SCIENTISTS REVERT HUMAN STEM CELLS TO PRISTINE STATE

Researchers at EMBL-EBI have resolved a long-standing challenge in stem cell biology by successfully 'resetting' human pluripotent stem cells to a fully pristine state, at point of their greatest developmental potential. The study, published in Cell, involved scientists from the UK, Germany and Japan and was led jointly by EMBL-EBI and the University of Cambridge.

Embryonic stem (ES) cells, which originate in early development, are capable of differentiating into any type of cell. Until now, scientists have only been able to revert 'adult' human cells (for example, liver, lung or skin) into pluripotent stem cells with slightly different properties that predispose them to becoming cells of certain types. Authentic ES cells have only been derived from mice and rats.

"Reverting mouse cells to a completely 'blank slate' has become routine, but generating equivalent naïve human cell lines has proven far more challenging," says Dr Paul Bertone, Research Group Leader at EMBL-EBI and a senior author on the study. "Human pluripotent cells resemble a cell type that appears slightly later in mammalian development, after the embryo has implanted in the uterus."

At this point, subtle changes in gene expression begin to influence the cells, which are then considered 'primed' towards a particular lineage. Although pluripotent human cells can be cultured from in vitro fertilised (IVF) embryos, until now there have been no human cells comparable to those obtained from the mouse.

Wiping cell memory

"For years, it was thought that we could be missing the developmental window when naïve human cells could be captured, or that the right growth conditions hadn't been found," Paul explains. "But with the advent of iPS cell technologies, it should have been possible to drive specialised human cells back to an earlier state, regardless of their origin -- if that state existed in primates."

Taking a new approach, the scientists used reprogramming methods to express two different genes, NANOG and KLF2, which reset the cells. They then maintained the cells indefinitely by inhibiting specific biological pathways. The resulting cells are capable of differentiating into any adult cell type, and are genetically normal.

The experimental work was conducted hand-in-hand with computational analysis.

"We needed to understand where these cells lie in the spectrum of the human and mouse pluripotent cells that have already been produced," explains Paul. "We worked with the EMBL Genomics Core Facility to produce comprehensive transcriptional data for all the conditions we explored. We could then compare reset human cells to genuine mouse ES cells, and indeed we found they shared many similarities."

Together with Professor Wolf Reik at the Babraham Institute, the researchers also showed that DNA methylation (biochemical marks that influence gene expression) was erased over much of the genome, indicating that reset cells are not restricted in the cell types they can produce. In this more permissive state, the cells no longer retain the memory of their previous lineages and revert to a blank slate with unrestricted potential to become any adult cell.

Unlocking the potential of stem cell therapies

The research was performed in collaboration with Professor Austin Smith, Director of the Wellcome Trust-Medical Research Council Stem Cell Institute.

"Our findings suggest that it is possible to rewind the clock to achieve true ground-state pluripotency in human cells," said Professor Smith. "These cells may represent the real starting point for formation of tissues in the human embryo. We hope that in time they will allow us to unlock the fundamental biology of early development, which is impossible to study directly in people."

The discovery paves the way for the production of superior patient material for translational medicine. Reset cells mark a significant advance for human stem cell applications, such as drug screening of patient-specific cells, and are expected to provide reliable sources of specialised cell types for regenerative tissue grafts.

SIMPLE METHOD TURNS HUMAN SKIN CELLS IN TO IMMUNE STRENGTHENING WHITE BLOOD CELLS

For the first time, scientists have turned human skin cells into transplantable white blood cells, soldiers of the immune system that fight infections and invaders. The work, done at the Salk Institute, could let researchers create therapies that introduce into the body new white blood cells capable of attacking diseased or cancerous cells or augmenting immune responses against other disorders

The work, as detailed in the journal Stem Cells, shows that only a bit of creative manipulation is needed to turn skin cells into human white blood cells.

"The process is quick and safe in mice," says senior author Juan Carlos Izpisua Belmonte, holder of Salk's Roger Guillemin Chair. "It circumvents long-standing obstacles that have plagued the reprogramming of human cells for therapeutic and regenerative purposes."

Those problems includes the long time -- at least two months -- and tedious laboratory work it takes to produce, characterize and differentiate induced pluripotent stem (iPS) cells, a method commonly used to grow new types of cells. Blood cells derived from iPS cells also have other obstacles: an inability to engraft into organs or bone marrow and a likelihood of developing tumors.

The new method takes just two weeks, does not produce tumors, and engrafts well.

"We tell skin cells to forget what they are and become what we tell them to be -- in this case, white blood cells," says one of the first authors and Salk researcher Ignacio Sancho-Martinez. "Only two biological molecules are needed to induce such cellular memory loss and to direct a new cell fate."

