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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.

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.

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.

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.

Channeling morphogenic signaling gradients intrinsic to intestinal epithelial stem cells, Nikolaev et al. (2020) optimized a three-dimensional microchip perfusion system that augments growth, maturation, and longevity of tubular intestinal enteroids. Reported in Nature, this system may ultimately pave the way to study human intestinal development and pathophysiology, perhaps for therapeutic discovery.

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 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 (

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.

Background In short bowel syndrome, luminal factors influence adaptation in which the truncated intestine increases villus lengths and crypt depths to increase nutrient absorption. No study has evaluated the effect of adaptation within the distal intestine after intestinal separation. We evaluated multiple conditions, including Igf1r inhibition, in proximal and distal segments after intestinal resection to evaluate the epithelial effects of the absence of mechanoluminal stimulation. Methods Short bowel syndrome was created in adult male zebrafish by performing a proximal stoma with ligation of the distal intestine. These zebrafish with short bowel syndrome were compared to sham-operated zebrafish. Groups were treated with the Igf1r inhibitor NVP-AEW541, DMSO, a vehicle control, or water for 2 weeks. Proximal and distal intestine were analyzed by hematoxylin and eosin for villus epithelial circumference, inner epithelial perimeter, and circumference. We evaluated BrdU+ cells, including costaining for β-catenin, and the microbiome was evaluated for changes. Reverse transcription quantitative polymerase chain reaction was performed for β-catenin, CyclinD1, Sox9a, Sox9b, and c-Myc. Results Proximal intestine demonstrated significantly increased adaptation compared to sham-operated proximal intestine, whereas the distal intestine showed no adaptation in the absence of luminal flow. Addition of the Igf1r inhibitor resulted in decreased adaption in the distal intestine but an increase in distal proliferative cells and proximal β-catenin expression. While some proximal proliferative cells in short bowel syndrome colocalized β-catenin and BrdU, the distal proliferative cells did not co-stain for β-catenin. Sox9a increased in the distal limb after division but not after inhibition with the Igf1r inhibitor. There was no difference in alpha diversity or species richness of the microbiome between all groups. Conclusion Luminal flow in conjunction with short bowel syndrome significantly increases intestinal adaption within the proximal intestine in which proliferative cells contain β-catenin. Addition of an Igf1r inhibitor decreases adaptation in both proximal and distal limbs while increasing distal proliferative cells that do not colocalize β-catenin. Igf1r inhibition abrogates the increase in distal Sox9a expression that otherwise occurs in short bowel syndrome. Mechanoluminal flow is an important stimulus for intestinal adaptation.

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.

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.

The intestinal epithelium is one of the most rapidly cycling tissues in the mammalian body, with the majority of its cells turning over every 4–8 days. Lineage-tracing studies have identified two distinct populations of intestinal stem cells (ISCs): the rapidly dividing crypt base columnar cells marked by Lgr5, and a loosely defined quiescent population of cells at the +4 cell position. In addition, recent studies indicate that both proliferative progenitors and theoretically terminally differentiated cells can “revert” to an ISC phenotype following depletion of the Lgr5+ population. The stem cell niche is maintained by a complex network of signaling molecules, including Wnt, Notch, epidermal growth factor, BMP, and Hedgehog. Gradients of these and other growth factors maintain homeostasis and assist in injury response. Understanding and leveraging these complex processes may further be the goal of creating tissue-engineered intestine (TEI) as a clinical therapy. TEI has been generated from multiple sources of stem/progenitor cells and demonstrated to contain all mature epithelial cell types, associated mesenchymal cells, and to recapitulate the digestive and absorptive capacity of the mature intestine.

