Stem Cell Transplants Help Kidney Damage

Stem Cell Transplants Help Kidney Damage
Tuesday, 15 February 2011

Transplanting autologous renal progenitor cells (RPCs), (kidney stem cells derived from self-donors), into rat models with kidney damage from pyelonephritis - a type of urinary infection that has reached the kidney - has been found to improve kidney structure and function.

The study, authored by a research team from the Tehran University of Medical Sciences, is published in the current issue of Cell Medicine.

"Advancements in stem cell therapies and tissue engineering hold great promise for regenerative nephrology," said Dr. Abdol-Mohammad Kajbafzadeh, corresponding author.

"Our RPC transplant study demonstrated benefits for pyelonephritis, a disease characterized by severe inflammation, renal function impairment and eventual scarring, and which remains a major cause of end-stage-renal disease worldwide."

The researchers divided 27 rats into three groups, two of which were modelled with an induced pyelonephritis in their right kidneys, while the third group did not have induced disease. RPCs were obtained from the diseased animals' left kidneys and injected into the right kidney six weeks later. Two weeks after injection, tubular atrophy was reduced. After four weeks, fibrosis was reduced and after sixty days, right renal tissue integrity was "significantly improved."

"We propose that kidney augmentation was mainly due to functional tissue regeneration following cellular transplantation," said Dr. Kajbafzadeh.

"Kidney-specific stem/progenitor cells might be the most appropriate candidates for transplantation because of their inherent organ-specific differentiation and their capacity to modulate tissue remodelling in chronic nephropathies."

The researchers concluded that because renal fibrosis is a common and ultimate pathway leading to end-stage renal disease, amelioration of fibrosis might be of major clinical relevance.

"Transplanting RPCs showed the potential for partial augmentation of kidney structure and function in pyelonephritis," said Dr. Kajbafzadeh.

"This is one of the first studies to demonstrate improved renal function after cell transplantation. The translation of this study into larger clinical models will be very relevant to validate the success of this small animal study." said Dr. Amit Patel, Section Editor Cell Medicine, Associate Professor of Surgery, University of Utah.

Source: Florida Science Communications
Contact: Randolph Fillmore

Reference:
Autografting of Renal Progenitor Cells Ameliorates Kidney Damage in Experimental Model of Pyelonephritis
Kajbafzadeh, A-M.; Elmi, A.; Talab, S. S.; Sadeghi, Z.; Emami, H.; Sotoudeh, M.
Cell Med. 1(3): 115-122; 2010
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Gene Regulation Mechanism Unique to Primates Discovered

Study finds long-known, but little-understood DNA elements serve important purpose
Thursday, 10 February 2011

Scientists have discovered a new way genes are regulated that is unique to primates, including humans and monkeys. Though the human genome – all the genes that an individual possesses – was sequenced 10 years ago, greater understanding of how genes function and are regulated is needed to make advances in medicine, including changing the way we diagnose, treat and prevent a wide range of diseases.

"It's extremely valuable that we've sequenced a large bulk of the human genome, but sequence without function doesn't get us very far, which is why our finding is so important," said Lynne E. Maquat, Ph.D., lead author of the new study published today in the journal Nature.

When our genes go awry, many diseases, such as cancer, Alzheimer's and cystic fibrosis can result. The study introduces a unique regulatory mechanism that could prove to be a valuable treatment target as researchers seek to manipulate gene expression – the conversion of genetic information into proteins that make up the body and perform most life functions – to improve human health.

The newly identified mechanism involves Alu elements, repetitive DNA elements that spread throughout the genome as primates evolved. While scientists have known about the existence of Alu elements for many years, their function, if any, was largely unknown.

Maquat discovered that Alu elements team up with molecules called long noncoding RNAs (lncRNAs) to regulate protein production. They do this by ensuring messenger RNAs (mRNAs), which take genetic instructions from DNA and use it to create proteins, stay on track and create the right number of proteins. If left unchecked, protein production can spiral out of control, leading to the proliferation or multiplication of cells, which is characteristic of diseases such as cancer.

"Previously, no one knew what Alu elements and long noncoding RNAs did, whether they were junk or if they had any purpose. Now, we've shown that they actually have important roles in regulating protein production," said Maquat, the J. Lowell Orbison Chair, professor of Biochemistry and Biophysics and director of the Center for RNA Biology at the University of Rochester Medical Center.