Belmonte's team developed the faster technique (called indirect lineage conversion) and previously demonstrated that these approaches could be used to produce human vascular cells, the ones that line blood vessels. Rather than reversing cells all the way back to a stem cell state before prompting them to turn into something else, such as in the case of iPS cells, the researchers "rewind" skin cells just enough to instruct them to form the more than 200 cell types that constitute the human body.

The technique demonstrated in this study uses a molecule called SOX2 to become somewhat plastic -- the stage of losing their "memory" of being a specific cell type. Then, researchers use a genetic factor called miRNA125b that tells the cells that they are actually white blood cells.

The researchers are now conducting toxicology studies and cell transplantation proof-of-concept studies in advance of potential preclinical and clinical studies.

"It is fair to say that the promise of stem cell transplantation is now closer to realization," Sancho-Martinez says.

Study co-authors include investigators from the Center of Regenerative Medicine in Barcelona, Spain, and the Centro de Investigacion Biomedica en Red de Enfermedades Raras in Madrid, Spain.

SCIENTISTS GENERATE FIRST HUMAN TISSUE IN L AB WITH STEM CELLS

Scientists used pluripotent stem cells to generate functional, three-dimensional human stomach tissue in a laboratory -- creating an unprecedented tool for researching the development and diseases of an organ central to several public health crises, ranging from cancer to diabetes.

Scientists at Cincinnati Children's Hospital Medical Center report Oct. 29 in Nature they used human pluripotent stem cells -- which can become any cell type in the body -- to grow a miniature version of the stomach. In collaboration with researchers at the University of Cincinnati College of Medicine, they used laboratory generated mini-stomachs (called gastric organoids) to study infection by H. pylori bacteria, a major cause of peptic ulcer disease and stomach cancer.

This first-time molecular generation of 3D human gastric organoids (hGOs) presents new opportunities for drug discovery, modeling early stages of stomach cancer and studying some of the underpinnings of obesity related diabetes, according to Jim Wells, PhD, principal investigator and a scientist in the divisions of Developmental Biology and Endocrinology at Cincinnati Children's.

It also is the first time researchers have produced 3D human embryonic foregut -- a promising starting point for generating other foregut organ tissues like the lungs and pancreas, he said.

"Until this study, no one had generated gastric cells from human pluripotent stem cells (hPSCs)," Wells said. "In addition, we discovered how to promote formation of three-dimensional gastric tissue with complex architecture and cellular composition."

This is important because differences between species in the embryonic development and architecture of the adult stomach make mouse models less than optimal for studying human stomach development and disease, Wells added.

Researchers can use human gastric organoids as a new discovery tool to help unlock other secrets of the stomach, such as identifying biochemical processes in the gut that allow gastric-bypass patients to become diabetes-free soon after surgery before losing significant weight. Obesity fueled diabetes and metabolic syndrome are an exploding public health epidemic. Until now, a major challenge to addressing these and other medical conditions involving the stomach has been a relative lack of reliable laboratory modeling systems to accurately simulate human biology, Wells explained.

The key to growing human gastric organoids was to identify the steps involved in normal stomach formation during embryonic development. By manipulating these normal processes in a petri dish, the scientists were able to coax pluripotent stem cells toward becoming stomach. Over the course of a month, these steps resulted in the formation of 3D human gastric organoids that were about 3mm (1/10th of an inch) in diameter. Wells and his colleagues also used this approach to identify what drives normal stomach formation in humans with the goal of understanding what goes wrong when the stomach does not form correctly.

Along with study first author Kyle McCracken, an MD/PhD graduate student working in Wells' laboratory, and Yana Zavros, PhD, a researcher at UC's Department of Molecular and Cellular Physiology, the authors report they were impressed by how rapidly H. pylori bacteria infected stomach epithelial tissues.

Within 24 hours, the bacteria had triggered biochemical changes to the organ, according to McCracken. The human gastric organoids faithfully mimicked the early stages of gastric disease caused by the bacteria, including the activation of a cancer gene called c-Met and the rapid spread of infection in epithelial tissues.

Another significant part of the team's challenge has been the relative lack of previous research literature on how the human stomach develops, the authors said. Wells said the scientists had to use a combination of published work, as well as studies from his own lab, to answer a number of basic developmental questions about how the stomach forms. Over the course of two years, this approach of experimenting with different factors to drive the formation of the stomach eventually resulted in the formation of 3D human gastric tissues in the petri dish.

