Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors

Wen, Wei; Zhang, Jianping; Xu, Jing; Su, Ruijun; Neises, Amanda; Ji, Guangzhen; Yuan, Weiping; Cheng, Tao; Zhang, Xiaobing

doi:10.1016/j.stemcr.2016.04.005

Cited by 53 publications

(53 citation statements)

References 66 publications

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“…These iPSCs were generated by lentiviral transduction of Yamanaka reprogramming factors together with BCL-XL ( 60 , 61 ). This result is in striking contrast to what we observed with integration-free iPSCs generated with episomal vectors ( 38 ), strongly suggesting that BCL-XL improves iPSC survival after nucleofection. To verify this assumption, we transduced three different integration-free iPSC lines with a lentiviral vector that expressed both BCL-XL and puromycin resistance gene (MOI = 0.1).…”

Section: Resultscontrasting

confidence: 99%

“…In addition, inhibiting caspase or TP53 with small molecules showed no obvious effects on the improvement of cell survival and editing efficiency. The superior effects of BCL-XL over BCL2 and MCL1 are reminiscent of our previous finding that BCL-XL is more potent than BCL2 or MCL1 in enhancing reprogramming of iPSCs from adult human peripheral blood cells ( 38 , 60 ).…”

Section: Discussionmentioning

confidence: 57%

“…The iPSC lines were generated from different anonymous adult donors by peripheral blood (PB) reprogramming using episomal vectors that express OCT4, SOX2, MYC, KLF4, and BCL-XL ( 37 , 38 , 45 ). Blood samples were obtained from the Tianjin Blood Center with the approval of the local research ethics committee.…”

Section: Methodsmentioning

confidence: 99%

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Highly efficient genome editing via CRISPR–Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression

et al. 2018

Nucleic Acids Research

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102

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Genome editing of human induced pluripotent stem cells (iPSCs) is instrumental for functional genomics, disease modeling, and regenerative medicine. However, low editing efficiency has hampered the applications of CRISPR–Cas9 technology in creating knockin (KI) or knockout (KO) iPSC lines, which is largely due to massive cell death after electroporation with editing plasmids. Here, we report that the transient delivery of BCL-XL increases iPSC survival by ∼10-fold after plasmid transfection, leading to a 20- to 100-fold increase in homology-directed repair (HDR) KI efficiency and a 5-fold increase in non-homologous end joining (NHEJ) KO efficiency. Treatment with a BCL inhibitor ABT-263 further improves HDR efficiency by 70% and KO efficiency by 40%. The increased genome editing efficiency is attributed to higher expressions of Cas9 and sgRNA in surviving cells after electroporation. HDR or NHEJ efficiency reaches 95% with dual editing followed by selection of cells with HDR insertion of a selective gene. Moreover, KO efficiency of 100% can be achieved in a bulk population of cells with biallelic HDR KO followed by double selection, abrogating the necessity for single cell cloning. Taken together, these simple yet highly efficient editing strategies provide useful tools for applications ranging from manipulating human iPSC genomes to creating gene-modified animal models.

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Section: Resultscontrasting

confidence: 99%

Section: Discussionmentioning

confidence: 57%

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Highly efficient genome editing via CRISPR–Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression

et al. 2018

Nucleic Acids Research

Self Cite

102

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“…To further evaluate whether our protocol can be used to model pathological germ cell development in vitro , we obtained six iPSC lines from four NOA patients and two male controls. Of these, five lines were derived from skin biopsy samples with classical retroviral infection, and one (i.e., P24) from peripheral blood cells using electroporation with non-integrated episomal plasmids to introduce core pluripotency factors ( Wen et al., 2016 ). Among four patients, two (P2369 and P2656) were diagnosed as NOA with Sertoli cell-only syndrome, whereas two (P2514 and P2618) had microdeletions at the AZFc locus in both parental fibroblasts and established iPSC lines, as detected by PCR-based analyses of Yq genomic markers (Yq sequence-tagged sites) ( Figures 5 A and S5 A).…”

Section: Resultsmentioning

confidence: 99%

In Vitro Modeling of Human Germ Cell Development Using Pluripotent Stem Cells

Zhao

Liang

et al. 2018

Stem Cell Reports

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SummaryDue to differences across species, the mechanisms of cell fate decisions determined in mice cannot be readily extrapolated to humans. In this study, we developed a feeder- and xeno-free culture protocol that efficiently induced human pluripotent stem cells (iPSCs) into PLZF+/GPR125+/CD90+ spermatogonium-like cells (SLCs). These SLCs were enriched with key genes in germ cell development such as MVH, DAZL, GFRα1, NANOS3, and DMRT1. In addition, a small fraction of SLCs went through meiosis in vitro to develop into haploid cells. We further demonstrated that this chemically defined induction protocol faithfully recapitulated the features of compromised germ cell development of PSCs with NANOS3 deficiency or iPSC lines established from patients with non-obstructive azoospermia. Taken together, we established a powerful experimental platform to investigate human germ cell development and pathology related to male infertility.

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“…Generation of iPSCs through an integration-free approach is still less efficient than another viral-based reprogramming system (Drozd et al 2015 ). However, integration-free iPSCs hold great potential for cell therapy and regenerative medicine (Wen et al 2016 ). In this study, we generated functional and expandable Epi-iPSCs using a non-viral integration-free system.…”

Section: Discussionmentioning

confidence: 99%

Optimization of episomal reprogramming for generation of human induced pluripotent stem cells from fibroblasts

Bang

Choi

Lee

et al. 2018

Animal Cells and Systems

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Generation of induced pluripotent stem cells (iPSCs) by defined factors (OCT4, SOX2, C-MYC, and KLF4) from various human primary cells has been reported. Human fibroblasts have been widely used as a cellular source in reprogramming studies over recent decades. The original method of iPSC generation uses retro- or lentivirus vectors that require integration of viral DNA into the target cells. The integration of exogenous genes encoding transcription factors (OCT4, SOX2, C-MYC, and KLF4) can be detected in iPSCs, raising concern about the risk of mutagenesis and tumor formation. Therefore, stem cell therapy would ideally require generation of integration-free iPSCs using non-integration gene delivery system such as Sendai virus, recombinant proteins, synthetic mRNA, and episomal vectors. Several groups have reported that episomal vectors are capable of reprogramming human fibroblasts into iPSCs. Although vector concentration and cell density are important in the episomal vector reprogramming method, optimization of this method for human fibroblasts has not been reported. In this study, we determined optimal conditions for generating integration-free iPSCs from human fibroblasts through the use of different concentrations of episomal vectors (OCT4/p53, SOX2/KLF4, L-MYC/LIN28A) and different plating cell density. We found that optimized vector concentration and cell density accelerate reprogramming and improve iPSC generation. Our study provides a detailed stepwise protocol for improved generation of integration-free iPSCs from human fibroblasts by transfection with episomal vectors.

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Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors

Cited by 53 publications

References 66 publications

Highly efficient genome editing via CRISPR–Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression

Highly efficient genome editing via CRISPR–Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression

In Vitro Modeling of Human Germ Cell Development Using Pluripotent Stem Cells

Optimization of episomal reprogramming for generation of human induced pluripotent stem cells from fibroblasts

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