From Pluripotency to Totipotency

Scientists discover mechanism that may lead to more efficient reprogramming of somatic cells

Wednesday, 05 August 2015

Human embryonic stem cells have the potential

to form in vitro neural tube-like structures of the

embryo. Credit: Inserm/Benchoua Alexandra.

While it is already possible to obtain in vitro pluripotent cells (i.e., cells capable of generating all tissues of an embryo) from any cell type, researchers from Maria-Elena Torres-Padilla's team from Institut de Génétique et de Biologie Moléculaire et Cellulaire, have pushed the limits of science even further. They managed to obtain totipotent cells with the same characteristics as those of the earliest embryonic stages and with even more interesting properties. Obtained in collaboration with Juanma Vaquerizas from the Max Planck Institute for Molecular Biomedicine (Münster, Germany), these results are published on 3rd of August in the journal Nature Structural & Molecular Biology.

Totipotency vs pluripotency

Just after fertilization, when the embryo is comprised of only 1 or 2 cells, cells are "totipotent", that is to say, capable of producing an entire embryo as well as the placenta and umbilical cord that accompany it. During the subsequent rounds of cell division, cells rapidly lose this plasticity and become "pluripotent". At the blastocyst stage (about thirty cells), the so-called "embryonic stem cells" can differentiate into any tissue, although they alone cannot give birth to a foetus anymore. Pluripotent cells then continue to specialise and form the various tissues of the body through a process called cellular differentiation.

For some years, it has been possible to re-programme differentiated cells into pluripotent ones, but not into totipotent cells. Now, the team of Maria-Elena Torres-Padilla has studied the characteristics of totipotent cells of the embryo and found factors capable of inducing a totipotent-like state.

“Totipotency is a much more flexible state than the pluripotent state and its potential applications are extraordinary”, says Maria-Elena Torres-Padilla, who led the study.

Looking for the keys of totipotency

When culturing pluripotent stem cells in vitro, a small amount of totipotent cells appear spontaneously; these are called "2C-like cells" (named after their resemblance to the 2-cell stage embryo). The researchers compared these cells to those present in early embryos in order to find their common characteristics and those that make them different from pluripotent cells. In particular, the teams found that the DNA was less condensed in totipotent cells and that the amount of the protein complex CAF1 was diminished. A closer look revealed that CAF1 – already known for its role in the assembly of chromatin (the organised state of DNA) – is responsible for maintaining the pluripotent state by ensuring that the DNA is wrapped around histones. Based on this hypothesis, the Torres-Padilla team was able to induce a totipotent state by inactivating the expression of the CAF1 complex, which led to chromatin reprogramming into a less condensed state.

A 2C-like cell (green) is different from an

embryonic stem cell (magenta). Credit:

IGBMC/Maria-Elena Torres-Padilla.

In order to carefully examine at a molecular level the similarities between 2-cell stage embryos, 2C-like cells and those induced by inactivating the CAF1 complex, the Torres-Padilla team then joined forces with the Vaquerizas laboratory to analyse, in a genome-wide fashion, the gene expression programmes of these cells. The scientists found that the induced, CAF1-depleted, totipotent cells overexpressed a significant amount of 2-cell stage embryo genes.

“One could imagine that if cells lose their ability to assemble chromatin, this would affect gene expression”, explains Cells-in-Motion PhD student Rocio Enriquez-Gasca of Juanma Vaquerizas’ lab, who performed the computational analyses of the work.

“So it was really exciting to realise that the resulting gene expression programme in fact significantly overlaps with that of early embryo, totipotent cells”.

Moreover, the teams found that specific classes of repetitive elements (repeated sequences of DNA that form around 50% of the mouse and human genomes) were also up-regulated in induced totipotent-like cells, a hallmark of the 2-cell embryo.

“The computational analysis of expression of repetitive elements is very challenging, since these are found many times in the genome”, says Juanma Vaquerizas.

“Now it is key to understand why these repetitive elements and gene expression programmes are both up-regulated in totipotent cells”.

These results provide new elements for the understanding of pluripotency and could increase the efficiency of reprogramming somatic cells to be used for applications in regenerative medicine.

Source: INSERM

Contact: Maria-Elena Torres-Padilla 

Reference:

Early embryonic-like cells are induced by down-regulation of replication-dependent chromatin assembly

Takashi Ishiuchi, Rocio Enriquez-Gasca, Eiji Mizutani, Ana Boškovi, Celine Ziegler-Birling, Diego Rodriguez-Terrones, Teruhiko Wakayama, Juan M. Vaquerizas & Maria-Elena Torres-Padilla

Nature Structural & Molecular Biology, 3 Aug 2015, doi:10.1038/nsmb.3066

.........

