Planarian Stem Cell Genes Provide Insight into Human Stem Cells

When I was an undergraduate, I cut up planarians in the laboratory and watched them regenerate. Planarians are a type of free-living flatworm that has an uncanny ability to regenerate. Cut them in half, and the tail half will grow a head and the head half will grow a tail. Cut them in half, and the left half will grow a new right side and the left half will grow a new left side. They are truly remarkable critters. How do these worms do this? It appears that the cells of this worm can de-differentiate and become like embryonic cells that originally made the damaged structures. Essentially, the bit of the worm that needs to regrow, recapitulates the developmental process that made it in the first place.

In this way, the bodies of planarians act like stem cells. Stem cells have the potential to regenerate tissues that have been irreparably damaged. One of the problems with stem cells is how to control them. Yet planarians have a genome full of genes that have human homologs. Therefore, planarians seem a logical choice as a model system to study stem cell behavior. Yet, until now, scientists have been unable to efficiently identify the genes that regulate the planarian stem cell system.

At the Whitehead Institute, Peter Reddien‘s lab has revealed some unique insights into planarian biology. These discoveries might help stem cell scientists deliver on a promising role in regenerative medicine. Published in the journal Cell Stem Cell, Reddien and his co-workers used a novel approach to identify and study those genes that control stem cell behavior in planarians. Perhaps unsurprisingly, at least one class of these genes has a counterpart in human embryonic stem cells.

Once injured, planarians (Schmidtea mediterranea) use stem cells, called cNeoblasts, to regrow missing tissues and organs. Within about a week after being injured, the worms have formed two complete planarians. These cNeoblasts are similar to embryonic stem cells in that they are “pluripotent,” which simply means that they have the capacity to form almost cell type in the body. In order to regrow damaged tissues, researchers want to be able to turn on pluripotency and then turn it off after cells have replaced the damaged or missing adult cells.

Reddien, associate professor of biology at MIT and a Howard Hughes Medical Institute (HHMI) Early Career Scientist, said this about his paper: “This is a huge step forward in establishing planarians as an in vivo system for which the roles of stem cell regulators can be dissected. In the grand scheme of things for understanding stem cell biology, I think this is a beginning foray into seeking general principles that all animals utilize. I’d say we’re at the beginning of that process.”

Dan Wagner, a postdoctoral research fellow in Reddien’s lab, and Reddien constructed a protocol to identify genes that regulate the differentiation and renewal of the stem cell population. After identifying genes active in cNeoblasts, Wagner exposed the planarians to ionizing radiation. This left only one surviving cNeoblast in each planarian. After this treatment, each cNeoblast can divide and form colonies of new cells that will differentiate into distinct cell types and divide at specific rates.

Now Wagner and his colleagues eliminated each of the active genes, one per planarian, and observed to determine the behavior of the surviving cNeoblasts without that missing gene. Because the cNeoblasts divide and differentiate at a reproducible rate, the research group could easily determine the role of each gene in cNeoblast behavior. If a colony cNeoblasts was missing a particular gene and had fewer stem cells than the controls, that gene plays in stem cell renewal. Conversely, if the colony had fewer differentiated cells than normal, then the missing gene played a role in differentiation.

Wagner explained, “Because it’s a quantitative method, we can now precisely measure the role of each gene in different aspects of stem cell function. Being able to measure stem cell activity with a colony is a great improvement over methods that existed before, which were much more indirect.”

This screen identified 10 genes that affect cNeoblast renewal, and two of these genes also play roles in cNeoblast renewal and differentiation. Three of the stem cell renewal genes are rather interesting because they code for proteins that are similar to components of Polycomb Repressive Complex 2 (PRC2). PRC2 is known to regulate stem cell biology in mammalian embryonic stem cells and other types of stem cells as well.

