Researchers grow embryonic humanized kidneys inside pigs for 28 days

Guangzhou Institutes of Biomedicine and Health researchers have successfully created chimeric embryos containing a combination of human and pig cells. When transferred into surrogate pig mothers, the developing humanized kidneys had normal structure and tubule formation after 28 days. This is the first time that scientists have been able to grow a solid humanized organ inside another species, though previous studies have used similar methods to generate human tissues such as blood or skeletal muscle in pigs. The work appears September 7 in the journal Cell Stem Cell.

The researchers focused on kidneys because they are one of the first organs to develop, and they're also the most commonly transplanted organ in human medicine.

"Rat organs have been produced in mice, and mouse organs have been produced in rats, but previous attempts to grow human organs in pigs have not succeeded," says senior author Liangxue of the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, and Wuyi University. "Our approach improves the integration of human cells into recipient tissues and allows us to grow human organs in pigs."

Integrating human stem cells into pig embryos has been a challenge because pig cells outcompete human cells and pig and human cells have different physiological needs. "We have been working on mechanisms to overcome the extremely low efficiency in interspecies chimera," says senior author Guangjin Pan of the Guangzhou Institutes of Biomedicine and Health. "We identified a couple of critical factors that enhance the formation of interspecies chimera by facilitating cell competition."

The team's technique depends on three key components:
 

• First, they created a niche within the pig embryo so that the human cells would not have to compete with pig cells by using CRISPR to genetically engineer a single-cell pig embryo so that it was missing two genes that are needed for kidney development.
• Second, the researchers engineered human pluripotent stem cells -- cells that have the potential to develop into any cell type -- to make them more amenable to integration and less likely to self-destruct by temporarily shutting down apoptosis. Then, they converted these cells into "naïve" cells resembling early human embryonic cells by culturing them in a special medium.
• Third, before implanting the developing embryos in surrogate sows, the researchers grew the chimeras in conditions that were optimized to provide unique nutrients and signals to both the human and pig cells, since these cells usually have disparate needs.

Altogether, the researchers transferred 1,820 embryos to 13 surrogate mothers. After either 25 or 28 days, they terminated gestation and extracted the embryos to assess whether the chimeras had successfully produced humanized kidneys.

The researchers collected five chimeric embryos for analysis (two at 25 days and three at 28 days post-implantation) and found that they had structurally normal kidneys for their stage of development and were composed of 50-60% human cells. At 25-28 days, the kidneys were in the mesonephros stage (the second stage of kidney development); they had formed tubules and buds of cells that would eventually become ureters connecting the kidney to the bladder.

The team also investigated whether human cells were contributing to other tissues throughout the embryos, which could have ethical implications, especially if abundant human cells were found in neural or germline tissues and the pigs were brought to term. They showed that human cells were mostly localized to the kidneys, whereas the remainder of the embryo was composed of pig cells.

"We found that if you create a niche in the pig embryo, then the human cells naturally go into these spaces," says senior author Zhen Dai of Guangzhou Institutes of Biomedicine and Health. "We saw only very few human neural cells in the brain and spinal cord and no human cells in the genital ridge, indicating that the human pluripotent stem cells did not differentiate into germ cells." This may be further prevented by knocking out further genes in the human pluripotent stem cells, which can be tested in future studies, the researchers say.

Now that they have optimized conditions for growing humanized kidneys in human-pig chimeras, the team wants to allow the kidneys to develop for a longer duration. They're also working to generate other human organs in pigs, including the heart and pancreas.

The long-term goal is to optimize this technology for human organ transplantation, but the researchers acknowledge the work will be complex and could take many years. Growing a fully functional humanized organ in a pig would require some additional steps because organs are composed of multiple types of cells and tissues. In this study, the researchers created a niche for only one subset of cells, which meant that the kidneys had pig-derived vascular cells, and this could cause organ rejection if they were used in a transplant scenario.

"Because organs are not composed of just one cell lineage, in order to have an organ where everything comes from the human, we would probably need to engineer the pigs in a much more complex way and that also brings some additional challenges," says senior author Miguel A. Esteban of Guangzhou Institutes of Biomedicine and Health.