The expression of genes that call for the development of proteins involves numerous steps, all of which are required to occur in a precise order to achieve the appropriate timing and amount of protein production. Each of these steps is regulated, and the pathway discovered is one of only a few pathways known to regulate mRNAs directly in the midst of the protein production process.

Regulating mRNAs is one of several ways cells control gene expression, and researchers from institutions and companies around the world are honing in on this regulatory landscape in search of new ways to manage and treat disease.

"This new mechanism is really a surprise. We continue to be amazed by all the different ways mRNAs can be regulated," according to Maquat.

Maquat and the study's first author, Chenguang Gong, a graduate student in the Department of Biochemistry and Biophysics at the Medical Center, found that long noncoding RNAs and Alu elements work together to trigger a process known as SMD (Staufen 1-mediated mRNA decay). SMD conditionally destroys mRNAs after they orchestrate the production of a certain amount of proteins, preventing the creation of excessive, unwanted proteins in the body that can disrupt normal processes and initiate disease.

Specifically, long noncoding RNAs and Alu elements recruit the protein Staufen-1 to bind to numerous mRNAs. Once an mRNA finishes directing a round of protein production, Staufen-1 works with another regulatory protein previously identified by Maquat, UPF1, to initiate the degradation or decay of the mRNA so that it cannot create any more proteins.

While the research fills in a piece of the puzzle as to how our genes operate, it also accentuates the overwhelming complexity of how our DNA shapes us and the many known and unknown players involved. Maquat and Gong plan on exploring the newly identified pathway in future research.

Source: University of Rochester Medical Center
Contact: Emily Boynton

Reference:
lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3′ UTRs via Alu elements
Chenguang Gong & Lynne E. Maquat
Nature Vol. 470, 284–288, (10 February 2011), doi:10.1038/nature09701
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Cell Reprogramming Leaves a 'Footprint' Behind

Cell Reprogramming Leaves a 'Footprint' Behind
Wednesday, 09 February 2011

Reprogramming adult cells to recapture their youthful "can-do-it-all" attitude appears to leave an indelible mark, found researchers at the Salk Institute for Biological Studies. When the team, led by Joseph Ecker, PhD., a professor in the Genomic Analysis Laboratory, scoured the epigenomes of so-called induced pluripotent stem cells base by base, they found a consistent pattern of reprogramming errors.

What's more, these incompletely or inadequately reprogrammed hotspots are maintained when iPS cells are differentiated into a more specialized cell type, providing what the researchers dubbed an iPS cell-specific signature.

"We can tell by looking at these hotspots whether a cell is an iPS cell or an embryonic stem cell," says Ecker.

"But we don't know yet what it means for their self-renewal or differentiation potential."

Their findings, published in the February 3, 2011, issue of Nature, confirm that iPS cells, which by all appearances look and act like embryonic stem cells, differ in certain aspects from their embryonic cousins, emphasizing that further research will be necessary before they can rightfully take embryonic stem cells' place.


The reprogramming process leaves

indelible marks in the methylation profile

of induced pluripotent stem cells.

Credit: Courtesy of Dr. Ryan Lister, Salk

Institute for Biological Studies.

The fact that reprogramming of somatic (body) cells does not pose the same ethical quandaries as working with stem cells isolated from embryos prompted scientists to develop iPS technology for human cells that are just as potent as human embryonic stem cells, with the hope that one day, iPS cell technology can be applied to regenerative medicine.
However, before cells derived from iPS cells can be used to repair tissue damaged through disease or injury, some remaining questions have to be solved.

"Embryonic stem cells are considered the gold standard for pluripotency," says Ecker.

"So we need to know whether — and if so, how — iPS cells differ from ES cells."

The reprogramming process, which turns back the clock and endows fully differentiated cells with pluripotent potential, is not a genetic transformation but an epigenomic one. The epigenome is what differentiates a fibroblast from a hepatocyte and a stem cell from a fully differentiated cell. With a few exceptions, every cell in our body contains the same genome, but epigenomic marks — tiny tags atop DNA that can tell your genes to turn on or off, to speak up or speak softly — determine a cell's gene expression profile and hence its fate.

While others have compared genomic bits and pieces between iPS and embryonic stem cells — and found small differences — the Salk researchers and their collaborators at the University of Wisconsin and the University of California, San Diego, set their sights higher.

