Study Reveals the Genetic Start-up of a Human Embryo

Study Reveals the Genetic Start-up of a Human Embryo

Thursday, 03 September 2015

An international team of scientists led from Sweden’s Karolinska Institutet has for the first time mapped all the genes that are activated in the first few days of a fertilised human egg. The study, which is being published in the journal Nature Communications, provides an in-depth understanding of early embryonic development in human – and scientists now hope that the results will help finding for example new therapies against infertility.

At the start of an individual’s life there is a single fertilised egg cell. One day after fertilisation there are two cells, after two days four, after three days eight and so on, until there are billions of cells at birth. The order in which our genes are activated after fertilisation has remained one of the last uncharted territories of human development.

Juha Kere is a Professor of Molecular Genetics at

Karolinska Institutet. Credit: Ulf Sirborn.

There are approximately 23,000 human genes in total. In the current study, scientists found that only 32 of these genes are switched on two days after fertilization, and by day three there are 129 activated genes. Seven of the genes found and characterised had not been discovered previously.

“These genes are the ‘ignition key’ that is needed to turn on human embryonic development. It is like dropping a stone into water and then watching the waves spread across the surface”, says principal investigator Juha Kere, professor at theDepartment of Biosciences and Nutrition at Karolinska Institutet and also affiliated to the SciLifeLab facility in Stockholm.

The researchers had to develop a new way of analysing the results in order to find the new genes. Most genes code for proteins but there are a number of repeated DNA sequences that are often considered to be so-called ‘junk DNA’, but are in fact important in regulating gene expression.

Treatment of infertility

In the current study, the researchers show that the newly identified genes can interact with the ‘junk DNA’, and that this is essential to the start of development.

Outi Hovatta is a Professor of Obstetrics and

Gynaecology at Karolinska Institutet. Credit:

Ulf Sirborn.

“Our results provide novel insights into the regulation of early embryonic development in human. We identified novel factors that might be used in reprogramming cells into so-called pluripotent stem cells for possible treatment of a range of diseases, and potentially also in the treatment of infertility”, says Outi Hovatta, professor at Karolinska Institutet’s Department of Clinical Science, Intervention and Technology, and a senior author.

The study was a collaboration between three research groups from Sweden and Switzerland that each provided a unique set of skills and expertise. The work was supported by the Karolinska Institutet Distinguished Professor Award, the Swedish Research Council, the Strategic Research Program for Diabetes funding at Karolinska Institutet, Stockholm County, the Jane & Aatos Erkko Foundation, the Instrumentarium Science Foundation, and the Åke Wiberg and Magnus Bergvall foundations. The computations were performed on resources provided by SNIC through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX).

Source: Karolinska Institutet

Contact: KI Press Office

Reference:

Novel PRD-like homeodomain transcription factors and retrotransposon elements in early human development

Virpi Töhönen, Shintaro Katayama, Liselotte Vesterlund, Eeva-Mari Jouhilahti, Mona Sheikhi, Elo Madissoon, Giuditta Filippini-Cattaneo, Marisa Jaconi, Anna Johnsson, Thomas R. Bürglin, Sten Linnarsson, Outi Hovatta and Juha Kere

Nature Communications, 3 September 2015, doi: 10.1038/NCOMMS9207

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Master Orchestrator of the Genome Is Discovered

Master Orchestrator of the Genome Is Discovered

Thursday, 07 May 2015

One of developmental biology’s most perplexing questions concerns what signals transform masses of undifferentiated cells into tremendously complex organisms, a process called ontogeny.

UB research suggests a new paradigm, visualized

in this diagram, for developmental global genome

programming by the nuclear FGFR1 protein.

New research by University at Buffalo scientists, published last week in PLOS ONE, provides evidence that it all begins with a single “master” growth factor receptor that regulates the entire genome.