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New computational method builds detailed maps of human tissues

Weill Cornell Medicine researchers have developed a computational method to map the architecture of human tissues in unprecedented detail. Their approach promises to accelerate studies on organ-scale cellular interactions and could enable powerful new diagnostic strategies for a wide range of diseases.

The method, published Oct. 31 in Nature Methods, grew out of the scientists' frustration with the gap between classical microscopy and modern single-cell molecular analysis. "Looking at tissues under the microscope, you see a bunch of cells that are grouped together spatially -- you see that organization in images almost immediately," said lead author Junbum Kim, a graduate student in physiology and biophysics at Weill Cornell Medicine. "Now, cell biologists have gained the ability to examine individual cells in tremendous detail, down to which genes each cell is expressing, so they're focused on the cells instead of focusing on the tissue structure," he said.

However, "it's crucial for researchers to learn more about the details of tissue structure; fundamental changes in the relationships between cells within a tissue drive both healthy and diseased organ function," said senior author Dr. Olivier Elemento, director of the Englander Institute for Precision Medicine and a professor of physiology and biophysics and of computational genomics in computational biomedicine at Weill Cornell Medicine.

Manually combining single cell data with maps of tissue structure is slow and tedious, though. Machine learning algorithms have shown some potential for automating the process, but they're limited by the data used to train them. To address that, Kim and his colleagues developed an unsupervised computational strategy, using a combination of single-cell gene expression profiles and cells' locations to define structural regions within a tissue.

Co-senior author Dr. André Rendeiro, a postdoctoral fellow at Weill Cornell Medicine during the study and currently a principal investigator at the Research Center for Molecular Medicine of the Austrian Academy of Sciences in Vienna, Austria, compares the new method to mapping a city such as New York: "One way to go about it would be to go to every intersection and count each kind of building: is it residential, is it commercial … is it a shop or restaurant?" Putting all of those data into one matrix, and the buildings' locations into another, one could then combine the two matrices and look for patterns.

"Essentially, we could start to make a general statement about where the different neighborhoods are and where their borders are based on the abundance of, say, residential versus commercial buildings -- just as anyone walking through the Upper East Side, Midtown or Downtown would do based on their observations," said Dr. Rendeiro.

The researchers used the new method to generate detailed maps of several types of tissues, identifying and quantifying new aspects of microanatomy -- the patterns that emerge at small scale when cells interact and that determine the ultimate function of tissue. Collaborating with a colleague at the University of North Carolina at Chapel Hill who studies lung disease, they also demonstrated that their technique could draw fine shades of distinction between different disease states in a tissue.

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New study reveals why HIV remains in human tissue even after antiretroviral therapy

Thanks to antiretroviral therapy, HIV infection is no longer the life sentence it once was. But despite the effectiveness of drugs to manage and treat the virus, it can never be fully eliminated from the human body, lingering in some cells deep in different human tissues where it goes unnoticed by the immune system.

Now, new research by University of Alberta immunologist Shokrollah Elahi reveals a possible answer to the mystery of why infected people can't get rid of HIV altogether.

Elahi and his team found that in HIV patients, killer T cells -- a type of white blood cells responsible for identifying and destroying cells infected with viruses -- have very little to none of a protein called CD73.

Because CD73 is responsible for migration and cell movement into the tissue, the lack of the protein compromises the ability of killer T cells to find and eliminate HIV-infected cells, explained Elahi.

"This mechanism explains one potential reason for why HIV stays in human tissues forever," he said, adding that the research also shows the complexity of HIV infection.

"This provides us the opportunity to come up with potential new treatments that would help killer T cells migrate better to gain access to the infected cells in different tissues."

After identifying the role of CD73 -- a three-year project -- Elahi turned his focus to understanding potential causes for the drastic reduction. He found it is partly due to the chronic inflammation that is common among people living with HIV.