ZenMaster

---

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

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

---

The ISSCR Issues Statement on Human Germ Line Genome Modification

The International Society for Stem Cell Research has released a statement calling for a moratorium on attempts to apply nuclear genome editing of the human germ line in clinical practice

Sunday, 22 March 2015

In a statement released on Thursday, the International Society for Stem Cell Research called for a moratorium on attempts at clinical application of nuclear genome editing of the human germ line to enable more extensive scientific analysis of the potential risks of genome editing and broader public discussion of the societal and ethical implications.

Technologies used to introduce changes into the DNA sequence of cells have advanced rapidly, making genome editing increasingly simple. Genome editing is feasible, not just in the somatic cells of an adult organism, but also in early embryos, as well as the gametes (sperm and egg) that carry the inheritable, germ line DNA. Research involving germ line nuclear genome editing has been performed to date in many organisms, including mice and monkeys, and applications to human embryos are possible.

The ISSCR statement raises significant ethical, societal and safety considerations related to the application of nuclear genome editing to the human germ line in clinical practice. Current genome editing technologies carry risks of unintended genome damage, in addition to unknown consequences. Moreover, consensus is lacking on what, if any, therapeutic applications of germ line genome modification might be permissible.

The statement calls for a moratorium on attempts to apply nuclear genome editing of the human germ line in clinical practice, as scientists currently lack an adequate understanding of the safety and potential long term risks of germ line genome modification. Moreover, the ISSCR asserts that a deeper and more rigorous deliberation on the ethical, legal and societal implications of any attempts at modifying the human germ line is essential if its clinical practice is ever to be sanctioned.

In calling for the above moratorium, the ISSCR is not taking a position on the clinical testing of mitochondrial replacement therapy, a form of germ line modification that entails replacing the mitochondria (found outside the nucleus) in the eggs of women at risk of transmitting certain devastating diseases to their children.

Source: International Society for Stem Cell Research

Contact: Michelle Quivey

.........

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/

---

Nuclear Pore Complex: How to Build Your Gate

New approach solves large molecular puzzles

Friday, 12 July 2013

At first glance, this may look like the latest view

from a telescope, but it was taken by looking not

out at the vastness of space, but into a human

cell. What could pass for stars or distant galaxies

are actually gates to the genome that is locked up

inside the cell's nucleus, as imaged by a super-

resolution microscope. By combining images of

thousands of these gates, called nuclear pore

complexes, scientists at the European Molecular

Biology Laboratory (EMBL) in Heidelberg,

Germany, have solved a decade-old controversy

over how the pieces that form each pore's ring are

arranged. Credit: EMBL/A.Szymborska. 

It's a parent's nightmare: opening a Lego set and being faced with 500 pieces, but no instructions on how to assemble them into the majestic castle shown on the box. Thanks to a new approach by scientists at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, researchers studying large sets of molecules with vital roles inside our cells can now overcome a similar problem.

In a study published online today in Science, the scientists used super-resolution microscopy to solve a decade-long debate about the structure of the nuclear pore complex, which controls access to the genome by acting as a gate into the cell's nucleus.

Like the bewildered parent staring at the image on the box, scientists knew the gate's overall shape, from electron tomography studies. And thanks to techniques like X-ray crystallography and single particle electron microscopy, they knew that the ring which studs the nucleus' wall and controls what passes in and out is formed by sixteen or thirty-two copies of a Y-shaped building block. They even knew that each Y is formed by nine proteins. But how the Y’s are arranged to form a ring was up for debate.

"When we looked at our images, there was no question: they have to be lying head-to-tail around the hole" says Anna Szymborska, who carried out the work.

To figure out how the Y’s were arranged, the EMBL scientists used fluorescent tags to label a series of points along each of the Y's arms and tail, and analysed them under a super-resolution microscope. By combining images from thousands of nuclear pores, they were able to obtain measurements of where each of those points was, in relation to the pore's centre, with a precision of less than a nanometre – a millionth of a millimetre. The result was a rainbow of rings whose order and spacing meant the Y-shaped molecules in the nuclear pore must lie in an orderly circle around the opening, all with the same arm of the Y pointing toward the pore's centre.

