Reprogramming Human Fibroblasts to Induced Pluripotent Stem Cells Using the GFP-Marked Lentiviral Vectors in the Chemically Defined Medium

Shao, Zhicheng; Cevallos, Ricardo R.; Hu, Kejin

doi:10.1007/978-1-0716-1084-8_7

Cited by 5 publications

(7 citation statements)

References 13 publications

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“…Two major reasons account for this discrepancy: (1) the profiling method here is the highly sensitive, unbiased, and comprehensive genome-wide RNA-seq, and Mah et al used microarray [15], which is limited by probe sets and low sensitivity; (2) they used gamma retroviruses to transduce human fibroblasts, while the current study used lentiviral vectors to deliver the OSKM transgenes. Retroviral transduction efficiency of human cells is very low [4], and in fact, the efficiency of their retroviral transduction was only 27.6% while we have routinely reached >90% of transduction of human fibroblasts with our lentiviral vectors [6,7,14,16].…”

Section: Genes In Nucleic Acidmentioning

confidence: 83%

“…The lentiviral reprogramming vectors were packaged using calcium/phosphate precipitation method, and the resulting virus particles were concentrated with PEI precipitation and subsequent centrifugation. The titers of the concentrated viruses were determined using flow cytometry taking advantage of the GFP co-expressed with each reprogramming factor as described before [6,7,14,16].…”

Section: Viral Transduction Of Human Fibroblastsmentioning

confidence: 99%

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A PIANO (Proper, Insufficient, Aberrant, and NO Reprogramming) Response to the Yamanaka Factors in the Initial Stages of Human iPSC Reprogramming

2020

IJMS

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Yamanaka reprogramming is revolutionary but inefficient, slow, and stochastic. The underlying molecular events for these mixed outcomes of induction of pluripotent stem cells (iPSC) reprogramming is still unclear. Previous studies about transcriptional responses to reprogramming overlooked human reprogramming and are compromised by the fact that only a rare population proceeds towards pluripotency, and a significant amount of the collected transcriptional data may not represent the positive reprogramming. We recently developed a concept of reprogramome, which allows one to study the early transcriptional responses to the Yamanaka factors in the perspective of reprogramming legitimacy of a gene response to reprogramming. Using RNA-seq, this study scored 579 genes successfully reprogrammed within 48 h, indicating the potency of the reprogramming factors. This report also tallied 438 genes reprogrammed significantly but insufficiently up to 72 h, indicating a positive drive with some inadequacy of the Yamanaka factors. In addition, 953 member genes within the reprogramome were transcriptionally irresponsive to reprogramming, showing the inability of the reprogramming factors to directly act on these genes. Furthermore, there were 305 genes undergoing six types of aberrant reprogramming: over, wrong, and unwanted upreprogramming or downreprogramming, revealing significant negative impacts of the Yamanaka factors. The mixed findings about the initial transcriptional responses to the reprogramming factors shed new insights into the robustness as well as limitations of the Yamanaka factors.

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Section: Genes In Nucleic Acidmentioning

confidence: 83%

Section: Viral Transduction Of Human Fibroblastsmentioning

confidence: 99%

A PIANO (Proper, Insufficient, Aberrant, and NO Reprogramming) Response to the Yamanaka Factors in the Initial Stages of Human iPSC Reprogramming

2020

IJMS

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“…All of our reprogramming factors are constructed to co-express GFP. 19 , 33 The established iPSCs have silenced all the multiple copies of the co-expressed GFP ( Figure S4 D).…”

Section: Resultsmentioning

confidence: 99%

“…We used our lentiviral vector (pLV-EF1a-AcGFP-P2A-) with the EF1a promoter to drive the expression of transgenes, as described previously. 19 , 33 To facilitate titration of the lentiviral preparations, we co-express GFP along the transgenes via a P2A peptide 5 except for the point mutation constructs, which were generated based on the original BRD3R plasmid from our human kinome library without GFP co-expression. 19 The plasmids used in this study are listed in Table S1 .…”

Section: Methodsmentioning

confidence: 99%

Attenuating iPSC reprogramming stress with dominant-negative BET peptides

et al. 2023

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“…The reprogramming of human fibroblasts has been described previously [ 4 , 5 , 61 ]. Briefly, human fibroblasts were transduced with lentiviral reprogramming factors OCT4, SOX2, KLF4, and MYC at a multiplicity of infection (MOI) of 8:5:5:3.…”

Section: Methodsmentioning

confidence: 99%

Quick, Coordinated and Authentic Reprogramming of Ribosome Biogenesis during iPSC Reprogramming

2020

Cells

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Induction of pluripotent stem cells (iPSC) by OCT4 (octamer-binding transcription factor 4), SOX2 (SR box 2), KLF4 (Krüppel-Like Factor 4), and MYC (cellular Myelocytomatosis, c-MYC or MYC) (collectively OSKM) is revolutionary, but very inefficient, slow, and stochastic. It is unknown as to what underlies the potency aspect of the multi-step, multi-pathway, and inefficient iPSC reprogramming. Mesenchymal-to-epithelial (MET) transition is known as the earliest pathway reprogrammed. Using the recently established concepts of reprogramome and reprogramming legitimacy, the author first demonstrated that ribosome biogenesis (RB) is globally enriched in terms of human embryonic stem cells in comparison with fibroblasts, the popular starting cells of pluripotency reprogramming. It is then shown that the RB network was reprogrammed quickly in a coordinated fashion. Human iPSCs also demonstrated a more robust ribosome biogenesis. The quick and global reprogramming of ribosome biogenesis was also observed in an independent fibroblast line from a different donor. This study additionally demonstrated that MET did not initiate substantially at the time of proper RB reprogramming. This quick, coordinated and authentic RB reprogramming to the more robust pluripotent state by the OSKM reprogramming factors dramatically contrasts the overall low efficiency and long latency of iPSC reprogramming, and aligns well with the potency aspect of the inefficient OSKM reprogramming.

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Reprogramming Human Fibroblasts to Induced Pluripotent Stem Cells Using the GFP-Marked Lentiviral Vectors in the Chemically Defined Medium

Cited by 5 publications

References 13 publications

A PIANO (Proper, Insufficient, Aberrant, and NO Reprogramming) Response to the Yamanaka Factors in the Initial Stages of Human iPSC Reprogramming

A PIANO (Proper, Insufficient, Aberrant, and NO Reprogramming) Response to the Yamanaka Factors in the Initial Stages of Human iPSC Reprogramming

Attenuating iPSC reprogramming stress with dominant-negative BET peptides

Quick, Coordinated and Authentic Reprogramming of Ribosome Biogenesis during iPSC Reprogramming

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