Meet Zhong Zhong and Hua Hua

The first monkey clones produced by method that made Dolly
Sunday, 28 January 2018

The first primate clones made by somatic cell nuclear transfer are two genetically identical long-tailed macaques born recently at the Chinese Academy of Sciences Institute of Neuroscience in Shanghai. Researchers named the newborns Zhong Zhong and Hua Hua - born eight and six weeks ago, respectively - after the Chinese adjective "Zhonghua," which means Chinese nation or people. The technical milestone, presented January 24 in the journal Cell, makes it a realistic possibility for labs to conduct research with customizable populations of genetically uniform monkeys.

"There are a lot of questions about primate biology that can be studied by having this additional model," says senior author Qiang Sun, Director of the Nonhuman Primate Research Facility at the Chinese Academy of Sciences Institute of Neuroscience.

"You can produce cloned monkeys with the same genetic background except the gene you manipulated. This will generate real models not just for genetically based brain diseases, but also cancer, immune, or metabolic disorders and allow us to test the efficacy of the drugs for these conditions before clinical use."

 CAPTION: This is a photograph of Zhong Zhong, one of the first two monkeys created by somatic cell nuclear transfer. CREDIT: Qiang Sun and Mu-ming Poo/Chinese Academy of Sciences.

Zhong Zhong and Hua Hua are not the first primate clones - the title goes to Tetra, a rhesus monkey born in 1999 through a simpler method called embryo splitting (Chan et al., Science 287, 317-319). This approach is how twins arise naturally but can only generate up to four offspring at a time. Zhong Zhong and Hua Hua are the product of somatic cell nuclear transfer (SCNT), the technique used to create Dolly the sheep over 20 years ago, in which researchers remove the nucleus from an egg cell and replace it with another nucleus from differentiated body cells. This reconstructed egg then develops into a clone of whatever donated the replacement nucleus.

 CAPTION: This is a photograph of Hua Hua, one of the first monkey clones made by somatic cell nuclear transfer. CREDIT: Qiang Sun and Mu-ming Poo/Chinese Academy of Sciences.

Differentiated monkey cell nuclei, compared to other mammals such as mice or cows, have proven resistant to SCNT. Sun and his colleagues overcame this challenge primarily by introducing epigenetic modulators after the nuclear transfer that switch on or off the genes that are inhibiting embryo development. The researchers found their success rate increased by transferring nuclei taken from fetal differentiated cells, such as fibroblasts, a cell type in the connective tissue. Zhong Zhong and Hua Hua are clones of the same macaque fetal fibroblasts. Adult donor cells were also used, but those clones only lived for a few hours after birth.

"We tried several different methods, but only one worked," says Sun.

"There was much failure before we found a way to successfully clone a monkey."

The first author Zhen Liu, a postdoctoral fellow, spent three years practicing and optimizing the SCNT procedure. He tested various methods to quickly and precisely remove the nuclear materials from the egg cell and promote the fusion of the nucleus-donor cell and enucleated egg. With the additional help of epigenetic modulators that re-activate the suppressed genes in the differentiated nucleus, he was able to achieve much higher rates of normal embryo development and pregnancy in the surrogate female monkeys.

"The SCNT procedure is rather delicate, so the faster you do it, the less damage to the egg you have, and Dr. Liu has a green thumb for doing this," says Muming Poo, a co-author on the study who directs the Institute of Neuroscience of CAS Center for Excellence in Brain Science and Intelligence Technology and helps to supervise the project.

"It takes a lot of practice. Not everybody can do the enucleation and cell fusion process quickly and precisely, and it is likely that the optimization of transfer procedure greatly helped us to achieve this success."

The researchers plan to continue improving the technique, which will also benefit from future work in other labs, and monitoring Zhong Zhong and Hua Hua for their physical and intellectual development. The babies are currently bottle fed and are growing normally compared to monkeys their age. The group is also expecting more macaque clones to be born over the coming months.

The lab is following strict international guidelines for animal research set by the US National Institutes of Health, but Sun and Poo encourage the scientific community to discuss what should or should not be acceptable practices when it comes to cloning of non-human primates.

"We are very aware that future research using non-human primates anywhere in the world depends on scientists following very strict ethical standards," Poo says.

This work was supported by grants from Chinese Academy of Sciences, the CAS Key Technology Talent Program, the Shanghai Municipal Government Bureau of Science and Technology, the National Postdoctoral Program for Innovative Talents and the China Postdoctoral Science Foundation.

Source: CELL PRESS

Contact: Joseph Caputo jcaputo@cell.com

Reference:

Cloning of macaque monkeys by somatic cell nuclear transfer

Zhen Liu, Yijun Cai, Yan Wang, Yanhong Nie, Chenchen Zhang, Yuting Xu, Xiaotong Zhang, Yong Lu, Zhanyang Wang, Muming Poo, Qiang Sun

Cell, DOI: http://dx.doi.org/10.1016/j.cell.2018.01.020

.........

