Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Summary

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Abstract

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies are: 1) the use of a 2A "self-cleavage" peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

Introduction

After forced expression of several transcription factors (i.e. OCT4, SOX2, MYC and KLF4), somatic cells can be reprogrammed to induced Pluripotent Stem Cells (iPSCs), which hold great promise for applications in regenerative medicine and cell replacement therapy^1-3. To date, diverse methods have been developed to increase the success rate of reprogramming^4-7. Viral vectors-induced reprogramming is widely used for efficient generation of iPSCs, because viral integration leads to a high-level, stable expression of the reprogramming factors. However, permanent integration of the vector DNA into the cell genome may induce insertional mutagenesis⁵. In addition, insufficient inactivation of reprogramming factors may disturb iPSCs differentiation⁸. As such, the use of iPSCs without integration of reprogramming factors is imperative, especially for use in cell therapy applications.

Episomal Vectors (EVs) are widely used in the generation of integration-free iPSCs. The most commonly used EV is a plasmid containing two elements, origin of viral replication (oriP) and EB Nuclear Antigen 1 (EBNA1), from the Epstein-Barr (EB) virus⁹. The oriP element promotes plasmid replication in mammalian cells, while the EBNA1 element tethers the oriP-containing plasmid DNA to the chromosomal DNA that allows for the partitioning of the episome during division of the host cell. In comparison to other integration-free approaches, including Sendai Virus (SV) and RNA transfection, EVs possess multiple advantages^5,6,10. As plasmid DNA, EVs can be readily produced and modified in house, making them extremely affordable. In addition, reprogramming with EV is a less labor-intensive process since a single transfection with EVs is sufficient for iPSC generation, whereas several RNA transfections are necessary for successful reprogramming.

Dermal fibroblasts have been used in many reprogramming studies. However, skin biopsy is not only an invasive and painful process, but also time-consuming for expanding cells to sufficient quantities for reprogramming. Of greater concern, skin cells of adult donors have often been exposed to long-term UV light radiation, which may lead to mutations associated with tumors, thus limiting the applications for iPSCs derived from skin fibroblasts^11,12. Recently, it has been reported that normal human skin cells accumulate somatic mutations and multiple cancer genes, including most of the key drivers of cutaneous squamous cell carcinomas, are under strong positive selection¹³.

In contrast to skin fibroblasts, peripheral blood (PB) cells are a preferable source of cells for reprogramming because 1) blood cells can be easily obtained through a minimally invasive process, 2) peripheral blood cells are the progeny of hematopoietic stem cells residing in bone marrow, thus protected from harmful radiation. Peripheral blood mononuclear cells (PB MNCs) can be collected in an hour from the buffy coat layer following a simple gradient centrifugation using Ficoll-Hypaque (1.077 g/mL). The obtained PB MNCs are composed of lymphocytes, monocytes and a few Hematopoietic Progenitor Cells (HPCs) ¹⁴. Although human T lymphocytes are one of the major cell types in PB, mature T cells contain rearrangements of the T cell receptor (TCR) genes and lack an intact genome thus limiting their potential for applications^15,16. However, rejuvenation of T cells via iPSC generation may have potential applications in Chimeric Antigen Receptor (CAR) T-cell therapy ^17-19. In comparison, HPCs have an intact genome and are readily reprogrammable. Although only 0.01 – 0.1% cells in peripheral circulation are HPCs, these cells can be expanded ex vivo in erythroid medium that favors proliferation of erythroid progenitor cells¹⁴.

In our previous study, we used the factor BCL-XL in addition to the Yamanaka factors (OCT4, SOX2, MYC and KLF4), which resulted in a 10x increase in PB reprogramming efficency²⁰. BCL-XL, also known as BCL2L1, is a potent inhibitor of cell death, by inhibiting activation of caspases^21,22. But, BCL-XL may also play an important role in maintaining pluripotency^21,22. Recently, we have further optimized our EV reprogramming system by separately expressing MYC and KLF4 with two vectors, which leads to an approximately 100x increase in reprogramming efficiency²³. Using this method, the reprogramming efficiency, defined by colony number divided by starting cell number at transfection, is 0.2 - 0.5% from healthy donors. As follows, we describe the detailed experimental procedure for generating integration-free iPSCs from PB.

