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.

A, Primed human stem cells: mRNA-iPSC line J-1 grown on CellStar-coated surface and in E8 medium. The cells have human iPSC morphology of being compact in size and in "shiny" colonies. B, Naïve human stem cells: J-1 iPSCs shown 2 days after switching to a medium similar to the Naïve Human Stem cell Medium (NHSM). Compared to primed stem cells in A, the naïve stem cells are more flat and transparent, with no spontaneous differentiation on the edges.

Tags: EpiSCs, ESC, Gafni et al., ground state naive, Hanna lab, human ground state naive pluripotent stem cells, iPSCs, mRNA iPSCs, naive ESCs, naive human stem cells, naive iPSCs, NHSM

We at Allele Biotech encourage you to help those in need

Typhoon Haiyan (Yolanda) has devastated vast areas in the Philippines and the local people are still suffering, particularly those who had minimum resources even before the disaster hit. Our company’s representatives’ recent trip to the country created a special bond between our employees and the people in the Philippines, and we will do what we can to help, including directly donate to agencies like the Philippine Red Cross. We encourage our clients, colleagues, and friends to do the same. Thank you.

Tags: haiyan, philippines, red cross, stem cell banking in the philippines, typhoon, Yolanda

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

New Allele Product of the Month: FP-nAb™ products for 100% pull-down

Tags: feeder-free, footprint free, high efficiency reprogramming, iPSC, iPSCs, Jiwu Wang, Luigi Warren, mRNA iPSC, mRNA reprogrammin, pluripotent, stem cell protocol, stem cells, xeno-free

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

New Product of the Month: Nano antibody Line including GFP-nAb and mNeonGreen-nAb for co-IP using fluorescent proteins as precipitation tag, nab your complexes like never before!

Human iPSC Commercial Service and Technology Licensing: mRNA-iPSCs made feeder-free, xeno-free, and footprint-free.

Tags: dome-shaped colonies, EpiSCs, ESC, ground state naive, human ground state naive pluripotent stem cells, iPSCs, mRNA iPSCs, naive ESCs, naive human stem cells, naive iPSCs, NHSM

Lab Skills You Stopped Being Proud Of

Molecular biologists who were in graduate school in the 90’s learned how to isolate plasmid DNA from E. coli cultures by a method called “boil-prep” during their first lab rotation. This process involved mixing the bacterial cell pellet in a little bit of detergent, salt and sucrose, dabbed with some fresh lysozyme, and then you are ready to cook, literally! Bacterial cell membranes are disrupted by boiling this soup in a beaker of water over a Bunsen Burner for one minute, and the debris (containing the broken cell membrane and attached chromosomal DNA) is collected by centrifugation in a microfuge at top speed for 10 minutes. Then comes the step that differentiates a true master of lab skills versus a rotation student—if you knew just the right amount of bacterial culture to begin with and handled the E coli pellet by the right techniques, a skillful lab person could collect nearly all the liquid without disturbing the pellet. Pouring out the plasmid-containing supernatant without dislodging the goo on the side/bottom of the tube was such a desirable skill that would not only give you your plasmid but also give you admiration from fellow lab members. That is, of course, if you were doing it before the mid-90’s, because after the introduction of miniprep spin columns by Qiagen, nobody, even the true masters of boil-preps (or its contemporary alkali prep that also involves pelleting by centrifugation and careful removal of tiny volume of liquids surrounding small pellets) would be showing off those skills any longer.

It is actually never easy or fun to collect liquid surrounding small amount of beads or pellets as you always have to struggle to remove as much liquid as possible while trying not to lose any of the beads

Some of the old-timers used to also be very proud of being able to pour a “sequencing gel” (a very thin ~40 cm x 30 cm polyacrylamide gel). I still remember the first time I reported to the second rotation lab at USC. After describing the lab research, the PI showed me around the lab and complained how “Sarah destroyed all my sequencing gel plates”. But consider this, in order to avoid any greasy spot on either plate, you needed to wash both of them fanatically if not religiously. Why? You would have just about a minute’s time to pour non-polymerized acrylamide without leaking from the sides or bubbles forming anywhere in the DNA running lanes, and then inserting a pair of paper-thin combs, all at a speed quicker than TEMED/AP-catalyzed acrylamide polymerization. Good thing that after capillary sequencing was invented, we all happily retired our sequencing-gel pouring skills with a collective sigh of relief.

Technology will always move forward, so will the skills lab researchers will be required to perfect. Using a spin column is very much a “skill-less” technique in contrast to collecting pellets and washing beads after centrifugation, but when there is a choice, people will chose the method that requires “less skills”, such as the spin-column format as the preferred platform for the new FP-nAb™ products.

BTW, like to have information on the spin column kit? Here it is: http://www.allelebiotech.com/gfp-nab-agarose-spin-kit-20-reactions/

Tags: agarose beads, camelid antibody, capture reagent, co-IP 101, GFP, gfp affinity tag, gfp antibody, GFP co-IP, GFP-nAb, magnetic beads, mNeonGreen-nAb, nano antibodies, nanobodies, polyacrylamide beads, RFP, spin column, Trap, VHH domain