RC17 hESCs or AST18 iPSCs (8 × 105 cells) were transfected with 1 μg of each of the 4 gRNA plasmids and GFPiPuroR (a GFP indicator plasmid) using Amaxa™ Human Stem Cell Nucleofector™ Kit 1 (Lonza) and programme B‐016 on Nucleofector® II (Lonza). 24 hr after transfection, puromycin (0.5 μg/ml, Sigma) selection (16 hr) was applied for cells on Matrigel (Thermo Fisher Scientific) or 1.0 μg/ml puromycin for cells on L521 (5 μg/ml). Clones were picked 8–14 days after transfection and expanded on L521‐coated 96‐well plates.
Amaxa human stem cell nucleofector kit 1
Manufactured by Lonza
Sourced in Germany
The Amaxa Human Stem Cell Nucleofector Kit 1 is a laboratory equipment product designed for the transfection of human stem cells. It facilitates the introduction of DNA, RNA, or other macromolecules into the cells using an electroporation-based technology.
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4 protocols using amaxa human stem cell nucleofector kit 1
1
Efficient Genome Editing of hESCs and iPSCs
2
Gene Editing of S2 FD PSCs Using IKBKAP Donor, gRNA, and Cas9
3
Efficient Genome Editing of Human iPSCs
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Human iPSCs were harvested using StemPro Accutase Cell Dissociation Reagent. Ten µg of a non-linearized donor vector, 5 µg of AAVS1 1L TALEN (Cat#. 35431, Addgene) and 5 µg of AAVS1 1R TALEN (Cat#. 35432, Addgene) were transduced with a Nucleofector II/Program B-016 (Cat#. AAD-1001N, Amaxa Biosystems, Nordrhein-Westfalen, Germany) and an Amaxa Human Stem Cell Nucleofector Kit 1 (Cat#. VAPH-5012, Lonza, Basel, Switzerland). The cells were treated with 50 µg/ml G-418 (Cat#. 4727878001, Roche Applied Science, Penzberg. Germany) for 10 days. EGFP-positive clones were picked up and expanded on iMatrix-511-coated plates in StemFit AK02N. The established clones were genotyped by PCR. PCR primers are shown in Table 1 .
4
Tracking Human iPSC-Derived Macrophage Localization
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To monitor the localization of human iPSC-derived macrophages, 3 x10 6 iPSC-M0 were labeled with PKH-26 red fluorescent dye (Sigma-Aldrich) and injected IP into eight-to ten-week-old uninjured Rag2 À/À γc À/À mice (51). Mice were harvested at various time points over the course of 16 days (3, 5, 7, 10, 12, 14, 16 days post macrophage injection). Harvested organs including liver, kidney, and spleen, and peritoneal lavage were examined under an Olympus UV microscope (Supplemental Figure 5A).
To monitor the homing of human iPSC-macrophages into the livers of CCl 4 -injured Rag2 À/À γc À/À mice, a piggyBac plasmid expressing tdTomato (Vector Builder) was utilized to transfect human iPSCs using Amaxa Human Stem Cell Nucleofector Kit 1 (Lonza) following manufacturer's protocols. Transfected cells were cultured in presence of Puromycin (0.5 μg/mL) for 9 days until all cells expressed tdTomato and then they used to generate human iPSC-M2 macrophages as described above. 20x10 6 human iPSC-M2 were injected IP during week 7 of the CCl 4 regimen into Rag2 À/À γc À/À mice. One week later, livers were harvested and evaluated for the presence of tdTomato-expressing human iPSC-M2 via histology.
To monitor the homing of human iPSC-macrophages into the livers of CCl 4 -injured Rag2 À/À γc À/À mice, a piggyBac plasmid expressing tdTomato (Vector Builder) was utilized to transfect human iPSCs using Amaxa Human Stem Cell Nucleofector Kit 1 (Lonza) following manufacturer's protocols. Transfected cells were cultured in presence of Puromycin (0.5 μg/mL) for 9 days until all cells expressed tdTomato and then they used to generate human iPSC-M2 macrophages as described above. 20x10 6 human iPSC-M2 were injected IP during week 7 of the CCl 4 regimen into Rag2 À/À γc À/À mice. One week later, livers were harvested and evaluated for the presence of tdTomato-expressing human iPSC-M2 via histology.
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