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Stem cell success
CINCINNATI—Organoids—mini versions of organs grown in a lab—represent a lot of untapped potential, both for the drug development industry and as a possible solution for the massive demand and limited supply of transplantable organs. These lab-grown organs also enable scientists to test compounds in human organs to determine toxicity without endangering human patients and allow them to better model disease development. And now the colon is the latest organ to be generated in miniature, as scientists at Cincinnati Children's Hospital Medical Center have successfully produced human embryonic colons in the lab using human pluripotent stem cells. The work was published in Cell Stem Cell in late June.
Researchers in the laboratory of Dr. James Wells, senior study investigator and director of the Cincinnati Children’s Pluripotent Stem Cell Center, have been publishing studies since 2009 detailing their efforts using human pluripotent stem cells (hPSCs) to grow embryonic-stage small intestines with a functional nervous system, as well as the antrum and fundus regions of the stomach (the lowermost and upper parts of the organ, respectively). As noted in the recent Cell Stem Cell paper, growing the colon has proved more difficult than other sections of the gastrointestinal tract.
A large part of that difficulty is due to the lack of data about how colons develop; without knowledge of embryonic germination of the organ, it's much harder to know how to properly program hPSCs to create a full organoid. To try and fill in the gaps, the team searched public databases for molecular markers of the hindgut—the part of the developing gut that becomes the entire large intestine, including the cecum, colon and rectum—in the adult colon, and molecularly and genetically screened developing hindgut tissues in animal models.
For their model of the colon, the researchers noted special AT-rich sequence-binding protein 2, or SATB2, as a definitive molecular marker for the hindgut in humans. This DNA-binding protein supports the structural organization of chromosomes in the nucleus of cells. The protein sequence for SATB2 is highly similar in humans, mice and frogs, and as such, the team hypothesized that molecular signals for the protein in frogs and mice could be used in developing human colon organoids expressing SATB2. Growth factor bone morphogenetic protein (BMP) was also found to play a role, being highly active in the region of the gut tube that expresses SATB2.
As the authors noted in the paper, “To develop a method for generating large intestinal organoids, we first identified SATB2 as a definitive marker of the presumptive large intestinal epithelium in frogs, mice and humans. Using SATB2 as a marker, we show that BMP signaling is required for specification of posterior gut endoderm of frogs and mice, consistent with the known role of BMP in posterior-ventral development (Kumar et al., 2003, Roberts et al., 1995, Sherwood et al., 2011, Tiso et al., 2002, Wills et al., 2008). Moreover, stimulation of BMP signaling in PSC-derived gut tube cultures for 3 days is sufficient to induce a posterior HOX code and the formation of SATB2-expressing colonic organoids. Human colonic organoids (HCOs) had a marker profile and cell types consistent with large intestine.”
Following these results, the team next decided to see how the gastrointestinal tissues might function in vivo, aided by collaborators from the Hospital's Division of Surgery, led by Dr. Michael Helmrath, a pediatric surgeon and director of the Surgical Research program. The lab-grown colonic organoids were transplanted into the kidney capsules of immunocompromised mice for six to 10 weeks and monitored for signs of posterior region enteroendocrine cells, which produce hormones found in the human colon.
After being transplanted, the organoids presented with the form, structures and molecular and cellular properties of the human colon. Specifically, the authors reported, “HCOs engrafted under the kidney capsule of immunocompromised mice and grown in vivo for 8–10 weeks maintained their regional identify, formed tissues with colonic morphology, contained colon-specific cell types, had zones of proliferation and differentiation and well-formed smooth muscle layers.”
Given the success of this work, the team is optimistic about how these organoids could be used in the future.
“Diseases affecting this region of the GI tract are quite prevalent and include ailments like colitis, colon cancer, irritable bowel syndrome, Hirschsprung’s disease and polyposis syndromes,” Wells said. “We’ve been limited in how we can study these diseases, including the fact that animal models like mice don’t precisely recreate human disease processes in the gastrointestinal tract. This system allows us to very effectively model human diseases and human development.”
Dr. Jorge Munera, first author for the paper and postdoctoral fellow in the Wells laboratory, added: “By exposing human colonic organoids to inflammatory triggers, we can now learn how the cell lining of the colon and the supporting cells beneath cooperate to respond to inflammation. This could be very relevant for patients with Crohn’s disease or ulcerative colitis. And because the microbiome, the organisms that live in our guts, are most concentrated in the colon, the organoids potentially could be used to model the human microbiome in health and disease.”
A few days prior to that, an international team—led by Dr. Takanori Takebe, a physician/investigator at Cincinnati Children’s Hospital Medical Center in the Division of Gastroenterology, Hepatology & Nutrition, and Dr. Barbara Treutlein of the Max Planck Institute for Evolutionary Anthropology—shared news of their discovery of new networks of genetic-molecular crosstalk that control the development of the liver, a finding that could boost efforts to produce healthy, viable liver tissue from hPSCs.
The work began with using single-cell RNA sequencing to monitor the way individual cells change when combined in a 3D microenvironment, a situation where hepatic (liver) cells, vascular cells and connective tissue cells all communicate. RNA sequencing generates a blueprint of genetic activity in all cell types, and produced for the research team a blueprint of active transcription factors as well as the signaling molecules and receptors in the different cells before and after they united to form liver tissue.
Among that communication is the crosstalk between VEGF, a signaling protein that cells produce to trigger angiogenesis, and KDR, a protein/receptor that communicates with VEGF to initiate formation of a blood supply for a developing liver. This line of communication was found to be critical to the process of liver tissue growth.
This analysis also enabled the team to compare the hPSC-generated liver tissue with regular human fetal and adult liver cells, which showed that the lab-grown liver buds presented with molecular and genetic profiles highly similar to those in natural liver cells. However, the gene expression landscape in the lab-grown liver buds did not completely match that of natural liver cells, the authors noted, adding that more “fine-tuning” is needed before these tissues are ready for clinical trials.
“There is still a lot left to learn about how to best generate a functioning human liver tissue in a dish; nevertheless, this a big step in that direction,” said Treutlein.