Evaluation of the Spanish population coverage of a prospective HLA haplobank of induced pluripotent stem cells.

BACKGROUND: iPSC (induced pluripotent stem cells) banks of iPSC lines with homozygous HLA (human leukocyte antigen) haplotypes (haplobanks) are proposed as an affordable and off-the-shelf approach to allogeneic transplantation of iPSC derived cell therapies. Cord blood banks offer an extensive source of HLA-typed cells suitable for reprogramming to iPSC. Several initiatives worldwide have been undertaken to create national and international iPSC haplobanks that match a significant part of a population. METHODS: To create an iPSC haplobank that serves the Spanish population (IPS-PANIA), we have searched the Spanish Bone Marrow Donor Registry (REDMO) to identify the most frequently estimated haplotypes. From the top ten donors identified, we estimated the population coverage using the criteria of zero mismatches in HLA-A, HLA-B, and HLA-DRB1 with different stringencies: high resolution, low resolution, and beneficial mismatch. RESULTS: We have calculated that ten cord blood units from homozygous donors stored at the Spanish cord blood banks can provide HLA-A, HLA-B, and HLA-DRB1 matching for 28.23% of the population. CONCLUSION: We confirm the feasibility of using banked cord blood units to create an iPSC haplobank that will cover a significant percentage of the Spanish and international population for future advanced therapy replacement strategies.

Construction and characterization of a minimized version of the HIV-1 pNL4-3 plasmid and its application for pseudotyping HIV-1 vectors.

The pUC-based pNL4-3 plasmid is the most widely used vector for in vitro manipulations of the HIV-1 proviral sequences. We have developed a minimal plasmid (pCHUS) based on pNL4-3, which may be useful to facilitate the design of HIV-based constructions. The strategy that has allowed us to construct pCHUS includes the following steps: (1) pNL4-3 digestion by using restriction sites contained within the long terminal repeats (LTRs), (2) recircularization of the fragment containing the pUC18 sequence, (3) amplification of the LTR region restored in the previous step, (4) double digestion of the products obtained in steps 2 and 3, (5) ligation of the fragment containing ColE1+Amp(R) with the LTR fragment, (6) linearization of the intermediate plasmid obtained, and (7) insertion of the fragment containing the proviral genome into the linearized vector. The pCHUS plasmid includes essential information for its replication and antibiotic selection in bacteria, but it lacks all the unnecessary sequences. Our results suggest that pCHUS may be more advantageous than pNL4-3 for in vitro manipulation of the HIV-1 proviral genome. In addition, we describe a potential application of this new vector for pseudotyping HIV-1 particles, using a single plasmid transfection, as a more helpful alternative to the traditionally used cotransfection method.