These data suggests that the mechanisms that control stem cells in planarians and mammals certainly share some similarities. This might even extend to the mechanisms by which cNeoblasts and embryonic stem cells maintain their naive developmental state. Such work might lead to more insights into stem cell biology that will allows better control and manipulation of stem cells, which will make their use in regenerative medicine much safer.

University of Georgia Lab Generates Blueprint for Stem Cell Responses to Signaling Molecules

What makes a stem cell a stem cell? This is not a trivial question, but an answer to this question is essential in order to understand how to make adult cells stem cells and how to find, and manipulate other stem cells in the body to amplify their healing properties.

Fortunately a great deal of work has been done in this area – genes expressed by stem cells under particular conditions. However, data from different labs tends to conflict with each other. What is a stem cell scientist to do?

From this morass of cacophony comes a very satisfying study from the University of Georgia at Athens, GA. This study, which comes from the laboratory of Stephen Dalton, professor of cellular biology, has generated a wiring diagram of sorts that describes how stem cells respond to external signaling molecules. In one paper, Dalton and his band of intrepid scientists have managed to reconcile several conflicting observations from many different labs.

This paper, which appeared in the March 2 edition of the journal Cell Stem Cell, can potentially provide stem cell scientists with the ability to control precisely the differentiation of particular stem cells into specific cell types. Dalton offered this assessment of his publication: ‘We can use the information from this study as an instruction book to control the behavior of stem cells. “We’ll be able to allow them to differentiate into therapeutic cell types much more efficiently and in a far more controlled manner.”

Many researchers have tended to view signaling in stem cells in an atomistic way. In other words, a single type of signaling molecule sets in motion a specific signal transduction pathway that culminates in maintaining or changing the fate of the stem cell. This, however, appears to be far too simplistic. In the Dalton paper, evidence is presented that several signaling molecules work together in complex ways to control a variety of molecular switches that specified is a stem cell continues to divide and renew itself, or becomes a specific cell type, such as a neuron, heart muscle or skin cell.

To paint of picture of our understanding of stem cell signaling before the publication of the Dalton paper, let us take the “Wnt” signaling molecule as an example. Approximately half the published studies presented evidence that Wnt signaling molecules drove stem cells to renew themselves and not differentiate, but remain in the naïve development state. For example:
1. Cai C, Zhu X. The Wnt/β-catenin pathway regulates self-renewal of cancer stem-like cells in human gastric cancer. Mol Med Report. 2012 Feb 21. doi: 10.3892/mmr.2012.802.
2. Miki T, Yasuda SY, Kahn M. Wnt/β-catenin signaling in embryonic stem cell self-renewal and somatic cell reprogramming. Stem Cell Rev. 2011 Nov;7(4):836-46.
3. Bisson I, Prowse DM. WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics. Cell Res. 2009 Jun;19(6):683-97.
4. Shimizu T, Kagawa T, Inoue T, Nonaka A, Takada S, Aburatani H, Taga T. Stabilized beta-catenin functions through TCF/LEF proteins and the Notch/RBP-Jkappa complex to promote proliferation and suppress differentiation of neural precursor cells. Mol Cell Biol. 2008 Dec;28(24):7427-41.

However, several other papers argued just the opposite. Instead Wnt drove stem cells to differentiate and not stay in the developmentally naïve state:
1. Davidson KC, et al., Wnt/β-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4. Proc Natl Acad Sci U S A. 2012 Mar 5.
2. Li HX, Luo X, Liu RX, Yang YJ, Yang GS. Roles of Wnt/beta-catenin signaling in adipogenic differentiation potential of adipose-derived mesenchymal stem cells. Mol Cell Endocrinol. 2008 Sep 10;291(1-2):116-24.
3. Munji RN, Choe Y, Li G, Siegenthaler JA, Pleasure SJ. Wnt signaling regulates neuronal differentiation of cortical intermediate progenitors. J Neurosci. 2011 Feb 2;31(5):1676-87.
4. Kirton JP, Crofts NJ, George SJ, Brennan K, Canfield AE. Wnt/beta-catenin signaling stimulates chondrogenic and inhibits adipogenic differentiation of pericytes: potential relevance to vascular disease? Circ Res. 2007 Sep 14;101(6):581-9

Could Wnt molecules drive cells to do both differentiate and remain in the naive state? According to Dalton the answer is yes and there is a simple reason why. Dalton’s research team showed that at low concentrations, Wnt signaling keeps the stem cell in its naive, developmental, pluripotent state. However, at higher concentrations, Wnt signaling does just the opposite and drives the stem cell to stop dividing and differentiate.