Having resolved this decade-old controversy, the scientists intend to delve deeper into the mysteries of the nuclear pore – determining whether the circle of Y’s is arranged clockwise or anticlockwise, studying it at different stages of assembly, looking at other parts of the pore, and investigating it in three dimensions.

"There's been a lot of interest from other groups, so we'll soon be looking into a number of other molecular puzzles, like the different 'machines' that allow a cell to divide, which are also built from hundreds of pieces," says Jan Ellenberg, who led the work.

The work was carried out in collaboration with John Briggs' group at EMBL, who helped adapt the image averaging algorithms from electron microscopy to super-resolution microscopy, and Volker Cordes at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, who provided antibodies and advice.

Source: European Molecular Biology Laboratory 

Contact: Sonia Furtado Neves

Reference:

Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging

Szymborska, A., Marco, A., Daigle, N., Cordes, V.C., Briggs, J.A.G. & Ellenberg, J.

Science Express on 11 July 2013. DOI: 10.1126/science.1240672

..........

ZenMaster

---

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

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

---

Enhancer RNAs Alter Gene Expression

New class of RNA molecules may be key emerging 'enhancer therapy'

Tuesday, 04 June 2013

In a pair of distinct but complementary papers, researchers at the University of California, San Diego School of Medicine and colleagues illuminate the functional importance of a relatively new class of RNA molecules. The work, published online this week in the journal Nature, suggests modulation of "enhancer-directed RNAs" or "eRNAs" could provide a new way to alter gene expression in living cells, perhaps affecting the development or pathology of many diseases.

Enhancers are sequences in the genome that act to boost or "enhance" the activity or expression of nearby genes. They "often behave in a cell-specific manner and play an important role in establishing a cell's identity and functional potential," said Christopher Glass, MD, PhD, a professor in the department of Medicine and Cellular and Molecular Medicine at UC San Diego and principal investigator of one of the papers.

Although enhancers have been recognized for more than 25 years, scientists have laboured to fully flesh out the breadth and complexity of what enhancers do and how they do it. In 2010, it was discovered that enhancers directed expression of RNA on a broad scale in neurons and macrophages, a type of immune system cell. Dubbed eRNAs, they were different from other classes of nuclear non-coding RNAs, and raised new questions about their potential roles in the functions of enhancers. The two Nature papers attempt to answer some of these questions.

In the first, principal investigator Glass and colleagues investigated a pair of related transcriptional repressors called Rev-Erb-alpha and Rev-Erb-beta (proteins with important roles in regulating the circadian rhythm in many cell types) in mouse macrophages. Using genome-wide approaches, they found that the Rev-Erb proteins repressed gene expression in macrophages primarily by binding to enhancers. Collaboration with researchers at the Salk Institute for Biological Studies revealed that the repressive function of Rev-Erbs was highly correlated with their ability to repress the production of eRNAs.

In the second paper, principal investigator Michael G. Rosenfeld, MD, a professor in the UC San Diego Department of Medicine and Howard Hughes Medical Institute investigator, and colleagues looked at oestrogen receptor binding in human breast cancer cells – and its impact on enhancer transcription. In contrast to the repressive functions of Rev-Erbs, oestrogen receptors (ERs) activate gene expression; but, like Rev-Erbs, they primarily function by also binding to enhancers. ER binding was shown to be associated with increases in enhancer-directed eRNAs in the vicinity of oestrogen-induced genes, and to exert roles on activation of coding target genes.

Both papers offer new evidence that eRNAs significantly contribute to enhancer activity, and therefore to expression of nearby genes.

"Because many broadly expressed genes that play key roles in essential cellular functions are under the control of cell-specific enhancers, the ability to affect enhancer function by knocking down eRNAs could potentially provide a new strategy for altering gene expression in vivo in a cell-specific manner," said Glass, noting that in his research, anti-sense oligonucleotides were developed in conjunction with Isis Pharmaceuticals, which suppressed enhancer activity and reduced expression in nearby genes.

Source: University of California - San Diego

Contact: Scott LaFee

References:

Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription

Michael T. Y. Lam, Han Cho, Hanna P. Lesch, David Gosselin, Sven Heinz, Yumiko Tanaka-Oishi, Christopher Benner, Minna U. Kaikkonen, Aneeza S. Kim, Mika Kosaka, Cindy Y. Lee, Andy Watt, Tamar R. Grossman, Michael G. Rosenfeld, Ronald M. Evans &Christopher K. Glass

Nature (2013), doi:10.1038/nature12209

Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation

Wenbo Li, Dimple Notani, Qi Ma, Bogdan Tanasa, Esperanza Nunez, Aaron Yun Chen, Daria Merkurjev, Jie Zhang, Kenneth Ohgi, Xiaoyuan Song, Soohwan Oh, Hong-Sook Kim, Christopher K. Glass & Michael G. Rosenfeld

Nature (2013), doi:10.1038/nature12210

.........