They scrutinized whole-genome DNA methylation profiles — methylation is one the best-studied and most important epigenetic tags — at single-base resolution in five iPS cell lines, along with the methylomes of embryonic stem cells and somatic cells and differentiated iPS cells and differentiated embryonic stem cells.

Reprogramming induces a remarkable wholesale reconfiguration of the DNA methylation pattern throughout the genome, returning partially methylated domains to a fully methylated state; reinstating so-called non-CG methylation; and reprogramming most un-methylated and methylated CG islands, which play a crucial role in regulation gene activity, to an embryonic stem cell-like state.

"Overall, this process results in an iPS cell methylation pattern that's very similar to that of embryonic stem cells," says postdoctoral researcher and co-first author Ryan Lister.

"But when we started to dig deeper, we discovered significant differences."

Their experiments revealed considerable variability between iPS cell lines, including a "memory" of their tissue of origin.

"Some marks carry over," explains Ecker.

"If iPS cells were derived from adipose tissue, we can see that they ‘remember’ some methylation marks from being a fat cell."

Furthermore, new methylation patterns not found in either embryonic stem cells or the tissues of origin were identified in the iPS cells, and many of the regions showing epigenomic changes were disrupted in all iPS lines studied.

But regardless of their individual history, iPS cells showed a common defect — hotspots near telomeres and centromeres that proved resistant to reprogramming. Averaging more than one million bases in length, these hotspots failed to acquire the non-CG methylation typical of embryonic stem cells.

"These regions are really signatures," explains postdoctoral researcher and co-first author Mattia Pelizzola.

"They are shared in iPS cells derived from different parental cells, by different research groups and using different methodologies. Moreover, these regions coincide with specific modifications of histones — proteins that are important to determine the accessibility and the activity of genomic regions — and the genes contained within these regions are less expressed."

However, when the researchers zoomed in closer, they found that the opposite held true for CG islands, short stretches of CG-rich DNA sequences that are typically found in the proximity of genes, where they may regulate gene activity.

"The consequence is that some genes within these areas seem to be silenced by the altered CG island methylation patterns in the iPS cells," says Lister.

"Conceivably, these changes could limit the potential fate of the iPS cells."

To gain a better understanding of the implications, they looked again at these regions after differentiating embryonic stem cells and iPS cells into trophoblasts, a standard cell differentiation assay. A subset of iPS cell-specific silencing marks was transmitted to differentiated cells at high frequency.

"They are not easily removed," says Lister, "and could be used as a diagnostic marker for incomplete reprogramming."

Adds Ecker: "Now that we know that these regions exist, we want to understand why these regions can't be reprogrammed to a more ES cell-like state."

Source: Salk Institute
Contact: Gina Kirchweger

Reference:
Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells
Ryan Lister, Mattia Pelizzola, Yasuyuki S. Kida, R. David Hawkins, Joseph R. Nery, Gary Hon, Jessica Antosiewicz-Bourget, Ronan O’Malley, Rosa Castanon, Sarit Klugman, Michael Downes, Ruth Yu, Ron Stewart, Bing Ren, James A. Thomson, Ronald M. Evans, & Joseph R. Ecker
Nature, 02 February 2011, doi:10.1038/nature09798
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For Stem Cells, A Way to Keep Score

For Stem Cells, A Way to Keep Score
Wednesday, 09 February 2011

Ever since researchers devised a recipe for turning adult cells into cells that look and act like embryonic stem cells, there has been lingering doubt in the field about just how close to embryonic stem cells each of those cell lines really is at a molecular and functional level. Now, researchers reporting in the February 4th issue of the journal Cell, have developed a systematic way to lay those doubts about quality to rest.

They have devised a method to quickly and comprehensively characterize those cells using a series of genomic assays, ultimately assigning a scorecard to each. The effort has also yielded genome-wide reference maps detailing the epigenetic and gene expression landscapes for several of the so-called induced pluripotent stem cells (iPS) and embryonic stem cell lines that can now be used for comparison by anyone studying an iPS or embryonic cell line. (Epigenetics refers to chemical modifications to DNA that can alter the expression of genes without changing the underlying sequence.)

