Episomal Reprogramming of Human Peripheral Blood Mononuclear Cells into Pluripotency
Peripheral blood is an easily accessible cell resource for reprogramming into pluripotency by episomal vectors. Here, we describe an approach for efficient generation of integration-free induced pluripotent stem cells (iPSCs) under feeder or feeder-free c
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Introduction By forced expression of Yamanaka factors (OCT4, SOX2, MYC, and KLF4), one can reprogram somatic cells into induced pluripotent stem cells (iPSCs) [1–5]. This advance has opened opportunities for personalized cell therapy, disease modeling, and drug screening [6–8]. Peripheral blood (PB) is widely used in medical diagnostics and is easily obtained. By gradient centrifugation, one can obtain peripheral blood mononuclear cells (PBMNCs) from fresh PB samples or existing stocks. For the derivation of clinicalgrade iPSCs, reprogramming methods that minimize the chances of insertional mutagenesis are preferred. The Sendai virus vectors reprogram PBMNCs at high efficiency [9]. However, the Sendai virus method is less affordable. In contrast, episomal reprogramming is an ideal and cost-effective method for creating clinical-grade and integration-free iPSCs. Transfecting EBNA1/ oriP-based episomal vectors expressing Yamanaka factors in PBMNCs by nucleofection can generate footprint-free iPSCs [10, 11]. We have optimized the episomal reprogramming
Kejin Hu (ed.), Nuclear Reprogramming: Methods and Protocols, Methods in Molecular Biology, vol. 2239, https://doi.org/10.1007/978-1-0716-1084-8_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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[12–14]. One can use our optimized protocol to obtain thousands of colonies from 1 mL of PB. CRISPR-Cas9 is a powerful genome-editing technology [15–17] and has been widely used for editing iPSCs [18, 19]. Conventionally, iPSCs are generated from somatic cells first, and then the iPSC genome is edited [20–23]. To facilitate and simplify the reprogramming and editing processes, we have established simultaneous reprogramming and genome editing of PB by one nucleofection of episomal vectors for both reprogramming and CRISPR-Cas9 editing [24]. In this chapter, we describe a protocol for the highly efficient episomal reprogramming of PB under feeder-dependent or feederfree conditions. In combination with the CRISPR-Cas9 system, we provide procedures for simultaneous reprogramming and geneediting of PB to generate genetically edited iPSCs by a single nucleofection.
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Materials Cells
1. Human peripheral blood is obtained from anonymous adult donors with no patient identification from the local blood center. 2. Mouse embryonic fibroblasts (MEFs) are prepared inhouse from E13.5 mouse embryos. 3. Feeder cells are derived by culturing MEFs to passage 4 or 5. MEFs are inactivated by treating with 10 μg/mL Mitomycin C for 3 h.
2.2
MNC Isolation
1. Ficoll-Paque Premium (1.077 g/mL). 2. DPBS, no calcium, no magnesium. 3. (Optional) Red Blood Cell Lysis Buffer (RBC lysis buffer): Dissolve 8.3 g NH4Cl and 1.0 g KHCO3 in 900 mL of water, and add 1.8 mL of 5% EDTA. Adjust the pH to 7.2–7.4. Bring the volume to 1.0 L with water. Filter-sterilize through a 0.2 μm filter. Store at 4 C, but warm up to room temperature before use.
2.3
Cell Culture
1. Erythroid culture medium: Stemline® II Hematopoietic Stem Cell Expansion Medium suppl
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