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

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Mitochondrial Donation: How Many Women Could Benefit?

Two and a Half Thousand Women Could Benefit from Mitochondrial Donation in the UK

Thursday, 29 January 2015

Almost 2,500 women of child-bearing age in the UK are at risk of transmitting mitochondrial disease to their children, according to the most recent estimates published today in the New England Journal of Medicine.

The research offers the most recent evidence yet of how many families could potentially be helped by new IVF techniques to prevent mitochondrial disease, which would be permitted by new regulations on which a vote in parliament is imminent.

Mitochondrial diseases are caused by inherited mutations in the DNA contained in mitochondria - tiny structures present in every cell that generate energy. Mitochondrial diseases can be devastating and particularly affect tissues that have high energy demands - brain, muscle (including heart), liver and kidney.

New IVF-based techniques have been developed which have the potential to prevent the transmission of serious mitochondrial disease. Known as 'mitochondrial donation' the techniques involve removing faulty mitochondria inherited from the mother and replacing them with the healthy mitochondria of another woman. The nuclear DNA, containing 99.9% of genetic material from the mother and father, remains unchanged.

Researchers at the Wellcome Trust Centre for Mitochondrial Research at Newcastle University, which will be the first to offer mitochondrial donation if parliament agrees to new regulations of the Human Fertilisation and Embryology Act (1990), have now calculated how many women have disease-causing mutations in their mitochondrial DNA in order to estimate how many could potentially benefit. The new regulations only allow for mitochondrial donation to prevent mitochondrial disease and set no precedent for genetic manipulation of nuclear DNA.

They calculate that 2,473 women in the UK, and 12,423 women in the US, aged between 15 and 44 years, are at risk of passing on potentially lethal mitochondrial DNA disease to their children. This equates to an average of 152 births per year in the UK, and 778 births per year in the US.

The estimates were made by identifying the number of women in North East England who are at risk of passing on mitochondrial disease to their children and extrapolating the figure to the rest of the UK, based on the relative number of women of child-bearing age in the North East compared to the UK as a whole. A similar method was used for the US figures. The study did not account for variance due to ethnicity or potentially different fertility rates in different parts of the UK.

Researchers also assessed the fertility of women with mitochondrial DNA mutations. To do this they compared fertility data from their patients' to data about the general population, obtained from the UK Office for National Statistics. They found that mitochondrial mutation has no statistically significant effect on fertility rate.

Dr Gráinne Gorman from the Wellcome Trust Centre for Mitochondrial Research at Newcastle University, and joint first author of the paper, said:

"Our estimate of how many women could benefit from mitochondrial donation is based on our data from North East England, where we have very detailed insight into how many women are affected. We are confident that there are a similar number of women across the UK at risk of passing on mitochondrial disease to their children."

Professor Doug Turnbull, Director of the Wellcome Trust Centre for Mitochondrial Research at Newcastle University, and an author of the paper, said:

"Our findings have considerable implications for all countries that are considering allowing mitochondrial donation techniques. In the UK we are waiting for parliament to decide whether to support these regulations. This would allow women who carry these mutations greater reproductive choice. "

Source: Wellcome Trust 

Contact: Clare Ryan 

Reference:

Mitochondrial Donation: How many women could benefit? 

Gráinne S. Gorman, John P. Grady, Yi Ng, Andrew M. Schaefer, Richard J. McNally, Patrick F. Chinnery, Patrick Yu Wai Man, Mary Herbert, Robert W. Taylor, Robert McFarland, and Doug M. Turnbull

New England Journal of Medicine, January 28, 2015 DOI: 10.1056/NEJMc1500960

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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

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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

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Three Parents and a Baby

Scientists advise caution with regard to artificial insemination method

Wednesday, 18 June 2014

Already a few dysfunctional mitochondria (in

yellow on top of the picture) could cause a

disease by overgrowing functional ones (in blue).

Credit: Illustration: Iain Johnston. 

Mitochondria are cell organelles located within animal and human cells. They produce energy for the organism, possess their own genetic material - mitochondrial DNA (mtDNA) - and are transmitted exclusively by the mother. Depending on their activity and tasks, different numbers of mitochondria are present in a cell - usually a few hundred to a thousand per body cell.