ZenMaster

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

http://cellnews-blog.blogspot.com/ or

https://www.facebook.com/CellNEWS.blog/

“Open” Stem Cell Chromosomes Reveal New Possibilities for Diabetes

Researchers map chromosomal changes that must take place before stem cells can be used to produce pancreatic and liver cells 

Thursday, 02 April 2015

These are pancreatic cells derived from

embryonic stem cells. Credit: UC San Diego

School of Medicine. 

Stem cells hold great promise for treating a number of diseases, in part because they have the unique ability to differentiate, specializing into any one of the hundreds of cell types that comprise the human body. Harnessing this potential, though, is difficult. In some cases, it takes up to seven carefully orchestrated steps of adding certain growth factors at specific times to coax stem cells into the desired cell type. Even then, cells of the intestine, liver and pancreas are notoriously difficult to produce from stem cells. Writing in Cell Stem Cell April 2, researchers at University of California, San Diego School of Medicine have discovered why.

It turns out that the chromosomes in laboratory stem cells open slowly over time, in the same sequence that occurs during embryonic development. It isn’t until certain chromosomal regions have acquired the “open” state that they are able to respond to added growth factors and become liver or pancreatic cells. This new understanding, say researchers, will help spur advancements in stem cell research and the development of new cell therapies for diseases of the liver and pancreas, such as type 1 diabetes.

“Our ability to generate liver and pancreatic cells from stem cells has fallen behind the advances we’ve made for other cell types,” said Maike Sander, MD, professor of paediatrics and cellular and molecular medicine and director of the Pediatric Diabetes Research Center at UC San Diego.

“So we haven’t yet been able to do things like test new drugs on stem cell-derived liver and pancreatic cells. What we have learned is that if we want to make specific cells from stem cells, we need ways to predict how those cells and their chromosomes will respond to the growth factors.”

Sander led the study, together with co-senior author Bing Ren, PhD, professor of cellular and molecular medicine at UC San Diego and Ludwig Cancer Research member.

Chromosomes are the structures formed by tightly wound and packed DNA. Humans have 46 chromosomes – 23 inherited from each parent. Sander, Ren and their teams first made maps of chromosomal modifications over time, as embryonic stem cells differentiated through several different developmental intermediates on their way to becoming pancreatic and liver cells. Then, in analysing these maps, they discovered links between the accessibility (openness) of certain regions of the chromosome and what they call developmental competence – the ability of the cell to respond to triggers like added growth factors.

“We’re also finding that these chromosomal regions that need to open before a stem cell can fully differentiate are linked to regions where there are variations in certain disease states,” Sander says.

In other words, if a person were to inherit a genetic variation in one of these chromosomal regions and his or her chromosome didn’t open up at exactly the right time, he or she could hypothetically be more susceptible to a disease affecting that cell type. Sander’s team is now working to further investigate what role, if any, these chromosomal regions and their variations play in diabetes.

Source: UCSD

Contact: Heather Buschman

Reference:

Epigenetic Priming of Enhancers Predicts Developmental Competence of hESC-Derived Endodermal Lineage Intermediates

Allen Wang, Feng Yue, Yan Li, Ruiyu Xie, Thomas Harper, Nisha A. Patel, Kayla Muth, Jeffrey Palmer, Yunjiang Qiu, Jinzhao Wang, Dieter K. Lam, Jeffrey C. Raum, Doris A. Stoffers, Bing Ren, Maike Sander

Cell Stem Cell, Volume 16, Issue 4, p386–399, 2 April 2015, DOI: http://dx.doi.org/10.1016/j.stem.2015.02.013

.........

ZenMaster

---

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

http://cellnews-blog.blogspot.com/

---

Scientists Pinpoint Molecule that Switches On Stem Cell Genes

Experiments placed Sox9 at the crux of a shift in gene expression associated with hair follicle stem cell identity

Friday, 20 March 2015

Stem cells can have a strong sense of identity. Taken out of their home in the hair follicle, for example, and grown in culture, these cells remain true to themselves. After waiting in limbo, these cultured cells become capable of regenerating follicles and other skin structures once transplanted back into skin. It's not clear just how these stem cells – and others elsewhere in the body – retain their ability to produce new tissue and heal wounds, even under extraordinary conditions.

New research at Rockefeller University has identified a protein, Sox9, which takes the lead in controlling stem cell plasticity. In a paper published March 18 in Nature, the team describes Sox9 as a "pioneer factor" that breaks ground for the activation of genes associated with stem cell identity in the hair follicle.

"We found that in the hair follicle, Sox9 lays the foundation for stem cell plasticity. First, Sox9 makes the genes needed by stem cells accessible, so they can become active. Then, Sox9 recruits other proteins that work together to give these "stemness" genes a boost, amplifying their expression," says study author Elaine Fuchs, Rebecca C. Lancefield Professor, Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Development.

