iPSCs
Picture Blog: Naive Human Pluripotent Stem Cells Regrown From Allele’s iPSCs
As we blogged a month ago, the Hanna lab recently published a paper in Nature describing that human ESCs or iPSCs, which typically resemble more of mouse EpiSCs (epiblast stem cells) than ground state mouse stem cells, could be converted to naïve pluripotent stem cells if grown in a stem cell medium that includes hLIF, JNKi, and p38i. The figure here shows that the reported system did perform well when we at Allele Biotech tested growing our banked iPSCs under similar conditions. The colonies grown in naive stem cell conditions (B) did become dome-shaped when cultured for longer period of time; when transferred back into regular stem cell medium, the once naive-looking iPSCs formed tighter and “cleaner” colonies than typical “primed” human iPSC colonies.
New Allele Biotech Publication on Stem Cells
Feeder-Free Reprogramming of Human Fibroblasts with Messenger RNA
Current Protocols in Stem Cell Biology • November 13, 2013
DOI: 10.1002/9780470151808.sc04a06s27
Authors: Luigi Warren, Jiwu Wang
This unit describes a feeder-free protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts by transfection of synthetic mRNA. The reprogramming of somatic cells requires transient expression of a set of transcription factors that collectively activate an endogenous gene regulatory network specifying the pluripotent phenotype. The necessary ectopic factor expression was first effected using retroviruses; however, as viral integration into the genome is problematic for cell therapy applications, the use of footprint-free vectors such as mRNA is increasingly preferred. Strong points of the mRNA approach include high efficiency, rapid kinetics, and obviation of a clean-up phase to purge the vector. Still, the method is relatively laborious and has, up to now, involved the use of feeder cells, which brings drawbacks including poor applicability to clinically oriented iPSC derivation. Using the methods described here, mRNA reprogramming can be performed without feeders at much-reduced labor and material costs relative to established protocols.
Allele iPSC Service and Technology Licensing Contact: http://www.allelebiotech.com/cell-line-and-culture-services/#ips-line
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Human Pluripotent Stem Cells Are Getting Naïve
In the field of stem cell studies there has been a long standing notion that human embryonic stem cells (ESCs) are not equivalent to mouse ESCs isolated from mouse inner cell mass of blastocyst. When induced pluripotent stem cells (iPSCs) were developed by the Yamanaka lab from human adult cells, they were found to be closer to human ESCs but not as “naïve” as the mouse ESCs.
To learn more, see the following key points about naïve stem cell:
The ground state of human iPSCs or ESCs remains the holy grail in stem cell research largely because of its conceptual value, and also because it was difficult to achieve. When mRNA reprogramming was first described by Warren et al. 2010, the hope was that the mRNA-iPSCs could be closer to ground state compared to virus-mediated iPSCs since mRNA-iPSCs had no issue with uncontrolled transgene expression or silencing. However, the human mRNA- iPSCs produced even with our current, much more potent mRNA mix did not grow in dome-shaped colonies like mouse ESCs, making us wonder whether that is achievable. A recent publication by the Hanna group showed that a ground state pluripotency could be achieved by simply growing cells in the presence of a few additional medium factors, mostly controlling signaling pathways. Since it has been shown that the currently available “primed” (not naïve) human iPSCs can already be derived into various tissue types, the practical impact of the new discovery might be more likely found in removing epigenetic memory after reprogramming, or line-to-line variations if a truly naïve state could be achieved.
Technically, any existing human ESCs or iPSCs could be converted to naïve stem cells, according to the new publication. And when the new medium system, termed NHSM for Naïve Human Stem Cell Medium, was applied to iPSCs, it was used 4 days after the start of the reprogramming run.
Key points about naïve stem cells:
1) Stem cells grow in dome-shaped colonies under 2i/LIF conditions.
2) Doubling time is around 14 h compared to 26 h of primed PSCs.
3) Up to 88% single-cell cloning efficiency in the presence of ROCK inhibitor.
4) OCT4 distal enhancer is used more than the proximal enhancer in naïve PSCs.
5) In cells from female donors, naïve iPSCs are at pre-X inactivation state.
6) More E-CADHERIN expression on the surface of naïve stem cells.
7) It is easier to perform gene targeting by homologous recombination in naïve PSCs.
8) Less H3K27me3 in development genes in naïve cells.
9) High efficiency of integration and chimaerism when naïve iPSCs were injected into mouse embryos.
Gafni et al. “Derivation of novel human ground state naive pluripotent stem cellsNature 2013” http://www.nature.com/nature/journal/vaop/ncurrent/full/nature12745.html
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Human iPSC Commercial Service and Technology Licensing: mRNA-iPSCs made feeder-free, xeno-free, and footprint-free.
Picture Blog: How Do You Like Your iPSCs, Clonal or Bulk-Conversion?
Reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs) is commonly considered a stochastic process, i.e. with randomness, which offers an excuse for the commonly seen low efficiency and low constancy of making iPSCs. We have demonstrated time and again that by using potent mRNA cocktails, the majority of the fibroblasts seeded in a well can be converted into pluripotent stage in a nearly synchronized manner (Warren et al. 2012, Warren and Wang 2013, and this Allele Picture Blog series). mRNA molecules can function robustly yet transiently while avoiding the need of entering the nucleus, a bottle-neck for all DNA-based vehicles.
Other researchers are used to the idea of clonal expansion partly because isolating iPSCs from “clones” was a common step during reprogramming using viruses or other low efficiency methods, even though those clones were not necessarily from single precursor cells. This week, the Allele iPSC team developed a new way of managing our mRNA reprogramming that allowed us to achieve clonal iPSCs that appear to be a lot purer and more likely true clones compared to previous reports, without compromising any of the main benefits of our protocol, e.g. feeder-free, xeno-free, footprint-free, very fast and highly efficient. This work is currently supported by an NIDA/NIH grant to Dr. Jiwu Wang at Allele Biotech.
Warren, Ni, Wang, and Guo, Scientific Reports, 2012
Warren and Wang, Current Protocols, 2013, in press
Picture Blog: mRNA Reprogramming for Human iPSCs without B18R!
Human induced pluripotent stem cells provide a great route towards personalized medicine and high accuracy drug screening. Allele Biotech has developed the most efficient method of making human iPSCs by using enhanced mRNAs, which have been adopted by leading pharmaceutical companies for clinical trials. The effects from medium-supplementing mRNAs are robust yet transient, and highly specific compared to both miRNAs (off-targets) and small molecules (unknown targets). To repress cellular immune response to introduced RNA molecules, viral protein B18R was previously used during mRNA reprogramming.
B18R is relatively expensive and inconvenient to use because it requires pre-aliquoting and -80C storage. The protocol has recently been dramatically improved at Allele through an NIDA-funded project. In our latest reprogramming run, all we needed to do was to include mRNA complex in the supplement during medium change for just a week without the need of adding any other type of molecules (such as B18R, miRNA, or chemicals) to help the mRNA mix, unlike all other known mRNA-reprogramming protocols. This advancement can make reprogramming human fibroblasts to footprint-free and xeno-free iPSCs a routine experiment for any lab to perform.
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