ZenMaster

---

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

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

---

Human Skin Cells Converted into Embryonic Stem Cells by Cloning

The breakthrough marks the first time human stem cells have been produced via nuclear transfer and follows several unsuccessful attempts by research groups worldwide

Thursday, 16 May 2013

Scientists at Oregon Health & Science University and the Oregon National Primate Research Center (ONPRC) have successfully reprogrammed human skin cells to become embryonic stem cells capable of transforming into any other cell type in the body. It is believed that stem cell therapies hold the promise of replacing cells damaged through injury or illness. Diseases or conditions that might be treated through stem cell therapy include Parkinson's disease, multiple sclerosis, cardiac disease and spinal cord injuries.

The research breakthrough, led by Shoukhrat Mitalipov, Ph.D., a senior scientist at ONPRC, follows previous success in transforming monkey skin cells into embryonic stem cells in 2007. This latest research will be published in the journal Cell online May 15 and in print June 6.

Scematic description of how to create 

stem cells from human skin. Credit: 

ONPRC. 

The technique used by Drs. Mitalipov, Paula Amato, M.D., and their colleagues in OHSU's Division of Reproductive Endocrinology and Infertility, Department of Obstetrics & Gynecology, is a variation of a commonly used method called somatic cell nuclear transfer, or SCNT. It involves transplanting the nucleus of one cell, containing an individual's DNA, into an egg cell that has had its genetic material removed. The unfertilized egg cell then develops and eventually produces stem cells.

"Our finding offers new ways of generating stem cells for patients with dysfunctional or damaged tissues and organs," says senior study author Shoukhrat Mitalipov.

"Such stem cells can regenerate and replace those damaged cells and tissues and alleviate diseases that affect millions of people."

"A thorough examination of the stem cells derived through this technique demonstrated their ability to convert just like normal embryonic stem cells, into several different cell types, including nerve cells, liver cells and heart cells. Furthermore, because these reprogrammed cells can be generated with nuclear genetic material from a patient, there is no concern of transplant rejection," explained Dr. Mitalipov.

"While there is much work to be done in developing safe and effective stem cell treatments, we believe this is a significant step forward in developing the cells that could be used in regenerative medicine."

Another noteworthy aspect of this research is that it does not involve the use of fertilized embryos, a topic that has been the source of a significant ethical debate.

The first step during SCNT is enucleation

or removal of nuclear genetic material

(chromosomal) from a human egg. An

egg is positioned with holding pipette 

(on the left) and egg's chromosomes are

visualized under polarized microscope.

A hole is made in the egg's shell (zone

pellucida) using a laser and a smaller

pipette (on the right) is inserted through

the opening. The chromosomes then

sucked in inside the pipette and slowly

removed from the egg. Credit: Cell,

Tachibana et al.. 

The Mitalipov team's success in reprogramming human skin cells came through a series of studies in both human and monkey cells. In the past, researchers have used SCNT to generate only mouse and monkey embryonic stem cells — immature cells that can develop into different types of specialized cells, from neurons to heart muscle cells. Most previous attempts failed to produce human SCNT embryos that could progress beyond the 8-cell stage, falling far short of the 150-cell blastocyst stage that could provide hESCs for clinical purposes. Until now, it was not clear which factors and protocols are important for promoting SCNT embryonic development. Previous unsuccessful attempts by several labs showed that human egg cells appear to be more fragile than eggs from other species. Therefore, known reprogramming methods stalled before stem cells were produced.

To solve this problem, the OHSU group studied various alternative approaches first developed in monkey cells and then applied to human cells. Through moving findings between monkey cells and human cells, the researchers were able to develop a successful method.

The key to this success was finding a way to prompt egg cells to stay in a state called "metaphase" during the nuclear transfer process. Metaphase is a stage in the cell's natural division process (meiosis) when genetic material aligns in the middle of the cell before the cell divides. The research team found that chemically maintaining metaphase throughout the transfer process prevented the process from stalling and allowed the cells to develop and produce stem cells.

This is a colony of human ESCs (upper

portion) extracted from a blastocyst

generated by SCNT Credit: Cell,

Tachibana et al.. 

