How you derive embryonic stem cells matters

A scientist named James Thompson was the first to successfully culture human embryonic stem cells in 1998. He didn’t know it then, but his technique isolated a specific type of embryonic stem cell (ESC) that had a “primed pluripotent state”.

There are actually two phases of pluripotency: naïve and primed. Naïve ESCs occur a step earlier in embryonic development (during the beginning of the blastocyst stage), and the naïve state can be thought of as the ground state of pluripotency. Primed ESCs on the other hand are more mature and while they can still become every cell type in the body, they are somewhat less flexible compared to naïve ESCs. If you want to learn more about naïve and primed ESCs, you can refer to this scientific review.

Scientists have developed methods to derive both naïve and primed human ESCs in culture and are attempting to use these cells for biomedical applications. However, a recent CIRM-funded study published in Cell Stem Cell, calls into question the quality of ESCs produced using these culturing methods and could change how lab-derived stem cells are used for stem cell transplant therapies and regenerative medicine.

Primed human embryonic stem cells (purple) identified by a green stem cell surface marker. (Image courtesy of UCLA)

Primed human embryonic stem cells (purple) identified by a green stem cell surface marker. (Image courtesy of UCLA)

Culturing methods erase stem cell memory

UCLA scientists discovered that some of the culturing methods used to propagate naïve ESCs actually erase important biochemical signatures that are essential for maintaining ESCs in a naïve state and for passing down genetic information from the embryo to the developing fetus.

When they studied naïve ESCs in culture, they focused on a naturally occurring process called DNA methylation. It controls which genes are active and which are silenced by adding chemical tags to certain stretches of DNA called promoters, which are responsible for turning genes on or off. This process is critical for normal development and keeping cells functional and healthy in adults.

UCLA scientists compared the DNA methylation state of the mature human blastocyst – the early-stage embryo and where naïve ESCs come from – to the methylation state of naïve ESCs generated in culture. They found that the methylation patterns in the blastocyst six days after fertilization were the same as the patterns found in the egg that it developed from. This discovery is contrary to previous beliefs that the DNA methylation patterns in eggs are lost a few hours after fertilization.

Amander Clark, the study’s lead author and UCLA professor explained in a UCLA news release:

Amander+Clark+headshot_68295d00-2717-4d5c-99f3-f791e6b6ebcf-prv

Amandar Clark, UCLA

“We know that the six days after fertilization is a very critical time in human development, with many changes happening within that period. It’s not clear yet why the blastocyst retains methylation during this time period or what purpose it serves, but this finding opens up new areas of investigation into how methylation patterns built in the egg affect embryo quality and the birth of healthy children.”

The group also discovered cultured naïve ESCs lack these important DNA methylation patterns seen in early-stage blastocysts. Current methods to derive naïve ESCs wipe their memory leaving them in an unstable state. This is an issue for researchers because some prefer the use of naïve ESCs over primed ESCs for their studies because naïve ESCs have more potential for experimentation.

“In the past three years, naïve stem cells have been touted as potentially superior to primed cells,” Clark said. “But our data show that the naïve method for creating stem cells results in cells that have problems, including the loss of methylation from important places in DNA. Therefore, until we have a way to create more stable naïve embryonic stem cells, the embryonic stem cells created for the purposes of regenerative medicine should be in a primed state in order to create the highest-quality cells for differentiation.”

How you derive embryonic stem cells matters

Now that this culturing problem has been identified, the UCLA group plans to develop new and improved methods for generating naïve ESCs in culture such that they retain their DNA methylation patterns and are more stable.

The hope from this research is that scientists will be able to produce stem cells that more closely resemble their counterparts in the developing human embryo and will be better suited for stem cell therapies and regenerative medicine applications.


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CIRM Creativity Student Long Nguyen Learns First-Hand about the Value of Scientific Research

This summer we’re sponsoring high school interns in stem cell labs throughout California as part of our annual Creativity Program. We asked those students to share their experiences through blog posts and videos.

Today, we hear from Long Nguyen, who has been busy at Stanford University’s Beckman Center for Molecular and Genetic Medicine.

Summer Reflections

Long Nguyen

It’s been a real pleasure spending the past eight weeks here at Stanford University. When I first walked into Beckman Center on June 9th, I did not know what to expect. There was a crowd of students all waiting, just as I was. I got my lab coat, my notebooks, and my bag. Frankly, I was anxious beyond imagination. At the time, I was still wondering to myself: “How did I get into this program? It’s inexplicable.” Those thoughts vanished as I stepped out of that room three hours later and headed to my workplace. I was confident and ready to start the new experience.

The beloved hood upon which I daringly cultured my cells!

The beloved hood upon which I daringly cultured my cells!

Learning about stem cells has made me more passionate about scientific research. I am glad to have been given this opportunity. Up to this point, I had only been exposed to textbooks upon textbooks—a dull methodology, as many may agree. The only hands-on experience I ever had were agarose gel electrophoresis and transformation of bacteria with an insulin-GFP reporter complex.

My experience here, however, has given me a strong foundation beyond the scope of these. Initially, I could not open a conical tube with one hand, and my pipetting was absolutely horrendous. I could not calculate simple dilutions for my working solutions. I even made the mistake of vacuum-aspirating over half of my cells during the second week. As time progressed, my culturing of stem cells improved considerably and I made few, if no, mistakes. I learned the background, the methodology, and the purpose of my work. These little details proved more important than they seemed, as they gave me a much clearer understanding of my work. Looking back, despite many, many errors, I learned to appreciate the value of science.

An interesting moment before a media change.

An interesting moment before a media change.

Prior to my experience, I had known little about stem cells: they were mentioned briefly in a page of my AP Biology textbook. I only knew that they differentiated into specific cell types to repair the body; there was no mention of iPSCs in the slightest. My knowledge of stem cells now is much more extensive. Regenerative medicine, wound healing, disease treatments—all that can be possible with stem cell research surprised me, to say the least. I have no doubts that this developing field will be a major game changer in the coming decades. The research is definitely something to respect. Being a part of ongoing research made me more aware of the problems that scientists, especially those in medicine, face in their attempts to do something, whether it be to cure scleroderma, to repair damaged neural connections, or to screen drugs with iPSC-derived cells. One thing is for sure: what I do now and what I expose myself to will be critical once I start planning for my future. Thanks go to Stanford’s faculty, SIMR 2014, CIRM, my peers, and my family, all of whom have supported me in my work.

My dear cells!

My dear cells!