Purification of signaling protein may boost tissue engineering

April 28, 2003

The purification of a powerful signaling molecule that coaxes cells to mature may also signal the beginning of a new era in tissue engineering.

In newly published studies in the journal Nature, researchers show that purified Wnt protein, long known as a potent trigger of development and cell proliferation, can also cause blood-forming stem cells to proliferate.

The discovery suggests novel ways to enhance stem cells to restore the blood-forming systems of cancer patients whose cells have been destroyed by chemotherapy.

The research on Wnt activation and its role promoting the development of blood-forming (hematopoietic) stem cells was reported in two related articles published online on April 28, 2003, in Nature.

Howard Hughes Medical Institute investigator Roel Nusse and Karl Willert at Stanford University School of Medicine led one team that reported that the addition of a lipid molecule is necessary for the activation of Wnt proteins. Co-authors on that article include Jeff Brown and Esther Danenberg, as well as researchers in the laboratory of Irving Weissman, also of Stanford Medical School. Weissman led a second group of scientists who reported in Nature that activated Wnt is crucial for self-renewal of hematopoietic stem cells. Lead authors on that paper were Tannishtha Reya and Andrew Duncan, now at Duke University Medical Center.

According to Nusse, researchers had not isolated active Wnt proteins before -- a long sought-after goal since the proteins play important roles in embryonic development and in controlling the proliferation of stem cells. Nusse and Harold Varmus, President of Memorial Sloan-Kettering Cancer Center and a member of the HHMI medical advisory board, were members of one of the research teams that discoverered Wnt genes in 1982, finding them as oncogenes activated in mouse breast cancer.

Stem cells are immature cells that have the capability to mature into a wide range of blood and tissue cells. They may to hold the key to restoring compromised immune systems and even regenerating tissues and organs damaged by disease or trauma. Even though such stem cells have been isolated, inducing them to proliferate for use in treatment has been only marginally successful.

"It has always been predicted that the Wnt proteins could act as growth factors that could be added to cells -- and without genetically changing the cells, directing them into a particular growth pathway," said Nusse. Central to this process, he said, has been purifying and characterizing the active form of Wnt proteins, which has proven especially frustrating.

"We now understand that this protein is modified by the attachment of a lipid after it is produced, which gives it a tendency to stick to cell membranes where it is active," said Nusse. "However, that means the protein also was not soluble and would stick to containers, so standard purification techniques didn't work."

Nusse and his colleagues developed techniques that used detergents to render the protein soluble, and also robust cell assays that would measure the biological activity of the protein. "It was a real challenge to get the methods to work," said Nusse, "requiring all kinds of biochemical tricks."

In their latest work, Nusse and his colleagues showed that one member of the Wnt protein family, mouse Wnt3a, is activated by the attachment of a lipid called palmitoyl to a particular amino acid on the protein. The lipid is necessary for activation, as is the presence of the attached amino acid, cysteine, which is conserved in all Wnt proteins, said Nusse. The researchers may well have found the key mechanism of activation for the multitude of Wnt proteins.

Next, Nusse, Weissman and their colleagues tested the effects of the activated Wnt protein on hematopoietic stem cells. They found that the protein greatly enhanced stem cell proliferation in the test tube, while maintaining the stem cells in their immature state. The researchers also observed that Wnt-treated stem cells retained their activity and were able to reconstitute the blood-forming systems of mice that had been irradiated to destroy their hematopoietic cells.

The accompanying Nature article by Reya, Duncan, Weissman and their colleagues demonstrated that the Wnt signaling pathway plays a crucial role in hematopoietic stem cell self-renewal. The researchers showed that the protein specifically affected by Wnt -- beta catenin -- is necessary for stem cell proliferation, as is the Wnt protein itself.

"With these studies, we can now imagine isolating and expanding a patient's stem cells using activated Wnt proteins before they are treated with chemotherapy which destroys their immune system," said Nusse. "Those proliferated cells could then provide a powerful way to restore the hematopoietic system. And since Wnt is a specific growth factor and doesn't fundamentally alter the nature of the cells, there is no danger that the cells will take on unwanted properties."

According to Nusse, over-activation of the Wnt signaling pathway due to genetic mutation has been implicated in some cancers. Thus, he said, discovery of the nature of Wnt activation in cells will enable researchers to mimic the cancer process experimentally, to study its mechanism.

Nusse and his colleagues are now studying other Wnt proteins to determine whether they require the same mechanism for activation and whether they, too, can trigger proliferation of other types of stem cells. In some ways, Nusse and his colleagues have a head start - in collaboration with HHMI investigator Jeremy Nathans at The Johns Hopkins University School of Medicine they have already identified the specific cell surface receptors, known as Frizzleds, to which the Wnt proteins bind to activate the cells.

"We're trying also to understand why cells respond to particular Wnt proteins by looking at the expression of the specific receptor for the Wnts," said Nusse.

Howard Hughes Medical Institute

Related Stem Cells Articles from Brightsurf:

SUTD researchers create heart cells from stem cells using 3D printing
SUTD researchers 3D printed a micro-scaled physical device to demonstrate a new level of control in the directed differentiation of stem cells, enhancing the production of cardiomyocytes.

More selective elimination of leukemia stem cells and blood stem cells
Hematopoietic stem cells from a healthy donor can help patients suffering from acute leukemia.

Computer simulations visualize how DNA is recognized to convert cells into stem cells
Researchers of the Hubrecht Institute (KNAW - The Netherlands) and the Max Planck Institute in Münster (Germany) have revealed how an essential protein helps to activate genomic DNA during the conversion of regular adult human cells into stem cells.

First events in stem cells becoming specialized cells needed for organ development
Cell biologists at the University of Toronto shed light on the very first step stem cells go through to turn into the specialized cells that make up organs.

Surprising research result: All immature cells can develop into stem cells
New sensational study conducted at the University of Copenhagen disproves traditional knowledge of stem cell development.

The development of brain stem cells into new nerve cells and why this can lead to cancer
Stem cells are true Jacks-of-all-trades of our bodies, as they can turn into the many different cell types of all organs.

Healthy blood stem cells have as many DNA mutations as leukemic cells
Researchers from the Princess Máxima Center for Pediatric Oncology have shown that the number of mutations in healthy and leukemic blood stem cells does not differ.

New method grows brain cells from stem cells quickly and efficiently
Researchers at Lund University in Sweden have developed a faster method to generate functional brain cells, called astrocytes, from embryonic stem cells.

NUS researchers confine mature cells to turn them into stem cells
Recent research led by Professor G.V. Shivashankar of the Mechanobiology Institute at the National University of Singapore and the FIRC Institute of Molecular Oncology in Italy, has revealed that mature cells can be reprogrammed into re-deployable stem cells without direct genetic modification -- by confining them to a defined geometric space for an extended period of time.

Researchers develop a new method for turning skin cells into pluripotent stem cells
Researchers at the University of Helsinki, Finland, and Karolinska Institutet, Sweden, have for the first time succeeded in converting human skin cells into pluripotent stem cells by activating the cell's own genes.

Read More: Stem Cells News and Stem Cells Current Events
Brightsurf.com is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com.