"Following extensive studies, we discovered that chronic inflammation results in increased levels of a type of RNA found in cells and in blood, called microRNAs," he explained. "These are very small types of RNA that can bind to messenger RNAs to block them from making CD73 protein. We found this was causing the CD73 gene to be suppressed."

The team's discovery also helps explain why people with HIV have a lower risk of developing multiple sclerosis, Elahi noted.

"Our findings suggest that reduced or eliminated CD73 can be beneficial in HIV-infected individuals to protect them against MS. Therefore, targeting CD73 could be a novel potential therapeutic marker for MS patients."

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Illuminating dark matter in human DNA

Researchers at University of California San Diego have produced a single-cell chromatin atlas for the human genome. Chromatin is a complex of DNA and protein found in eukaryotic cells; regions of chromatin at key gene regulatory elements appear in open configurations within certain cell nuclei. Precisely delineating these accessible chromatin regions in cells of different human tissue types would be a major step toward understanding the role of gene regulatory elements (non-coding DNA) in human health or disease.

The findings are published online in the November 12, 2021, issue of Cell.

For scientists, the human genome, popularly called the "book of life," is mostly unwritten. Or at least unread. While science has famously put an (approximate) number to all of the protein-coding genes required to build a human being, approximately 20,000+, that estimation does not really begin to explain how exactly the construction process works or, in the case of disease, it might go awry.

"The human genome was sequenced 20 years ago, but interpreting the meaning of this book of life continues to be challenging," said Bing Ren, PhD, director of the Center for Epigenomics, professor of cellular and molecular medicine at UC San Diego School of Medicine and a member of the Ludwig Institute for Cancer Research at UC San Diego.

"A major reason is that the majority of the human DNA sequence, more than 98 percent, is non-protein-coding, and we do not yet have a genetic code book to unlock the information embedded in these sequences."

Put another way, it's a bit like knowing chapter titles but with the rest of the pages still blank.

Efforts to fill in the blanks are broadly captured in an ongoing international effort called the Encyclopedia of DNA Elements (ENCODE), and include the work of Ren and colleagues. In particular, they have investigated the role and function of chromatin, a complex of DNA and proteins that form chromosomes within the nuclei of eukaryotic cells.

DNA carries the cell's genetic instructions. The major proteins in chromatin, called histones, help tightly package the DNA in a compact form that fits within the cell nucleus. (There are roughly six feet of DNA tucked into each cell nucleus and approximately 10 billion miles in each human body.) Changes in how chromatin bundles up DNA are associated with DNA replication and gene expression.

After working with mice, Ren and collaborators turned their attention to a single-cell atlas of chromatin in the human genome.

They applied assays to more than 600,000 human cells sampled from 30 adult human tissue types from multiple donors, then integrated that information with similar data from 15 fetal tissue types to reveal the status of chromatin at approximately 1.2 million candidate cis-regulatory elements in 222 distinct cell types.

"One of the initial challenges was identifying the best experimental conditions for such a diverse set of sample types, particularly given each tissue's unique makeup and sensitivity to homogenization," said study co-author Sebastian Preissl, PhD, associate director for Single Cell Genomics at UC San Diego Center for Epigenomics, a collaborative research center that carried out the assays.

Cis-regulatory elements are regions of non-coding DNA that regulate transcription (copying a segment of DNA into RNA) of neighboring genes. Transcription is the essential process that converts genetic information into action.

"Studies in the last decade have established that sequence variations in non-coding DNA are a key driver in multi-genic traits and diseases in human populations, such as diabetes, Alzheimer's' disease and autoimmune diseases," said study co-author Kyle J. Gaulton, PhD, assistant professor in the Department of Pediatrics at UC San Diego School of Medicine.

"A new paradigm that helps explain how these noncoding variants contribute to diseases posits that these sequence alterations disrupt function of transcriptional regulatory elements and lead to dysregulation of gene expression in disease-relevant cell types, such as neurons, immune cells or epithelial cells," said co-first author Kai Zhang, PhD, a postdoctoral fellow in the Department of Cellular and Molecular Medicine. "A major barrier to unlocking the function of noncoding risk variants, however, is the lack of cell-type-specific maps of transcriptional regulatory elements in the human genome."