Introduction: Splenectomy is common after trauma or hematologic disease, and alters immune protection against pathogens, which may lead to fulminant infection with high mortality. Yet the spleen has demonstrable regenerative capacity and cells might be recovered and reimplanted at the time of injury or excision to avoid these risks. Methods: Tissue-engineered spleen (TESp) was generated from ActinGFP mice (mTESp) or human donor spleen (hTESp) through implantation of spleen organoid units (spleen OU), in NOD/SCID mice with concurrent splenectomy, on a biodegradable scaffold. Explants were evaluated and blood smears were obtained to investigate the presence of target cells or Howell-Jolly bodies, which are erythrocyte sequelae of asplenia. Results: TESp was generated from mouse (mTESp) and human (hTESp) donor cells with essential splenic components: red and white pulp with trabeculae. mTESp and hTESp demonstrated green fluorescent protein- or lamin-positive costaining with proliferating cell nuclear antigen, CD4, and CD11c, identifying proliferative donor cells and key immune components of the spleen of donor origin. Animals with hTESp and mTESP combined with splenectomy had significantly fewer Howell-Jolly bodies on blood smears than controls. Conclusion: TESp from mouse and human donor cells can be generated by 4 weeks and contains donor immune cells identified by CD4 and CD11c. TESp reduces postsplenectomy erythrocyte inclusions, indicating possible function. Impact Statement Overwhelming postsplenectomy infection is rare but highly mortal. Tissue-engineered spleen (TESp) was generated from murine (mTESp) and human (hTESp) donors and appeared histologically similar to native spleen. Both mTESp and hTESp demonstrated proliferative cells of donor spleen origin. Importantly, functional cells were demonstrated on imaging with a corresponding reduction in the number of erythrocyte inclusions in blood smears that are typically identified in patients with asplenia and indicate a lack of clearance by functional spleen tissue. Taken together, these findings indicate that this approach might be clinically relevant as a future human therapy.

Models for enteric neuropathies, in which intestinal nerves are absent or injured, are required to evaluate possible cell therapies. However, existing options, including transgenic mice, are variable and fragile. Here immunocompromised mice were implanted with human pluripotent stem cell–derived tissue-engineered small intestine 10 weeks prior to a second survival surgery in which enteric nervous system precursor cells, or saline controls, were injected into the human intestinal organoid–derived tissue-engineered small intestine and analyzed 4 weeks later. Human intestinal organoid–derived tissue-engineered small intestine implants injected with saline as controls illustrated formation of intestinal epithelium and mesenchyme without an enteric nervous system. Second surgical introduction of human pluripotent stem cell–generated enteric nervous system precursors into developing human intestinal organoid–derived tissue-engineered small intestine implants resulted in proliferative migratory neuronal and glial cells, including multiple neuronal subtypes, and demonstrated function in contractility assays.

Building complex tissues requires the development of innovative interdisciplinary engineering solutions. In this Forum, the INTENS Consortium discuss experimental considerations and challenges for generating a tissue-engineered intestine for the treatment of short bowel syndrome, taking into account cell source, scaffold choice, and design strategy for achieving proper assembly and function.

Background R-spondins, including R-spondin 1 (RSPO1), are a family of Wnt ligands that help to activate the canonical Wnt/β-catenin pathway, which is critical for intestinal epithelial cell proliferation and maintenance of intestinal stem cells. This proliferation underpins the epithelial expansion, or intestinal adaptation (IA), that occurs following massive bowel resection and short bowel syndrome (SBS). The purpose of this study was to identify if recombinant human RSPO1 (rhRSPO1) could be serially administered to SBS zebrafish to enhance cellular proliferation and IA. Methods Adult male zebrafish were assigned to four groups: sham + PBS, SBS + PBS, sham + rhRSPO1, and SBS + rhRSPO1. Sham fish had a laparotomy alone. SBS fish had a laparotomy with distal intestinal ligation and creation of a proximal stoma. Fish were weighed at initial surgery and then weekly. rhRSPO1 was administered post-operatively following either a one- or two-week dosing schedule with either 3 or 5 intraperitoneal injections, respectively. Fish were harvested at 7 or 14 days with intestinal segments collected for analysis. Results Repeated intraperitoneal injection of rhRSPO1 was feasible and well tolerated. At 7 days, intestinal epithelial proliferation was increased by rhRSPO1. At 14 days, SBS + rhRSPO1 fish lost significantly less weight than SBS + PBS fish. Measurements of intestinal surface area were not increased by rhRSPO1 administration but immunofluorescent staining for β-catenin and gene expression for cyclin D1 was increased. Conclusions Intraperitoneal injection of rhRSPO1 decreased weight loss in SBS zebrafish with increased β-catenin + cells and cyclin D1 expression at 14 days, indicating improved weight maintenance might result from increased activation of the canonical Wnt pathway., Highlights • rhRSPO1 decreased weight loss in SBS zebrafish. • rhRSPO1 increased intestinal cell epithelial proliferation at 7 days. • rhRSPO1 increased β-catenin + cells at 14 days in the intestine of SBS zebrafish. • rhRSPO1 increased cyclin D1 expression at 14 days in the intestine of SBS zebrafish.

