Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential.

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease.

The enteric nervous system (ENS) is the largest component of the autonomic nervous system, with neuron numbers surpassing those present in the spinal cord. The ENS has been called the 'second brain' given its autonomy, remarkable neurotransmitter diversity and complex cytoarchitecture. Defects in ENS development are responsible for many human disorders including Hirschsprung disease (HSCR). HSCR is caused by the developmental failure of ENS progenitors to migrate into the gastrointestinal tract, particularly the distal colon. Human ENS development remains poorly understood owing to the lack of an easily accessible model system. Here we demonstrate the efficient derivation and isolation of ENS progenitors from human pluripotent stem (PS) cells, and their further differentiation into functional enteric neurons. ENS precursors derived in vitro are capable of targeted migration in the developing chick embryo and extensive colonization of the adult mouse colon. The in vivo engraftment and migration of human PS-cell-derived ENS precursors rescue disease-related mortality in HSCR mice (Ednrb(s-l/s-l)), although the mechanism of action remains unclear. Finally, EDNRB-null mutant ENS precursors enable modelling of HSCR-related migration defects, and the identification of pepstatin A as a candidate therapeutic target. Our study establishes the first, to our knowledge, human PS-cell-based platform for the study of human ENS development, and presents cell- and drug-based strategies for the treatment of HSCR.

Diffuse Anaplastic Wilms Tumor in a Child With LAMA2 -related Muscular Dystrophy.

Laminin alpha-2-related muscular dystrophy ( LAMA2 -MD), caused by mutations in the LAMA2 gene, is inherited in an autosomal recessive manner. There is no known association of LAMA2 -MD with cancer predisposition. We present a 4-year-old female with LAMA2 -MD and Children's Oncology Group stage III diffuse anaplastic Wilms tumor (DAWT). Given our patient's comorbidities, it was essential to tailor her adjuvant chemotherapy by omitting vincristine and doxorubicin to avoid the potential worsening of her neuromuscular dysfunction and cardiomyopathy. This report illustrates the sporadic occurrence of 2 rare events in our patient and highlights the successful risk-adapted management of DAWT based on the pathophysiology of LAMA2 -MD.

NH2-terminal deletion of specific phosphorylation sites on PHOX2B disrupts the formation of enteric neurons in vivo.

Mutations in the paired-like homeobox 2 b (PHOX2B) gene are associated with congenital central hypoventilation syndrome (CCHS), which is a rare condition in which both autonomic dysregulation with hypoventilation and an enteric neuropathy may occur. The majority of patients with CCHS have a polyalanine repeat mutation (PARM) in PHOX2B, but a minority of patients have nonpolyalanine repeat mutations (NPARMs), some of which have been localized to exon 1. A PHOX2B-Y14X nonsense mutation previously generated in a human pluripotent stem cell (hPSC) line results in an NH2-terminus truncated product missing the first 17 or 20 amino acids, possibly due to translational reinitiation at an alternate ATG start site. This NH2-terminal truncation in the PHOX2B protein results in the loss of two key phosphorylation residues. Though the deletion does not affect the potential for PHOX2BY14X/Y14X mutant hPSC to differentiate into enteric neural crest cells (ENCCs) in culture, it impedes in vivo development of neurons in an in vivo model of human aganglionic small intestine.NEW & NOTEWORTHY A mutation that affects only 17-20 NH2-terminal amino acids in the paired-like homeobox 2 b (PHOX2B) gene hinders the subsequent in vivo establishment of intestinal neuronal cells, but not the in vitro differentiation of these cells.

Broad-spectrum antibiotics alter the microbiome, increase intestinal fxr, and decrease hepatic steatosis in zebrafish short bowel syndrome.