However, we must avoid viewing Wnt signaling in a linear fashion because Wnt does not work alone. Other signaling molecules, such as fibroblast growth factor (FGF2), Activin A, and insulin-like growth factor (IGF), work with Wnt to modify stem cell behavior. If that doesn’t make things complicated enough, these signaling pathways can amplify or inhibit each other to cause what would be a two-fold increase under one set of conditions to become a 10-fold increase under another distinct set of conditions. The timing of cell signaling (when the cells are given the signaling molecule) also plays a crucial role with respect to the outcome.

Dalton remarked on his findings: “One of the things that surprised us was how all of the pathways ‘talk’ to each other. You can’t do anything to the IGF pathway without affecting the FGF2 pathway, and you can’t do anything to FGF2 without affecting Wnt. It’s like a house of cards; everything is totally interconnected.”

In another example, when activated, the PI3K/Akt signaling pathway maintains stem cell self-renewal, and it does so by inhibiting Raf/Mek/Erk and Wnt signal transduction pathways. The PI3K/Akt pathway also drives another signal transduction pathways called the “Activin A/Smad2,3” pathway to promote self-renewal, and this is mediated by stimulating the expression of a gene long known to be essential for stem cell self-renewal called Nanog. However, at low levels of PI3K/Akt signaling, the Wnt pathway is activated an, in combination with the Smad2,3 pathway, promotes differentiation.

Why is it that the Smad2,3 signaling proteins promote stem cell self-renewal and differentiation? When PI3K/Akt signaling decreases, the Wnt signal transduction pathway teams up with the Raf/Mek/Erk signal transduction pathway, which was suppressed by PI3K/Akt. Together, these two pathways target the protein kinase Gsk3β, which drives cells to differentiate. Thus, the signal to self-renew or differentiate revolves around Smad2,3 and the state of this signaling pathway determines if the stem cell differentiates of continues in its naïve developmental state, self-renewing with abandon.

This paper is the result of five years of generating hypotheses, testing them, and then revising the hypotheses in light of new data. This painstaking process was continued until the discrepancies were properly resolved. Fortunately, these data can provide scientists with a better grasp of that first step that stem cells might take as they differentiate. Furthermore, Dalton is quite confident that the same approach can be used to dissect and elucidate the molecular events that underlie other developmental steps that occur as the cells in an embryo divide and differentiate into more specific cell types.

Dalton sounded a hopeful note: “Hopefully this type of approach will give us a greater understanding of cells and how they can be manipulated so that we can progress much more rapidly toward the routine use of stem cells in therapeutic settings.” Dalton said.

Marion Zatz, who is chief of the Developmental and Cellular Processes Branch in the Division of Genetics and Developmental Biology at the National Institutes of Health (NIH), oversees stem cell biology grants awarded by the NIH (which partially supported Dalton’s work). Zatz made this comment about Dalton’s paper: “This work addresses one of the biggest challenges in stem cell research—figuring out how to direct a stem cell toward becoming a specific cell type. In this paper, Dr. Dalton puts together several pieces of the puzzle and offers a model for understanding how multiple signaling pathways coordinate to steer a stem cell toward differentiating into a particular type of cell. This framework ultimately should not only advance a fundamental understanding of embryonic development, but facilitate the use of stem cells in regenerative medicine.”

Dalton’s paper is truly a remarkable achievement that will allow a deeper and more accurate understanding of stem cell biology and development.