The cellular and molecular mechanisms that underpin regeneration of the human lung are unknown, and the study of lung repair has been impeded by the necessity for reductionist models that may exclude key components. We hypothesized that multicellular epithelial and mesenchymal cell clusters or lung organoid units (LuOU) could be transplanted to recapitulate proximal and distal cellular structures of the native lung and airways. Transplantation of LuOU resulted in the growth of tissue-engineered lung (TELu) that contained the necessary cell types consistent with native adult lung tissue and demonstrated proliferative cells at 2 and 4 weeks. This technique recapitulated important elements of both mouse and human lungs featuring key components of both the proximal and distal lung regions. When LuOU were generated from whole lung, TELu contained key epithelial and mesenchymal cell types, and the origin of the cells was traced from both ActinGFP and SPCGFP donors to indicate that the cells in TELu were derived from the transplanted LuOU. Alveolar epithelial type 2 cells (AEC2s), club cells, ciliated cells marked by beta-tubulin IV, alveolar epithelial type I cells, Sox-2-positive proximal airway progenitors, p63-positive basal cells, and CGRP-positive pulmonary neuroendocrine cells were identified in the TELu. The mesenchymal components of peribronchial smooth muscle and nerve were identified with a CD31-positive donor endothelial cell contribution to TELu vasculature. TELu successfully grew from postnatal tissues from whole murine and human lung, distal murine lung, as well as murine and human trachea. These data support a model of postnatal lung regeneration containing the diverse cell types present in the entirety of the respiratory tract.

Background The pediatric intestinal microbiome is impacted by many factors, including age, diet, antibiotics, and environment. We hypothesized that in operative patients, alterations to antibiotics and mechanoluminal stimulation would demonstrate measurable changes in the intestinal microbiome and that microbial diversity would be reduced without normal mechanoluminal stimulation and with prolonged antibiotic treatment. Methods Bacterial 16s rRNA was extracted from swabbed samples of 43 intestines from 29 patients, aged 5 days to 13 years old. Swabs were obtained during initial resection or later stoma closure. Samples were compared using phylogenetic diversity whole tree alpha diversity and unweighted UniFrac distance beta diversity and by comparing significantly different taxonomic groups. Results Microbial community structure varied significantly between obstructive and inflammatory diseases (P = .001), with an effect size of 0.99 (0.97, 1.00). This difference persisted even 6 weeks after return to health. Family Enterobacter and Clostridiaceae predominated in patients with necrotizing enterocolitis or focal intestinal perforation; patients with an obstructive pathology had an abundance of Bacteroides. Comparison of UniFrac distance between paired proximal and distal intestines demonstrated that paired samples were significantly closer than any other comparison. Conclusion In infants, inflammatory and ischemic intestinal pathologies treated with prolonged courses of antibiotics durably alter the intestinal mucosal microbiome. Diversion of mechanoluminal stimulation, however, does not.

The authors highlight the speciality field of regenerative medicine and its application to health care. Academic pediatric surgeons have been the early pioneers here sharing exciting discovery and the opportunities for research enterprise. An overview of current and future therapeutics is provided for the reader.

