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.

Keywords: Sendai viral reprogramming; episomal reprogramming; human induced pluripotent stem cells (hiPSCs); reprogramming.

FIGURE 1. PBNC isolation, expansion, reprogramming, and hiPSC colony picking.

A) CPT tube after centrifugation. B) Appearance of PBMCs at various days during expansion and after Sendai viral transduction. C) Emerging cell clusters and colonies, ranging from mostly non-hiPSC like (left) to mostly hiPSC-like (right). D) Preparation and use of the colony cutting tool E) Reprogramming well with multiple colonies that are promising but not yet large enough to pick (blue circle), ready to be picked now (green circles), or larger than ideal for picking (red circles). Undifferentiated areas are identifiable by the characteristic small size and nuclear morphology with multiple prominent nucleoli under 10 or 20x Phase contrast microscopy (insert=20x). An area with gross differentiation is marked by a chevron. Colonies are cut as shown by the dotted lines using a sharp tool (D). F) During expansion, grossly differentiated areas (chevrons) must be removed before the cells are passaged.

FIGURE 2. hiPSC clone expansion.

A) Typical appearance of hiPSCs one day after plating on LN-521 (4x; insert=20x). B) Typical appearance of hiPSCs four days after plating on LN-521 (4x). C) Typical appearance of hiPSCs four days after plating on Matrigel™ (4x). D) Typical appearance of hiPSCs five days after plating on LN-511-E8. This culture is ready to be split now (4x). E) Appearance of hiPSC colonies during dissociation and detachment induced by the Versene+TrypLE-Select passaging method. The dissociation agent should be removed now to avoid excessive dissociation and to limit the production of completely dissociated single cells (left=4x, right=10x).

FIGURE 3. hiPSC clone expansion on different medium-matrix-combinations.

hiPSC can be expanded efficiently using various medium-matrix combinations. Some lines perform better in certain medium-matrix combinations. The colonies in this figure were stained using the chromogenic alkaline phosphatase staining protocol described in UNIT 1C.12 (Manos, Ratanasirintrawoot, Loewer, Daley, & Schlaeger, 2011).

FIGURE 4. Confocal immunofluorescence analysis of hiPSC marker expression.

A) hiPSCs stained with the indicated immunofluorescence FMO (fluorescence-minus-one) staining cocktails. B) hiPSCs stained with the complete immunofluorescence staining cocktail. Inserts show zoomed-in areas to better show the expected subcellular localization of the various stains.

FIGURE 5. Quantification of residual Sendai viral RNA by Q-RT-PCR-analysis

SeV-FAM (left) and ACTB-VIC (right) TaqMan RT-qPCR data plots. (1) positive sample (assay run in duplicate). (2) example of a negative sample assay well with unspecific background signal (note the lack of a linear component). Higher background signals are sometimes seen with water-control DNA-negative samples or with PCR assay wells that were incompletely sealed. The slope of the increase of the signal that is expected for a perfect PCR reaction (2-fold increase per cycle) is shown for the SeV-FAM (3) and ACTB (4) plots as dotted lines. The Cq thresholds (thick horizontal lines) should be set close to the mid-range of the linear component of the curves of positive samples and above the background signal (2), usually to around 1,000–3,000 relative light units.