Human Intestinal Stem Cell Breakthrough for Regenerative Medicine

ScienceDaily (Sep. 4, 2011) — Human colon stem cells have been identified and grown in a petri dish in the lab for the first time. This achievement, made by researchers of the Colorectal Cancer Lab at the Institute for Research in Biomedicine (IRB Barcelona) and published in Nature Medicine, is a crucial advance towards regenerative medicine.

Throughout life, stem cells of the colon regenerate the inner layer of our large intestine in a weekly basis. For decades scientists had evidences of the existence of these cells yet their identity remained elusive. Scientists led by the ICREA Professor and researcher at the Institute for Research in Biomedicine (IRB Barcelona) Eduard Batlle discovered the precise location of the stem cells in the human colon and worked out a method that allows their isolation and in vitro expansion, that is their propagation in lab-plates (petri dishes).

Growing cells outside the body generally requires providing the cells in a petri dish with the right mix of nutrients, growth factors and hormones. But in the same way that each of the more than 200 types of cells in our body differs from the others so too do optimal growing conditions for them in the lab. Consequently, human adult stem cell culture in labs has been practically impossible until now.

Batlle's team has also established the conditions for maintain living human colon stem cells (CoSCs) outside of the human body: "This is the first time that it has been possible to grow single CoSCs in lab-plates and to derive human intestinal stem cell lines in defined conditions in a lab setting," explains the IRB Barcelona researcher Peter Jung, first author of the study together with Toshiro Sato, from the University Medical Center Utrecht in The Netherlands.

The development, published by Batlle's research group in the journal Nature Medicine, arrives after more than 10 years of intense research focused on the characterization of the biology of the intestinal stem cells and its connection with cancer. The research has been made possible by close collaboration between Batlle's team and the group led by Hans Clevers at the Hubretcht Institute and University Medical Center Utrecht in The Netherlands, and María A. Blasco at the Spanish National Cancer Research Centre in Madrid (Spain).

"For years, scientists all over the world have been trying to grow intestinal tissue in lab-plates; testing different conditions; using different nutritive media. But because the vast majority of cells in this tissue are in a differentiated state in which they do not proliferate, they survived only for a few days," explains Jung. "The aim of this study was to find a way to identify and select individual CoSCs and to grow them while maintaining their undifferentiated and proliferative state in lab conditions. Thus, we would be able to model how they grow -- in number -- and differentiate into normal intestinal epithelial cells in lab-plates," continues Jung. The scientific community now has a defined 'recipe' for isolating CoSCs and deriving stable CoSCs lines, which have the capacity to grow undifferentiated for months. In fact, "now we can maintain stem cells in a plate up to 5 months or we can induce these cells to differentiate artificially, as they do inside our bodies."

"This achievement opens up an exciting new area of research with the potential to bring about a huge breakthrough in regenerative medicine," says Jung. Regenerative medicine -- or the idea of repairing the body by developing new tissues and organs as the old ones wear out -- involves growing new cells from patients into tissues and organs in a lab. However, the main element for making regenerative medicine a reality, namely adult stem cells, are just starting to be understood. "Now that guidelines for growing and maintaining colon stem cells in the lab are in place, we have an ideal platform that could help the scientific community to determine the molecular bases of gastrointestinal cell proliferation and differentiation. It is also suspected that alterations in the biology of CoSCs are at origin of several diseases affecting the gastrointestinal tract, such as colorectal cancer or Crohn's disease, an autoimmune and inflammatory disorder. Our discovery also paves the way to start exploring this exciting field," finishes Jung.

Rapid Method of Assembling New Gene-Editing Tool Could Revolutionize Genetic Research

ScienceDaily (Apr. 9, 2012) — Development of a new way to make a powerful tool for altering gene sequences should greatly increase the ability of researchers to knock out or otherwise alter the expression of any gene they are studying. The new method allows investigators to quickly create a large number of TALENs (transcription activator-like effector nucleases), enzymes that target specific DNA sequences and have several advantages over zinc-finger nucleases (ZFNs), which have become a critical tool for investigating gene function and potential gene therapy applications.

"I believe that TALENs and the ability to make them in high throughput, which this new technology allows, could literally change the way much of biology is practiced by enabling rapid and simple targeted knockout of any gene of interest by any researcher," says J. Keith Joung, MD, PhD, associate chief for Research in the Massachusetts General Hospital (MGH) Department of Pathology and co-senior author of the report that will appear in Nature Biotechnology and has received advance online release.

TALENs take advantage of TAL effectors, proteins naturally secreted by a plant bacteria that are able to recognize specific base pairs of DNA. A string of the appropriate TAL effectors can be designed to recognize and bind to any desired DNA sequence. TALENs are created by attaching a nuclease, an enzyme that snips through both DNA strands at the desired location, allowing the introduction of new genetic material. TALENs are able to target longer gene sequences than is possible with ZFNs and are significantly easier to construct. But until now there has been no inexpensive, publicly available method of rapidly generating a large number of TALENs.

The method developed by Joung and his colleagues -- called the FLASH (fast ligation-based automatable solid-phase high-throughput) system -- assembles DNA fragments encoding a TALEN on a magnetic bead held in place by an external magnet, allowing automated construction by a liquid-handling robot of DNA that encodes as many as 96 TALENs in a single day at a cost of around $75 per TALEN. Joung's team also developed a manual version of FLASH that would allow labs without access to robotic equipment to construct up to 24 TALEN sequences a day. In their test of the system in human cells, the investigators found that FLASH-assembled TALENs were able to successfully induce breaks in 84 of 96 targeted genes known to be involved in cancer or in epigenetic regulation.

"Finding that 85 to 90 percent of FLASH-assembled TALENs have very high genome-editing activity in human cells means that we can essentially target any DNA sequence of interest, a capability that greatly exceeds what has been possible with other nucleases," says Jeffry D. Sander, PhD, co-senior author of the FLASH report and a fellow in Joung's laboratory. "The ability to make a TALEN for any DNA sequence with a high probability of success changes the way we think about gene-altering technology because now the question isn't whether you can target your gene of interest but rather which genes do you want to target and alter."

The research team also found that the longer a TALEN was, the less likely it was to have toxic effects on a cell, which they suspect may indicate that shorter TALENs have a greater probability of binding to and altering unintended gene sites. Joung notes that this supports the importance of designing longer TALENs for future research and potential therapeutic applications.