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

  • Negin Hosseini Rouzbahani Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  • Saeid Kaviani Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
  • Mohammad Vasei Cell Based Therapies Research Center, Digestive Diseases Research Institute, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
  • Masoud Soleimani Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
  • kayhan Azadmanesh Department of Virology, Pasteur Institute of Iran, Tehran, Iran
  • Mohammad Hossein Nicknam Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran AND Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran
Keywords: Chemokines, CRISPR-cas systems, Flow cytometry, Gene editing, Transfection


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.



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How to Cite
Hosseini Rouzbahani N, Kaviani S, Vasei M, Soleimani M, Azadmanesh kayhan, Nicknam MH. Generation of CCR5-ablated Human Induced Pluripotent Stem Cells as a Therapeutic Approach for Immune-mediated Diseases. Iran J Allergy Asthma Immunol. 18(3):310-319.
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