Human Embryonic Stem Cell Communication Network Discovered

Cells use a variety of mechanisms to talk to each other. These signaling pathways are called “signal transduction” pathways, and they vary extensively from one cell type to another.

Therefore, it should be no surprise that human embryonic stem cells signal to each other. The precise signal transduction pathway that human embryonic stem cells use to communicate with each other is the subject of a research project from a laboratory in Singapore.

Human embryonic stem cells or hESCs can differentiate into any adult cell type. The factors that keep hESCs in their pluripotent state are of interest to stem cell scientists because they might allow them to better direct the differentiation of hESCs or even grow them in culture better.

Cell-to-cell communication is vitally important to multicellular organisms. The coordinated development of tissues in the embryo that culminate in the formation of specific organs requires that cells receive signals and respond accordingly. If there are errors in these signals, the cells will respond differently and the embryo will either be grossly abnormal, or the cell might divide uncontrollably to make a tumor.

Human ESCs communicate by means of a signal transduction pathway known as the extracellular regulated kinase or ERK pathway.  The ERK signal transduction pathway begins with the binding of a growth factor receptor by a growth factor.  These growth factors are almost always bound to the extracellular matrix, which is the goo that surrounds cells and provides a structure in which the cells live.  The binding of the receptor causes the receptor to pair with another copy of itself, and that activates the bits of the receptor found inside the cell (tyrosine kinase domain for the interested).  The activated receptor attaches phosphates to itself, which causes particular proteins to find and bind the receptor, which recruits particular proteins to the cell membrane.  One of the recruited proteins is a protein kinase called RAF.  RAF attaches phosphate groups to the protein kinase MEK, and MEK attaches phosphate groups to the protein kinase ERK.  Once ERK has a phosphate attached to it, it can move into the nucleus and regulate transcription factors involved in the control of gene expression.  Thus a phenomenon that began at the cell membrane culminates in a change in gene expression.


Stem cell scientists a A*STAR’s Genomic Institute of Singapore and the Max Planck Institute of Molecular Genetics (MPIMG) in Berlin, Germany studied how genetic information is accessed in hESCs. To do this they mapped the kinase interactions across the entire human genome (kinases are enzymes that attach phosphate groups to other molecules) and discovered that ERK2, a protein that belongs to the ERK signal transduction pathway targets important sites such as non-coding genes, and histones, cell cycle, metabolism, and stem cell-specific genes.

The ERK signaling pathway involves an additional protein called ELK1 that interacts with ERK2. However, this research team discovered that ELK1 has a second, totally opposite function. At genomic sites not targeted by ERK signaling, ELK1 silences genetic information, which keeps the cell in its undifferentiated state.

ELK1 Interaction with ERK2

The authors propose a model that integrates this bi-directional control to keep the cell in the stem cell state, in which genes necessary for differentiation are repressed by ELK1 that is not associated with ERK2, and cell-cycle, translation and other pluripotency genes are activated by ELK1 in association with ERK2 or ERK2 plus other transcription factors.

Model of the Transcriptional Regulatory Network of ERK2 Signaling in hESCsTranscription factors such as ELK1 link ERK2 to sequence-specific regulation of gene expression. ERK2 and ELK1 colocalization defines three distinct modules that target different sets of genes. In this model, combinatorial binding of ERK2 and ELK1 with transcription factors, chromatin regulators, and the basal transcriptional machinery integrates external signaling into the cell-type-specific regulatory network. In hESCs, ERK2 and ELK1 participate in the regulation of pluripotency and self-renewal pathways, whereas differentiation genes are repressed.
Model of the Transcriptional Regulatory Network of ERK2 Signaling in hESCsTranscription factors such as ELK1 link ERK2 to sequence-specific regulation of gene expression. ERK2 and ELK1 colocalization defines three distinct modules that target different sets of genes. In this model, combinatorial binding of ERK2 and ELK1 with transcription factors, chromatin regulators, and the basal transcriptional machinery integrates external signaling into the cell-type-specific regulatory network. In hESCs, ERK2 and ELK1 participate in the regulation of pluripotency and self-renewal pathways, whereas differentiation genes are repressed.

First author Jonathan Göke from Stem Cell and Developmental Biology at the GIS said, “The ERK signaling pathway has been known for many years, but this is the first time we are able to see the full spectrum of the response in the genome of stem cells. We have found many biological processes that are associated with this signaling pathway, but we also found new and unexpected patterns such as this dual-mode of ELK1. It will be interesting to see how this communication network changes in other cells, tissues, or in disease.”

A co-author of this study, Martin Vingron said, “A remarkable feature of this study is, how information was extracted by computational means from the data.”

Professor Ng Huck Hui, managing author of this paper, added, “This is an important study because it describes the cell’s signaling network and its integration into the general regulatory network. Understanding the biology of embryonic stem cells is a first step to understanding the capabilities and caveats of stem cells in future medical applications.”

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.