This year’s International Society for Stem Sell Research (ISSCR) meeting was held across the Pacific Ocean in Japan. Despite the long travel and unfamiliar culture, or maybe because of it, we convinced Stanford University’s Jonathan Davila to take copious notes and report back on all the conference happenings. Read all about Jon’s experiences in the Land of the Rising Sun below:
Having never been to Japan before, I wasn’t sure what to expect during this trip. Would my travels resemble an episode of No Reservations with Anthony Bourdain, or more like the Bill Murray character from Lost in Translation? It was actually a mix of both. For foodies like me, Japan is a culinary Mecca, having the most restaurants with Michelin Stars, but ordering food in another language can be an adventure; like the time I asked our waiter for “one second” to review the menu, and instead got “one hot sake”. Not exactly a horrible outcome, but clearly my communication with locals was lacking.
I also got completely lost with the little things like the correct way to take an escalator or eat breakfast. There seems to be a specific protocol for everything and I was unable to perform even the most remedial tasks without standing out like a tourist. All jokes aside, these experiences are what made for a great trip. Yokohama was a great host city, with some of the friendliest and most hospitable locals I have ever met during my travels. I must say Domo Arigato, Japan.
Immediately upon landing at Narita airport, I was thrown right into conference mode when the shuttle to Yokohama was filled with the “Who’s Who” of stem cell biology including Irv Weissman (Stanford), Rudolf Jaenish (Whitehead) and Arturo Alvarez Bulla (UCSF) just to mention a few. It felt like a red carpet movie premier featuring stem cell biologists. With that cast I knew I was going to be in for a treat once the talks began.
Reprogramming Factors in iPSC Formation
Rudy Jaenisch, Whitehead Institute
Dr. Jaenisch’s main theme was to determine if iPSC formation by reprogramming factors is a stochastic or a deterministic event. To address this question and avoid the huge variability in gene expression amongst different cells, his group is assaying mRNA expression in individual cells either by mRNA FISH or Fluidigm. They found that the reprogramming factors form a regulatory network that need to activate their downstream effectors in a sequential manner for adequate reprogramming to occur. He indicated that some of these downstream genes seem to be great predictors for successful iPSC formation. These genes are ESRRB, UTF, Lin28 and DPPA2. The second part of his talk consisted of new methods for gene targeting. His lab is currently using Talens to integrate small oligos into specific regions of the genome in order to add or repair specific mutations. The take home message was to make sure to use the adequate controls for all in vitro disease modeling. He emphasized the importance of having identical genetic backgrounds when studying genetic diseases, thus, the importance of gene targeting for disease modeling.
Enhancing Naïve Stem Cells with Small Molecules
Austin Smith, Wellcome Trust Centre for Stem Cell Research
Dr. Smith showed some interesting results, expanding on his story about enhancing what he calls naïve stem cells with two small molecules. He is using the MEK inhibitor, PD0325901, and the GSK3 inhibitor, Chir99021, to enhance this naïve state. The use of this 2i system reduces the mosaic population within the ES colonies, creating a homogenous population that does not spontaneously differentiate as readily. His talk went into the details of the pathways that are regulated by these small molecules and showed that ESRRB can replace the GSK inhibitor. One other interesting bit of information was that his lab can rebuild pluripotency in unipotent primordial germ cells when grown in Lif plus the 2i molecules.
Comparing Embryonic Stem Cells and Induced Pluripotent Stem Cells
Kazutoshi Takahashi, Kyoto University
Dr. Takahashi stood in for Shinya Tamanaka and gave a great talk regarding the differences between ES and iPS cells. They have analyzed gene expression, DNA methylation status and exon sequencing on over 50 different ES and iPS lines. They conclude that there are small clonal variations across individual ES and iPS lines; but, there is no sharp contrast across all the samples assayed. In other words the small differences found are attributed to line to line variation, implying that there are good and bad lines, but as a whole ES and iPS cells are virtually the same. Also, he showed that even within iPS lines there is a level of variability. For example, the neuronal differentiation capacity of specific iPSCs varies amongst subclones of a specific iPS line. This illustrates that there must be epigenetic differences between subclones that arises under normal culture conditions.
Identifying and Characterizing Long Non-coding RNAs
John Rinn, Harvard University
Dr. Rinn declared his lab to be RNA hunters. Spearheading a movement to identify all lnc-RNAs, Rinn and his lab have taken on the task not only to identify these elusive molecules but also to characterize their biological roles. His talk was more of a preview of things to come than a complete story regarding one of these lnc-RNAs. He showed expression data for a few of these molecules, but then started to explain the importance of performing RNA-FISH to actually see these RNA molecules. He showed that most of these are in the nucleus and are specifically localized at discrete focal points on the chromatin. He theorizes that lncRNAs primarily work in cis regulating the chromatin state of the immediate surroundings of their transcription site by serving as a scaffold or landing strip for chromatin remodelers. Expect to see some crazy stuff from his lab in the near future because they have fifty different transgenic animals for some of the lncRNAs that they have identified. He gave a small taste of things to come by showing that some of the transgenic animals have phenotypes in neuronal differentiation.
Induced Neuronal Reprogramming by miRNAs
Jerry Crabtree, Stanford University
Dr. Crabtree presented a very elegant story about the role of microRNAs in induced neuronal (iN) reprogramming. He showed that over-expression of mir-124 along with mir-9* in fibroblast is sufficient to obtain Map2+ cells. These microRNAs when combined with transcription factors, Ascl1, Myt1L and NeuroD2 converted fibroblast to neurons exhibiting mature action potentials. This reprogramming is in portion mediated by the repression of BAF53a during iN formation by these two microRNAs. BAF53a is a subunit within Swi/Snf-like neural-progenitor-specific BAF (npBAF) complexes. For complete reprogramming of post-mitotic neurons, BAF53a needs to be replaced by the homologous BAF53b subunit within the neuron-specific BAF (nBAF) complexes. He also showed that BAF53a seems to block the polycomb repressive complex from binding and repressing specific genes, among these are cell cycle regulators. Once these microRNAs mediate the shift of BAF53a to BAF53b, polycomb can repress several cell cycle genes and create a chromatin landscape that will be conductive for post-mitotic iN formation.