Inherited mitochondrial disorders or so-called mitochondropathies occur in about one of 10,000 humans throughout the world. Diseases such as diabetes, stroke, cardiac defects, epilepsy, or muscle weakness may originate from mitochondrial defects. Inherited mitochondrial disorders have been incurable so far. Therefore, efforts are now being made to enable women with this disease to bear healthy children by means of nuclear transfer.

Mitochondria multiply at different rates

Jörg Burgstaller, a scientist and member of Gottfried Brem's research group at the Vetmeduni Vienna, has been working for several years on the genetics of mitochondria. It was known before that different types of mitochondria within a cell can proliferate at different rates. However, it was not known whether this is a singular phenomenon or if these cases occur more frequently.

Burgstaller investigated this in four newly bred mouse models which carried different mixtures of mitochondria whose DNA were related to each other to a differing extent.

This meant no health problem for the mice since all mtDNAs are were fully functional.

The outcome was: the more distantly two types of mitochondria within an egg cell were related, the more frequently a growth advantage was noted in favour of one of the two types of mitochondria. When two different mtDNAs were equally common in cells of an organ at the time of birth, one type was completely lost after a while. One mitochondria variant had thus achieved a growth advantage compared to the other variant and superseded the latter. This effect was almost non-existent in genetically very similar mitochondria within the cells; the ratio between the two types of mitochondria was not altered in that case.

The effect is of significance in reproduction medicine

Burgstaller's results may have effects on the planned introduction of the so called "Three-Parent Baby" in Great Britain. Experts take the cell nucleus of one human egg cell whose mitochondria have a defect and place it in an egg cell with "healthy" mitochondria. The baby resulting from this procedure has three parents, namely the mother whose cell nucleus is used, the mother whose mitochondria are involved, and the father whose sperm inseminated the egg cell.

However, this method raises the following problem: in every nuclear transfer, a small number of defective mitochondria are transferred into the healthy egg cell.

"So far it was believed that this minimal 'contamination' is of no consequence for the baby. However, our data show that the effect may have dramatic consequences on the health of the offspring. If the mitochondria of both mothers are genetically very different, it may have the same effects seen in the mouse model," says Burgstaller who developed the theory together with co-author Joanna Poulton, Professor of Mitochondrial Genetics at the John Radcliffe Hospital in Oxford.

"One mitochondrial type may be able to assert itself against the other. If the assertive one happens to carry the defective mtDNA, the benefit of the therapy would be jeopardized."

The solution to the "Three-Parent Baby"-problem

Burgstaller and his colleagues suggest the following solution to the problem: the mtDNA of both mothers, i.e. the donor of the nucleus and the donor of the mitochondria, should be analysed in advance and aligned to each other. So called "matching haplotypes" could prevent the dangerous effect. In the future the effect may even be utilized in a targeted manner to suppress defective mtDNA.

Source: University of Veterinary Medicine – Vienna 

Contact: Joerg Burgstaller

Reference:

mtDNA Segregation in Heteroplasmic Tissues Is Common In Vivo and Modulated by Haplotype Differences and Developmental Stage

Joerg Patrick Burgstaller, Iain G. Johnston, Nick S. Jones, Jana Albrechtová, Thomas Kolbe, Claus Vogl, Andreas Futschik, Corina Mayrhofer, Dieter Klein, Sonja Sabitzer, Mirjam Blattner, Christian Gülly, Joanna Poulton, Thomas Rülicke, Jaroslav Piálek, Ralf Steinborn and Gottfried Brem

Cell Reports. DOI:10.1016/j.celrep.2014.05.020 

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First Disease-specific Human Embryonic Stem Cell Line by Nuclear Transfer

Major step toward cell-based therapies for life-threatening diseases

Tuesday, 29 April 2014

Using somatic cell nuclear transfer, a team of scientists led by Dr. Dieter Egli at the New York Stem Cell Foundation (NYSCF) Research Institute and Dr. Mark Sauer at Columbia University Medical Center has created the first disease-specific embryonic stem cell line with two sets of chromosomes.

Embryonic stem cells from an adult with type 1

diabetes were created by transferring the nucleus

from a skin cell of the patient into a donor oocyte.

Credit: Bjarki Johannesson, NYSCF. 