“The finding provides a new level of understanding of the fundamental aspects of how organisms develop,” says senior author Michal K. Stachowiak, PhD, professor in the Department of Pathology and Anatomical Sciences in the UB School of Medicine and Biomedical Sciences and senior author. He also directs the Stem Cell Engraftment and In Vivo Analysis Facility and the Stem Cell Culture and Training Facility at the Western New York Stem Cell Culture and Analysis Center at UB.

“Our research shows how a single growth factor receptor protein moves directly to the nucleus in order to program the entire genome,” he said.

Michal Stachowiak, PhD, Professor, Department

of Pathology and Anatomical Sciences. 

The research challenges a long-held supposition in biology that specific types of growth factors only functioned at a cell’s surface. For two decades, Stachowiak’s team has been intrigued by the possibility that growth factors function from within the nucleus, a point, he says, this current paper finally proves.

A more advanced understanding of how organisms form, based on this work, has the potential to significantly enhance the understanding and treatment of cancers, which result from uncontrolled development as well as congenital diseases, the researchers say. The new research also will contribute to the understanding of how stem cells work.

This work was conducted on mouse embryonic stem cells, not human cells.

Organizing ‘this cacophony of genes’

“We’ve known that the human body has almost 30,000 genes that must be controlled by thousands of transcription factors that bind to those genes,” Stachowiak said, “yet we didn’t understand how the activities of genes were coordinated so that they properly develop into an organism.”

“Now we think we have discovered what may be the most important player, which organizes this cacophony of genes into a symphony of biological development with logical pathways and circuits,” he said.

At the centre of the discovery is a single protein called nuclear Fibroblast Growth Factor Receptor 1 (nFGFR1).

“FGFR1 occupies a position at the top of the gene hierarchy that directs the development of multicellular animals,” said Stachowiak.

The FGFR1 gene is known to govern gastrulation, occurring in early development, where the three-layered embryonic structure forms. It also plays a major role in the development of the central and peripheral nervous systems and the development of the body’s major systems, including muscles and bones.

To study how nuclear FGFR1 worked, the UB team used genome-wide sequencing of mouse embryonic stem cells programmed to develop cells of the nervous system, with additional experiments in which nuclear FGFR1 was either introduced or blocked. The researchers found that the protein was responsible, either alone or with so-called partner nuclear receptors, for ensuring that embryonic stem cells develop into differentiated cells. By targeting thousands of genes, it controls the development of the major points of growth in the body (known as axes) as well as neuronal and muscle development.

The research shows that nuclear FGFR1 binds to promoters of genes that encode transcription factors, the proteins that control which genes are turned on or off in the genome.

“We found that this protein works as a kind of ‘orchestration factor,’ preferably targeting certain gene promoters and enhancers. The idea that a single protein could bind thousands of genes and then organize them into a hierarchy, that was unknown,” Stachowiak said.

“Nobody predicted it.”

Sequencing advances

The discovery that a single protein can exert such a global genomic function stems from recent advances in DNA sequencing technologies, which allow for the sequencing of a complex genome in just hours.

“NextGen DNA sequencing allows us to analyse millions of DNA sequences selected by the interacting protein,” Stachowiak said.

In the UB research, the DNA sequencing data were processed by the supercomputer at the university’s Center for Computational Research (CCR). Stachowiak and his colleagues then spent weeks aligning these data to the genome and conducting further analyses.

“We imposed nuclear FGFR1 on every little corner of genome,” he said.

“The computer spit out which genes are affected by nuclear FGFR1: it was an enormously complex network of genome activity.”

They found that the protein binds to genes that make neurons and muscles as well as to an important oncogene, TP63, which is involved in a number of common cancers.

Other studies in Stachowiak’s laboratory demonstrate that these interactions also take place in the human genome, controlling function and possibly underlying diseases like schizophrenia. Targeting of the nuclear FGFR1 allows for the reactivation of neural development in the adult brain in preclinical studies and thus, Stachowiak says, may offer unprecedented opportunity for regenerative medicine. Nuclear accumulation of nuclear FGFR1 may be altered in some cancer cells, and thus could become a focus in cancer therapy, he added.  