The advance is critical for the future use of iPS cells in the study of disease, for cell-based drug screening and as a renewable source of cells for transplantation medicine, the researchers say. Overall, the news is quite positive.

"The exciting thing is that this tells us something fundamental about what these cells are," said Kevin Eggan of Harvard University and the Howard Hughes Medical Institute.

"It appears that the version 1.0 reprogramming technology is actually pretty good."

In a careful comparison of 20 previously derived human embryonic stem cell lines and 12 human iPS cell lines, the new method does turn up some variation. But the variation observed for many iPS cell lines falls within that seen for embryonic stem cells as well. It appears that the quality of many of those iPS cell lines is high, depending in part on what they might be used for.

"There are different grades of quality," said Alexander Meissner of the Broad Institute and Harvard University.

"A particular line might be bad for one purpose but great for an alternative one. This gives us a more refined view."

"Beauty will be in the eye of the beholder," Eggan added. Certain lines might be really good at making one type of tissue, but not as good for making another. Now researchers will have a more accessible way to make that determination reliably without having to rely on guesswork.

Eggan and Meissner report that they were able to assess the similarities and differences among lines and to use those assessments to determine which lines should be avoided for a particular purpose. They were also able to predict the efficiency with which a given iPS cell line used in an independent study would differentiate into motor neurons.

There are methods for assessing iPS cell quality today, but those are rather time consuming and would not be suitable as iPS cell technology is scaled up for broader use. The gold standard assay today involves injecting cells into an immune compromised mouse and allowing those cells to grow and divide for one to three months to see whether they successfully differentiate into all the cell types.

"It takes months and it's fairly subjective," Meissner said.

The new method takes just one or two weeks to complete and is much more quantitative. It's possible the assays could be made to go even faster, the researchers said, particularly if industry were to devise a user-friendly kit. Ultimately, this series of assays or other methods along the same lines might be used clinically.

In addition to its practical use for selecting the best cells for a particular task, the findings and scoring method will also offer new insights into the cells themselves, the researchers said.

"We can begin to get to the bottom of the differences among cell lines and their root causes," Eggan said.

Source: Cell Press
Contact: Elisabeth Lyons

Reference:
Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines
Christoph Bock, Evangelos Kiskinis, Griet Verstappen, Hongcang Gu, Gabriella Boulting, Zachary D. Smith, Michael Ziller, Gist F. Croft, Mackenzie W. Amoroso, Derek H. Oakley, Andreas Gnirke, Kevin Eggan, Alexander Meissner
Cell, Volume 144, Issue 3, 439-452, 4 February 2011, 10.1016/j.cell.2010.12.032
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Multiple Genome Sequencing Yields Detailed Map of Structural Variants Behind Our Genetic Differences

Boston College bioinformatics lab's analysis supports 1000 Genomes Project
Wednesday, 09 February 2011

Analyzing billions of pieces of genetic data collected from people around the world, Boston College biologist Gabor Marth and his research team are playing an integral role in the global effort to sequence 1000 genomes and move closer to understanding in fine detail how genetics influence human health and development.

The most comprehensive map to date of genomic structural variants – the layer of our DNA that begins to distinguish us from one another – has been assembled by analyzing 185 human genomes, Marth and co-authors from the 1000 Genomes Project team report in the Feb. 3 edition of the journal Nature.

The complexity of the 1000 Genome Project draws on a range of expertise in the Marth bioinformatics lab, which receives volumes of data produced by other project teams using DNA sequencing technology, stores the data, and then analyzes it using proprietary computer software programs the Marth lab has developed.

"The tools we have developed are being used to discover a biological reality that we could not see before," said Marth, an associate professor of biology whose group is one of the lead analytics units for the 1000 Genomes Project.

"There are many challenges and the work is very exciting."

The goal is to understand the genetic make up of the earth's population by analyzing genome data from as many as 2,500 individuals in order to provide new insights into the development of the human race and to understand the links between the genome and human health.

"We are working with some of the world's best research groups," said Marth, joined as a co-author on the paper with his BC colleagues Research Assistant Professor Chip Stewart and doctoral candidates Deniz Kural and Jiantao Wu.

"There are engineering, mathematical, and algorithmic challenges at every level," Marth added.

"We work to make sure our computational tools are performing well, make continuous improvements and process data in a timely fashion to send to our colleagues around the world."