Pediatric surgeons are ideal allies for the translation of basic science including stem cell therapies. In the spirit of Robert E. Gross, of applying creative solutions to pediatric problems with technical expertise, we describe the impending cellular therapies that may be derived from stem and progenitor cells. Understanding the types and capabilities of stem and progenitor cells is important for pediatric surgeons to join and facilitate progress for babies. We are developing an induced pluripotent stem cell therapy for enteric neuropathies such as Hirschsprung disease that might be helpful for children in the near future. Our goals, which we hope to share with other surgeons and scientists, include working to establish safe clinical trials and meeting regulatory standards in a thoughtful way that balances patients need and unknown risks.

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.

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)., National Institutes of Health (U.S.) (Grant R01 RHL115145A)

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 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.

Hirschsprung disease (HD), a neurocristopathy characterized by failed migration of neural crest cells to the distal colon, requires surgical resection of the aganglionic segment. Advances in stem cell and regenerative medicine research have opened the possibility to treat HD less invasively using enteric nervous system (ENS) cell replacement therapy. This article reviews the progress to date of culturing and delivering ENS stem cells in various in vitro and in vivo models, as well as review the available evidence of functionality of the transplant-derived cells. Potential areas of future study are identified, and application of conditions other than HD is briefly discussed.

Tissue engineering is a promising potential candidate for treating intestinal failure resulting from necrotizing enterocolitis. This requires the acquisition, preparation and implantation of autologous organoid units. This may be affected by the complexities of periods of storage of viable donor tissue and delayed implantation. This chapter addresses the development, methodology, and application of tissue-engineered intestine in the experimental and clinical setting. Tissue engineering has the potential of avoiding the inherent toxicities of intestinal transplantation and prolonged immunosuppression.

Short bowel syndrome (SBS) is a morbid and mortal condition characterized in most patients by insufficient intestinal surface area. Current management strategies are inadequate, but tissue-engineered small intestine (TESI) offers a potential therapy. A barrier to translation of TESI is the generation of scalable mucosal surface area to significantly increase nutritional absorption. Fibroblast growth factor 10 (Fgf10) is a critical growth factor essential for the development of the gastrointestinal tract. We hypothesized that overexpression of Fgf10 would improve the generation of TESI. Organoid units, the multicellular donor tissue that forms TESI, were derived from Rosa26(rtTA/+), tet(o)Fgf10/(-) or Fgf10(Mlc-nlacZ-v24) (hereafter called Fgf10(lacZ)) mice. These were implanted into the omentum of NOD/SCID γ-chain-deficient mice and induced with doxycycline in the case of tet(o)Fgf10/(-). Resulting TESI were explanted at 4 weeks and studied by histology, quantitative RT-PCR and immunofluorescence. Four weeks after implantation, Fgf10 overexpressing TESI was larger and weighed more than the control tissues. Within the mucosa, the villus height was significantly longer and crypts contained a greater percentage of proliferating epithelial cells. A fully differentiated intestinal epithelium with enterocytes, goblet cells, enteroendocrine cells and Paneth cells was identified in the Fgf10-overexpressing TESI, comparable to native small intestine. β-Galactosidase expression was found in both the epithelium and the mesenchyme of the TESI derived from the Fgf10(LacZ) duodenum. However, this was not the case with TESI generated from jejunum and ileum. We conclude that Fgf10 enhances the formation of TESI.