Short bowel syndrome (SBS) is associated with changes in the intestinal microbiome and marked local and systemic inflammation. There is also a late complication of SBS, intestinal failure associated liver disease (IFALD) in which hepatic steatosis progresses to cirrhosis. Most patients with SBS arrive at massive intestinal resection after a contaminating intraabdominal catastrophe and have a history of exposure to broad-spectrum antibiotics. We therefore investigated whether the administration of broad-spectrum antibiotics in conjunction with SBS in zebrafish (ZF) would replicate these systemic effects observed in humans to identify potentially druggable targets to aid in the management of SBS and resulting IFALD. In zebrafish with SBS, broad-spectrum antibiotics altered the microbiome, decreased inflammation, and reduced the development of hepatic steatosis. After two weeks of broad-spectrum antibiotics, these fish exhibited decreased alpha diversity, with less variation in microbial community composition between SBS and sham fish. Additionally, administration of broad-spectrum antibiotics was associated with decreased expression of intestinal toll-like receptor 4 (tlr4), increased expression of the intestinal gene encoding the Farnesoid X receptor (fxr), decreased expression of downstream hepatic cyp7a1, and decreased development of hepatic steatosis. SBS in zebrafish reproducibly results in increased epithelial surface area as occurs in human patients who demonstrate intestinal adaptation, but antibiotic administration in zebrafish with SBS reduced these gains with increased cell death in the intervillus pocket that contains stem/progenitor cells. These alternate states in SBS zebrafish might direct the development of future human therapies.NEW & NOTEWORTHY In a zebrafish model that replicates a common clinical scenario, systemic effects of the administration of broad-spectrum antibiotics in a zebrafish model of SBS identified two alternate states that led to the establishment of fat accumulation in the liver or its absence. Broad-spectrum antibiotics given to zebrafish with SBS over 2 wk altered the intestinal microbiome, decreased intestinal and hepatic inflammation, and decreased hepatic steatosis.

Grading TESI: Crypt and villus formation in tissue-engineered small intestine alters with stem/progenitor cell source.

The small intestine has a remarkable ability to enhance its absorptive and digestive surface area through the formation of villi, a process known as villification. We sought to learn whether developing mouse and human tissue-engineered small intestine (TESI) followed known developmental biology routes to villification, such as Sonic hedgehog (SHH)/Indian hedgehog (IHH) and bone morphogenetic protein 4 (BMP4)/forkhead box F1 (FOXF1) signaling to identify targets to enhance the development of TESI. After generating TESI from prenatal and postnatal stem cell sources, we evaluated the effect of cell source derivation on villification with a grading scheme to approximate developmental stage. χ2 analysis compared the prevalence of TESI grade from each stem cell source. RNAscope probes detected genes known to direct villification and the development of the crypt-villus axis in mouse and human development. These were compared in TESI derived from various pluripotent and progenitor cell donor cell types as well as native human fetal and postnatal tissues. Prenatal and pluripotent cell sources form mature villus and crypt-like structures more frequently than postnatal donor sources, and there are alternate routes to villus formation. Human TESI recapitulates epithelial to mesenchymal crosstalk of several genes identified in development, with fetal and pluripotent donor-derived TESI arriving at villus formation following described developmental patterns. However, postnatal TESI is much less likely to form complete villus-crypt patterns and demonstrates alternate SHH/IHH and BMP4/FOXF1 signaling patterns. Grading TESI and other cellular constructs may assist discoveries to support future human therapies.NEW & NOTEWORTHY The small intestine can enhance its absorptive and digestive surface area through a process known as villification. Tissue-engineered small intestine achieves mature villification at varying levels of success between differing sources. We have developed a consistent grading schema of morphology and characterized it across multiple developmental pathways, allowing objective comparison between differing constructs and sources.

Thermal Injection Measurement (TIM) Adds Accuracy to Injections and Can Assess Cold Chain Management.

BACKGROUND: Inaccurate assessment of injected drug delivery may increase cost and morbidity or reduce efficacy. Yet currently most injections are evaluated solely by the formation of a visible wheal that might not truly estimate the actual area of effect. We hypothesized that thermal injection measurement (TIM) might verify appropriate temperature at the time of injection, as required for some temperature-sensitive vaccines and provide more accurate information about the area of delivery. METHODS: 0.1 mL of either iced (n = 11) or room temperature (n = 17) methylene blue solution was injected subcutaneously in mice under anesthesia and photos taken with an iPhone 7 built-in camera and Thermal Seek Camera phone plug-in. After 5 min, true values were determined at necropsy. RESULTS: TIM was closer in value to the measured area at necropsy than the area of the visualized skin wheal at both ice temperature and room temperature. The difference between the true value and thermal area assessment of iced solution averaged 0.15 cm2 as compared with the difference between the true value and wheal size, which averaged 0.27 cm2 (P = 0.04). At room temperature, this was maintained for thermal and visible wheal differences, 0.23 cm2 and 0.65 cm2, respectively (P = 0.0006). CONCLUSIONS: TIM can assess temperature at the time of injection and is more accurate than visual inspection. TIM could be applied to colorless injections and areas that are hard to visualize such as scar. As a portable phone plug-in, it might be a useful adjunct to aid the evaluation of injected drug delivery including in resource-limited settings.

