Generation of CCR5-ablated Human Induced Pluripotent Stem Cells as a Therapeutic Approach for Immune-mediated Diseases

Negin Hosseini Rouzbahani , Saeid Kaviani , Mohammad Vasei , Masoud Soleimani , Kayhan Azadmanesh , Mohammad Hossein Nicknam 
Source: Allergy Asthma Immunol Res.
Publication Date: (2019)
Issue: 18(3): 310-319
Research Area:
Stem Cells
Regenerative medicine
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Nucleofector® I/II/2b

The nucleofection procedure was performed based on the manufacturer’s instruction [Human Stem Cell Nucleofector Kit 1 (Lonza, cat. no. VPH-5012)]. According to the manufacturer manual: the efficiency of nucleofection is drastically influenced by the cell type, the cell source, the cell viability, confluence and number of cell passage. Thus, providing a unique optimized protocol with predefined conditions i.e. one program (electrical parameter) which is guaranteed to work with any hiPSCs clone is not possible. For our PMSC-hiPSCs clones, it is recommended to first determine the optimal program. In brief, on the day of nucleofection, PMSC-hiPSCs were pre-treated with 10 µmol/L Rho kinase inhibitor (Y-27632) (R&D Systems, EMD Millipore) for 2-3 hours before nucleofection. To prepare a single-cell suspension, the culture was treated with collagenase IV (Sigma). Detached PMSC-hiPSCs were collected and centrifuged at 100 x g for 3 min at RT. The hiPSCs pellet was suspended and dispensed (8 x 105 cells) into the seven separated tubes, and spin down in low-speed centrifugation. Then, ready-to-use nucleofection solutions and 7 µg of fluorescent protein-expressing plasmid DNA were added into the Nucleocuvette containing the cell suspension and mixed by tapping gently. Finally, to find the best nucleofection program,  six different programs of A-012, A-13, A-023, A-027, A-033, and B-016 were applied. Nucleofected PMSChiPSCs were recovered and transferred onto the feeder layers. On the following day, nucleofection efficiency was monitored using Flow cytometry analysis. Also, the expression of GFP was examined to find and mark the nucleofected PMSC-hiPSCs by fluorescence microscope (Figure 4). Then, PMSC-hiPSCs were cultured at 37°C until distinct colonies appear large enough for colony picking. About 70 clear single colonies were picked under sterile conditions. The picked up single colonies were cultured and propagated, separately.


C-C chemokine receptor type 5 (CCR5) is a receptor for some pro-inflammatory chemokines which plays important roles in immunological disorder and host responses to infectious agents. Additionally, the prognosis of some immune-mediated diseases in the people who are naturally carrying the CCR5 32bp deletions is optimistic. However, the clinical application of CCR5 32bp mutant cells is very limited due to the rare availability of donors who are homozygous for CCR5 D32. The transfection efficiency of nucleofected placental mesenchymal stem cells derived - human induced pluripotent stem cells (PMSC-hiPSCs) was examined through the evaluation of green fluorescent protein (GFP) expression using flow cytometry. The nucleofected clonal populations were selected using colony picking. The CCR5 gene disrupted clonal populations were evaluated and confirmed by PCR and Sanger sequencing methods. Also, off-target sites were evaluated by the "Loss of a primer binding site" technique. The results of the flow cytometry revealed that among the six applied nucleofection programs for PMSC-iPSCs, the program of A-033 has achieved the best transfection efficiency (27.7%). PCR and then sequencing results confirmed the CCR5 gene was disrupted in two clonal populations of 16 (D6) and 62 (D20) by the Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR/Cas9) system. The "Loss of a primer binding site" technique showed that no exonic off-target mutations were induced in both CCR5 gene disrupted clonal populations. We establish a CRISPR/Cas9 mediated CCR5 ablated PMSC-hiPSCs without detectable off-target damage. This approach can provide a stable supply of autologous/allogeneic CCR5-disrupted PMSC-hiPSCs that might be a feasible approach for the treatment of immune-mediated diseases.