Protocol

All of the human PB samples were obtained from anonymous adult donors with no identification information available from Tianjin Blood Center with approval of the local research ethics committee. 1. Endo-free Plasmid Preparation Use a commercial Plasmid Purification Maxi Kit to extract episomal vectors from E. coli according to manufacturer's protocol. For the final step, substitute TE buffer with endotoxin-free sterile water to dissolve the DNA pellet. Measur…

Representative Results

Using this protocol, we can obtain hundreds of colonies from 1 x 105 nucleofected PB MNCs (Figures 1A and 1B). The reprogramming efficiency is approximately 0.2 – 0.5% and the colonies express pluripotency markers (Figures 1C and 1D). iPSCs generated using the described protocol are integration-free and have the ability to form teratoma composing the 3 germ layers (Figures 1E and 1F</st…

Discussion

Acquiring blood samples from healthy donors or patients is convenient and noninvasive, making it an attractive cell source for basic research and clinical cell therapy. Here we have described a protocol for highly efficient generation of integration-free iPSCs from peripheral blood samples. This reproducible and affordable approach should benefit the iPSC field.

We have reported that there are two critical factors responsible for the highly efficient PB reprogramming³⁰. One is equim…

Materials

Hematopoietic Stem Cell Expansion Medium	Sigma	S0192	Store at 4 °C
Human stem cell factor (SCF)	Peprotech	300-07	Store at -20 or -80°C
Interleukin-3 (IL-3)	Peprotech	AF-200-03	Store at -20 or -80°C
Eryrthropoietin (EPO)	Peprotech	100-64	Store at -20 or -80°C
Insulin growth factor-1 (IGF-1)	Peprotech	100-11	Store at -20 or -80°C
Dexamethasone	Sigma	D4902	Store at -20 or -80°C
1-thioglycerol (MTG)	Sigma	M6145	Store at -20 or -80°C
DMEM/F12 medium	Gibco	112660-012	Store at 4 °C
L-glutamine (100x)	Gibco	25030-081	Store at -20 °C
Penicillin/Streptomycin (100x)	Gibco	15140-122	Store at -20 °C
Non-essential amino acids solution (100x)	Gibco	11140-050	Store at 4 °C
Fibroblast growth factor 2 (FGF2)	Peprotech	100-18B	Store at -20 or -80°C
ITS (100x)	Gibco	41400-045	Store at 4 °C
Ascorbic acid	Sigma	49752	Store at -20 °C
DMEM (high glucose) medium	Thermo	SH30243.01B	Store at 4 °C
FBS	Hyclone	SV30087.01	Store at -40 °C
Ficoll	GE Healthcare, SIGMA	17-5442-02	Store at RT
Trehalose	Sigma	T9531	Store at 4 °C
DMSO	Sigma	D2650	Store at RT, protect from light
Endofree Plasmid Maxi Kit(10)	Qiagen	12362	Store at RT
IMDM	Gibco	21056-023	Store at 4 °C
Human CD34+ Cell Nucleofection Kit	Lonza	VPA-1003	Store at RT, nucleofection buffer and supplement buffer should be stored at 4 °C
Sodium butyrate	Sigma	B5887	Store at -20 or -80°C
ROCK inhibitor Y27632	STEMGENT	04-0012-10	Store at -20 °C
Essential 8 basal medium (E8)	Gibco	A15169-01	Store at 4 °C, the supplement should be stored at -20 or -80°C
Matrigel	BD	354277	Store at -20 or -80°C
2x EasyTaq PCR SuperMix(+dye)	TransGen Biotech	AS111	Store at -20 °C
Cell detachment solution	STEMGENT	01-0006	Store at -20 °C, Accutase as a cell detachment solution to obtain a single cell suspension
DAPI	Sigma	D9542-1MG	Store at 4 °C or -20 °C
Anti-nanog-AF488	BD	560791	Store at 4 °C, primary antibody used for Immunofluorescence, dilute 1/100 when use
Anti-OCT4	abcam	ab19857	Store at 4 °C, primary antibody used for Immunofluorescence, dilute 1/100 when use
AF488 donkey anti-mouse IgG	Invitrogen	A21202	Store at 4 °C, secondary antibody used for Immunofluorescence,  dilute 1/500 when use
PE anti-human TRA-1-60-R Antibody	Biolegend	330610	Store at 4 °C, antibody used for flow cytometry
eFluor 570-conjugated anti-SSEA4	eBioscience	41-8843	Store at 4 °C, antibody used for flow cytometry
Isotype antibody	eBioscience	11-4011	Store at 4 °C, antibody used for flow cytometry
Alkaline Phosphatase Detection Kit	SiDanSai	1102-100	Store at 4 °C
Genomic DNA Extraction Kit	TIANGEN	DP304-02	Store at RT
Trypan Blue solution	Sigma	T8154	Store at RT
Flow cytometry cell analyzer	BD	LSRII	for flow cytometry analysis
Spinning disk confocal microscope (SDC)	PerkinElmer	UltraVIEW VOX	for confocal imaging
Nucleofection device	Lonza	Nucleofector 2b	for the nucleofection of PB MNC
ImageQuant LAS-4010	GE		take photo of AP staining in bulk