Remnant Intestinal Length Defines Intestinal Adaptation and Hepatic Steatosis: Two Zebrafish Models.

BACKGROUND: Short bowel syndrome (SBS) is a condition that results from inadequate intestinal absorptive capacity, usually after the loss of functional intestine. We have previously developed a severe model of SBS in zebrafish that demonstrated increased intestinal adaptation (IA) and epithelial proliferation in SBS zebrafish. However, many children with SBS do not have this extreme intestinal loss. Therefore, in this study, we developed a variation of this model to evaluate the effects of increasing intestinal length on IA and the complications of SBS. MATERIALS AND METHODS: After Institutional Animal Care and Use Committee approval, adult male zebrafish were assigned to three groups: sham (n = 30), S1-SBS (n = 30), and S3-SBS (n = 30). Sham surgery included ventral laparotomy alone. S1-SBS surgery consisted of laparotomy with creation of a proximal stoma at S1 (jejunostomy equivalent) and ligation at S4. S3-SBS surgery had stoma creation at S3 (ileostomy equivalent) and the same ligation. Fish were harvested at 14 d. Markers of IA were measured from proximal intestinal segments, and the liver was analyzed for development of hepatic steatosis. RESULTS: At 14 d, S3-SBS fish lost less weight than S1-SBS and had increased markers of IA compared with sham fish, which were decreased compared with S1-SBS fish. S3-SBS fish had decreased proximal intestinal inflammation compared with S1-SBS fish. S1-SBS fish developed extensive hepatic steatosis. Although S3-SBS fish have increased hepatic steatosis compared with sham fish, it is decreased compared with S1-SBS. CONCLUSIONS: Longer remnant intestine decreases the extent of IA, inflammation, and hepatic steatosis in a zebrafish model of SBS.

Wnt signaling inhibition by monensin results in a period of Hippo pathway activation during intestinal adaptation in zebrafish.

Intestinal adaptation (IA) is a critical response to increase epithelial surface area after intestinal loss. Short bowel syndrome (SBS) may follow massive intestinal resection in human patients, particularly without adequate IA. We previously validated a model in zebrafish (ZF) that recapitulates key SBS pathophysiological features. Previous RNA sequencing in this model identified upregulation of genes in the Wnt and Hippo pathways. We therefore sought to identify the timeline of increasing cell proliferation and considered the signaling that might underpin the epithelial remodeling of IA in SBS. SBS was created in a ZF model as previously reported and compared with sham fish with and without exposure to monensin, an ionophore known to inhibit canonical Wnt signaling. Rescue of the monensin effects was attempted with a glycogen synthase kinase 3 inhibitor that activates wnt signaling, CHIR-99021. A timeline was constructed to identify peak cellular proliferation, and the Wnt and Hippo pathways were evaluated. Peak stem cell proliferation and morphological changes of adaptation were identified at 7 days. Wnt inhibition diminished IA at 2 wk and resulted in activation of genes of the Wnt/ß-catenin and Yes-associated protein (YAP)/Hippo pathway. Increased cytoplasmic YAP was observed in monensin-treated SBS fish. Genes of the WASP-interacting protein (WIP) pathway were elevated during Wnt blockade. In conclusion, cellular proliferation and morphological changes accompany SBS even in attempted Wnt blockade. Wnt/ß-catenin, YAP/Hippo pathway, and WIP pathway genes increase during early Wnt blockade. Further understanding of the effects of Wnt and YAP pathway signaling in proliferating stem cells might enrich our knowledge of targets to assist IA. NEW & NOTEWORTHY Intestinal adaptation is a critical response to increase epithelial surface area after large intestinal losses. Inhibition of Wnt/ß-catenin signaling impairs intestinal adaptation in a zebrafish model of short bowel syndrome. There is a subsequent upregulation in genes of the Yes-associated protein/Hippo and WIP pathway. These may be targets for future human therapies, as patients are salvaged by the compensation of increased intestinal epithelial surface area through successful intestinal adaptation.