“This seminal discovery lends new perspectives to the origin, nature and treatment of a variety of human disease,” Stachowiak concluded.

Source: University at Buffalo, New York

Contact: Ellen Goldbaum

Reference:

Global Developmental Gene Programing Involves a Nuclear Form of Fibroblast Growth Factor Receptor-1 (FGFR1)

Christopher Terranova,Sridhar T. Narla, Yu-Wei Lee, Jonathan Bard, Abhirath Parikh, Ewa K. Stachowiak, Emmanuel S. Tzanakakis, Michael J. Buck, Barbara Birkaya, Michal K. Stachowiak

PLoS ONE 2015 10(4):e0123380, doi:10.1371/journal.pone.0123380

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First Steps in Basic Biological Process That Could be Harnessed to Make Therapeutic Cells

First Steps in Basic Biological Process That Could be Harnessed to Make Therapeutic Cells

Friday, 17 April 2015

Pioneer factor binding DNA on nucleosome is

shown. Credit: Ken Zaret, Ph.D., Perelman

School of Medicine, University of Pennsylvania.

Understanding the molecular signals that guide early cells in the embryo to develop into different types of organs provides insight into how tissues regenerate and repair themselves. By knowing the principles that underlie the intricate steps in this transformation, researchers will be able to make new cells at will for transplantation and tissue repair in such situations as liver or heart disease.

Now, investigators at the Perelman School of Medicine at the University of Pennsylvania are able to explain how cell identity changes occur at the very beginning of the process.

"During my scientific life, I've been fascinated by how early cells make 'decisions' to turn on one genetic program and exclude others," says Kenneth S. Zaret, PhD, director of the Institute for Regenerative Medicine and a professor of Cell and Developmental Biology. Zaret and postdoctoral fellow Abdenour Soufi, PhD, led a team that describes this research, which appeared online this week ahead of print in Cell. Soufi is now at the MRC Centre for Regenerative Medicine, University of Edinburgh.

What they found could be applied to guiding cells to fates proposed by scientists for a wide variety of biomedical contexts, for example, to better understand molecular changes in the early embryo after fertilization, when one cell type morphs into another. Another application could be to directly change one cell type into another for therapeutic purposes, for example transforming a skin cell directly into a liver, blood, or heart cell.

Pioneer factor binding chromosomes is shown.

Credit: Kenneth S. Zaret, Ph.D., Perelman

School of Medicine, University of Pennsylvania.

Tightly Packed

DNA in each cell is two meters long and 20 atoms wide. All of this genetic material needs to be wound into the nucleus in each of the 14 trillion cells in the human body. This is done by coiling DNA around chromosomal proteins to make repeating units of nucleosomes. These units are further compacted into a structure called chromatin, to make the entire DNA fit into the nucleus of the cell. How proteins that regulate gene expression search through the nucleosomes to find their sites of action on DNA has been a mystery.

Nobel Prize winner Shinya Yamanaka from Kyoto University found that turning on four gene regulatory proteins in mouse skin cells can convert these into embryonic-like stem cells called induced pluripotent stem cells, or iPS cells. The special gene regulatory proteins that make iPS cells, called Oct4, Sox2, KIf4, and c-Myc, are normally active in the early embryo and are collectively known as the Yamanaka factors.

Building on this knowledge, the Zaret lab compared the nucleosome and chromatin targeting activities of the Yamanaka factors. To elicit cell programming or reprogramming, the gene regulatory factors must be able to engage genes that are silenced and not meant for expression in the original cell type. These silenced genes are typically embedded in tightly coiled, "closed" chromatin that is covered by nucleosomes. Transcription factors with the highest reprogramming activity have the necessary ability to interact with their target sites on closed nucleosome DNA. These transcription proteins are called "pioneer factors" because they initiate molecular changes in closed chromatin.

