Stem cell researchers have been pretty amped since last month’s presidential decision to bulldoze funding restrictions on human embryonic stem cells. We know the Scripps Institute’s Center for Regenerative Medicine even threw a “Stem Cell Freedom Party” to celebrate. We weren’t able to attend unfortunately, but we’re sure there was some crazy behavior. After all, after navigating eight years of red tape, even the most even-keeled researcher can build up more frustration than during a late night session at Kinko’s.
The promising field of regenerative medicine that has goals of one day programming stem cells to be useful therapies for repairing tissues like the neural tissue in a damaged spinal cord or for treating debilitating diseases like Parkinson’s must first dial in which factors are instrumental in human stem cell maintenance and differentiation, a task that will definitely keep the community busy with late nights for the foreseeable future. Like many areas of developmental biology, epigenetic factors including DNA methylation, non-coding RNAs, histone modifications and histone variants are emerging as a central players.
Epigenetic Screening in ESCs and NSCs
Chris Ricupero, a graduate student in the Hart Lab at Rutgers’ Stem Cell Research Center was directly impacted by the recent stem cell policy changes. “As a NSF fellow, I wasn’t allowed to touch any of the lab’s non-federally funded cell lines.” Ricupero, who has been looking at the epigenetic state of rat neural clones at various points in development, is now getting ready to add human cell lines into the mix.
The Hart Lab is focused on developing tools to facilitate transplanting neural stem cells for spinal cord injuries. “In broad terms, we are interested in how and when neural stem cells decide to become a neuron or a glial cell and how flexible or rigid that initial decision is,” said Ricupero
Recently Ricupero has been working on identifying key histone modifications that mark stem cell states. “Looking at the chromatin state of these restricted precursor cells is a way to look upstream of phenotypic differences that have not been played out yet. These cells may already have there fate determined before any obvious changes to mRNA levels.”
A Leaner, Meaner, and Sensitive ChIP
Using chromatin immunoprecipitation (ChIP) with an assortment of antibodies against histone modifications, Ricupero has managed to identify key mods that associate with different levels of stem cell commitment. But with a large number of potential modifications to profile and biological variation, stem cell researchers like Ricupero have a full plate considering many ChIP protocols can last a few days.
Lately Ricupero has been blasting through ChIP pulldowns using a magnetic bead-based kit from Invitrogen (MAGnify™ ChIP System) that streamlines the process considerably. “I did about 30 IPs last night. I typically wouldn’t do more than 12 and even that is a multi-day procedure.”
ChIP has been an invaluable weapon in the chromatin biologist’s toolkit for years, but the protocol has been pushed further in recent years as the technologies used downstream of it become more advanced, and the research scenarios in which it is used have become more demanding. High throughput sequencing has replaced gels and PCR, and researchers, especially in the stem cell community, need more out of less material.
“Using this new ChIP system, we can drastically reduce the input cell amount required per IP which allows us to look at more histone modification profiles in a given sample of differentiated cell types that are difficult to expand,” explained Ricupero. As in many studies, sample heterogeneity can mask many interesting findings. “Being able to work with a small, homogenous cell population is key.”
Screening for the Restricted
Ricupero and his colleagues at Rutgers hope to tease out the key chromatin marks that will aid in screening cell types prior to engaging in the differentiation process.
“We may be able to save time by only choosing clones that have the potential to become either neurons or glia before we undergo days or weeks of differentiation protocols. This may be especially important in human stem cells which we are starting to investigate.”
“If we learn key chromatin states specific neurons or glia, engineering cells to specific fates using epigenetic modifiers may be possible. There are labs already doing this with specific HDAC inhibitors.”