Differential epithelial growth in tissue-engineered larynx and trachea generated from postnatal and fetal progenitor cells.

Postnatal organ-specific stem and progenitor cells are an attractive potential donor cell for tissue-engineering because they can be harvested autologous from the recipient and have sufficient potential to regenerate the tissue of interest with less risk for ectopic growth or tumor formation compared to donor cells from embryonic or fetal sources. We describe the generation of tissue-engineered larynx and trachea (TELT) from human and mouse postnatal organoid units (OU) as well as from human fetal OU. Mouse TELT contained differentiated respiratory epithelium lining large lumens, cartilage and smooth muscle. In contrast, human postnatal TE trachea, formed small epithelial lumens with rare differentiation, in addition to smooth muscle and cartilage. Human fetal TELT contained the largest epithelial lumens with all differentiated cell types as well as smooth muscle and cartilage. Increased epithelial cytokeratin 14 was identified in both human fetal and postnatal TELT compared to native trachea, consistent with regenerative basal cells. Cilia in TELT epithelium also demonstrated function with beating movements. While both human postnatal and fetal progenitors have the potential to generate TELT, there is more epithelial growth and differentiation from fetal progenitors, highlighting fundamental differences in these cell populations.

Short-term and long-term human or mouse organoid units generate tissue-engineered small intestine without added signalling molecules.

NEW FINDINGS: What is the central question of this study? Tissue-engineered small intestine was previously generated in vivo by immediate implantation of organoid units derived from both mouse and human donor intestine. Although immediate transplantation of organoid units into patients shows promise as a potential future therapy, some critically ill patients might require delayed transplantation. What is the main finding and its importance? Unlike enteroids, which consist of isolated intestinal crypts, short- and long-term cultured organoid units are composed of epithelial and mesenchymal cells derived from mouse or human intestine. Organoid units do not require added signalling molecules and can generate tissue-engineered intestine in vivo. ABSTRACT: Mouse and human postnatal and fetal organoid units (OUs) maintained in either short-term culture (2 weeks) or long-term culture (from 4 weeks up to 3 months) without adding exogenous growth factors were implanted in immunocompromised mice to form tissue-engineered small intestine (TESI) in vivo. Intestinal epithelial stem and neuronal progenitor cells were maintained in long-term OU cultures from both humans and mice without exogenous growth factors, and these cultures were successfully used to form TESI. This was enhanced with OUs derived from human fetal tissues. Organoid unit culture is different from enteroid culture, which is limited to epithelial cell growth and requires supplementation with R-Spondin, noggin and epidermal growth factor. Organoid units contain multiple cell types, including epithelial, mesenchymal and enteric nervous system cells. Short- and long-term cultured OUs derived from mouse and human intestine develop into TESI in vivo, which contains key components of the small intestine similar to native intestine.

Fibroblast Growth Factors in the Gastrointestinal Tract: Twists and Turns.

Fibroblast growth factors (FGFs) are a family of conserved peptides that play an important role in the development, homeostasis, and repair processes of many organ systems, including the gastrointestinal tract. All four FGF receptors and several FGF ligands are present in the intestine. They play important roles in controlling cell proliferation, differentiation, epithelial cell restitution, and stem cell maintenance. Several FGFs have also been proven to be protective against gastrointestinal diseases such as inflammatory bowel diseases or to aid in regeneration after intestinal loss associated with short bowel syndrome. Herein, we review the multifaceted actions of canonical FGFs in intestinal development, homeostasis, and repair in rodents and humans. Developmental Dynamics 246:344-352, 2017. © 2016 Wiley Periodicals, Inc.

Short bowel syndrome results in increased gene expression associated with proliferation, inflammation, bile acid synthesis and immune system activation: RNA sequencing a zebrafish SBS model.