"We found that pioneer protein activity relates simply to the ability of a transcription factor to adapt to particular areas of DNA building blocks on the nucleosome surface. This was an 'aha moment' of simplicity," recalls Zaret.

The Wiggle Factor

The DNA-binding domain (DBD) of pioneer factors allows the protein to recognize its target site on a segment of nucleosome DNA, where part of the DNA structure is occluded by proteins associated with chromosomes. The initial targeting of this DNA by pioneers in closed, silent chromatin allows the pioneer factor to initiate expression of silent genes in a given cell, enabling conversion of one cell type to another.

Zaret and Soufi found that the pioneer factors have an adaptable DBD that wiggles in a special fashion. The Yamanaka factors – Oct4, Sox2, and Klf4 all have the wiggle, and thus act as pioneers, while c-Myc is more rigid and is aided by a pioneer. The wiggle allows the pioneer factor to adapt physically to the shape of the DNA molecule that is in a complex with chromosomal proteins.

"We showed that Oct4, Sox2, and Klf4, but not c-Myc, could function as pioneers during reprogramming by virtue of their ability to target 'closed' chromatin sites that are 'naïve' in that they lack chemical modifications that active parts of the DNA might have," explains Zaret.

To check for the generality of the principles they found, the team looked to other studies and found that the same mechanisms applied to other examples where gene regulatory proteins act as pioneers during cellular reprograming, such as in the creation of neurons from skin cells.

"We all want to know how to transform one cell into another," says Zaret.

"Now we understand how that happens at the first step. We are working on how the pioneer gene regulatory proteins physically open up the chromatin to loosen it, in preparation for gene activity. These points are fundamentally important for understanding tissue development, cell regeneration, and for making designer cells."

Source: Penn Medicine

Contact: Karen Kreeger

Reference:

Pioneer Transcription Factors Target Partial DNA Motifs on Nucleosomes to Initiate Reprogramming

Abdenour Soufi, Meilin Fernandez Garcia, Artur Jaroszewicz, Nebiyu Osman, Matteo Pellegrini, Kenneth S. Zaret

Cell, published online: April 16, 2015, DOI: http://dx.doi.org/10.1016/j.cell.2015.03.017

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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

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Genome's Tale of 'Conquer and Enslave'

Genome's Tale of 'Conquer and Enslave'

Saturday, 21 February 2015

Toronto scientists uncovered how viral remnants helped shape control of our genes.

If genes were lights on a string of DNA, the genome would appear as an endless flicker, as thousands of genes come on and off at any given time. Tim Hughes, a Professor at the University of Toronto's Donnelly Centre, is set on figuring out the rules behind this tightly orchestrated light-show, because when it fails, disease can occur.

This is Professor Tim Hughes of University of

Toronto's Donnelly Centre for Cellular and

Biomolecular Research. Credit: University of

Toronto.

Genes are switched on or off by proteins called transcription factors. These proteins bind to precise sites on the DNA that serve as guideposts, telling transcription factors that their target genes are nearby.

In their latest paper, published in Nature Biotechnology, Hughes and his team did the first systematic study of the largest group of human transcription factors, called C2H2-ZF.

Despite their important roles in development and disease, these proteins have been largely unexplored because they posed a formidable challenge for researchers.

C2H2-ZF transcription factors count over 700 proteins - around three per cent of all human genes! To make matters more complicated, most human C2H2-ZF proteins are very different from those in other organisms, like those in mice. This means that scientists could not apply insights gained from animal studies to human C2H2-ZFs.

Hughes' team found something remarkable: the reason C2H2-ZFs are so abundant and diverse - which makes them difficult to study - is that many of them evolved to defend our ancestral genome from damage caused by the notorious "selfish DNA."

Selfish DNA are bits of parasitic DNA whose only purpose is to multiply, a kind of virus for our genome. They seize a cell's resources to make copies of themselves, which they insert randomly across the genome - causing harmful mutations along the way.

