Researchers Engineer Miniature Human Livers in the Lab

Researchers Engineer Miniature Human Livers in the Lab
Sunday, 31 October 2010

Researchers at the Institute for Regenerative Medicine at Wake Forest University Baptist Medical Center have reached an early, but important, milestone in the quest to grow replacement livers in the lab. They are the first to use human liver cells to successfully engineer miniature livers that function – at least in a laboratory setting – like human livers. The next step is to see if the livers will continue to function after transplantation in an animal model.

The ultimate goal of the research, which will be presented Sunday at the annual meeting of the American Association for the Study of Liver Diseases in Boston, is to provide a solution to the shortage of donor livers available for patients who need transplants. Laboratory-engineered livers could also be used to test the safety of new drugs.

"We are excited about the possibilities this research represents, but must stress that we're at an early stage and many technical hurdles must be overcome before it could benefit patients," said Shay Soker, Ph.D., professor of regenerative medicine and project director.

"Not only must we learn how to grow billions of liver cells at one time in order to engineer livers large enough for patients, but we must determine whether these organs are safe to use in patients."

Pedro Baptista, PharmD, Ph.D., lead author on the study, said the project is the first time that human liver cells have been used to engineer livers in the lab.

"Our hope is that once these organs are transplanted, they will maintain and gain function as they continue to develop," he said.

To engineer the organs, the scientists used animal livers that were treated with a mild detergent to remove all cells (a process called de-cellularization), leaving only the collagen "skeleton" or support structure. They then replaced the original cells with two types of human cells: immature liver cells known as progenitors, and endothelial cells that line blood vessels.

The cells were introduced into the liver skeleton through a large vessel that feeds a system of smaller vessels in the liver. This network of vessels remains intact after the de-cellularization process. The liver was next placed in a bioreactor, special equipment that provides a constant flow of nutrients and oxygen throughout the organ.

After a week in the bioreactor system, the scientists documented the progressive formation of human liver tissue, as well as liver-associated function. They observed widespread cell growth inside the bioengineered organ.

The ability to engineer a liver with animal cells had been demonstrated previously. However, the possibility of generating a functional human liver was still in question.

The researchers said the current study suggests a new approach to whole-organ bioengineering that might prove to be critical not only for treating liver disease, but for growing organs such as the kidney and pancreas. Scientists at the Wake Forest Institute for Regenerative Medicine are working on these projects, as well as many other tissues and organs, and also working to develop cell therapies to restore organ function.

Bioengineered livers could also be useful for evaluating the safety of new drugs.

"This would more closely mimic drug metabolism in the human liver, something that can be difficult to reproduce in animal models," said Baptista.

Source: Wake Forest University Baptist Medical Center
Contact: Karen Richardson
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ZenMaster

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Is the Shape of a Genome as Important as Its Content?

Study shows structure exposes genes to regulation and chromosomal crosstalk
Friday, 29 October 2010

The laboratory of Ken-ichi Noma, Ph.D., an assistant

professor at the Wistar Institute, has produced the

first detailed structure of the fission yeast genome.

The researchers demonstrate how the physical

structure of the genome itself helps cells regulate

and control gene expression. Credit: Ken-ichi Noma,

Ph.D./The Wistar Institute.

If there is one thing that recent advances in genomics have revealed, it is that our genes are interrelated, "chattering" to each other across separate chromosomes and vast stretches of DNA. According to researchers at The Wistar Institute, many of these complex associations may be explained in part by the three-dimensional structure of the entire genome. A given cell's DNA spends most of its active lifetime in a tangled clump of chromosomes, which positions groups of related genes near to each other and exposes them to the cell's gene-controlling machinery. This structure, the researchers say, is not merely the shape of the genome, but also a key to how it works.

Their study, published online as a featured article in the journal Nucleic Acids Research, is the first to combine microscopy with advanced genomic sequencing techniques, enabling researchers to literally see gene interactions. It is also the first to determine the three-dimensional structure of the fission yeast genome, S. pombe. Applying this technique to the human genome may provide both scientists and physicians a completely new framework from which to better understand genes and disease, the researchers say.

"People are familiar with the X-shapes our chromosomes form during cell division, but what they may not realize is that DNA only spends a relatively small amount of time in that conformation," said Ken-ichi Noma, Ph.D., an assistant professor in Wistar's Gene Expression and Regulation program and senior author of the study.

"Chromosomes spend the majority of their time clumped together in these large, non-random structures, and I believe these shapes reflect various nuclear processes such as transcription."

To map both individual genes and the overall structure of the genome, Noma and his colleagues combined next generation DNA sequencing with a technique called chromosome conformation capture (3C). They then used fluorescent probes to pinpoint the exact location of specific genes through a microscope. With these data, the researchers were able to create detailed three-dimensional computer models of the yeast genome.

Using this novel approach, the researchers can view genes as they interact with each other. Noma and his colleagues can view where highly active genes are located, or see if genes that are turned on and off together also reside near each other in the three-dimensional structure of the genome. In total, the Wistar researchers also studied 465 so-called gene ontology groups – groups of genes that share a related purpose in the cell, such as structure or metabolism.

