Bone marrow or stem cell transplantation
Centre de Recherche en Transplantation et Immunologie UMR 1064, Inserm, Nantes, France; Institut de Transplantation Urologie Néphrologie, Nantes, France, iPSC corefacility, University of Nantes; INSERM UMS 016; CNRS 3556; CHU de Nantes, France
Kidney transplanted patients with long term graft survival harbor a higher frequency of B lymphocytes with regulatory properties. These “regulatory” B lymphocytes prevent effector T cell proliferation through the Granzyme B (GzmB) molecule (Chesneau et al., JASN 2015). Our aim was to generate regulatory B lymphocytes from human Induced Pluripotent Stem (hIPS) cells with a repressible GzmB expression as a tool to better understand their function and ultimately to translate them toward clinical use. Starting with Embryonic Stem cells (hES) as a proof of concept, and following a well-established protocol (French et al., Stem Cells Dev. 2015), we succeed in generating a small proportion (1,5%) of B cells characterized as pre-B cells. To optimize this protocol we find that both increasing the number of CD34+ cells and adding cytokines for the 42 days of B cell differentiation, increase the amount of differentiated B cells by 2 fold. In order to perform a Knock Out (KO) of the endogenous GzmB expression, we validate our plasmid Cas9/sgRNA targeting GzmB on 293T cells with high cut efficiency (33%) and ready to use on hES cells. An expression cassette composed of the GzmB gene under control of a Tet-Off system is inserted in the AAVS1 locus of hES GzmB KO. In this system, we expect a constitutive expression of GzmB by B cells differentiated from those hES cells, while addition of doxycycline will abrogate its expression. This tool will allow deeply studying GzmB B lymphocytes function and future translation toward clinical uses.