Almost half the human genome is made of selfish DNA, which probably came from ancient retro-viruses which, similar to modern counterparts, inserted their DNA into the host's genome. When this happens in an egg or sperm, the viral DNA gets passed on to the next generation, and the selfish DNA is then known as endogenous retro-elements (EREs).

Evolutionary biologists believe that selfish DNA was instrumental in making genomes bigger, giving natural selection additional DNA material to tinker with.

But Hughes' data suggest that EREs took centre stage in an evolutionary arms race, and that this change spawned C2H2-ZFs, a new group of proteins.

It is an enthralling tale of "conquer and enslave," one that stretches from before mammals existed to the present day.

Hughes says that C2H2-ZFs initially evolved to switch off EREs. As new EREs invaded the genome of our lizard-like ancestor, new C2H2-ZFs arose to prevent them from disrupting gene function.

This would explain how the C2H2-ZF came to be so abundant but also why they are so diverse among different organisms.

"What I think was not appreciated until this study is that retro-elements are really a driving force in the evolution of transcription factors themselves. All mammals have a whole bunch of custom transcription factors that came about to silence the EREs," says Hughes, who is also a Professor in U of T's Department of Molecular Genetics and a Senior Fellow of the Canadian Institute for Advanced Research.

"But the EREs and these new transcription factors are different even for different vertebrates."

These EREs are now harmless because they are millions of years old. Over time they accumulated mutations, which pepper the genome at a constant rate, and, as a result, lost their ability to multiply and move around.

The C2H2-ZFs, on the other hand, took on new jobs.

C2H2-ZF proteins began using the EREs scattered across the genome as DNA docking sites, from which they could take control of nearby genes. The conquered EREs were finally enslaved.

Hughes describes a neat example of this process. One C2H2-ZF family member, a transcription factor called ZNF189 evolved to silence an ancient retro-element, known as LINE L2, which is a staggering 100 million years old. L2 is now inactive but ZNF189 still binds to it because it uses L2 remnants to reach other genes.

Relics of L2 sequences happen to be near genes that drive brain and heart development. And so ZNF189 could take on a new role in shaping these organs, an arrangement preserved by natural selection because it was beneficial to the embryo.

ZFN189 likely puts "breaks" on the "brain genes", similar to its ancient role with L2. But in heart cells, it may actually turn genes on because it misses the part that makes the "off switch."

This story is a beautiful example of just how malleable genomes are at the hands of evolution.

Source: University of Toronto

Contact: Jovana Drinjakovic

Reference:

C2H2 zinc finger proteins greatly expand the human regulatory lexicon

Najafabadi HS, Mnaimneh S, Schmitges FW, Garton M, Lam KN, Yang A, Albu M, Weirauch MT, Radovani E, Kim PM, Greenblatt J, Frey BJ, Hughes TR

Nat Biotechnol. 2015 Feb 18; doi:10.1038/nbt.3128

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New Technology Directly Reprograms Skin Fibroblasts for a New Role

Penn study has implications for new skin disease treatments

Tuesday, 16 December 2014

Dermal fibroblasts are directly reprogrammed to

pigmented melanocytes by three transcription

factors (SOX10, MITF and PAX3).

Credit: Ruifeng Yang, Perelman School of

Medicine, University of Pennsylvania. 

As the main component of connective tissue in the body, fibroblasts are the most common type of cell. Taking advantage of that ready availability, scientists from the Perelman School of Medicine at the University of Pennsylvania, the Wistar Institute, Boston University School of Medicine, and New Jersey Institute of Technology have discovered a way to repurpose fibroblasts into functional melanocytes, the body's pigment-producing cells. The technique has immediate and important implications for developing new cell-based treatments for skin diseases such as vitiligo, as well as new screening strategies for melanoma. The work was published this week in Nature Communications.

The new technique cuts out a cellular middleman. Study senior author Xiaowei "George" Xu, MD, PhD, an associate professor of Pathology and Laboratory Medicine, explains.

