Production of iPSCs from a small volume of cryopreserved human umbilical cord blood buffy coat under “gmp-compliant” conditions

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Background & Aim An iPSC bank derived from cord blood selected on the basis of human leukocyte antigens (HLA) requires the development and validation of protocols to generate iPSC lines under good manufacturing practice (GMP) conditions. Working towards this goal, we aim to create clinically compliant GMP grade iPSCs from small volumes of banked cord blood (CB) buffy coat samples for potential clinical use. Methods, Results & Conclusion We have established a protocol to generate iPSC lines from a minimum of 50 µl of cryopreserved CB buffy coat using defined medium and reagents manufactured under GMP. This allows us to use only segments or pilot tube of the stored CB unit without compromising the whole CB unit. Thawed CB cells were washed and an erythroblast cell population expanded in Animal Component Free (ACF) erythroblast growth medium prior to reprograming. Up to 0.11% reprogramming efficiency was obtained. iPSC colonies formed on Vitronectin coated plates were manually picked and passaged on the same matrix in defined Xeno-free E8 Flex medium. Karyotype integrity of the generated iPSC lines was confirmed with SNP Illumina Infinium CoreExome-24 v1.1 array at 0.50Mb resolution. Flow cytometry was used to show that iPSCs expressed the known stemness markers SSEA-3, SSEA-4, TRA-1-81 and TRA-1-60R but lacked expression of SSEA-1. In vitro monolayer differentiation experiments indicated that CB derived iPSCs could differentiate into cells representing the 3 germline lineages: beating cardiac cells (mesoderm), neuronal cells (ectoderm) and endodermal precursor cells. All derived cell types demonstrated the appropriate lineage markers by FACS or immunochemistry. The established iPSC line was passaged for >45 times under current culture conditions to demonstrate the stability of karyotype integrity and pluripotency potential of the cells, consistent with the earlier passages. Differentiation into cardiomyocytes indicates that differentiation capacity is similar to the earlier passages. We have demonstrated that GMP-compliant iPSC lines can be reproducibly and stability generated from small volumes of cryopreserved umbilical cord blood. By using the protocols established through this work we will create clinically compliant cell lines that will facilitate clinical research and the creation of an Australian CB-derived iPSC “haplobank” for cellular therapies.