"Without Sox9, this process never happens, and hair follicle stem cells cannot survive."

Sox9 is a type of protein called a transcription factor, which can act like a volume dial for genes. When a transcription factor binds to a segment of DNA known as an enhancer, it cranks up the activity of the associated gene. Recently, scientists identified a less common, but more powerful version: the super-enhancer. Super-enhancers are much longer pieces of DNA, and host large numbers of cell type-specific transcription factors that bind cooperatively. Super-enhancers also contain histones, DNA-packaging proteins, which harbour specific chemical groups – epigenetic marks – that make genes they are associated with accessible so they can be expressed.

Using an epigenetic mark associated specifically with the histones of enhancers, first author Rene Adam, a graduate student in the lab, and colleagues, identified 377 of these high-powered gene-amplifying regions in hair follicle stem cells. The majority of these super-enhancers were bound by at least five transcription factors, often including Sox9. Then, they compared the stem cell super-enhancers to those of short-lived stem cell progeny, which have begun to choose a fate, and so lost the plasticity of stem cells. These two types of cells shared only 32 percent of their super-enhancers, suggesting these regions played an important role in skin cell identity. By switching off super-enhancers associated with stem cell genes, these genes were silenced while new super-enhancers were being activated to turn on hair genes.

To better understand these dynamics, the researchers took a piece of a super-enhancer, called an epicentre, where all the stem cell transcription factors bind, and they linked it to a gene that glowed green whenever the transcription factors were present. In living mice, all the hair follicle stem cells glowed green, but surprisingly, the green gene turned off when the stem cells were taken from the follicle and placed in culture. When they put the cells back into living skin, the green glow returned.

Another clue came from experiments performed by Hanseul Yang, another student in the lab. By examining the new super-enhancers that were gained when the stem cells were cultured, they learned that these new super-enhancers bound transcription factors that were known to be activated during wound-repair. When they used one of these epicentres to drive the green gene, the green glow appeared in culture, but not in skin. When they wounded the skin, then the green glow switched on.

Researchers made stem cells fluoresce green (at

the base of hair follicles above) by labelling their

super-enhancers, regions of the genome bound

by gene-amplifying proteins. It appears one such

protein, Sox9, leads the activation of super-

enhancers that boost genes associated with stem

cell plasticity. Credit: Laboratory of

Mammalian Cell Biology and Development at

the Rockefeller University/Nature. 

"We were learning that some super-enhancers are specifically activated in the stem cells within their native niche, while other super-enhancers specifically switch on during injury," explained Adam.

"By shifting epicentres, you can shift from one cohort of transcription factors to another to adapt to different environments. But we still needed to determine what was controlling these shifts."

The culprit turned out to be Sox9, the only transcription factor expressed in both living tissue and culture. Further experiments confirmed Sox9's importance by showing, for example, that removing it spelled death for stem cells, while expressing it in the epidermis gave the skin cells features of hair follicle stem cells. These powers seemed to be special to Sox9, placing it atop the hierarchy of transcription factors in the stem cells. Sox9 is one of only a few pioneer factors known in biology which can initiate such dramatic changes in gene expression.

"Importantly, we link this pioneer factor to super-enhancer dynamics, giving these domains a 'one-two punch' in governing cell identity. In the case of stem cell plasticity, Sox9 appears to be the lead factor that activates the super-enhancers that amplify genes associated with stemness," Fuchs says.

"These discoveries offer new insights into the way in which stem cells choose their fates and maintain plasticity while in transitional states, such as in culture or when repairing wounds."

Source: Rockefeller University

Contact: Wynne Parry

Reference:

Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice

Rene C. Adam, Hanseul Yang, Shira Rockowitz, Samantha B. Larsen, Maria Nikolova, Daniel S. Oristian, Lisa Polak, Meelis Kadaja, Amma Asare, Deyou Zheng, and Elaine Fuchs

Nature, March 18, 2015, doi:10.1038/nature14289

.........

ZenMaster

---

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

http://cellnews-blog.blogspot.com/

---

NIH-Supported Researchers Map Epigenome of More Than 100 Tissue and Cell Types

NIH-Supported Researchers Map Epigenome of More Than 100 Tissue and Cell Types

Thursday, 19 February 2015

Much like mapping the human genome laid the foundations for understanding the genetic basis of human health, new maps of the human epigenome may further unravel the complex links between DNA and disease. The epigenome is part of the machinery that helps direct how genes are turned off and on in different types of cells.

Researchers supported by the National Institutes of Health Common Fund's Roadmap Epigenomics Program have mapped the epigenomes of more than 100 types of cells and tissues, providing new insight into which parts of the genome are used to make a particular type of cell. The data, available to the biomedical research community, can be found at the National Center for Biotechnology Information website.