Ren said the new findings identify disease-trait-relevant cell types for 240 multi-genic traits and diseases, and annotate the risk of noncoding variants.

"We believe that this resource will greatly facilitate the study of mechanism across a broad spectrum of human diseases for many years to come."

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Major advance enables study of genetic mutations in any tissue

For the first time, scientists are able to study changes in the DNA of any human tissue, following the resolution of long-standing technical challenges by scientists at the Wellcome Sanger Institute. The new method, called nanorate sequencing (NanoSeq), makes it possible to study how genetic changes occur in human tissues with unprecedented accuracy.

The study, published today (28 April) in Nature, represents a major advance for research into cancer and ageing. Using NanoSeq to study samples of blood, colon, brain and muscle, the research also challenges the idea that cell division is the main mechanism driving genetic changes. The new method is also expected to allow researchers to study the effect of carcinogens on healthy cells, and to do so more easily and on a much larger scale than has been possible up until now.

The tissues in our body are composed of dividing and non-dividing cells. Stem cells renew themselves throughout our lifetimes and are responsible for supplying non-dividing cells to keep the body running. The vast majority of cells in our bodies are non-dividing or divide only rarely. They include granulocytes in our blood, which are produced in the billions every day and live for a very short time, or neurons in our brain, which live for much longer.

Genetic changes, known as somatic mutations, occur in our cells as we age. This is a natural process, with cells acquiring around 15-40 mutations per year. Most of these mutations will be harmless, but some of them can start a cell on the path to cancer.

Since the advent of genome sequencing in the late twentieth century, cancer researchers have been able to better understand the formation of cancers and how to treat them by studying somatic mutations in tumour DNA. In recent years, new technologies have also enabled scientists to study mutations in stem cells taken from healthy tissue.

But until now, genome sequencing has not been accurate enough to study new mutations in non-dividing cells, meaning that somatic mutation in the vast majority of our cells has been impossible to observe accurately.

In this new study, researchers at the Wellcome Sanger Institute sought to refine an advanced sequencing method called duplex sequencing1. The team searched for errors in duplex sequence data and realised that they were concentrated at the ends of DNA fragments, and had other features suggesting flaws in the process used to prepare DNA for sequencing.

They then implemented improvements to the DNA preparation process, such as using specific enzymes to cut DNA more cleanly, as well as improved bioinformatics methods. Over the course of four years, accuracy was improved until they achieved fewer than five errors per billion letters of DNA.

Dr Robert Osborne, an alumnus of the Wellcome Sanger Institute who led the development of the method, said: "Detecting somatic mutations that are only present in one or a few cells is incredibly technically challenging. You have to find a single letter change among tens of millions of DNA letters and previous sequencing methods were simply not accurate enough. Because NanoSeq makes only a few errors per billion DNA letters, we are now able to accurately study somatic mutations in any tissue."

The team took advantage of NanoSeq's improved sensitivity to compare the rates and patterns of mutation in both stem cells and non-dividing cells in several human tissue types.

Surprisingly, analysis of blood cells found a similar number of mutations in slowly dividing stem cells and more rapidly dividing progenitor cells2. This suggested that cell division is not the dominant process causing mutations in blood cells. Analysis of non-dividing neurons and rarely dividing cells from muscle also revealed that mutations accumulate throughout life in cells without cell division, and at a similar pace to cells in the blood.

Dr Federico Abascal, the first author of the paper from the Wellcome Sanger Institute, said: "It is often assumed that cell division is the main factor in the occurrence of somatic mutations, with a greater number of divisions creating a greater number of mutations. But our analysis found that blood cells that had divided many times more than others featured the same rates and patterns of mutation. This changes how we think about mutagenesis and suggests that other biological mechanisms besides cell division are key."

The ability to observe mutation in all cells opens up new avenues of research into cancer and ageing, such as studying the effects of known carcinogens like tobacco or sun exposure, as well as discovering new carcinogens. Such research could greatly improve our understanding of how lifestyles choices and exposures to carcinogens can lead to cancer.

