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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Researchers Grow Cardiac Tissue on 'Spider Silk' Substrate

Researchers Grow Cardiac Tissue on 'Spider Silk' Substrate

Saturday, 11 April 2015

Genetically engineered fibres of the protein spidroin, which is the construction material for spider webs, has proven to be a perfect substrate for cultivating heart tissue cells, Moscow Institute of Physics and Technology researchers found. They discuss their findings in an article that has recently come out in the journal PLOS ONE.

These are heart tissue cells grown on a matrix,

stained with fluorescent markers. Credit: ©

Alexander Teplenin et al./PLOS ONE.

The cultivation of organs and tissues from a patient's cells is the leading edge of medical research - regenerative methods can solve the problem of transplant rejection. However, it’s quite a challenge to find a suitable frame, or substrate, to grow cells on. The material should be non-toxic and elastic and should not be rejected by the body or impede cell growth. A group of researchers led by Professor Konstantin Agladze, who heads the Laboratory of the Biophysics of Excitable Systems at MIPT, works on cardiac tissue engineering. The group has been cultivating fully functional cardiac tissues, able to contract and conduct excitation waves, from cells called cardiomyocytes. Previously, the group used synthetic polymeric nano-fibres but recently decided to assay another material – electro-spun fibres of spidroin, the cobweb protein. Cobweb strands are incredibly light and durable. They're five times stronger than steel, twice more elastic than nylon, and are capable of stretching a third of their length. The structure of spidroin molecules that make up cobweb drag lines is similar to that of the silk protein, fibroin, but is much more durable.

This is a spidroin fibre matrix captured with a

microscope. Credit: Alexander Teplenin et al./

PLOS ONE.

Researchers would normally use artificial spidroin fibre matrices as a substrate to grow implants like bones, tendons and cartilages, as well as dressings. Professor Agladze's team decided to find out whether a spidroin substrate derived from genetically modified yeast cells can serve to grow cardiac cells.

For this purpose, they seeded isolated neonatal rat cardiomyocytes on fibre matrices. During the experiment, the researchers monitored the growth of the cells and tested their contractibility and the ability to conduct electric impulses, which are the main features of normal cardiac tissue.

The monitoring, carried out with the help of a microscope and fluorescent markers, showed that within three to five days a layer of cells formed on the substrate that were able to contract synchronously and conduct electrical impulses just like the tissue of a living heart would.

"We can answer positively all questions we put at the beginning of this research project," Professor Agladze says.

"Cardiac tissue cells successfully adhere to the substrate of recombinant spidroin; they grow forming layers and are fully functional, which means they can contract co-ordinately."

Source: Moscow Institute of Physics and Technology

Contact: Stanislav Goryachev

Reference:

Functional Analysis of the Engineered Cardiac Tissue Grown on Recombinant Spidroin Fiber Meshes

Alexander Teplenin, Anna Krasheninnikova, Nadezhda Agladze, Konstantin Sidoruk, Olga Agapova, Igor Agapov, Vladimir Bogush, Konstantin Agladze

PLOS ONE, March 23, 2015, DOI:10.1371/journal.pone.0121155

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“Open” Stem Cell Chromosomes Reveal New Possibilities for Diabetes

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

Thursday, 02 April 2015

These are pancreatic cells derived from

embryonic stem cells. Credit: UC San Diego

School of Medicine. 

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

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

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

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

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

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

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

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

Source: UCSD

Contact: Heather Buschman

Reference:

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

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

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

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Stem Cells Age-discriminate Organelles to Maintain Stemness

Stem Cells Age-discriminate Organelles to Maintain Stemness

Thursday, 02 April 2015

Tissue stem cells, that continuously renew our tissues, can divide asymmetrically to produce two types of daughter cells. One will be the new stem cell, whereas the other will give rise to the differentiating cells of the tissue.

A study jointly leads by laboratories in the Institute of Biotechnology, University of Helsinki and Massachusetts Institute of Technology (MIT) investigated whether stem cells may also use asymmetric cell division to reduce accumulation of cellular damage. Damage build-up can cause stem cell exhaustion that result in reduced tissue renewal and aging.

Human mammary stem-like cell apportions

aged mitochondria asymmetrically between

daughter cells. Mitochondria were labelled age-

selectively red 51 hours prior to imaging, leaving

mitochondria that are younger unlabelled. The

daughter cell that will become the new stem cell

(bottom left) receives only few old mitochondria.

Credit: Julia Döhla. 

Researchers developed a novel approach to follow cellular components, such as organelles, age-selectively during cell division. Scientists in David Sabatini's lab studied stem-like cells (SLCs) from cultures of immortalized human mammary epithelial cells. These SLCs were chosen because they express genes associated with the stem-cell state (referred to as stemness), are able to form structures known as mammospheres in culture. To track the destinations of subcellular components during cell division, the researchers, led by former postdoctoral scientist Pekka Katajisto, tagged the components – including lysosomes, mitochondria, Golgi apparatus, ribosomes, and chromatin – with a fluorescent protein that glows when hit by a pulse of ultraviolet light.

"We found that stem cells segregate their old mitochondria to the daughter cell that will differentiate, whereas the new stem cell will receive only young mitochondria" says Pekka Katajisto, a Group leader and Academy research fellow at BI.

By tracing the movements of the glowing organelles, the researchers were able to demonstrate that while the normal epithelial cells distributed all of the tagged components symmetrically to daughter cells, the SLCs localized their older mitochondria distinctly and passed on the lion's share of them to the daughter cells headed for differentiation. The researchers ultimately found that the number of older mitochondria in those cells was roughly six times that in daughter cells whose fate was to remain as stem cells.

Mitochondria appear to be particularly important for stem cells, as other analysed organelles were not similarly age-discriminated, and since inhibition of normal mitochondrial quality control pathways stopped their age-selective segregation.

"There is a fitness advantage to renewing your mitochondria," says David Sabatini, Professor at MIT and Whitehead Institute.

"Stem cells know this and have figured out a way to discard their older components."

“While the mechanism used by stem cells to recognize the age of their mitochondria remains unknown, forced symmetric apportioning of aged mitochondria resulted in loss of stemness in all of the daughter cells," says Katajisto.

"This suggests that the age-selective apportioning of old and potentially damaged organelles may be a way to fight stem cell exhaustion and aging," says Katajisto who now runs a lab at the Institute of Biotechnology at University of Helsinki.

Katajisto laboratory is now exploring how old mitochondria differ from old, and whether this phenomenon occurs in other cell types beyond the human mammary stem-like cells examined here as well as in in vivo.

Source: University of Helsinki

Contact: Pekka Katajisto

Reference:

Asymmetric apportioning of aged mitochondria between daughter cells is required for stemness 

Pekka Katajisto, Julia Döhla, Christine Chaffer, Nalle Pentinmikko, Nemanja Marjanovic, Sharif Iqbal, Roberto Zoncu, Walter Chen, Robert A. Weinberg, David M. Sabatini

Science April 2, .2015, DOI:10.1126/science.1260384

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