Reference epigenomes are available for more than 100 cell and tissue types.
Credit: Image courtesy of Nature and Roadmap Epigenomics Consortium. 

"This represents a major advance in the ongoing effort to understand how the 3 billion letters of an individual's DNA instruction book are able to instruct vastly different molecular activities, depending on the cellular context," said NIH Director Francis Collins, M.D., Ph.D.

"This outpouring of data-rich publications, produced by a remarkable team of creative scientists, provides powerful momentum for the rapidly growing field of epigenomics."

Researchers from the NIH Common Fund's Roadmap Epigenomics Program published a description of the epigenome maps in the journal Nature. More than 20 additional papers, published in Nature and Nature-associated journals, show how these maps can be used to study human biology.

"What the Roadmap Epigenomics Program has delivered is a way to look at the human genome in its living, breathing nature from cell type to cell type," said Manolis Kellis, Ph.D., professor of computer science at the Massachusetts Institute of Technology, Cambridge, and senior author of the paper.

Understanding epigenomics

Almost all human cells have identical genomes that contain instructions on how to make the many different cells and tissues in the body. During the development of different types of cells, regulatory proteins turn genes on and off and, in doing so, establish a layer of chemical signatures that make up the epigenome of each cell. In the Roadmap Epigenomics Program, researchers compared these epigenomic signatures and established their differences across a variety of cell types. The resulting information can help us understand how changes to the genome and epigenome can lead to conditions such as Alzheimer's disease, cancer, asthma, and foetal growth abnormalities.

An epigenomic signature can be made on the

genome in two ways, both of which play a role in

turning genes off or on. The first occurs when

chemical tags called methyl groups are attached

to a DNA molecule directly (DNA methylation).

The second occurs when a variety of chemical

tags attach to the tails of histone proteins that

package DNA (histone modifications). Credit:

Image courtesy of John Stamatoyannopoulos

and Rae Senarighi. 

The value of epigenomic data

Researchers can now take data from different cell types and directly compare them.

"Today, sequencing the human genome can be done rapidly and cheaply, but interpreting the genome remains a challenge," said Bing Ren, Ph.D., professor of cellular and molecular medicine at the University of California, San Diego, and co-author of the Nature paper and several of the associated papers.

"These 111 reference epigenome maps are essentially a vocabulary book that helps us decipher each DNA segment in distinct cell and tissue types. These maps are like snapshots of the human genome in action."

"This is the most comprehensive catalogue of epigenomic data from primary human cells and tissues to date," said Lisa Helbling Chadwick, Ph.D., project team leader and a program director at the National Institute of Environmental Health Sciences (NIEHS), part of NIH.

"This coordinated effort, along with uniform data processing, makes it much easier for researchers to make direct comparisons across the entire data set."

"Researchers from the 88 projects supported by the program, including those from this recent series of papers, have propelled the development of new epigenomic technologies," said John Satterlee, Ph.D., co-coordinator of the Roadmap Epigenomics Program, and program director at the National Institute on Drug Abuse (NIDA), part of NIH. Satterlee added that the work of this program has served as a foundation for continued exploration of the human epigenome through the International Human Epigenome Consortium.

"With this increased understanding of the full epigenome, and the datasets available to the entire scientific community, the NIH Common Fund is striving to catalyse future research, to aid the understanding of how epigenomics plays a role in human diseases, with the expectation that further studies will identify early indications of disease and targets for therapeutics," said James Anderson, M.D., Ph.D., director of NIH Division of Program Coordination, Planning, and Strategic Initiatives that oversees the NIH Common Fund.

NIDA, NIEHS, and the National Institute on Deafness and Other Communication Disorders are co-administrators of the NIH Common Fund's Epigenomics Program.

Source: National Institute of Environmental Health Sciences

Contact: Robin Mackar

Reference:

Integrative analysis of 111 reference human epigenomes

Roadmap Epigenomics Consortium

Nature, (2015); doi: 10.1038/nature14248

.........

ZenMaster

---

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

http://cellnews-blog.blogspot.com/

---

Scientists find that SCNT Derived Cells and iPS Cells are Similar

Scientists find that SCNT Derived Cells and iPS Cells are Similar

Thursday, 06 November 2014

A team led by New York Stem Cell Foundation (NYSCF) Research Institute scientists conducted a study comparing induced pluripotent stem (iPS) cells and embryonic stem cells created using somatic cell nuclear transfer (SCNT). The scientists found that the cells derived from these two methods resulted in cells with highly similar gene expression and DNA methylation patterns. Both methods also resulted in stem cells with similar amounts of DNA mutations, showing that the process of turning an adult cell into a stem cell introduces mutations independent of the specific method used. This suggests that both methods of producing stem cells need to be further investigated before determining their suitability for the development of new therapies for chronic diseases.

The NYSCF Research Institute is one of the only laboratories in the world that currently pursues all forms of stem cell research including SCNT and iPS cell techniques for creating stem cells. The lack of laboratories attempting SCNT research was one of the reasons that the NYSCF Research Institute was established in 2006.

