Two novel synthetic small molecules can be added to human stem cell culture that each individually prevent stem cell death, according to a new study from The Scripps Research Institute. The scientists also discovered that stem cells’ microenvironment plays an important role in their survival in culture, even without these novel compounds.
"This paper addresses a long-standing mystery," says Scripps associate professor Sheng Ding, Ph.D. “Scientists have been puzzled by why human embryonic stem cells die at a critical step in the culture process. In addition to posing a question in fundamental biology, this created a huge technical challenge in the lab.”
Dr. Ding is also senior author of the research paper, which appears in current issue of Proceedings of the National Academy of Sciences.
In the process of growing stem cells in culture, scientists must split off cells from their cell colonies. At this point in the process, however, human embryonic stem cells die unless the scientists take extraordinary care that this does not happen. However, mouse embryonic stem cells, which share much basic biology with human embryonic stem cells, do not pose the same difficulties in the laboratory. They can usually be split off from a colony and go on to survive and thrive.
To explore these issues, the scientists started by screening a library of chemical compounds to see if they could find small molecules that could be added to the human embryonic stem cell culture to promote the cells' survival. They found two novel compounds (Thiazovivin and Pyrintegrin) that worked to protect the cells, promoting human embryonic stem cell survival by more than 30 fold.
Next, using the two new survival-promoting small molecules as clues, the scientists set out to understand the biological mechanism behind the cells' survival or demise. By examining cell growth in the presence and absence of the compounds, the team found that the key factor was a protein on the cell surface called e-cadherin, which mediates interactions among cells and between cells and the extracellular matrix.
The researchers observed that when human embryonic stem cells are cut out from the colony, this key protein is disrupted and then internalized within the cell. Without e-cadherin on the cell surface, cell signaling between the cells and their environment is disrupted and the cells quickly die. Both chemical compounds identified by the study, however, protected e-cadherin from damage.
“While in the past people have often talked about the proteins in cell nucleus as regulating stem cell function, our study puts the focus on a different area,” explains Dr. Ding. “E-cadherin is a protein on the cell surface that is very important to cell survival and cell growth.”
In further experiments, the scientists found that the key difference between human and mouse embryonic stem cells lay not only within the cells themselves but also in and controlled by their microenvironment. Transferring human embryonic stem cells into a mouse embryonic stem cell microenvironment, they found that human cells were more likely to survive in this environment even without the survival-promoting compounds.
Moreover, when the scientists chemically induced human embryonic stem cells back to an earlier stage of development, which had an extracellular environment similar to mouse embryonic stem cells conventionally used in the laboratory, there were also no longer problems growing them in culture.
“This validated our mechanistic investigations from a different angle,” says Dr. Ding, “showing that we had dissected out a very core regulatory mechanism. My lab currently uses the novel small molecules indentified in this study on a routine basis.”