"This is a remarkable accomplishment by the Mitalipov lab that will fuel the development of stem cell therapies to combat several diseases and conditions for which there are currently no treatments or cures," said Dr. Dan Dorsa, Ph.D., OHSU Vice President for Research.

"The achievement also highlights OHSU's deep reproductive expertise across our campuses. A key component to this success was the translation of basic science findings at the OHSU primate centre paired with privately funded human cell studies."

One important distinction is that while the method might be considered a technique for cloning stem cells, commonly called therapeutic cloning, the same method would not likely be successful in producing human clones otherwise known as reproductive cloning. Several years of monkey studies that utilize somatic cell nuclear transfer have never successfully produced monkey clones. It is expected that this is also the case with humans. Furthermore, the comparative fragility of human cells as noted during this study is a significant factor that would likely prevent the development of clones.

"Our research is directed toward generating stem cells for use in future treatments to combat disease," added Dr. Mitalipov.

"While nuclear transfer breakthroughs often lead to a public discussion about the ethics of human cloning, this is not our focus, nor do we believe our findings might be used by others to advance the possibility of human reproductive cloning."

Source: Oregon Health & Science University 

Contact: Jim Newman and Mary Beth O'Leary (Cell Press)

Reference:

Human embryonic stem cells derived by somatic nuclear transfer

Masahito Tachibana, Paula Amato, Michelle Sparman, Nuria Marti Gutierrez, Rebecca Tippner-Hedges, Hong Ma, Eunju Kang, Alimujiang Fulati, Hyo-Sang Lee, Hathaitip Sritanaudomchai, Keith Masterson, Janine Larson, Deborah Eaton, Karen Sadler-Fredd, David Battaglia, David Lee, Diana Wu, Jeffrey Jensen, Phillip Patton, Sumita Gokhale, Richard L. Stouffer, Don Wolf, Shoukhrat Mitalipov

Cell, 15 May 2013, doi.org/10.1016/j.cell.2013.05.006, 2013

.........

ZenMaster

---

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

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

---

How to Make Stem Cells

Nuclear reprogramming moves a step forward

Monday, 29 October 2012

The idea of taking a mature cell and removing its identity (nuclear reprogramming) so that it can then become any kind of cell, holds great promise for repairing damaged tissue or replacing bone marrow after chemotherapy. Hot on the heels of his recent Nobel prize Dr John B. Gurdon has published today in BioMed Central's open access journal Epigenetics & Chromatin research showing that histone H3.3 deposited by the histone-interacting protein HIRA is a key step in reverting nuclei to a pluripotent type, capable of being any one of many cell types.

All of an individual's cells have the same DNA, yet these cells become programmed, as the organism matures, into different types such as heart, or lung or brain. To achieve this different genes are more or less permanently switched off in each cell lineage. As an embryo grows, after a certain number of divisions, it is no longer possible for cells which have gone down the pathway to become something else. For example heart cells cannot be converted into lung tissue, and muscle cells cannot form bone.

One way to reprogram DNA is to transfer the nucleus of a mature cell into an unfertilized egg. Proteins and other factors inside the egg alter the DNA switching some genes on and other off until it resembles the DNA of a pluripotent cell. However there seem to be some difficulties with this method in completely wiping the cell's 'memory'.

One of the mechanisms regulating the activation of genes is chromatin and in particular histones. DNA is wrapped around histones and alteration in how the DNA is wound changes which genes are available to the cell. In order to understand how nuclear reprogramming works Dr Gurdon's team transplanted a mouse nucleus into a frog oocyte (Xenopus laevis). They added fluorescently tagged histones by microinjection, so that they could see where in the cell and nucleus these histones collected.

Prof Gurdon explained:

"Using real-time microscopy it became apparent that from 10 hours onwards H3.3 (the histone involved with active genes) expressed in the oocyte became incorporated into the transplanted nucleus. When we looked in detail at the gene Oct4, which is known to be involved in making cells pluripotent, we found that H3.3 was incorporated into Oct4, and that this coincided with the onset of transcription from the gene."

Prof Gurdon's team also found that HIRA, a protein required to incorporate H3.3 into chromatin, was also required for nuclear reprogramming.

Dr Steven Henikoff, from the Fred Hutchinson Cancer Research Center, commented:

"Manipulating the H3.3 pathway may provide a way to completely wipe a cell's 'memory' and produce a truly pluripotent cell. Half a century after showing that cells can be reprogrammed this research provides a link to the work of Shinya Yamanaka (who shared the prize), and suggests that chromatin is a sticking point preventing artificially induced reprogramming being used routinely in the clinic."