Wells emphasized importance of basic research for the eventual success of this project, adding, "This milestone would not have been possible if it hadn't been for previous studies from many other basic researchers on understanding embryonic organ development."

HUMAN GENOME WAS SHAPED BY AN EVOLUTIONARY ARM RACE WITH ITSELF

New findings by scientists at the University of California, Santa Cruz, suggest that an evolutionary arms race between rival elements within the genomes of primates drove the evolution of complex regulatory networks that orchestrate the activity of genes in every cell of our bodies

The arms race is between mobile DNA sequences known as "retrotransposons" (a.k.a. "jumping genes") and the genes that have evolved to control them. The UC Santa Cruz researchers have, for the first time, identified genes in humans that make repressor proteins to shut down specific jumping genes. The researchers also traced the rapid evolution of the repressor genes in the primate lineage.

Their findings, published September 28 in Nature, show that over evolutionary time, primate genomes have undergone repeated episodes in which mutations in jumping genes allowed them to escape repression, which drove the evolution of new repressor genes, and so on. Furthermore, their findings suggest that repressor genes that originally evolved to shut down jumping genes have since come to play other regulatory roles in the genome.

"We have basically the same 20,000 protein-coding genes as a frog, yet our genome is much more complicated, with more layers of gene regulation. This study helps explain how that came about," said Sofie Salama, a research associate at the UC Santa Cruz Genomics Institute who led the study.

Retrotransposons are thought to be remnants of ancient viruses that infected early animals and inserted their genes into the genome long before humans evolved. Now they can only replicate themselves within the genome. Depending on where a new copy gets inserted into the genome, a jumping event can disrupt normal genes and cause disease. Often the effect is neutral, simply adding to the overall size of the genome. Very rarely the effect might be advantageous, because the added DNA can itself be a source of new regulatory elements that enhance gene expression. But the high probability of deleterious effects means natural selection favors the evolution of mechanisms to prevent jumping events.

Scientists estimate that jumping genes or "transposable elements" account for at least 50 percent of the human genome, and retrotransposons are by far the most common type.

"There have been successive waves of retrotransposon activity in primate evolution, when a transposable element changed to become expressed and replicated itself throughout the genome until something turned it off," Salama said. "We've discovered a major mechanism by which the genome is able to shut down these mobile DNA elements."

The repressors identified in the new study belong to a large family of proteins known as "KRAB zinc finger proteins." These are DNA-binding proteins that repress gene activity, and they constitute the largest family of gene-regulating proteins in mammals. The human genome has over 400 genes for KRAB zinc finger proteins, and about 170 of them have emerged since primates diverged from other mammals.

According to Salama, her team's findings support the idea that expansion of this family of repressor genes occurred in response to waves of retrotransposon activity. Because repression of a jumping gene also affects genes located near it on the chromosome, the researchers suspect that these repressors have been co-opted for other gene-regulatory functions, and that those other functions have persisted and evolved long after the jumping genes the repressors originally turned off have degraded due to the accumulation of random mutations.

"The way this type of repressor works, part of it binds to a specific DNA sequence and part of it binds other proteins to recruit a whole complex of proteins that creates a repressive landscape in the genome. This affects other nearby genes, so now you have a potential new layer of regulation available for further evolution," Salama said.

KRAB zinc finger proteins are the subject of intensive research as scientists try to sort out their many regulatory roles within the genome. The idea that they are involved in repression of jumping genes is not new--previous studies by other researchers have shown that these proteins silence jumping genes in mouse embryonic stem cells. But until now, no one had been able to demonstrate that the same thing occurs in human cells.

The UC Santa Cruz team developed a novel assay to test whether a particular KRAB zinc finger protein could shut down certain jumping genes. The first authors of the paper, postdoctoral researcher Frank Jacobs and graduate student David Greenberg, came up with the strategy of testing primate retrotransposons in non-primate cells by using mouse embryonic stem cells that contain a single human chromosome. In the environment of a mouse cell, jumping genes that were repressed in primate cells became active. Greenberg then developed an assay for testing individual zinc finger proteins for their ability to turn off a primate jumping gene in the mouse cell environment.

"We did all our tests in mouse cells because they lack all of the primate zinc finger proteins, so when you put primate retrotransposons into a mouse cell they're all active," Salama explained.

The results demonstrated that two human proteins called ZNF91 and ZNF93 bind and repress two major classes of retrotransposons (known as SVA and L1PA) that are currently or recently active in primates. Assistant research scientist Benedict Paten directed graduate student Ngan Nguyen in a painstaking analysis of primate genomes, including the reconstruction of ancestral genomes, which showed that ZNF91 underwent structural changes 8 to 12 million years ago that enabled it to repress SVA elements.