"We do not yet know which technique will allow scientists to create the best cells for new cellular therapies," said Susan L. Solomon, NYSCF CEO and co-founder.

"It is critical to pursue both SCNT and iPS cell techniques in order to accelerate research and bring new treatments to patients."

While both techniques result in pluripotent stem cells, or cells that can become any type of cell in the body, the two processes are different. SCNT consists of replacing the nucleus of a human egg cell or oocyte with the nucleus of an adult cell, resulting in human embryonic stem cells with the genetic material of the adult cell. In contrast, scientists create iPS cells by expressing a few key genes in adult cells, like a skin or blood cell, causing the cells to revert to an embryonic-like state. These differences in methods could, in principle, result in cells with different properties. Advances made earlier this year by NYSCF Research Institute scientists that showed that human embryonic stem cells could be derived using SCNT revived that debate.

"Our work shows that we now have two methods for the generation of a patient's personal stem cells, both with great potential for the development of treatments of chronic diseases. Our work will also be welcome news for the many scientists performing basic research on iPS cells. It shows that they are likely working with cells that are very similar to human embryonic stem cells, at least with regard to gene expression and DNA methylation. How the finding of mutations might affect clinical use of stem cells generated from adult cells is the subject of an ongoing debate," said Dr. Dieter Egli, NYSCF Senior Research Fellow, NYSCF - Robertson Investigator, Assistant Professor in Pediatrics & Molecular Genetics at Columbia University, and senior author on the paper.

The study, published today in Cell Stem Cell, compared cell lines derived from the same sources using the two differing techniques, specifically contrasting the frequency of genetic coding mutations seen and measuring how closely the stem cells matched the embryonic state through the analysis of DNA methylation and of gene expression patterns. The scientists showed that both methods resulted in cell types that were similar with regard to gene expression and DNA methylation patterns. This suggested that both methods were effective in turning a differentiated cell into a stem cell.

The scientists also showed that cells derived using both SCNT and iPS techniques showed similar numbers of genetic coding mutations, implying that neither technique is superior in that regard. A similar number of changes in DNA methylation at imprinted genes (genes that are methylated differentially at the maternal versus the paternal allele) were also found. It is important to note that both types of techniques led to cells that had more of these aberrations than embryonic stem cells derived from an unfertilized human oocyte, or than embryonic stem cells derived from leftover IVF embryos. These findings suggest that a small number of defects are inherent to the generation of stem cells from adult differentiated cells and occur regardless of the method used.

Source: New York Stem Cell Foundation

Contact: David McKeon

Reference:

Comparable Frequencies of Coding Mutations and Loss of Imprinting in Human Pluripotent Cells Derived by Nuclear Transfer and Defined Factors

Bjarki Johannesson, Ido Sagi, Athurva Gore, Daniel Paull, Mitsutoshi Yamada, Tamar Golan-Lev, Zhe Li, Charles LeDuc, Yufeng Shen, Samantha Stern, Nanfang Xu, Hong Ma, Eunju Kang, Shoukhrat Mitalipov, Mark V. Sauer, Kun Zhang, Nissim Benvenisty, Dieter Egli

Cell Stem Cell, Volume 15, Issue 5, p634–642, 6 November 2014, DOI: http://dx.doi.org/10.1016/j.stem.2014.10.002

.........

ZenMaster

---

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

http://cellnews-blog.blogspot.com/

---

Some Stem Cell Methods Closer to "Gold Standard" than Others

Nuclear transfer appears superior for creating embryonic stem cells

Thursday, 03 July 2014

Researchers around the world have turned to stem cells, which have the potential to develop into any cell type in the body, for potential regenerative and disease therapeutics.

Now, for the first time, researchers at the Salk Institute, with collaborators from Oregon Health & Science University and the University of California, San Diego, have shown that stem cells created using two different methods are far from identical. The finding could lead to improved avenues for developing stem cell therapies as well as a better understanding of the basic biology of stem cells.

The researchers discovered that stem cells created by moving genetic material from a skin cell into an empty egg cell — rather than coaxing adult cells back to their embryonic state by artificially turning on a small number of genes — more closely resemble human embryonic stem cells, which are considered the gold standard in the field.

Joseph R. Ecker, Professor, Genomic Analysis

Laboratory. Credit: Courtesy of the Salk

Institute for Biological Studies. 

"These cells created using eggs' cytoplasm have fewer reprogramming issues, fewer alterations in gene expression levels and are closer to real embryonic stem cells," says co-senior author Joseph R. Ecker, professor and director of Salk's Genomic Analysis Laboratory and co-director of the Center of Excellence for Stem Cell Genomics. The results of the study were published today in Nature.

