Creating Intestinal Organoids In Vitro

Scientists in Boston say they have developed a new method for generating groups of intestinal cells that can be used, among others, to make disease models in the lab to test treatments for diseases affecting the gastrointestinal system. Using human induced pluripotent stem cells, their approach combined a variety of techniques that enabled the development of three-dimensional groups of intestinal cells (organoids) in vitro, which can expand disease treatment testing in the lab using human cells.

The study (“Generation of mesenchyme free intestinal organoids from human induced pluripotent stem cells”), published online in Nature Communications, provides a novel platform to improve drug screenings and uncover novel therapies to treat a variety of diseases impacting the intestine, such as inflammatory bowel disease, colon cancer, and cystic fibrosis, according to the researchers.

“Efficient generation of human induced pluripotent stem cell (hiPSC)-derived human intestinal organoids (HIOs) would facilitate the development of in vitro models for a variety of diseases that affect the gastrointestinal tract, such as inflammatory bowel disease or cystic fibrosis. Here, we report a directed differentiation protocol for the generation of mesenchyme-free HIOs that can be primed towards more colonic or proximal intestinal lineages in serum-free defined conditions. Using a CDX2eGFP iPSC knock-in reporter line to track the emergence of hindgut progenitors, we follow the kinetics of CDX2 expression throughout directed differentiation, enabling the purification of intestinal progenitors and robust generation of mesenchyme-free organoids expressing characteristic markers of small intestinal or colonic epithelium. We employ HIOs generated in this way to measure CFTR function using cystic fibrosis patient-derived iPSC lines before and after correction of the CFTR mutation, demonstrating their future potential for disease modeling and therapeutic screening applications,” the investigators wrote.

The team at the Center for Regenerative Medicine (CReM) of Boston University and Boston Medical Center used donated hiPSCs, which are created by reprogramming adult cells into a primitive state. For this study, these cells were pushed to differentiate into intestinal cells using specific growth factors in order to create organoids in a gel. This new protocol allowed the cells to develop without mesenchyme, which typically in other protocols, provides support for the intestinal epithelial cells to grow. By taking out the mesenchyme, the researchers could study exclusively epithelial cells, which make up the intestinal tract.

In addition, using CRISPR technology, the researchers were able to modify and create a novel iPSC stem cell line that glowed green when differentiated into intestinal cells. This allowed the researchers to follow the process of how intestinal cells differentiate in vitro.“Generating organoids in our lab allows us to create more accurate disease models, which are used to test treatments and therapies targeted to a specific genetic defect or tissue—and it’s all possible without harming the patient,” said Gustavo Mostoslavsky, MD, PhD, co-director of CReM and faculty member in the gastroenterology section at Boston Medical Center. “This approach allows us to determine what treatments could be most effective, and which are ineffective, against a disease.”

Using this new protocol, the researchers generated intestinal organoids from iPSCs containing a mutation that causes cystic fibrosis, which typically affects several organs, including the gastrointestinal tract. Using CRISPR technology, the researchers corrected the mutation in the intestinal organoids. The intestinal organoids with the mutation did not respond to a drug while the genetically corrected cells did respond, demonstrating their future potential for disease modeling and therapeutic screening applications.

The protocol developed in this study provides strong evidence to continue using human iPSCs to study development at the cellular level, tissue engineering and disease modeling in order to advance the understanding, and possibilities, of regenerative medicine, noted Mostoslavsky.

“I hope that this study helps move forward our collective understanding about how diseases impact the gastrointestinal tract at the cellular level,” said Mostoslavsky, who also is associate professor of medicine and microbiology at Boston University School of Medicine. “The continual development of novel techniques in creating highly differentiated cells that can be used to develop disease models in a lab setting will pave the way for the development of more targeted approaches to treat many different diseases.”

Power Of One: Losing Single Night’s Sleep Raises Alzheimer’s Risk

UPPSALA, Sweden — The “all-nighter,” in which students stay up all night studying or completing school work, has become synonymous with the modern college experience. While it’s obviously unhealthy to stay up all night frequently, the occasional all-nighter isn’t typically considered detrimental to one’s well being. Now, quite surprisingly, a new study conducted at Uppsala University in Sweden has found some alarming evidence to the contrary. When young, completely healthy men went just one night without sleeping, their blood showed elevated levels of the protein tau, a biomarker for Alzheimer’s disease. Tau proteins are found in neurons, but they tend to become entangled and build up in the brains of those diagnosed with Alzheimer’s. While Alzheimer’s doesn’t actually reveal itself until old age, this accumulation of tau in the brain actually starts decades before any symptoms show themselves.
Moreover, this isn’t the first time that lack of sleep has been connected to tau, various prior studies with older adults had found that poor sleep habits lead to increased tau levels. Head trauma has also been identified as a cause of elevated tau levels.

“Many of us experience sleep deprivation at some point in our lives due to jet lag, pulling an all-nighter to complete a project, or because of shift work, working overnights or inconsistent hours,” says study author Jonathan Cedernaes, MD, PhD, from Uppsala University, in a release. “Our exploratory study shows that even in young, healthy individuals, missing one night of sleep results in a slight increase in the level of tau in blood. This suggests that over time, similar types of sleep disruption could potentially have detrimental effects.”

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This study consisted of 15 healthy men, all of a normal weight, with an average age of 22. On a typical night, each man reported getting around seven to nine hours of sleep.
The research portion of the study consisted of two phases. During each phase, participants were given the exact same meals and activity schedules while residing in a sleep clinic for a full two days and nights. Additionally, blood samples were taken each evening and morning during both phases. During the first experimental section, participants were allowed to get a good night’s sleep during both evenings. Then, for the second phase, they were able to sleep the first night but had to stay up all night the second time around. During the sleep deprivation night, lights were kept on and the men could play games, watch movies, or just talk to each other to pass the time. After just one night of sleep deprivation, the men saw their tau levels increase by an average of 17%. For comparison, tau levels only increased by an average of 2% following a normal night of adequate sleep.

“It’s important to note that while accumulation of tau in the brain is not good, in the context of sleep loss, we do not know what higher levels of tau in blood represent,” Cedernaes explains. “When neurons are active, release of tau in the brain is increased. Higher levels in the blood may reflect that these tau proteins are being cleared from the brain or they may reflect an overall elevation of the concentration of tau levels in the brain.”

Future studies are needed to investigate this further, as well as to determine how long these changes in tau last, and to determine whether changes in tau in blood reflects a mechanism by which recurrent exposure to restricted, disrupted or irregular sleep may increase the risk of dementia,” he adds. “Such studies could provide key insight into whether interventions targeting sleep should begin at an early age to reduce a person’s risk of developing dementia or Alzheimer’s disease.”
Researchers say that this study was a bit held back by its small size, as well as the fact that it only included young, male study subjects. In the future, larger studies including older participants, as well as women, should be the next step in confirming these findings.