Locally Sourced: What Comes First, Division or Differentiation?

Every year, high school seniors face the daunting task of deciding what to do after they graduate. Those who chose to further their education must decide where to go to college. After that, more questions arise. What will you major in? What clubs will you join? What internships will you pursue? Most students do not have immediate answers. Rather, most students begin this journey with uncertainty.


However, as they mature and continue the college path, undecided students find their place. In addition to their passions and interests, college students’ decisions are influenced by myriad factors including friends, family, and communities. Some students are motivated by the needs of society—as engineers become more desired, for example, more college students choose to major in engineering to fill the demand. Stem cells do not go to college, but they engage in similar mechanisms of differentiation and division as college students do.


A recent study led by Valentina Greco, a professor of genetics at Yale University, along with researchers Kailin Mesa and Katherine Cockburn, on stem cell self-renewal and homeostasis in the mammalian epidermis has shown that stem cells behave to maintain balance and fulfill demands. Their findings were upheld during experiments of natural stem cell loss as well as during forced cell loss, reaffirming their proposed mechanism.


Skin deep


Skin can be divided into three main layers: epidermis, dermis, and hypodermis. The epidermis is the outermost and visible layer and can be subdivided into the suprabasal and basal layers. The suprabasal layer includes dead skin cells that shed naturally. Beneath the suprabasal layer is the basal layer, where undifferentiated, multipotent stem cells await their opportunity to differentiate and replace old skin cells. Stem cells and students share this “multipotency” ability, whereby they have the power to follow many pathways in life.


The Greco Lab questioned whether differentiation drove division, division drove differentiation, or something else entirely. “Whether the drive to renew by external signals was constant or cued was unknown” said Cockburn.  In embryonic development, stem cells proliferate upstream, meaning that division in the bottom-most layer of stem cells leads to overcrowding in the middle layer, forcing the middle layer stem cells to differentiate upwards and enter the suprabasal layer in a cued manner. Here, division drives differentiation. However, as the Greco lab showed, the story of stem cell behavior in adult cells is different than that of embryonic cells.


A balancing act


The study began with live imaging of a large region of mouse epidermis; the team continually revisited the same region to track and create a complete record of all cell fates over time. The two possible “fates” were the decisions of the stem cells to either differentiate or divide. “Every twelve hours for seven days, activity was recorded to form a detailed map of events—differentiation or division, when and where,” said Cockburn. The team recorded 1,527 divisions and 1,540 differentiation events from six regions across five mouse test subjects.


The researchers wondered whether stem cell control was occurring rapidly or slowly and to what stimulus. The data collected showed a significant difference between the two fates over an initial twelve-hour period; there were more stem cells differentiating and leaving the basal layer than there were stem cells dividing to replace the loss. This showed that a balance is not reached instantaneously. Over seven days, however, the two cell fates occurred at nearly equivalent frequencies with one division event for every one differentiation event; this is suggestive of a slow stem cell control mechanism, whereby cells respond to their neighbors’ fates with a one to two-day lag to regain the original balance. Lastly, the difference between cells differentiating and dividing within a small region of tissue was significantly smaller than would be expected if this was an autonomous mechanism—neighboring cells must be communicating.


Now consider how much friends influence each other’s decisions. Do they have more of an influence than a stranger living across the globe? Stem cells in this study are found to respond more frequently to their closest neighbors’ decisions than that of more distantly located cells. This conclusion was drawn from the fact that neighbor pairs had opposite fates a significant number of times. “Every time a stem cell exited, we saw a direct neighbor dividing to replace it” said Cockburn. This coordination decreased as pairs at greater distances were observed. In effect, stem cell self-renewal and differentiation appeared to be balanced through local compensation of cell fates.


Who is driving whom?


Homeostasis is the tendency of the body to maintain an internal balance. Systems will behave to counteract, or correct, imbalances. In this case, the Greco Lab sought to define the homeostatic mechanism by which the body corrects imbalances created when epidermal stem cells differentiate. To confirm that differentiation is the homeostatic driver for cell division, and not vice versa, the team used an epidermal tape stripping method, which involves the removal of the outermost layers of the skin on a mouse, promoting stem cell differentiation to replace the lost cells. The team labeled and tracked stem cells following tape stripping and compared the results to a control set. They noted the significant increase in the number of cells moving upstream from the basal to the suprabasal epidermal layers, signifying differentiation.


Signs of cell division events in the form of visually distinguishable mitotic spindles appeared subsequent to the differentiation, confirming the previous results. The stem cells in this scenario are responding to supply and demand once again. When the researchers removed cells in the outermost suprabasal layer of the skin, some stem cells in the basal layer differentiated to move up, followed by other stem cells dividing to replace the ones that differentiated.


Supply and demand


Maintenance of homeostasis throughout life requires coordination, balance, active preservation, and constant communication. Without homeostasis, the body does not function optimally. Imagine a queue of people waiting on line to go onto a rollercoaster. The person at the head of the line moves to take his turn on the ride. Naturally, the person who was next up will move forward to fill that spot. This cycle continues down the line until everyone has moved forward a single spot. Now imagine that after the head of the line goes onto the rollercoaster the next person on line does not notice. Now, the queue cannot move forward. People on line become anxious, and disorder overcomes homeostasis. Fortunately, stem cells are alert and responsive to their surroundings and continually supply the demands of the body—division compensating for differentiation.


The team then wondered how exactly local differentiation drove subsequent cell division. Cell division takes place at the end of the cell cycle, after the cell has passed through the G1, S, and G2 phases. In G1, the cell begins to grow but continues its normal functions. In S, DNA is copied so that the two daughter cells that come from the division will each have a copy of the original parent DNA. In G2, the cell continues to grow and prepares proteins necessary for mitosis. The Greco Lab hypothesized that when stem cells differentiate out and up into a higher layer, they leave physical room for their neighbors to grow and progress towards cell division. The researchers used a fluorescent tag to make it easier to identify the boundaries of each stem cell and measure their individual areas over the course of seven days. They observed that cells divide after about two days of significant cell growth. The time of largest growth occurred while a neighbor differentiated. The researchers concluded that the spatial changes involved in differentiation are likely cues that promote neighboring division.


Loss by lasers


Lastly, the researchers tested whether artificial loss of stem cells was sufficient to drive local self-renewal. They used lasers to remove stem cells and tracked the fates of the neighboring cells. They saw a net excess of neighboring divisions following cell loss, which became balanced after a matter of three days, exhibiting the same results of compensatory self-renewal that were observed in previous tests of natural stem cell loss. This is consistent with the model that the departure of stem cells from the basal layer is sufficient to induce local division. All that the cells need to divide is an appropriate amount of space.


While the Greco Lab has drawn many profound conclusions regarding the homeostatic mechanisms of stem cells, there is still much to uncover in light of this study. Now that stem cell behavior has been proven to be different in embryonic versus adult development, the next step is to determine why this disparity occurs and at what point the mechanism changes in the body. Also, how cell sensing is incredibly localized rather than due to a global, density-dependent process is unclear. Other future work will consider stem cell self-renewal in the context of pathological conditions. “We are interested in how the relationship [between differentiation and division of neighboring pairs] changes in cancer with over proliferation” said Cockburn. Overall, the work of the Greco Lab provides evidence of homeostatic stem cell behavior that will give rise to a multipotency of applications in pathology.