The researchers report in Nature the generation of a map of structural variants – those pieces of genetic code that are the base layer of instructions, also known as the genotype, that ultimately determine our outward appearances and characteristics, or phenotypes. The new map is built upon a range of structural variants, including 22,025 deletions, or missing pieces of DNA, and 6,000 insertions, pieces of DNA that have been added along the evolutionary journey, and tandem duplications.

The analysis has produced new insights into genetic selection, the introduction of large structural variants into DNA and structural variant "hotspots" formed by common biological mechanisms, the team reports in Nature. The map will play a crucial role in sequencing-based association studies, where this new understanding of human variation is applied to unlocking new ways to use the genome to understand the world's population and to inform the life and medical sciences.

"The eventual goal of studying the genotype is so we can understand how the specific genetic make-up of an individual is responsible for an individual phenotype, such as height or weight or susceptibility to disease," said Marth.

"The specific question of the 1000 Genome Project is how much divergence, or how much genetic variation, exists within different populations. That is the question we are trying to unravel."

Source: Boston College
Contact: Ed Hayward

Reference:
Mapping copy number variation by population-scale genome sequencing
Ryan E. Mills, Klaudia Walter, Chip Stewart, Robert E. Handsaker, Ken Chen, Can Alkan, Alexej Abyzov, Seungtai Chris Yoon, Kai Ye, R. Keira Cheetham, Asif Chinwalla, Donald F. Conrad, Yutao Fu, Fabian Grubert, Iman Hajirasouliha, Fereydoun Hormozdiari, Lilia M. Iakoucheva, Zamin Iqbal, Shuli Kang, Jeffrey M. Kidd, Miriam K. Konkel, Joshua Korn, Ekta Khurana, Deniz Kural, Hugo Y. K. Lam, Jing Leng, Ruiqiang Li, Yingrui Li, Chang-Yun Lin, Ruibang Luo, Xinmeng Jasmine Mu, James Nemesh, Heather E. Peckham, Tobias Rausch, Aylwyn Scally, Xinghua Shi, Michael P. Stromberg, Adrian M. Stütz, Alexander Eckehart Urban, Jerilyn A. Walker, Jiantao Wu, Yujun Zhang, Zhengdong D. Zhang, Mark A. Batzer, Li Ding, Gabor T. Marth, Gil McVean, Jonathan Sebat, Michael Snyder, Jun Wang, Kenny Ye, Evan E. Eichler, Mark B. Gerstein, Matthew E. Hurles, Charles Lee, Steven A. McCarroll, Jan O. Korbel & 1000 Genomes Project
Nature, 03 February 2011, 470, 59–65, doi:10.1038/nature09708
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MicroRNA Cocktail Helps Turn Skin Cells into Stem Cells

New technique removes several hurdles in generating induced pluripotent stem (iPS) cells, smoothing the way for disease research and drug development.
Monday, 07 February 2011

Stem cells are ideal tools to understand disease and develop new treatments; however, they can be difficult to obtain in necessary quantities. In particular, generating induced pluripotent stem (iPS) cells can be an arduous task because reprogramming differentiated adult skin cells into iPS cells requires many steps and the efficiency is very low – researchers might end up with only a few iPS cells even if they started with a million skin cells. A team at Sanford-Burnham Medical Research Institute (Sanford-Burnham) set out to improve this process. In a paper published February 1 in The EMBO Journal, the team identified several specific microRNAs (miRNAs) that are important during reprogramming and exploited them to make the transition from skin cell to iPS cell more efficient.

"We identified several molecular barriers early in the reprogramming process and figured out how to remove them using miRNA," said Tariq Rana, Ph.D., director of the RNA Biology program at Sanford-Burnham and senior author of the study.

"This is significant because it will enhance our ability to use iPS cells to model diseases in the laboratory and search for new therapies."

"Our study not only presents new mechanistic insights about the role of non-coding RNAs during somatic cell reprogramming but also provides proof of principle using microRNAs as great enhancers for iPS cell generation," added Zhonghan Li, graduate student and first author of the study.

miRNAs are small strands of genetic material that may play a major role in many diseases by gumming up protein production. In this study, Dr. Rana and his colleagues observed that three groups of miRNAs, including two known individually as miR-93 and miR-106b, are activated as part of a defence mechanism that occurs when cells are stressed by the standard skin cell reprogramming process. Digging deeper, they determined that miR-93 and miR-106b target two proteins called Tgfbr2 and p21, which slow up the path to iPS cells by halting the cell cycle – the cell's process of duplicating its DNA and dividing into two identical "daughter" cells – and promoting cell death.

