A severe de novo methylation of episomal vectors by human ES cells

Kameda, Takashi; Smuga-Otto, Kim; Thomson, James A.

doi:10.1016/j.bbrc.2006.08.175

Cited by 20 publications

(11 citation statements)

References 73 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2E). OriP was reported to increase the transfection efficiency and plasmid stability in hPSCs if cotransfected with an RNA expressing the EBNA protein (41). The encoded tracrRNA and crRNA both corresponded to the mature, processed forms as they exist in N. meningitidis cells (14).…”

Section: Resultsmentioning

confidence: 99%

Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis

Hou

Zhang

Propson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

624

451

View full text Add to dashboard Cite

Significance Genome engineering in human pluripotent stem cells holds great promise for biomedical research and regenerative medicine, but it is very challenging. Recently, an RNA-guided nuclease system called clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) has been applied to genome engineering, greatly increasing the efficiency of genome editing. Here, using a CRISPR-Cas system identified in Neisseria meningitidis , which is distinct from the commonly used Streptococcus pyogenes system, we demonstrate efficient genome engineering in human pluripotent stem cells. Our study could have a tremendous impact in regenerative medicine.

show abstract

Section: Resultsmentioning

confidence: 99%

Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis

Hou

Zhang

Propson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

624

451

View full text Add to dashboard Cite

show abstract

“…The reprogramming efficiency with this method, however, was prohibitively low (∼3 iPSC colonies from ∼1×10 6 input fibroblasts). This low reprogramming efficiency was likely due to the loss of oriP/EBNA-1 vectors (>25% per cell generation) and DNA methylation-mediated transgene silencing during the first two weeks post-transfection, a time window required for successful iPSC generation [23]. Thus, small molecules that could either accelerate reprogramming process, or reduce episomal vector loss and transgene silencing during the first two weeks post-transfection, were expected to improve episomal reprogramming.…”

Section: Resultsmentioning

confidence: 99%

Efficient Feeder-Free Episomal Reprogramming with Small Molecules

et al. 2011

View full text Add to dashboard Cite

Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promises, human iPSCs will ideally be free of exogenous DNA (footprint-free), and be derived and cultured in chemically defined media free of feeder cells. Currently, methods are available to enable efficient derivation of footprint-free human iPSCs. However, each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery, but the process was inefficient and required feeder cells. Here, we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901, GSK3β inhibitor CHIR99021, TGF-β/Activin/Nodal receptor inhibitor A-83-01, ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover, we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts, adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs.

show abstract

“…H9 human ES cells with a stably transfected EBNA protein (H9-EBNA) that enables episomal replication of exogenous plasmid containing an Orip site were maintained in defined medium-TeSR medium [53,54] containing 50 ng/ml G418. For the transfection, H9-EBNA cells were dissociated by Dispase (Invitrogen) and seeded onto Matrigel-coated six-well plates.…”

Section: Methodsmentioning

confidence: 99%

Conserved Regulation of MAP Kinase Expression by PUF RNA-Binding Proteins

et al. 2007

View full text Add to dashboard Cite

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.

show abstract

A severe de novo methylation of episomal vectors by human ES cells

Cited by 20 publications

References 73 publications

Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis

Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis

Efficient Feeder-Free Episomal Reprogramming with Small Molecules

Conserved Regulation of MAP Kinase Expression by PUF RNA-Binding Proteins

Contact Info

Product

Resources

About