"When the chromosomes come together, they fold into positions that bring genes from different chromosomes near each other," Noma said.

"This positioning allows the processes that dictate how and when genes are read to operate efficiently on multiple genes at once."

This structure is not merely an accident of chemical attractions within and among the chromosomes – although that is certainly a part of the larger whole – but an arrangement guided by other molecules in the cell to create a mega-structure that dictates genetic function, Noma says. He envisions a scenario where accessory molecules, such as gene-promoting transcription factors, bind to DNA and contribute to the ultimate structure of the genome as the chromosomes fold together.

"I believe we are looking at a new way to visualize both the genome itself and the movements of all the various molecules that act on the genome," Noma said.

According to the Wistar scientists, their techniques are scalable to the human genome, even though fission yeast only has three chromosomes. In fact, the researchers found signs of "transcription factories" – clusters of related genes that are read, or "transcribed," at discrete sites – which have been proposed to exist in mammals.

Source: The Wistar Institute
Contact: Greg Lester

Reference:
Mapping of long-range associations throughout the fission yeast genome reveals global genome organization linked to transcriptional regulation
Hideki Tanizawa, Osamu Iwasaki, Atsunari Tanaka, Joseph R. Capizzi, Priyankara Wickramasinghe, Mihee Lee, Zhiyan Fu, and Ken-ichi Noma
Nucl. Acids Res., published online October 28, 2010, doi:10.1093/nar/gkq955
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
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Researchers Generate iPS Cells to Further Treatments for Lung Diseases

Researchers Generate iPS Cells to Further Treatments for Lung Diseases
Friday, 29 October 2010

A team of researchers from Boston University's Center for Regenerative Medicine and the Pulmonary Center have generated 100 new lines of human induced pluripotent stem cells (iPSC) from individuals with lung diseases, including cystic fibrosis and emphysema. The new stem cell lines could possibly lead to new treatments for these debilitating diseases. The findings, which appear in the current issue of Stem Cells, demonstrate the first time lung disease-specific iPSC have been created in a lab.

iPSCs are derived by reprogramming adult cells into a primitive stem cell state. This process results in the creation of cells that are similar to embryonic stem cells in terms of their capability to differentiate into different types of cells, including endoderm cells that can give rise to liver and lung tissue.

"iPSCs solve many major hurdles currently impacting embryonic stem cell research," said Darrell Kotton, the study's lead author and associate professor of medicine and pathology and laboratory medicine at Boston University's School of Medicine (BUSM).

iPSCs do not require embryos, and the process used to cultivate iPSCs is easier than the techniques used to obtain embryonic stem cells. iPSCs are genetically identical to the patient's cells and potentially can be transplanted back without rejection.

"In a laboratory dish, these cells have the ability to multiply indefinitely so that researchers have more time to investigate the diseased cell and correct its genes," said Kotton.

The study involved patients with different forms of lung disease – cystic fibrosis, alpha-1 antitrypsin deficiency-related emphysema, scleroderma (SSc) and sickle cell disease. The patients underwent skin biopsies and donated tissue samples, which the research team used to cultivate adult stem cells. Using a Boston University-patented vector in the form of a virus, named the Stem Cell Cassette (STEMCCA), the researchers were able to reprogram the skin cells into the primitive pluripotent stem cells known as iPSCs.

"The STEMCCA vector is proving invaluable for reprogramming cells from a variety of species, and this is the first report of the 'humanized' version of our vector for use in reprogramming human cells," said Gustavo Mostoslavsky, a co-author of the study and assistant professor of medicine at BUSM. Together Kotton and Mostoslavsky co-direct the new Boston University Center for Regenerative Medicine (CReM).

To test the differentiation power of the iPSCs, the team showed that the stem cells multiplied and could be differentiated into endoderm tissue, the natural precursor cells of the lung, the primary organ destroyed by the diseases cystic fibrosis and emphysema.

"We hope to build a bank of stem cells that could be used to help treat the two most common forms of inherited lung disease, cystic fibrosis and alpha-1 antitrypsin deficiency," said Kotton.

The next step, he said, is to correct the genetic mutations responsible for causing cystic fibrosis, emphysema and other lung diseases.

Source: Boston University Medical Center
Contact: Gina DiGravio

Reference:
Generation of Transgene-Free Lung Disease-Specific Human Induced Pluripotent Stem Cells Using a Single Excisable Lentiviral Stem Cell Cassette
Aba Somers, Jyh-Chang Jean, Cesar A. Sommer, Amel Omari, Christopher C. Ford, Jason A. Mills, Lei Ying, Andreia Gianotti Sommer, Jenny M. Jean, Brenden W. Smith, Robert Lafyatis, Marie-France Demierre, Daniel J. Weiss, Deborah L. French, Paul Gadue, George J. Murphy, Gustavo Mostoslavsky and Darrell N. Kotton
Stem Cells Vol. 28, Issue 10, pp. 1728–1740, October 2010, DOI: 10.1002/stem.495
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ZenMaster

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