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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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
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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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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/

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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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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/

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