Human embryonic stem cells (hESCs) are directly pulled from unused embryos discarded from in-vitro fertilization, but ethical and logistical quandaries have restricted their access. In the United States, federal funds have limited the use of hESCs so researchers have turned to other methods to create stem cells. Most commonly, scientists create induced pluripotent stem (iPS) cells by starting with adult cells (often from the skin) and adding a mixture of genes that, when expressed, regress the cells to a pluripotent stem-cell state. Researchers can then coax the new stem cells to develop into cells that resemble those in the brain or in the heart, giving scientists a valuable model for studying human disease in the lab.

Over the past year, a team at OHSU built upon a technique called somatic cell nuclear transfer (the same that is used for cloning an organism, such as Dolly the sheep) to transplant the DNA-containing nucleus of a skin cell into an empty human egg, which then naturally matures into a group of stem cells.

Shoukhrat Mitalipov, Ph.D., Oregon Health &

Science University, led a team that found that a

process called "somatic cell nuclear transfer" is

much better and more accurate at

reprogramming human skin cells to become

embryonic stem cells. Credit: Oregon Health &

Science University.

Ecker, holder of the Salk International Council Chair in Genetics, teamed up with Shoukhrat Mitalipov, developer of the new technique and director of the Center for Embryonic Cell and Gene Therapy at OHSU, and UCSD assistant professor Louise Laurent to carry out the first direct comparison of the two approaches. The scientists created four lines of nuclear transfer stem cells all using eggs from a single donor, along with seven lines of iPS cells and two lines of the gold standard hESCs. All cell lines were shown to be able to develop into multiple cell types and had nearly identical DNA content contained within them.

But when they looked closer at the cells, the researchers spotted some differences: the patterns of methylation — chemical flags that are added to genes to control their expression — varied between the cell lines. This indicates a difference in how and when genes, despite having identical sequences, might be expressed. The methylation of nuclear transfer cells more closely resembled hESCs than the iPS cells did. And when the investigators looked at patterns of actual gene expression — by measuring the levels of particular RNA strands produced by each cell — the differences continued. Once again, nuclear transfer cells had RNA levels closer to embryonic cells, making them more accurate for basic research and therapeutic studies.

"Both the DNA methylation and gene expression data show that nuclear transfer does a better job at erasing the signature of the original skin cell," says Laurent, who is a co-senior author of the paper.

"If you believe that gene expression is important, which we do, then the closer you get to the gene expression patterns of embryonic stem cells, the better," Ecker says.

"Right now, nuclear transfer cells look closer to the embryonic stem cells than do the iPS cells."

Ecker doesn't expect labs to race to make the switch to nuclear transfer protocols — after all, the method falls within those restricted for federal funding. But he thinks the new observation likely holds lessons that could help improve the protocols for making iPS cells.

"What this is telling us is that you can use the standard mix of genes and they do a pretty good job of creating iPS cells," Ecker says.

"But they're not perfect. The material in an egg does a better job than just those four genes alone."

If researchers can pin down what it is within an egg that drives the production of pluripotent stem cells, they may be able to integrate that knowledge into iPS methods to improve stem cell therapy for disease.

"At this point, nuclear transfer stem cells combine the key advantages of both hESCs and iPS cells and, as such, are ideal for clinical applications in regenerative therapy," adds Mitalipov.

Other researchers on the study were Ryan C. O'Neil, Yupeng He, Matthew D. Schultz, Manoj Heriharan, Joseph R. Nery, and Rosa Castanon of the Salk Institute for Biological Studies; Hong Ma, Brittany Daughtry, Masahito Tachibana, Eunju Kang, Rebecca Tippner-Hedges, Riffat Ahmed, Nuria Marti Gutierrez, Crystal Van Dyken, Alimujiang Fulati, Atsushi Sugawara, Michelle Sparman, Paula Amato and Don P. Wolf of Oregon Health & Science University; Robert Morey, Karen Sabatini and Rathi D. Thiagarajan of the University of California, San Diego; and Sumita Gokhale of the Boston University School of Medicine.

Source: Salk Institute for Biological Studies

Contact: Kristina Grifantini

Reference:

Abnormalities in human pluripotent cells due to reprogramming mechanisms

Hong Ma, Robert Morey, Ryan C. O'Neil, Yupeng He, Brittany Daughtry, Matthew D. Schultz, Manoj Hariharan, Joseph R. Nery, Rosa Castanon, Karen Sabatini, Rathi D. Thiagarajan, Masahito Tachibana, Eunju Kang, Rebecca Tippner-Hedges, Riffat Ahmed, Nuria Marti Gutierrez, Crystal Van Dyken, Alim Polat, Atsushi Sugawara, Michelle Sparman, Sumita Gokhale, Paula Amato, Don P.Wolf, Joseph R. Ecker, Louise C. Laurent & Shoukhrat Mitalipov

Nature (2014), doi:10.1038/nature13551

.........