A further benefit of the NanoSeq method is the relative ease with which samples can be collected. Rather than taking biopsies of tissue, cells can be collected non-invasively, such as by scraping the skin or swabbing the throat.

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Metabolic changes in fat tissue in obesity associated with adverse health effects

 Researchers at the Obesity Research Unit of the University of Helsinki have found that obesity clearly reduces mitochondrial gene expression in fat tissue, or adipose tissue. Mitochondria are important cellular powerplants which process all of our energy intake. If the pathways associated with breaking down nutrients are lazy, the changes can often have health-related consequences.

A total of 49 pairs of identical twins discordant for body weight participated in the study conducted at the University of Helsinki: their body composition and metabolism were studied in detail, and biopsies from adipose and muscle tissue were collected. Multiple techniques for analysing the genome-wide gene expression, the proteome and the metabolome were used in the study.

The study was recently published in the journal Cell Reports Medicine.

According to the findings, the pathways responsible for mitochondrial metabolism in adipose tissue were greatly reduced by obesity. Since mitochondria are key to cellular energy production, their reduced function can maintain obesity. For the first time, the study also compared the effects of obesity specifically on the mitochondria in muscle tissue in these identical twin pairs: muscle mitochondria too were found to be out of tune, but the change was less distinct than in adipose tissue.

The study provided strong evidence of a connection between the low performance of adipose tissue mitochondria and a proinflammatory state. Furthermore, the findings indicate that metabolic changes in adipose tissue are associated with increased accumulation of fat in the liver, prediabetic disorders of glucose and insulin metabolism as well as cholesterol.

"If mitochondria, the cellular powerplants, are compared to the engine of a car, you could say that the power output decreases as weight increases. A low-powered mitochondrial engine may also generate toxic exhaust fumes, which can cause a proinflammatory state in adipose tissue and, consequently, the onset of diseases associated with obesity," says Professor Kirsi Pietiläinen from the Obesity Research Unit, University of Helsinki.

"What was surprising was that the mitochondrial pathways in muscle had no association with these adverse health effects," Pietiläinen adds.

Obesity also affected amino acid metabolism

In the study, changes in mitochondrial function were also seen in amino acid metabolism. The metabolism of branched-chain amino acids, which are essential to humans, was weakened in the mitochondria of both adipose tissue and muscle tissue.

"This finding was of particular significance because the reduced breakdown of these amino acids and the resulting heightened concentration in blood have also been directly linked with prediabetic changes and the accumulation of liver fat in prior twin studies," says Pietiläinen.

Obesity, with its numerous associated diseases, is a common phenomenon that is continuously increasing in prevalence. While lifestyle influence the onset of obesity, genes also have a significant role.

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Silk fibers improve bioink for 3D-printed artificial tissues and organs

 How do you test, in early-stage research, whether a potential pharmaceutical effectively targets a human tumor, organ, or some other part of the body? How do you grow a new hand or some other body part? Researchers are in the early stages of using 3D cell printing technology to make developments like these happen. A standard way -- currently unavailable -- to fix the cells in place after printing would help researchers avoid having to 'reinvent the wheel' in every new investigation.

In a study recently published in Materials Today Bio, researchers from Osaka University have used silk nanofibers obtained by mechanical disintegration to enhance the printing process without damaging the cells or cell assemblies. An attractive point of silk for this application is that silk is believed to be a safe material for humans. This development will help bring 3D cell printing research out of the laboratory and into real-world biomedical use.

To obtain the fibers, the researchers started with virgin silk, then removed the protein sericin from it because this protein causes inflammation in patients. Next, the researchers ground the remaining biocompatible material into nanofibers. The fibers can be sterilized -- without damaging them -- for medical use, with common laboratory equipment.

"Our silk fibers are excellent additives to bioink cell printing media," says lead author Shinji Sakai. "They are compatible with many media, such as those containing gelatin, chitosan, or hyaluronic acid, giving them a broad range of potential applications."