As reported today in Nature, the scientists derived embryonic stem cells by adding the nuclei of adult skin cells to unfertilized donor oocytes using a process called somatic cell nuclear transfer (SCNT). Embryonic stem cells were created from one adult donor with type 1 diabetes and a healthy control. In 2011, the team reported creating the first embryonic cell line from human skin using nuclear transfer when they made stem cells and insulin-producing beta cells from patients with type 1 diabetes. However, those stem cells were triploid, meaning they had three sets of chromosomes, and therefore could not be used for new therapies.

The investigators overcame the final hurdle in making personalized stem cells that can be used to develop personalized cell therapies. They demonstrated the ability to make a patient-specific embryonic stem cell line that has two sets of chromosomes (a diploid state), the normal number in human cells. Reports from 2013 showed the ability to reprogram foetal fibroblasts using SCNT; however, this latest work demonstrates the first successful derivation by SCNT of diploid pluripotent stem cells from adult and neonatal somatic cells.

"From the start, the goal of this work has been to make patient-specific stem cells from an adult human subject with type 1 diabetes that can give rise to the cells lost in the disease," said Dr. Egli, the NYSCF scientist who led the research and conducted many of the experiments.

"By reprograming cells to a pluripotent state and making beta cells, we are now one step closer to being able to treat diabetic patients with their own insulin-producing cells."

"I am thrilled to say we have accomplished our goal of creating patient-specific stem cells from diabetic patients using somatic cell nuclear transfer," said Susan L. Solomon, CEO and co-founder of NYSCF.

"I became involved with medical research when my son was diagnosed with type 1 diabetes, and seeing today's results give me hope that we will one day have a cure for this debilitating disease. The NYSCF laboratory is one of the few places in the world that pursues all types of stem cell research. Even though many people questioned the necessity of continuing our SCNT work, we felt it was critical to advance all types of stem-cell research in pursuit of cures. We don't have a favourite cell type, and we don't yet know what kind of cell is going to be best for putting back into patients to treat their disease."

The research is the culmination of an effort begun in 2006 to make patient-specific embryonic stem cell lines from patients with type 1 diabetes. Ms. Solomon opened NYSCF's privately funded laboratory on March 1, 2006, to facilitate the creation of type 1 diabetes patient-specific embryonic stem cells using SCNT. Initially, the stem cell experiments were done at Harvard and the skin biopsies from type 1 diabetic patients at Columbia; however, isolation of the cell nuclei from these skin biopsies could not be conducted in the federally funded laboratories at Columbia, necessitating a safe-haven laboratory to complete the research. NYSCF initially established its lab, now the largest independent stem cell laboratory in the nation, to serve as the site for this research.

In 2008, all of the research was moved to the NYSCF laboratory when the Harvard scientists determined they could no longer move forward, as restrictions in Massachusetts prevented their obtaining oocytes. Dr. Egli left Harvard University and joined NYSCF; at the same time, NYSCF forged a collaboration with Dr. Sauer who designed a unique egg-donor program that allowed the scientists to obtain oocytes for the research.

"This project is a great example of how enormous strides can be achieved when investigators in basic science and clinical medicine collaborate. I feel fortunate to have been able to participate in this important project," said Dr. Sauer. Dr. Sauer is vice chair of the Department of Obstetrics and Gynecology, professor of obstetrics and gynaecology, and chief of reproductive endocrinology at Columbia University Medical Center and program director of assisted reproduction at the Center for Women's Reproductive Care.

Patients with type 1 diabetes lack insulin-producing beta cells, resulting in insulin deficiency and high blood-sugar levels. Therefore, producing beta cells from stem cells for transplantation holds promise as a treatment and potential cure for type 1 diabetes. Because the stem cells are made using a patient's own skin cells, the beta cells for replacement therapy would be autologous, or from the patient, matching the patient's DNA.

Generating autologous beta cells using SCNT is only the first step in developing a complete cell replacement therapy for type 1 diabetes. In type 1 diabetes, the body's immune system attacks its own beta cells; therefore, further work is underway at NYSCF, Columbia, and other institutions to develop strategies to protect existing and therapeutic beta cells from attack by the immune system, as well as to prevent such attack.

The technique described in the report published today can also be translated for use in the development of personalized autologous cell therapies for many other diseases and conditions including Parkinson's disease, macular degeneration, multiple sclerosis, and liver diseases and for replacing or repairing damaged bones.

