Older may not mean better when it comes to human embryonic stem cell lines

Older, established human embryonic stem cell lines, including those approved for federal research funding under former President George W. Bush, differ from newly derived human embryonic stem cell lines, according to a study by UCLA stem cell researchers.

The finding, by scientists with the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, highlights the importance of continuing to derive new stem cell lines so that researchers can better understand the ability of these cells to make every cell in the human body, said the study's senior author, Amander Clark, an assistant professor of molecular, cell and developmental biology in the UCLA Division of Life Sciences.

"It is critical to find out the characteristics that result in the highest-quality pluripotent stem cell lines that we can make," Clark said. "It is possible that we have not set the bar high enough yet for embryonic stem cells or induced pluripotent stem cells. We now know that established lines are different from newly derived lines, and now we have to find out how important that is."

The study was published Nov. 30 in the early online edition of the peer-reviewed journal Human Molecular Genetics. 

The study looked at the first six new human embryonic stem cell lines Clark's research team developed at UCLA, from 2009 to 2011. These lines have since been accepted by the National Institute of Health's embryonic stem cell registry, which was established by an executive order of President Obama in March 2009. Acceptance into the registry allows the UCLA lines to be used in federally funded research projects.

In the study, Clark examined X-chromosome inactivation, a process by which normal female cells shut off one of their two X chromosomes during embryonic development. The mechanism of X-chromosome inactivation is a large physical marker that is easy to visualize in individual cells. Clark wanted to compare this specific molecular signature in the established embryonic stem cell lines with what occurs when new embryonic stem cell lines are derived from human blastocysts.

The older, established lines examined in the study came from a group of stem cell lines derived prior to 2001. Scientists have known for many years that the majority of these established stem cell lines had already undergone X-chromosome inactivation, Clark said, and her work confirmed this. However, Clark found that with the progression of time, the molecular signature in these lines no longer reflected the normal process of X-chromosome inactivation.

Normally, the X chromosome is inactivated by non-coding RNA and a special form of chromatin in female cells. In abnormal states — like those found in the older, established human embryonic stem cells — the X chromosome is inactive, but this process is not regulated by the non-coding RNA, and the chromatin is different.

"The classic signature is gone, so something else is regulating X-chromosome inactivation in the established cell lines," Clark said. "It will be important not only to find out what that is, but also to discover what else is changing in the nucleus that we cannot see, regardless of whether the cell line is male or female."

The new cell lines generated by Clark's research team and used in the study were derived from human embryos donated to UCLA's Broad Stem Cell Research Center by couples who had previously undergone in vitro fertilization to overcome infertility.  The couples no longer planned to store or use their frozen embryos for reproductive purposes and had decided not to donate the embryos to others for reproductive use. 

The human embryos were transferred from the fertility clinic to the derivation lab at UCLA in frozen vials and were then thawed by Clark's research team. After six to seven days of development, the embryos — or blastocysts — contained a cluster of cells known as the inner cell mass. The inner cell mass is the source of new embryonic stem cell lines. 

Clark's lab examined the new human embryonic stem cell lines three to four weeks after growth from the inner cell mass and found that both X chromosomes were still active in many cells, making them more like the cells from the original inner cell mass and less like the cells from the established stem cell lines.

Slowly, however, with time in a culture and cryo-preservation — which is how the lines are ultimately stored — one X chromosome is inactivated and the cell lines become identical to the older, established lines, including abnormal X-chromosome inactivation, Clark said.

The question, Clark said, is whether the first cells to grow out from the inner cell mass are of a higher quality, and therefore the ones researchers should be aspiring to use for research and potentially therapeutically.

"It may prove to be important to stabilize these cells at that very young state, one that's closest in identity to the inner cell mass," Clark said. "And then we can ask whether these cells give the best quality when differentiated into clinical cell types."

Keeping both X chromosomes active will also be important in modeling diseases such as Rett syndrome.

Going forward, Clark will study human embryonic stem cells in three states: lines in which the X chromosome is inactivated by normal means; lines in which the chromosome is inactivated abnormally; and lines in which both X chromosomes remain active. Clark will seek to understand the differentiation potential of each of the three states.

"Our data highlights the importance of maintaining human embryonic stem cell derivation efforts," the study states. "Gold standard human embryonic stem cell lines should be the benchmark for all human pluripotent stem cell research. Developing new experimental approaches aimed at sustaining human pluripotent nuclei in an epigenetic state closer in identity to the day six or seven day human blastocyst is to work towards a more robust gold standard."

The three-year study was funded first by UCLA's Broad Stem Cell Research Center and the California Institute of Regenerative Medicine, followed by the National Institutes of Health and the National Institute of General Medical Sciences — after the UCLA lines were approved for use in federally funded research.

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University of California, Los Angeles