BACKGROUND: Much of the morbidity associated with short bowel syndrome (SBS) is attributed to effects of decreased enteral nutrition and administration of total parenteral nutrition (TPN). We hypothesized that acute SBS alone has significant effects on gene expression beyond epithelial proliferation, and tested this in a zebrafish SBS model. METHODS: In a model of SBS in zebrafish (laparotomy, proximal stoma, distal ligation, n = 29) or sham (laparotomy alone, n = 28) surgery, RNA-Seq was performed after 2 weeks. The proximal intestine was harvested and RNA isolated. The three samples from each group with the highest amount of RNA were spiked with external RNA controls consortium (ERCC) controls, sequenced and aligned to reference genome with gene ontology (GO) enrichment analysis performed. Gene expression of ctnnb1, ccnb1, ccnd1, cyp7a1a, dkk3, ifng1-2, igf2a, il1b, lef1, nos2b, saa1, stat3, tnfa and wnt5a were confirmed to be elevated in SBS by RT-qPCR. RESULTS: RNA-seq analysis identified 1346 significantly upregulated genes and 678 significantly downregulated genes in SBS zebrafish intestine compared to sham with Ingenuity analysis. The upregulated genes were involved in cell proliferation, acute phase response signaling, innate and adaptive immunity, bile acid regulation, production of nitric oxide and reactive oxygen species, cellular barrier and coagulation. The downregulated genes were involved in folate synthesis, gluconeogenesis, glycogenolysis, fatty-acid oxidation and activation and drug and steroid metabolism. RT-qPCR confirmed gene expression differences from RNA-Sequencing. CONCLUSION: Changes of gene expression after 2 weeks of SBS indicate complex and extensive alterations of multiple pathways, some previously implicated as effects of TPN. The systemic sequelae of SBS alone are significant and indicate multiple targets for investigating future therapies.

Marked stem/progenitor cell expansion occurs early after murine ileostomy: a new model.

BACKGROUND: Improving treatment for short bowel syndrome requires a better understanding of how intestinal adaptation is affected by factors like mechanoluminal stimulation. We hypothesized that in mice, luminal diversion via an ileostomy would drive adaptive changes similar to those seen in human intestine after diversion while offering the opportunity to study the immediate events after resection that precede intestinal adaptation. MATERIALS AND METHODS: With Institutional Animal Care and Use Committee approval, a distal ileostomy with a long distal Hartman's was created in 9- to 14-week-old C57/B6 mice (n = 8). Control mice only had a midline laparotomy without stoma formation (n = 5). A rim of tissue from the proximal stoma was resected as a historical control for the proximal segment. Postoperatively, mice received a high-protein liquid diet and water ad libitum. On day 3, tissue from both the proximal and distal limbs were collected for histologic and RNA analysis. Morphometric measures, immunofluorescent antigen detection, and RNA expression were compared with Student paired t-tests with a P value < 0.05 considered significant. RESULTS: At 3 d, survival for mice with an ileostomy was 87% and average weight loss was 12.5% of initial weight compared to 6.05% for control mice. Compared to the distal limb, the proximal limb in mice with an ileostomy demonstrated significantly taller villi with deeper and wider crypts. The proximal limb also had decreased expression of intestinal stem cell markers lgr5, bmi1, sox9, and ascl2. Fewer goblet and enteroendocrine cells per hemivillus were also noted in the proximal limb. In control mice, none of these measures were significant between proximal and distal ileum except for villus height. CONCLUSIONS: This new murine ileostomy model allows study of intestinal adaptation without intestinal anastomosis, which can be technically challenging and morbid.

Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.

Short bowel syndrome (SBS) is a devastating condition in which insufficient small intestinal surface area results in malnutrition and dependence on intravenous parenteral nutrition. There is an increasing incidence of SBS, particularly in premature babies and newborns with congenital intestinal anomalies. Tissue-engineered small intestine (TESI) offers a therapeutic alternative to the current standard treatment, intestinal transplantation, and has the potential to solve its biggest challenges, namely donor shortage and life-long immunosuppression. We have previously demonstrated that TESI can be generated from mouse and human small intestine and histologically replicates key components of native intestine. We hypothesized that TESI also recapitulates native small intestine function. Organoid units were generated from mouse or human donor intestine and implanted into genetically identical or immunodeficient host mice. After 4 wk, TESI was harvested and either fixed and paraffin embedded or immediately subjected to assays to illustrate function. We demonstrated that both mouse and human tissue-engineered small intestine grew into an appropriately polarized sphere of intact epithelium facing a lumen, contiguous with supporting mesenchyme, muscle, and stem/progenitor cells. The epithelium demonstrated major ultrastructural components, including tight junctions and microvilli, transporters, and functional brush-border and digestive enzymes. This study demonstrates that tissue-engineered small intestine possesses a well-differentiated epithelium with intact ion transporters/channels, functional brush-border enzymes, and similar ultrastructural components to native tissue, including progenitor cells, whether derived from mouse or human cells.