Experiments with ZNF 93, which shuts down L1PA retrotransposons, provided a striking illustration of the arms race between jumping genes and repressors. The researchers found that, while it is good at shutting down many L1PA elements, there is one subset of a recently evolved lineage of L1PA that has lost a short section of DNA that includes the ZNF93 binding site. Without the binding site, these jumping genes evade repression by ZNF93. Interestingly, when the researchers put the missing sequence back into one of these genes and put it in a mouse cell without ZNF93, they found that it was better at jumping. So even though the sequence helps with jumping activity, losing it gives the jumping gene an advantage in primates by allowing it to escape repression by ZNF93.

"That's kind of the icing on the cake for aficionados of molecular evolution, because it demonstrates that this is a never-ending race," Salama said. "KRAB zinc finger proteins are a rare class of proteins that is rapidly expanding and evolving in mammalian genomes, which makes sense because the transposable elements are themselves continually evolving to escape repression."

Corresponding author David Haussler, professor of biomolecular engineering and director of the UC Santa Cruz Genomics Institute, said the study involved close collaboration between his group's "wet lab," directed by Salama, and the "dry lab" where researchers under Paten's direction used the computational tools of genome bioinformatics to reconstruct the evolutionary history of primate genomes. Haussler, a Howard Hughes Medical Institute investigator who has used his background in computer science to do pioneering work in genomics, said he established the wet lab to enable just this kind of collaboration.

"Both parts were integral to this study, and there was a lot of back and forth between them. This paper shows how important it is to integrate computational and experimental approaches to fundamental scientific problems, such as how and why we continuously evolve to be more complex," Haussler said.

HUMAN MILK FAT IMPROVES GROWTH IN PREMATURE INFANTS

For premature infants, adequate growth while in the neonatal intensive care unit is an indicator of better long-term health and developmental outcomes. Researchers at the USDA/ARS Children's Nutrition Research Center at Baylor College of Medicine and Texas Children's Hospital have now successfully incorporated a cream supplement into premature infants' diets that improved their growth outcomes in the NICU. The report appears in the Journal of Pediatrics

For premature babies who weigh less than 1,000 grams (about 2 pounds, 2 ounces), one of the problems is that their lungs and other organs are still developing when they are born. If the infant gains weight and increases in length at a good rate while in the NICU, this helps improve their outcomes," said Dr. Amy Hair, assistant professor of pediatrics at Baylor, neonatologist at Texas Children's Hospital and first author of the study.

Previous research has shown that an exclusive human milk diet protects the intestines of premature infants and supports their growth. This diet consists of mothers' own breast milk or donor human milk, as well as a fortifier consisting of protein and minerals made from the donor milk.

In this study, researchers sought a way to optimize this growth in very small infants (those who weigh between 750 and 1,250 grams) who need additional calories. Because infants are already receiving enough protein from the fortifier, another way to help them grow is by giving them fat. One of the byproducts of pasteurizing donor milk is milk fat, also referred to as a cream supplement.

In this study, researchers compared the growth outcomes of infants who received the exclusive human milk diet and the cream supplement to infants who received just the exclusive human milk diet. They found that infants in the cream group had better growth outcomes in terms of weight and length than infants in the control group.

"This is a natural way to give them fat. Previously, we would add oils or infant formula to help premature babies grow, but we can now use a natural source from donor milk," said Hair.

Hair noted that because the growth was both in weight and length, this growth is likely lean mass, consisting of bone and muscle growth.

"You want to see babies growing in both weight and length," said Hair.

She also noted that the volume of milk given to these infants cannot change to help them grow because their stomach and intestine can only tolerate a certain amount of feedings.

"You cannot give them more volumes of milk. Especially if they have lung problems, they have to have a certain volume of milk. This is a way to add calories but not change the volume of milk," she said.

Since November 2013, the NICU at Texas Children's Hospital has changed its protocol to add this cream supplement to the diet of infants who weigh less than 1,500 grams.

"This also emphasizes the importance of donating excess breast milk that your baby doesn't need to a milk bank. It can help nourish our tiniest and most vulnerable infants," said Hair.

Texas Children's was the first hospital in the world to add human milk-based cream to the diets of very low birth weight infants.

In addition to adding cream to the diets of premature infants, since 2009, Texas Children's has significantly reduced its rates of necrotizing enterocolitis, one of the most devastating and potentially fatal diseases a neonate can face, by implementing a human milk feeding protocol for all infants weighing less than 3.3 pounds.

"Texas Children's strives to be a leader in human milk feeding, because we know it impacts outcomes," said Hair.