ZenMaster

---

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

http://cellnews-blog.blogspot.com/

---

Reconstructing the Life History of a Single Cell

Cell's unique mutations used to trace history back to its origins in the embryo

Sunday, 29 June 2014

Researchers have developed new methods to trace the life history of individual cells back to their origins in the fertilised egg. By looking at the copy of the human genome present in healthy cells, they were able to build a picture of each cell's development from the early embryo on its journey to become part of an adult organ.

Reconstruction of early cell divisions of two

mouse embryos: Each white-filled large circle

represents an individual cell from a mouse

embryo and the unique combination of

mutations in its DNA. Each mutation is

represented by a number and those highlighted in

yellow were acquired in the most recent cell

division. The smaller colour-filled circles are the

final sets of cells taken from different parts of

the mouse and there are an unknown number of

cell generations between each set of cells and its

closest identifiable precursor cell. [DOI:

10.1038/nature13448]

During the life of an individual, all cells in the body develop mutations, known as somatic mutations, which are not inherited from parents or passed on to offspring. These somatic mutations carry a coded record of the lifetime experiences of each cell.

By looking at the numbers and types of mutations in a cell's DNA, researchers were able to assess whether the cell had divided a few times or many times and detect the imprints, known as signatures, of the processes of DNA damage and repair that the cells had been exposed to during the life of the individual. Furthermore, comparing each cell's mutations with those of other cells in the body enabled scientists to map out a detailed tree of development from the fertilised egg.

"With this novel approach, we can peer back into an organism's development," says Dr Sam Behjati, first author from the Wellcome Trust Sanger Institute.

"If we can better understand how normal, healthy cells mutate as they divide over a person's lifetime, we will gain a fundamental insight into what can be considered normal and how this differs from what we see in cancer cells."

The team looked at mouse cells from the stomach, small bowel, large bowel and prostate. The single cells were grown to produce enough DNA to be sequenced accurately. Eventually, single-cell sequencing technology will develop so that this type of experiment can be conducted using just one cell. However, the tiny amounts of DNA in single cells mean that mutation data are not currently precise enough to reconstruct accurate lineages.

The researchers recorded differences in the numbers of mutations in cells from the different tissues studied, likely attributable to differences in rates of cell division. Moreover, different patterns of mutation were found in cells from different tissues, suggesting that they have been exposed to different processes of DNA damage and repair, reflecting different lifetime experiences.

This experiment used healthy mice. If mutation rates are similar in human cells, these techniques could be used to provide an insight into the life histories of normal human cells.

"The adult human body is composed of 100 million million cells, all of which have originated from a single fertilised egg," says Professor Mike Stratton, senior author and Director of the Sanger Institute.

"Much more extensive application of this approach will allow us to provide a clear picture of how adult cells have developed from the fertilised egg. Furthermore, by looking at the numbers and types of mutation in each cell we will be able to obtain a diary, writ in DNA, of what each healthy cell has experienced during its lifetime, and then explore how this changes in the range of human diseases."

Source: Wellcome Trust Sanger Institute 

Contact: Mark Thomson

Reference:

Genome sequencing of normal cells reveals developmental lineages and mutational processes

Sam Behjati, Meritxell Huch, Ruben van Boxtel, Wouter Karthaus, David C. Wedge, Asif U. Tamuri, Iñigo Martincorena, Mia Petljak, Ludmil B. Alexandrov, Gunes Gundem, Patrick S. Tarpey, Sophie Roerink, Joyce Blokker, Mark Maddison, Laura Mudie, Ben Robinson, Serena Nik-Zainal, Peter Campbell, Nick Goldman, Marc van de Wetering, Edwin Cuppen, Hans Clevers & Michael R. Stratton

Nature 2014, DOI: 10.1038/nature13448

.........

ZenMaster

---

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

http://cellnews-blog.blogspot.com/

---

Many Bodies Prompt Stem Cells to Change

Rice University scientists apply new theory to learn how and why cells differentiate

Monday, 16 June 2014

How does a stem cell decide what path to take? In a way, it's up to the wisdom of the crowd.

The DNA in a pluripotent stem cell is bombarded with waves of proteins whose ebb and flow nudge the cell toward becoming blood, bone, skin or organs. A new theory by scientists at Rice University shows the cell's journey is neither a simple step-by-step process nor all random.

Peter Wolynes, left, and Bin Zhang of Rice

University tested their new method to analyse

large gene networks to begin to understand how

stem cells differentiate. Credit: Jeff Fitlow/Rice

University. 

Theoretical biologist Peter Wolynes and postdoctoral fellow Bin Zhang set out to create a mathematical tool to analyse large, realistic gene networks. As a bonus, their open-access study to be published this week by the Proceedings of the National Academy of Sciences helped them understand that the process by which stem cells differentiate is a many-body problem.