Not only does this finding reveal more about the genetic underpinnings of iPS cell formation, but the researchers took advantage of this new information to speed up the process. When they added extra miR-93 and miR-106b to skin cells, Tgfbr2 and p21 were blocked, more cells survived, and iPS cells were more readily obtained.

"In some respects, this work may be regarded as a landmark contribution to the field of stem cell biology in general and cellular reprogramming in particular," said Evan Y. Snyder, M.D., Ph.D., director of Sanford-Burnham's Stem Cells and Regenerative Biology program.

"Up until now, cellular differentiation and de-differentiation has focused principally on the expression of genes; this work indicates that the strategic non-expression of genes may be equally important. The work has demonstrated that miRNAs do function in the reprogramming process and that the generation of iPSCs can be greatly enhanced by modulating miRNA action. In addition to helping us generate better tools for the stem cell field, such findings inevitably facilitate our understanding of normal and abnormal stem cell behaviour during development and in disease states."

Source: Sanford-Burnham Medical Research Institute
Contact: Josh Baxt

Reference:
Small RNA-mediated regulation of iPS cell generation
Li Z, Yang CS, Nakashima K, Rana TM
The EMBO Journal. February 1, 2011, doi:10.1038/emboj.2011.2
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Researchers Develop Safer Way to Make Induced Pluripotent Stem Cells

Researchers Develop Safer Way to Make Induced Pluripotent Stem Cells
Monday, 07 February 2011

Researchers at Johns Hopkins have found a better way to create induced pluripotent stem (iPS) cells — adult cells reprogrammed with the properties of embryonic stem cells — from a small blood sample. This new method, described last week in Cell Research, avoids creating DNA changes that could lead to tumour formation.

"These iPS cells are much safer than ones made with previous technologies because they don't involve integrating foreign viruses that can potentially lead to uncontrolled, cancerous cell growth," says Linzhao Cheng, Ph.D., an associate professor of medicine in the Division of Hematology and a member of the Johns Hopkins Institute of Cell Engineering.

"This is important if iPS cells are to be used as therapies one day."

Cheng says the higher-quality iPS cells will also be more reliable in research studies, "since we don't have to worry about extra genetic changes associated with previous technologies interfering with study results."

Johns Hopkins researchers created the safer iPS cells by transferring a circular piece of DNA into blood cells from anonymous donors to deliver the needed genetic components. The traditional way is to use viruses to carry DNA into a cell's genome. Unlike the viral methods, the circular DNA the Hopkins team used is designed to stay separate from the host cell's genome. After the iPS cells formed, the circular DNA delivered into the blood cells was gradually lost.

Using about a tablespoon of human adult blood or umbilical cord blood, the researchers grew the blood cells in the lab for eight to nine days. The researchers then transferred the circular DNA into the blood cells, where the introduced genes turned on to convert the blood cells to iPS cells within 14 days.

The research group verified conversion from mature blood cells to iPS cells by testing their ability to behave like stem cells and differentiate into other cell types, such as bone, muscle or neural cells. They also looked at the DNA from a dozen iPS cell lines to make sure there were no DNA rearrangements.

Cheng says the new method is also more efficient than the traditional use of skin cells to make iPS cells.

"After a skin biopsy, it takes a full month to grow the skin cells before they are ready to be reprogrammed into iPS cells, unlike the blood cells that only need to grow for eight or nine days," says Cheng.

"The time it takes to reprogram the iPS cells from blood cells is also shortened to two weeks, compared to the month it takes when using skin cells."

Cheng says "this easy method of generating integration-free human iPS cells from blood cells will accelerate their use in both research and future clinical applications."

Source: Johns Hopkins Medical Institutions
Contact: Vanessa McMains
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UCLA Researchers Eliminate Major Roadblock in Regenerative Medicine

New 'cocktails' support long-term maintenance of human embryonic stem cells
Monday, 07 February 2011

In regenerative medicine, large supplies of safe and reliable human embryonic stem (hES) cells are needed for implantation into patients, but the field has faced challenges in developing cultures that can consistently grow and maintain clinical-grade stem cells.

