]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) c...
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