"Through direct reprogramming, we do not have to go through the pluripotent stem cell stage, but directly convert fibroblasts to melanocytes. So these cells do not have tumourigenicity."

Changing a cell from one type to another is hardly unusual. Nature does it all the time, most notably as cells divide and differentiate themselves into various types as an organism grows from an embryo into a fully-functional being. With stem cell therapies, medicine is learning how to tap into such cell specialization for new clinical treatments. But controlling and directing the process is challenging. It is difficult to identify the specific transcription factors needed to create a desired cell type. Also, the necessary process of first changing a cell into an induced pluripotent stem cell (iPSC) capable of differentiation, and then into the desired type, can inadvertently create tumours.

Xu and his colleagues began by conducting an extensive literature search to identify 10 specific cell transcription factors important for melanocyte development. They then performed a transcription factor screening assay and found three transcription factors out of those 10 that are required for melanocytes: SOX10, MITF, and PAX3, a combination dubbed SMP3.

"We did a huge amount of work," says Xu.

"We eliminated all the combinations of the other transcription factors and found that these three are essential."

The researchers first tested the SMP3 combination in mouse embryonic fibroblasts, which then quickly displayed melanocytic markers. Their next step used a human-derived SMP3 combination in human foetal dermal cells, and again melanocytes (human-induced melanocytes, or hiMels) rapidly appeared. Further testing confirmed that these hiMels indeed functioned as normal melanocytes, not only in cell culture but also in whole animals, using a hair-patch assay, in which the hiMels generated melanin pigment. The hiMels proved to be functionally identical in every respect to normal melanocytes.

Xu and his colleagues anticipate using their new technique in the treatment of a wide variety of skin diseases, particularly those such as vitiligo for which cell-based therapies are the best and most efficient approach.

The method could also provide a new way to study melanoma.

By generating melanocytes from the fibroblasts of melanoma patients, Xu explains: "we can screen not only to find why these patients easily develop melanoma, but possibly use their cells to screen for small compounds that can prevent melanoma from happening."

Perhaps most significantly, say the researchers, is the far greater number of fibroblasts available in the body for reprogramming compared to tissue-specific adult stem cells, which makes this new technique well-suited for other cell-based treatments.

Source: University of Pennsylvania School of Medicine

Contact: Karen Kreeger

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Human Skin Cells Reprogrammed Directly into Brain Cells

Human Skin Cells Reprogrammed Directly into Brain Cells

Wednesday, 22 October 2014

Scientists have described a way to convert human skin cells directly into a specific type of brain cell affected by Huntington's disease, an ultimately fatal neurodegenerative disorder. Unlike other techniques that turn one cell type into another, this new process does not pass through a stem cell phase, avoiding the production of multiple cell types, the study's authors report.

Scientists have described a way to convert

human skin cells directly into a specific type of

brain cell affected by Huntington's disease, an

ultimately fatal neurodegenerative disorder.

Unlike other techniques that turn one cell type

into another, this new process does not pass

through a stem cell phase, avoiding the

production of multiple cell types, the study's

authors report. The researchers, at Washington

University School of Medicine in St. Louis,

demonstrated that these converted cells survived

at least six months after injection into the brains

of mice and behaved similarly to native cells in

the brain. Human skin cells (top) can be

converted into medium spiny neurons (bottom)

with exposure to the right combination of

microRNAs and transcription factors, according

to work by Andrew Yoo and his research team. 

Credit: Yoo lab.

The researchers, at Washington University School of Medicine in St. Louis, demonstrated that these converted cells survived at least six months after injection into the brains of mice and behaved similarly to native cells in the brain.

"Not only did these transplanted cells survive in the mouse brain, they showed functional properties similar to those of native cells," said senior author Andrew S. Yoo, PhD, assistant professor of developmental biology.

"These cells are known to extend projections into certain brain regions. And we found the human transplanted cells also connected to these distant targets in the mouse brain. That's a landmark point about this paper."