Source: BioMed Central 

Contact: Hilary Glover

Reference:

HIRA dependent H3.3 deposition is required for transcriptional reprogramming following nuclear transfer to Xenopus oocytes

Jerome Jullien, Carolina Astrand, Emmanuelle Szenker, Nigel Garrett, Genevieve Almouzni and John Gurdon 

Epigenetics & Chromatin (in press)

.........

ZenMaster

---

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

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

---

Researchers Reprogram Brain Cells to Become Heart Cells

Researchers Reprogram Brain Cells to Become Heart Cells Saturday, 09 July 2011

 

For the past decade, researchers have tried to reprogram the identity of all kinds of cell types. Heart cells are one of the most sought-after cells in regenerative medicine because researchers anticipate that they may help to repair injured hearts by replacing lost tissue. Now, researchers at the Perelman School of Medicine at the University of Pennsylvania are the first to demonstrate the direct conversion of a non-heart cell type into a heart cell by RNA transfer. Working on the idea that the signature of a cell is defined by molecules called messenger RNAs (mRNAs), which contain the chemical blueprint for how to make a protein, the investigators changed two different cell types, an astrocyte (a star-shaped brain cell) and a fibroblast (a skin cell), into a heart cell, using mRNAs.

James Eberwine, PhD, the Elmer Holmes Bobst Professor of Pharmacology, Tae Kyung Kim, PhD, post-doctoral fellow, and colleagues report their findings online this week in the Proceedings of the National Academy of Sciences. This approach offers the possibility for cell-based therapy for cardiovascular diseases.

"What's new about this approach for heart-cell generation is that we directly converted one cell type to another using RNA, without an intermediate step," explains Eberwine.

tCardiomyocyte (center), showing

protein distribution (green and red

colors) indicative of a young

cardiomyocyte. Credit: Tae Kyung Kim,

PhD, Perelman School of Medicine,

University of Pennsylvania.

The scientists put an excess of heart cell mRNAs into either astrocytes or fibroblasts using lipid-mediated transfection, and the host cell does the rest. These RNA populations (through translation or by modulation of the expression of other RNAs) direct DNA in the host nucleus to change the cell's RNA populations to that of the destination cell type (heart cell, or tCardiomyocyte), which in turn changes the phenotype of the host cell into the destination cell.

The method the group used, called Transcriptome Induced Phenotype Remodeling, or TIPeR, is distinct from the induced pluripotent stem cell (iPS) approach used by many labs in that host cells do not have to be dedifferentiated to a pluripotent state and then re-differentiated with growth factors to the destination cell type. TIPeR is more similar to prior nuclear transfer work in which the nucleus of one cell is transferred into another cell where upon the transferred nucleus then directs the cell to change its phenotype based upon the RNAs that are made. The tCardiomyocyte work follows directly from earlier work from the Eberwine lab, where neurons were converted into tAstrocytes using the TIPeR process.

The team first extracted mRNA from a heart cell, then put it into host cells. Because there are now so many more heart-cell mRNAs versus astrocyte or fibroblast mRNAs, they take over the indigenous RNA population. The heart-cell mRNAs are translated into heart-cell proteins in the cell cytoplasm. These heart-cell proteins then influence gene expression in the host nucleus so that heart-cell genes are turned on and heart-cell-enriched proteins are made.

To track the change from an astrocyte to heart cell, the team looked at the new cells' RNA profile using single cell microarray analysis; cell shape; and immunological and electrical properties. While TIPeR-generated tCardiomyocytes are of significant use in fundamental science it is easy to envision their potential use to screen for heart cell therapeutics, say the study authors. What's more, creation of tCardiomyoctes from patients would permit personalized screening for efficacy of drug treatments; screening of new drugs; and potentially as a cellular therapeutic.

Source: University of Pennsylvania School of Medicine
Contact: Karen Kreeger  

Reference:
Transcriptome transfer provides a model for understanding the phenotype of cardiomyocytes

Tae Kyung Kim, Jai-Yoon Sul, Nataliya B. Peternko, Jae Hee Lee, Miler Lee, Vickas V. Patel, Junhyong Kim, and James H. Eberwine

PNAS July 5, 2011, July 5, 2011, doi: 10.1073/pnas.1101223108

.........


ZenMaster

---

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

---