The main purpose of the fibers was to ensure that the cells in the bioink retained their 3D positioning after printing without damaging the cells. The fibers fulfill this purpose by enhancing the integrity of the bioink and minimizing the damaging high mechanical stresses often placed on cells during printing.

"Various mechanical experiments say the same thing: the nanofibers enhanced the properties of the printing media," explains Professor Sakai. "For example, Young's modulus -- a measure of stiffness -- increased several-fold and remained enhanced for over a month."

The fibers help printed configurations retain their structural integrity after printing. For example, a nose-shaped configuration retained its shape only when printed with bioink containing the silk fibers. Over 85% of the cells in the bioink remained alive after a week in the printed bioink with or without the added fibers, indicating that adding the fibers did not damage the cells.

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Trial drug can significantly block early stages of COVID-19 in engineered human tissues

SARS-CoV-2 coronavirus illustration

An international team led by University of British Columbia researcher Dr. Josef Penninger has found a trial drug that effectively blocks the cellular door SARS-CoV-2 uses to infect its hosts.

The findings, published today in Cell, hold promise as a treatment capable of stopping early infection of the novel coronavirus that, as of April 2, has affected more than 981,000 people and claimed the lives of 50,000 people worldwide.

The study provides new insights into key aspects of SARS-CoV-2, the virus that causes COVID-19, and its interactions on a cellular level, as well as how the virus can infect blood vessels and kidneys.

"We are hopeful our results have implications for the development of a novel drug for the treatment of this unprecedented pandemic," says Penninger, professor in UBC's faculty of medicine, director of the Life Sciences Institute and the Canada 150 Research Chair in Functional Genetics at UBC.

"This work stems from an amazing collaboration among academic researchers and companies, including Dr. Ryan Conder's gastrointestinal group at STEMCELL Technologies in Vancouver, Nuria Montserrat in Spain, Drs. Haibo Zhang and Art Slutsky from Toronto and especially Ali Mirazimi's infectious biology team in Sweden, who have been working tirelessly day and night for weeks to better understand the pathology of this disease and to provide breakthrough therapeutic options."

ACE2 -- a protein on the surface of the cell membrane -- is now at centre-stage in this outbreak as the key receptor for the spike glycoprotein of SARS-CoV-2. In earlier work, Penninger and colleagues at the University of Toronto and the Institute of Molecular Biology in Vienna first identified ACE2, and found that in living organisms, ACE2 is the key receptor for SARS, the viral respiratory illness recognized as a global threat in 2003. His laboratory also went on to link the protein to both cardiovascular disease and lung failure.

While the COVID-19 outbreak continues to spread around the globe, the absence of a clinically proven antiviral therapy or a treatment specifically targeting the critical SARS-CoV-2 receptor ACE2 on a molecular level has meant an empty arsenal for health care providers struggling to treat severe cases of COVID-19.

"Our new study provides very much needed direct evidence that a drug -- called APN01 (human recombinant soluble angiotensin-converting enzyme 2 -- hrsACE2) -- soon to be tested in clinical trials by the European biotech company Apeiron Biologics, is useful as an antiviral therapy for COVID-19," says Dr. Art Slutsky, a scientist at the Keenan Research Centre for Biomedical Science of St. Michael's Hospital and professor at the University of Toronto who is a collaborator on the study.

In cell cultures analyzed in the current study, hrsACE2 inhibited the coronavirus load by a factor of 1,000-5,000. In engineered replicas of human blood vessel and kidneys -- organoids grown from human stem cells -- the researchers demonstrated that the virus can directly infect and duplicate itself in these tissues. This provides important information on the development of the disease and the fact that severe cases of COVID-19 present with multi-organ failure and evidence of cardiovascular damage. Clinical grade hrsACE2 also reduced the SARS-CoV-2 infection in these engineered human tissues.

"Using organoids allows us to test in a very agile way treatments that are already being used for other diseases, or that are close to being validated. In these moments in which time is short, human organoids save the time that we would spend to test a new drug in the human setting," says Núria Montserrat, ICREA professor at the Institute for Bioengineering of Catalonia in Spain.

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