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