Standard culture systems use mouse "feeder" cells and media containing bovine sera to cultivate and maintain hES cells, but such animal product–based media can contaminate the cells. And because of difficulties in precise quality control, each batch of the medium can introduce new and unwanted variations.



Feedback. Representation of the feedback

system control scheme used by UCLA

researchers to identify an optimal combination

and concentration of small-molecule inhibitors

to support the long-term quality and

maintenance of hES cells in culture.

Credit: UCLA.

Now, a team of stem cell biologists and engineers from UCLA has identified an optimal combination and concentration of small-molecule inhibitors to support the long-term quality and maintenance of hES cells in feeder-free and serum-free conditions. The researchers used a feedback system control (FSC) scheme to innovatively and efficiently select the small-molecule inhibitors from a very large pool of possibilities.
The research findings, published in the journal Nature Communications, represent a major advance in the quest to broadly transition regenerative medicine from the benchtop to the clinic.

"What is significant about this work is that we've been able to very rapidly develop a chemically defined culture medium to replace serum and feeders for cultivating clinical-grade hES cells, thereby removing a major roadblock in the area of regenerative medicine," said Chih-Ming Ho, the Ben Rich–Lockheed Martin Professor at the UCLA Henry Samueli School of Engineering and Applied Science and a member of the National Academy of Engineering.

Unlike current animal product–based media, the new medium is a "defined" culture medium — one in which every component is known and traceable. This is important for clinical applications and as drugs or cells enters the world of regulatory affairs, including good manufacturing practice compliance and Food and Drug Administration supervision.

"It is also the first defined medium to allow for long term single-cell passage," said the paper's senior author, Hong Wu, the David Geffen Professor of Molecular and Medical Pharmacology at the David Geffen School of Medicine at UCLA and a researcher with UCLA's Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research.


Stem cells in new defined culture.

Microscopic image of human

embryonic stem cells in the new

defined culture created by UCLA

researchers. Credit: UCLA.


Single-cell passaging — a process in which hES cells are dissociated into single cells and subcultured through single-cell–derived colonies — is important in overcoming the massive cell death associated with hES cell dissociation during routine passage, and it allows for genetic manipulation at the clonal level.

"Although other studies have demonstrated growth of hES cells under defined media formulations and/or on defined surfaces, to the best of our knowledge, this is the first study that combines defined cultures with routine single-cell passaging, which plays an important role in supplying a large mass of clinically applicable cells," said Hideaki Tsutsui, a UCLA postdoctoral scholar and lead author of the study.

"Thus, our hES cell culture system, guided by the FSC technique, will bring hES cells one step closer to clinical therapies."

Initially, the very large number of small molecules in the culture medium and their unknown synergistic effects made it difficult for researchers to assess the proper concentration of each for achieving long-term expansion of hES cells. The major challenge was to find the best way to sort out those molecules and rapidly determine the best combinatorial concentrations.

The breakthrough, ultimately, was the product of a close interdisciplinary collaboration.

Tsutsui, then a UCLA Engineering graduate student, and Bahram Valamehr, then a graduate student at the Geffen School of Medicine, started working on the project two years ago. Armed with biological readouts and analyses of stem cells mastered in Hong Wu's laboratory through the lab's extensive accomplishments in stem cell research, Tsutsui and Valamehr used the FSC scheme — developed previously by Ho's group to search for optimal drug combinations for viral infection inhibition and cancer eradication — to facilitate the rapid screening of a very large number of possibilities.

Working together, the team was able to discover a unique combination of three small-molecule inhibitors that supports long-term maintenance of hES cell cultures through routine single-cell passaging.

"There are certain research projects biologists can dream about, and we know we can eventually get there, but we don't have the capacity to achieve them in a timely manner, especially in a study like this," Wu said.

"It would have taken 10 graduate students another 10 years to test all the possible combinations of molecules. Having an opportunity to collaborate with the engineering school has been invaluable in making this dream a reality."

"This is the best example of demonstrating the strength and potential of interdisciplinary collaborations," said Ho, who is also director of the Center for Cell Control at UCLA Engineering and a senior author of the paper.

"Engineers and biologists working side by side can accomplish a mission impossible."

Source: University of California, Los Angeles
Contact: Wileen Wong Kromhout
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