A Protocol To Generate Integration-Free Induced Pluripotent Stem Cells From Peripheral Blood

Here we present our work of establishing induced pluripotent stem cells (iPSCs) from peripheral blood cells via an integration-free protocol.Peripheral blood is the most convenient tissue obtained from patients, and the hematopoietic stem cells and progenitor cells are relatively prone to iPSCs induction. Although appeared as a rare population in blood, the number of blood progenitors effectively increased after one-week enrichment culture. We first isolated peripheral blood mono-nuclear cells (PBMNC) from whole blood by density gradient centrifugation, then we introduced exogenous transgenes with non-integratable episomal vectors into the PBMNC after a 6 to 7 days’ enrichment culture. Colonies of transgene-free iPSCs emerged after 4 weeks post’ induction. Individual colonies were picked, expanded, subjected to further analyses for pluripotency. All iPSC lines we derived from patients behaved similarly to human embryonic stem cells, with normal karyotype and no transgene integration, and most importantly, they maintained original genetic defects from their parental patient samples, In summary, using this protocol, we successfully established 64 transgene-free iPSCs cell lines from five male patients with infertility. Non-obstructive azoospermia (NOA) is one of the most serious infertile diseases, and can be caused by both environmental factors and genetic defects. Y chromosome region called microdeletion at the azoospermia factor (AZF) is the most common genetic defect that highly correlates with incidence of NOA. So far no effective therapeutics has been developed for this disease. Since no murine homologues of genes that contribute to NOA in the AZF region were found, disease models in murine system have not been established. Instead, in vitro differentiation of pluripotent stem cells provides an alternative model to investigate the developmental processes in human. Therefore, the iPSCs we obtained here could be used to dissect the underlying pathology of NOA and human spermatogenesis.