Reprogramming Fibroblasts to Induced Neural Precursor Cells
Ernesto Lujan, Stanford University
Dr. Lujan from Marius Wernig’s lab presented their most recent studies involving the reprogramming of mouse fibroblasts to an induced neural precursor cell (iNPC). Lujan showed that the transcription factor combination of Brn2, Sox2 and FoxG1 was sufficient to transform fibroblast to a tripotent iNPC. These cells have the capacity to differentiate and form electrophysiological mature neurons, astrocytes and oligodendrocytes that can engraft into mouse brains. What made this study interesting is the therapeutic potential of these cells for autologous transplantation into patients. Unfortunately, they have not got this to work in human cells yet but they are currently working on that.
miRNA role in Induced Pluripotent Stem Cell Formation
Robert Judson, UC San Francisco
Dr Judson from Robert Blelloch’s lab gave a very elegant talk elucidating microRNAs that are involved in iPSC formation. Among some of the microRNAs that they found to be involved in this process was mir-294. They over-expressed this microRNA and showed that it enhances the reprogramming efficiency. They also have identified multiple of its targets during reprogramming. Not surprisingly, siRNA targeting of these genes also can increase the reprogramming efficiency. What was truly interesting was the fact that the effects of the siRNA treatment on the targets of mir-294 were synergistic when several of the siRNAs were pooled together. This reinforces the idea that microRNAs most likely target multiple genes which are involved in parallel pathways.
Harvesting Cultured Cells as a Monolayer Sheath
Teruo Okano, Tokyo Women’s Medical University
Okano’s group has developed a cell culturing system that allows for the removal of cultured cells as a monolayer sheath by lowering the culturing temperature and changing the hydrophobicity of the plate surface, this circumvents enzymatic dissociation to a single cell suspension. His videos illustrating this technique were extremely cool. He clearly showed that transplantation of these cells, particularly myoblasts, as a monolayer sheath instead of single cells increase survival after transplantation. Okano mentioned that this transplantation technique has been used in several clinical trials in Japan with a good success rate. One of the examples he gave was a transplantation of myoblast cells onto the heart of a patient with cardio-myopathy. Multiple of these monolayers can be placed on top of each other to obtain thicker cell preparations for transplantation. Not surprisingly there was a limit to the amount of layers they can stratisfy prior to transplantation. If the preparation was too thick the outer layer would die do to the lack of nutrients. They overcame this limitation by allowing two to three days between transplations of the different layers. This allowed time for vascularization to occur in the lower layers of the transplanted tissue and provide nutrients to the outer layers added later. This talk really started to show us the potential of 3D tissue engineering.
Using Blastocyst Complementation to Grow Tissues in Different Species
Hiromitsu Nakauchi, University of Tokyo
Dr. Nakauchi was the true winner of the most interesting talk award and everyone was talking about this after the meeting. His group has been able to do blastocyst complementation between mouse and rat species. Interesting enough, but what made his talk so interesting was the fact that they can make rat pancreas in Pdx-/- mice which normally do not develop pancreas. These new pancreas are made solely of rat cells. Right away this opens the question of how can we start to grow human tissues in other species. With this question in mind, Nakauchi’s group took a similar approach and used somatic nuclear transfer to develop genetically altered pigs that lack pancreas. This defect can be rescued when they perform blastocyst complementation experiments with wild type pig cells. They also have been able to develop pig chimera lines to be able to maintain an expandable population of these defective animals. Hiromitsu stated that they are waiting for approval to start performing blastocyst complementation experiments with human iPSCs and these pig lines but as of now those experiments are illegal in Japan. There were two very interesting questions that were brought up during the Q&A session following the talk. This is when this talk started sounding like a bad scifi movie. First, how to avoid having humanized pig brains in these chimeras and secondly, how to avoid non-desired pig cells that would form the vascularization of these “human” pancreas. Both of these questions Nakauchi said were easy to address. He stated that his group has already made pigs that have defects in vascularization and would solely use the donor cells to fix the defect but did not expand on this topic. I guess that will be his next big article. The issue with human-pig brains he said he can be resolved by introducing a kill gene under the control of the promoter of a neuronal gene. I believe that is easier said than done but one thing is for sure we are living in a time where all kind of things are possible.
Generating Optic Cups from Human Embryonic Stem Cells
Yoshiki Sasai, RIKEN
Continuing with the trend of generating 3D tissue structures, Dr. Sasai presented some of his most recent research in which he describes a method of generating optic cups from self-organizing hESC cultures. He elaborated on how ESCs have an intrinsic capacity to spontaneously differentiate towards a neural lineage when grown in the absence of serum. When these cells are dissociated and re-plated some of them can self organize and form optic cups that contain stratified neural cells. He showed that they are able to see this self-organizing behavior in both human and mouse ESCs. Interestingly, the optic cups formed from the mESCs where significantly smaller than the hESCs ones. This shows species specific differences. What blew my mind with this story was the fact that the self organizing capacity seen in these cells was an endogenous trait, independent of any exogenous cues. I recommend you read a little more about this story, which was published in Cell Stem Cell, June 2012
**EpiGenie would like to give a huge “Thank You” to Jonathan Davila, PhD who is a Postdoc in the Marius Wernig lab at Stanford University.