"Many-body" refers to physical systems that involve interactions between large numbers of particles. Scientists assume these many bodies conspire to have a function in every system, but the "problem" is figuring out just what that function is. In the new work, these bodies consist not only of the thousands of proteins expressed by embryonic stem cells but also DNA binding sites that lead to feedback loops and other "attractors" that prompt the cell to move from one steady state to the next until it reaches a final configuration.

To test their tool, the researchers looked at the roles of eight key proteins and how they rise and fall in number, bind and unbind to DNA and degrade during stem cell differentiation. Though the interactions may not always follow a precise path, their general pattern inevitably leads to the desired result for the same reason a strand of amino acids will inevitably fold into the proper protein: because the landscape dictates that it be so.

Wolynes called the new work a "stylized," simplified model meant to give a general but accurate overview of how cell networks function. It's based on a theory he formed in 2003 with Masaki Sasai of Nagoya University but now takes into account the fact that not one but many genes can be responsible for even a single decision in a cellular process.

An overview of the stem cell gene network gives

a sense of the complex process involved in cell

differentiation, as transcription factors and

protein complexes influence and loop back upon

each other. Rice University researchers found

that stem cell differentiation can be defined as a

many-body problem as they developed a

theoretical system to analyse large gene

networks. Credit: Bin Zhang/Rice University.

"This is what Bin figured out, that one could generalize our 2003 model to be much more realistic about how several different proteins bind to DNA in order to turn it on or off," Wolynes said.

A rigorous theoretical approach to determine the transition pathways and rates between steady states was also important, Zhang said.

"This is crucial for understanding the mechanism of how stem cell differentiation occurs," he said.

Wolynes said that because the stem cell is stochastic – that is, its fate is not pre-determined – "we had to ask why a gene doesn't constantly flip randomly from one state to another state. This paper for the first time describes how we can, for a pretty complicated circuit, figure out there are only certain periods during which the flipping can occur, following a well-defined transition pathway."

In previous models of gene networks, "Instead of focusing on proteins actually binding to DNA, they just say, 'Well, there's a certain high level of this protein or low level of that protein,'" Wolynes said.

"At first, that sounds easier to study because you can measure how much protein you've got. But you don't always know if it is bound. It has become increasingly clear that the rate of protein binding to DNA plays an important role in gene expression, particularly in eukaryotic systems."

The notion that many-body effects even existed in cells began in 1942 when British scientist C.H. Waddington established the idea of an epigenetic landscape for stem cells as a way to describe why pluripotent cells in embryos are destined to turn into bone, muscle and all the other parts of the body – but don't turn back. Waddington compared the cells' paths to marbles rolling to the bottom of a valley.

That concept rang true to Wolynes. His energy landscape theory has become key to understanding protein folding, although that theory sees the landscape as a funnel rather than a valley.

"Waddington said that as a cell develops into an embryo and beyond, it becomes many different kinds of cells," Wolynes said.

"Those cells might branch off and differentiate further, but they don't typically go back to the original state and start over.”

"His analogy – the idea of falling down through a valley – kicked around for a long time, but it was hard to make it mathematically precise. In his time, they didn't know about DNA," Wolynes said.

In both energy and epigenetic landscapes, Wolynes said, the steady state at the bottom is an attractor.

"It means wherever you start from, you end up attracted to that same place," he said.

"In genetic networks, things like steadily oscillating patterns can also be considered attractors."

Once biologists began to understand genetic switches in DNA, the whole picture became more complicated, he said.

"The landscape now has to incorporate the active parts of DNA that are trying to decide whether to turn this gene on or that gene off. In the '50s, we learned how genes made decisions on the basis of their production of proteins. These proteins then act back on the same genes in a kind of feedback loop."

The loops allow genes to remain active for far longer than it would take a protein simply to bind or unbind to a section of DNA. In the researchers' equations, the loops become attractors that help regulate transformation of the cell and can be mapped onto the many-body landscape.

Analysing the coupled dynamics of all these chemical reactions in a cell could be done by brute force, he said, but the computational cost would be enormous. So the Rice team decided to take a big-picture approach based on Wolynes' earlier work. It so happened that the resulting theoretical models of embryonic stem cells matched nicely with what experimentalists had seen in their studies.

For example, the models explained the fluctuations experimentalists had observed in the expression of a master regulator, a protein called Nanog, and its important role in maintaining a cell's pluripotency. Stem cells move from one steady state to the next on their journeys; in their calculations, they found a much higher level of Nanog gene expression in what they called SC1, the basic stem cell, than in SC2, a stem cell that had moved to the second steady state. This matched what experiments had measured, the researchers said.

"This is still just a beginning," Wolynes said.

"We're looking at embryonic stem cells now, but someday we want to treat the complete developmental program of organisms with hundreds of genes. We can see how these mathematics can scale up to that regime."

Source: Rice University 

Contact: David Ruth

.........

ZenMaster

---

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

http://cellnews-blog.blogspot.com/

---