As part of the work, the scientists systematically analysed the factors that affect stem-cell derivation after SCNT. The reprogramming of skin cells from a type 1 diabetes patient by SCNT has long been sought, but has been challenging to achieve because of logistical difficulties in obtaining human oocytes for research, as well as an incomplete understanding of the biology of human oocytes.

The scientists found that the addition of specific chemicals, called histone deacetylase inhibitors, and an efficient protocol for human oocyte activation were critical to achieving development to the stage at which embryonic stem cells are derived. These findings are consistent with the 2013 report by Tachibana and colleagues that used foetal cells. Though the authors of the 2013 paper also performed studies with cells of an infant with Leigh syndrome, they did not demonstrate that diploid pluripotent stem cells could be derived from these cells. Because foetal cells are less mature than the cells after birth, it was critical to determine if diploid pluripotent stem cells could be derived from the cells of both infants and adults.

As an additional optimization of the SCNT protocol, the scientists found that it was important to maintain the integrity of the plasma membrane during manipulation, and that to do so, the agent used in the manipulations had to be at a low dose. The scientists applied this optimized protocol to skin cells of a male new-born and the cells of the adult patient with type 1 diabetes. From these two cell lines, the scientists produced a total of four SCNT-derived embryonic stem cell lines. All cell lines were diploid and could give rise to neurons, pancreatic cells, and cartilage, as well as various other cell types, demonstrating their pluripotency. Importantly, the cells of the type 1 diabetes patient also gave rise to insulin-producing beta cells.

Therefore, this is the first report of the derivation of diploid pluripotent stem cells from a patient. And together with a paper published this month in Cell Stem Cell by Chung et al., it is also the first report of diploid embryonic stem cell lines derived from a human after birth.

Dr. Nissim Benvenisty and his laboratory at Hebrew University of Jerusalem collaborated on this report by demonstrating that the cells produced were, in fact, embryonic stem cells by using microarrays to perform gene expression analysis of the cells.

Dr. Rudolph Leibel, a co-author and co-director with Dr. Robin Goland of the Naomi Berrie Diabetes Center, where aspects of these studies were conducted, said:

"This accomplishment is the product of an ongoing inter-institutional collaboration across scientific and clinical disciplines, supported by thoughtful philanthropy. The resulting technical and scientific insights bring closer the promise of cell replacement for a wide range of human disease."

NYSCF continues pursuing SCNT research despite many scientific obstacles and in light of the advent of induced pluripotent stem (iPS) cells, as it is not yet clear which type of stem cells will prove best for personalized treatments. Many thought that iPS cells, first created from human cells in 2007, would replace the need for patient-specific embryonic stem cells because they allow patient- and disease-specific stem cell lines to be generated by genetically reprogramming adult cells into becoming pluripotent cells. However, it is not clear how similar iPS cells are to naturally occurring embryonic stem cells, which remain the gold standard, and what will be the preferred cell type for therapies.

Though it is now possible to derive stem cell lines with a patient's genotype using iPS technology, the generation of stem cells using oocytes may have an advantage for use in cell replacement for diseases such as type 1 diabetes. The generation of pluripotent stem cell lines by SCNT uses human oocytes, while iPS cells use recombinant DNA, RNA, or chemicals, each of which requires its own safety testing and approval for clinical use. Human oocytes are already used routinely around the world to generate clinically relevant cells. The generation of pluripotent stem cell lines using human oocytes may therefore be particularly suitable for the development of cell-replacement therapies. Therefore, this work brings the scientists a significant step closer to this goal.

Drs. Mitsutoshi Yamada and Bjarki Johannesson, postdoctoral fellows at the NYSCF Research Institute, were the co-first authors of the paper.

Source: New York Stem Cell Foundation

Contact: David McKeon

Reference:

Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells

Mitsutoshi Yamada, Bjarki Johannesson, Ido Sagi, Lisa Cole Burnett, Daniel H. Kort, Robert W. Prosser, Daniel Paull, Michael W. Nestor, Matthew Freeby, Ellen Greenberg, Robin S. Goland, Rudolph L. Leibel, Susan L. Solomon, Nissim Benvenisty, Mark V. Sauer& Dieter Egli

Nature 28 April 2014, doi:10.1038/nature13287

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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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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

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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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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)

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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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