Fibroblast growth factor 10 alters the balance between goblet and Paneth cells in the adult mouse small intestine.

Intestinal epithelial cell renewal relies on the right balance of epithelial cell migration, proliferation, differentiation, and apoptosis. Intestinal epithelial cells consist of absorptive and secretory lineage. The latter is comprised of goblet, Paneth, and enteroendocrine cells. Fibroblast growth factor 10 (FGF10) plays a central role in epithelial cell proliferation, survival, and differentiation in several organs. The expression pattern of FGF10 and its receptors in both human and mouse intestine and their role in small intestine have yet to be investigated. First, we analyzed the expression of FGF10, FGFR1, and FGFR2, in the human ileum and throughout the adult mouse small intestine. We found that FGF10, FGFR1b, and FGFR2b are expressed in the human ileum as well as in the mouse small intestine. We then used transgenic mouse models to overexpress Fgf10 and a soluble form of Fgfr2b, to study the impact of gain or loss of Fgf signaling in the adult small intestine. We demonstrated that overexpression of Fgf10 in vivo and in vitro induces goblet cell differentiation while decreasing Paneth cells. Moreover, FGF10 decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2, and Sox9. FGF10 inhibited Hes1 expression in vitro, suggesting that FGF10 induces goblet cell differentiation likely through the inhibition of Notch signaling. Interestingly, Fgf10 overexpression for 3 days in vivo and in vitro increased the number of Mmp7/Muc2 double-positive cells, suggesting that goblet cells replace Paneth cells. Further studies are needed to determine the mechanism by which Fgf10 alters cell differentiation in the small intestine.

FGF10 controls the patterning of the tracheal cartilage rings via Shh.

During embryonic development, appropriate dorsoventral patterning of the trachea leads to the formation of periodic cartilage rings from the ventral mesenchyme and continuous smooth muscle from the dorsal mesenchyme. In this work, we have investigated the role of two crucial morphogens, fibroblast growth factor 10 and sonic hedgehog, in the formation of periodically alternating cartilaginous and non-cartilaginous domains in the ventral mesenchyme. Using a combination of gain- and loss-of-function approaches for FGF10 and SHH, we demonstrate that precise spatio-temporal patterns and appropriate levels of expression of these two signaling molecules in the ventral area are crucial between embryonic day 11.5 and 13.5 for the proper patterning of the cartilage rings. We conclude that the expression level of FGF10 in the mesenchyme has to be within a critical range to allow for periodic expression of Shh in the ventral epithelium, and consequently for the correct patterning of the cartilage rings. We propose that disturbed balances of Fgf10 and Shh may explain a subset of human tracheomalacia without tracheo-esophageal fistula or tracheal atresia.

Vitrification preserves murine and human donor cells for generation of tissue-engineered intestine.

