Xeno-Free Reprogramming of Peripheral Blood Mononuclear Erythroblasts on Laminin-521.

ABSTRACT

Translating human induced pluripotent stem cell (hiPSC)-derived cells and tissues into the clinic requires streamlined and reliable production of clinical-grade hiPSCs. This article describes an entirely animal component-free procedure for the reliable derivation of stable hiPSC lines from donor peripheral blood mononuclear cells (PBMCs) using only autologous patient materials and xeno-free reagents. PBMCs are isolated from a whole blood donation, from which a small amount of patient serum is also generated. The PBMCs are then expanded prior to reprogramming in an animal component-free erythroblast growth medium supplemented with autologous patient serum, thereby eliminating the need for animal serum. After expansion, the erythroblasts are reprogrammed using either cGMP-grade Sendai viral particles (CytoTune™ 2.1 kit) or episomally replicating reprogramming plasmids (Epi5™ kit), both commercially available. Expansion of emerging hiPSCs on a recombinant cGMP-grade human laminin substrate is compatible with a number of xeno-free or chemically defined media (some available as cGMP-grade reagents), such as E8, Nutristem, Stemfit, or mTeSR Plus. hiPSC lines derived using this method display expression of expected surface markers and transcription factors, loss of the reprogramming agent-derived nucleic acids, genetic stability, and the ability to robustly differentiate in vitro to multiple lineages. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1 Isolating peripheral blood mononuclear cells using CPT tubes Support Protocol 1 Removal of clotting factors to produce serum from autologous plasma collected in Basic Protocol 1 Basic Protocol 2 PBMC expansion in an animal-free erythroblast expansion medium containing autologous serum Basic Protocol 3 Reprogramming of expanded PBMCs with Sendai viral reprogramming particles Alternate Protocol Reprogramming of expanded PBMCs with episomal plasmids Basic Protocol 4 Picking, expanding, and cryopreserving hiPSC clones Support Protocol 2 Testing Sendai virus kit-reprogrammed hiPSC for absence of Sendai viral RNA Support Protocol 3 Testing Epi5 kit-reprogrammed hiPSC for absence of episomal plasmid DNA Support Protocol 4 Assessing the undifferentiated state of human pluripotent stem cell cultures by multi-color immunofluorescent staining and confocal imaging Support Protocol 5 Coating plates with extracellular matrices to support hiPSC attachment and expansion.