The work appears Oct. 22 in the journal Neuron.

The investigators produced a specific type of brain cell called medium spiny neurons, which are important for controlling movement. They are the primary cells affected in Huntington's disease, an inherited genetic disorder that causes involuntary muscle movements and cognitive decline usually beginning in middle-adulthood. Patients with the condition live about 20 years following the onset of symptoms, which steadily worsen over time.

The research involved adult human skin cells, rather than more commonly studied mouse cells or even human cells at an earlier stage of development. In regard to potential future therapies, the ability to convert adult human cells presents the possibility of using a patient's own skin cells, which are easily accessible and won't be rejected by the immune system.

Scientists have described a way to convert

human skin cells directly into a specific type of

brain cell affected by Huntington's disease, an

ultimately fatal neurodegenerative disorder.

Unlike other techniques that turn one cell type

into another, this new process does not pass

through a stem cell phase, avoiding the

production of multiple cell types, report

researchers at Washington University School of

Medicine in St. Louis. The investigators,

including Andrew Yoo, PhD, (from left) Michelle

Richner and Matheus Victor, demonstrated that

these converted cells survived at least six months

after injection into the brains of mice and

behaved similarly to native cells in the brain. 

Credit: Daniel Abernathy.

To reprogram these cells, Yoo and his colleagues put the skin cells in an environment that closely mimics the environment of brain cells. They knew from past work that exposure to two small molecules of RNA, a close chemical cousin of DNA, could turn skin cells into a mix of different types of neurons.

In a skin cell, the DNA instructions for how to be a brain cell, or any other type of cell, are neatly packed away, unused. In past research published in Nature, Yoo and his colleagues showed that exposure to two microRNAs called miR-9 and miR-124 altered the machinery that governs packaging of DNA. Though the investigators still are unravelling the details of this complex process, these microRNAs appear to be opening up the tightly packaged sections of DNA important for brain cells, allowing expression of genes governing development and function of neurons.

Knowing exposure to these microRNAs alone could change skin cells into a mix of neurons, the researchers then started to fine tune the chemical signals, exposing the cells to additional molecules called transcription factors that they knew were present in the part of the brain where medium spiny neurons are common.

"We think that the microRNAs are really doing the heavy lifting," said co-first author Matheus B. Victor, a graduate student in neuroscience.

"They are priming the skin cells to become neurons. The transcription factors we add then guide the skin cells to become a specific subtype, in this case medium spiny neurons. We think we could produce different types of neurons by switching out different transcription factors."

Yoo also explained that the microRNAs, but not the transcription factors, are important components for the general reprogramming of human skin cells directly to neurons. His team, including co-first author Michelle C. Richner, senior research technician, showed that when the skin cells were exposed to the transcription factors alone, without the microRNAs, the conversion into neurons wasn't successful.

The researchers performed extensive tests to demonstrate that these newly converted brain cells did indeed look and behave like native medium spiny neurons. The converted cells expressed genes specific to native human medium spiny neurons and did not express genes for other types of neurons. When transplanted into the mouse brain, the converted cells showed morphological and functional properties similar to native neurons.

To study the cellular properties associated with the disease, the investigators now are taking skin cells from patients with Huntington's disease and reprogramming them into medium spiny neurons using the approach described in the new paper. They also plan to inject healthy reprogrammed human cells into mice with a model of Huntington's disease to see if this has any effect on the symptoms.

Source: Washington University School of Medicine

Contact: Julia Evangelou Strait

Reference:

Generation of human striatal neurons by microRNA-dependent direct conversion of fibroblasts

Victor MB, Richner M, Hermanstyne TO, Ransdell JL, Sobieski C, Deng PY, Klyachko VA, Nerbonne JM, Yoo AS

Neuron. Oct. 22, 2014, Volume 84, Issue 2, p311–323

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'Master Switch' for Myelination in Human Brain Stem Cells is Identified

Finding is key to developing MS treatments using stem cells

Tuesday, 01 July 2014

The identification of the transcription factor,

SOX10, in human brain cells, brings researchers

closer to the goal of treating multiple sclerosis

(MS) by transplanting into patients the brain cells

that make myelin. Credit: Jing Wang. 