BACKGROUND: Short bowel syndrome causes significant morbidity and mortality. Tissue-engineered intestine may serve as a viable replacement. Tissue-engineered small intestine (TESI) has previously been generated in the mouse model from donor cells that were harvested and immediately reimplanted; however, this technique may prove impossible in children who are critically ill, hemodynamically unstable, or septic. We hypothesized that organoid units (OU), multicellular clusters containing epithelium and mesenchyme, could be cryopreserved for delayed production of TESI. METHODS: OU were isolated from <3 wk-old mouse or human ileum. OU were then cryopreserved by either standard snap freezing or vitrification. In the snap freezing protocol, OU were suspended in cryoprotectant and transferred directly to -80°C for storage. The vitrification protocol began with a stepwise increase in cryoprotectant concentration followed by liquid supercooling of the OU solution to -13°C and nucleation with a metal rod to induce vitrification. Samples were then cooled to -80°C at a controlled rate of -1°C/min and subsequently plunged into liquid nitrogen for long-term storage. OU from both groups were maintained in cryostorage for at least 72 h and thawed in a 37°C water bath. Cryoprotectant was removed with serial sucrose dilutions and OU were assessed by Trypan blue assay for post-cryopreservation viability. Via techniques previously described by our laboratory, the thawed murine or human OU were either cultured in vitro or implanted on a scaffold into the omentum of a syngeneic or irradiated Nonobese Diabetic/Severe Combined Immunodeficiency, gamma chain deficient adult mouse. The resultant TESI was analyzed by histology and immunofluorescence. RESULTS: After cryopreservation, the viability of murine OU was significantly higher in the vitrification group (93 ± 2%, mean ± standard error of the mean) compared with standard freezing (56 ± 6%) (P < 0.001, unpaired t-test, n = 25). Human OU demonstrated similar viability after vitrification (89 ± 2%). In vitro culture of thawed OU produced expanding epithelial spheres supported by a layer of mesenchyme. TESI was successfully generated from the preserved OU. Hematoxylin and eosin staining demonstrated a mucosa composed of a simple columnar epithelium whereas immunofluorescence staining confirmed the presence of both progenitor and differentiated epithelial cells. Furthermore, beta-2-microglobulin confirmed that the human TESI epithelium originated from human cells. CONCLUSIONS: We demonstrated improved multicellular viability after vitrification over conventional cryopreservation techniques and the first successful vitrification of murine and human OU with subsequent TESI generation. Clinical application of this method may allow for delayed autologous implantation of TESI for children in extremis.

FGF9-Pitx2-FGF10 signaling controls cecal formation in mice.

Fibroblast growth factor (FGF) signaling to the epithelium and mesenchyme mediated by FGF10 and FGF9, respectively, controls cecal formation during embryonic development. In particular, mesenchymal FGF10 signals to the epithelium via FGFR2b to induce epithelial cecal progenitor cell proliferation. Yet the precise upstream mechanisms controlling mesenchymal FGF10 signaling are unknown. Complete deletion of Fgf9 as well as of Pitx2, a gene encoding a homeobox transcription factor, both lead to cecal agenesis. Herein, we used mouse genetic approaches to determine the precise contribution of the epithelium and/or mesenchyme tissue compartments in this process. Using tissue compartment specific Fgf9 versus Pitx2 loss of function approaches in the gut epithelium and/or mesenchyme, we determined that FGF9 signals to the mesenchyme via Pitx2 to induce mesenchymal Fgf10 expression, which in turn leads to epithelial cecal bud formation.

Tissue engineering the small intestine.

Short bowel syndrome (SBS) results from the loss of a highly specialized organ, the small intestine. SBS and its current treatments are associated with high morbidity and mortality. Production of tissue-engineered small intestine (TESI) from the patient's own cells could restore normal intestinal function via autologous transplantation. Improved understanding of intestinal stem cells and their niche have been coupled with advances in tissue engineering techniques. Originally described by Vacanti et al of Massachusetts General Hospital, TESI has been produced by in vivo implantation of organoid units. Organoid units are multicellular clusters of epithelium and mesenchyme that may be harvested from native intestine. These clusters are loaded onto a scaffold and implanted into the host omentum. The scaffold provides physical support that permits angiogenesis and vasculogenesis of the developing tissue. After a period of 4 weeks, histologic analyses confirm the similarity of TESI to native intestine. TESI contains a differentiated epithelium, mesenchyme, blood vessels, muscle, and nerve components. To date, similar experiments have proved successful in rat, mouse, and pig models. Additional experiments have shown clinical improvement and rescue of SBS rats after implantation of TESI. In comparison with the group that underwent massive enterectomy alone, rats that had surgical anastomosis of TESI to their shortened intestine showed improvement in postoperative weight gain and serum B12 values. Recently, organoid units have been harvested from human intestinal samples and successfully grown into TESI by using an immunodeficient mouse host. Current TESI production yields approximately 3 times the number of cells initially implanted, but improvements in the scaffold and blood supply are being developed in efforts to increase TESI size. Exciting new techniques in stem cell biology and directed cellular differentiation may generate additional sources of autologous intestinal tissue for direct translation to human therapy.