Scientists at the University at Buffalo have identified the single transcription factor or "master switch" that initiates the critical myelination process in the brain. The research will be published online in Proceedings of the National Academy of Sciences (PNAS) on June 30.

The identification of this factor, SOX10, in human brain cells, brings researchers closer to the goal of treating multiple sclerosis (MS) by transplanting into patients the brain cells that make myelin.

"Now that we have identified SOX10 as an initiator of myelination, we can work on developing a viral or pharmaceutical approach to inducing it in MS patients," says Fraser Sim, PhD, senior author on the paper and assistant professor in the UB Department of Pharmacology and Toxicology in the School of Medicine and Biomedical Sciences.

"If we could create a small molecule drug that would switch on SOX10, that would be therapeutically important," he adds.

Stem cell therapy is seen as having dramatic potential for treating MS, but there are key obstacles, especially the length of time it takes for progenitor cells to turn into oligodendrocytes, the brain's myelin-making cells.

Using currently available methods, Sim explains, it can take as long as a year to generate a sufficient number of human oligodendrocyte cells to treat a single MS patient.

That's partly because there are so many steps: the skin or blood cell must be turned into induced pluripotent stem cells, which can differentiate into any other type of cell and from which neural progenitor cells can be produced. Those progenitor cells then must undergo differentiation to oligodendrocyte progenitors that are capable of ultimately producing the oligodendrocytes.

"Ideally, we'd like to get directly to oligodendrocyte progenitors," says Sim.

"The new results are a stepping stone to the overall goal of being able to take a patient's skin cells or blood cells and create from them oligodendrocyte progenitors," he says.

Using foetal (not embryonic) brain stem cells, the UB researchers searched for transcription factors that are absent in neural progenitor cells and switched on in oligodendrocyte progenitor cells.

While neural progenitor cells are capable of producing myelin, they do so very poorly and can cause undesirable outcomes in patients, so the only candidate for transplantation is the oligodendrocyte progenitor.

"The ideal cell to transplant is the oligodendrocyte progenitor cell," Sim says.

"The question was, could we use one of these transcription factors to turn the neural progenitor cell into an oligodendrocyte progenitor cell?"

To find out, they looked at different characteristics, such as mRNA expression, protein and whole gene expression and functional studies.

"We narrowed it down to a short list of 10 transcription factors that were made exclusively by oligodendrocyte progenitor cells," says Sim.

"Among all 10 factors that we studied, only SOX10 was able to make the switch from neural progenitor to oligodendrocyte progenitor cell," says Sim.

In addition, the UB researchers found that SOX10 could expedite the transformation from oligodendrocyte progenitor cell to differentiation as an oligodendrocyte, the myelin-producing cell and the ultimate treatment goal for MS.

"SOX10 facilitates both steps," says Sim.

That's tantalizing, he says, because one of the biggest problems with MS is that cells get stuck in the step between the oligodendrocyte progenitor cell and the oligodendrocyte.

"In MS, first the immune system attacks the brain, but the brain is unable to repair itself effectively," explains Sim.

"If we could boost the regeneration step by facilitating formation of oligodendrocytes from progenitor cells, then we might be able to keep patients in the relapsing remitting stage of MS, a far less burdensome stage of disease than the later, progressive stage."

Sim is also an investigator with other scientists at UB and the University of Rochester on the $12.1 million New York State Stem Cell Science award led by SUNY Upstate Medical Center. The research will test the safety and effectiveness of implanting stem cells that can reproduce myelin into the central nervous system of MS patients.

Source: University at Buffalo 

Contact: Ellen Goldbaum

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

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