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

Summary Acquired or congenital disruption in enteric nervous system (ENS) development or function can lead to significant mechanical dysmotility. ENS restoration through cellular transplantation may provide a cure for enteric neuropathies. We have previously generated human pluripotent stem cell (hPSC)-derived tissue-engineered small intestine (TESI) from human intestinal organoids (HIOs). However, HIO-TESI fails to develop an ENS. The purpose of our study is to restore ENS components derived exclusively from hPSCs in HIO-TESI. hPSC-derived enteric neural crest cell (ENCC) supplementation of HIO-TESI establishes submucosal and myenteric ganglia, repopulates various subclasses of neurons, and restores neuroepithelial connections and neuron-dependent contractility and relaxation in ENCC-HIO-TESI. RNA sequencing identified differentially expressed genes involved in neurogenesis, gliogenesis, gastrointestinal tract development, and differentiated epithelial cell types when ENS elements are restored during in vivo development of HIO-TESI. Our findings validate an effective approach to restoring hPSC-derived ENS components in HIO-TESI and may implicate their potential for the treatment of enteric neuropathies., Graphical Abstract, Highlights • ENCC implantation restores enteric glial and neural subpopulations in HIO-TESI • ENCC differentiate into diverse neuronal subtypes and synapse with luminal ECC • ENCC-HIO-TESI demonstrates neuron-dependent contractility and relaxation • Early in vivo ENCC implantation alters the developing HIO-TESI transcriptome, Human intestinal organoid and enteric neural crest cell co-culture restores enteric nervous system (ENS) function. Schlieve and colleagues developed an in vivo approach to establish ENS elements in tissue-engineered small intestine that demonstrates neuron-dependent functional integration. This method could be applied to other organ systems and represent a future cellular therapy for human enteric neuropathies.

Liver disease affects large numbers of patients, yet there are limited treatments available to replace absent or ineffective cellular function of this crucial organ. Donor scarcity and the necessity for immunosuppression limit one effective therapy, orthotopic liver transplantation. But in some conditions such as inborn errors of metabolism or transient states of liver insufficiency, patients may be salvaged by providing partial quantities of functional liver tissue. After transplanting multicellular liver organoid units composed of a heterogeneous cellular population that includes adult stem and progenitor cells, both mouse and human tissue-engineered liver (TELi) form in vivo. TELi contains normal liver components such as hepatocytes with albumin expression, CK19-expressing bile ducts and vascular structures with α-smooth muscle actin expression, desmin-expressing stellate cells, and CD31-expressing endothelial cells. At 4 weeks, TELi contains proliferating albumin-expressing cells and identification of β2-microglobulin-expressing cells demonstrates that the majority of human TELi is composed of transplanted human cells. Human albumin is detected in the host mouse serum, indicating in vivo secretory function. Liquid chromatography/mass spectrometric analysis of mouse serum after debrisoquine administration is followed by a significant increase in the level of the human metabolite, 4-OH-debrisoquine, which supports the metabolic and xenobiotic capability of human TELi in vivo. Implanted TELi grew in a mouse model of inducible liver failure.

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.

Background Signaling by fibroblast growth factor is critical for epithelial proliferation, differentiation, and the development of many organs, including the intestine. Fibroblast growth factor 10 and fibroblast growth factor 2c are upregulated after massive bowel resection during intestinal adaptation. This pathway is conserved highly. We hypothesized that inhibition of fibroblast growth factor signaling would impair intestinal adaptation in the zebrafish model of short bowel syndrome and allow insight into the negative regulation of this pathway. Methods Short bowel syndrome equivalent to a high jejunostomy was generated in adult male hsp70:dnfgfr1-GFP zebrafish, wildtype fish exposed to tyrosine-kinase inhibitor, and wildtype fish in absence of tyrosine-kinase inhibitor. Heat shock in hsp70:dnfgfr1-GFP fish decreases fgf 1 expression. Parameters including weight, proliferation, and differentiation were evaluated after harvest in experimental and control groups. Results Although short bowel syndrome zebrafish lost more weight relative to sham zebrafish in both groups, heat shock fish with short bowel syndrome lost more weight compared with non-heat shock fish with short bowel syndrome. In the non-heat shock controls, the villus epithelial perimeter increased in short bowel syndrome compared with sham fish, but this did not occur in heat shock fish. Non-heat shock fish with short bowel syndrome fish had significantly increased Bromodeoxyuridine(+) proliferative cells per hemivillus compared with non-heat shock-sham, while heat shock-short bowel syndrome had a more substantial increase in Bromodeoxyuridine(+) cells compared with HS-sham. Non-heat shock-short bowel syndrome demonstrated a significantly increased percentage of Alcian blue(+) goblet cells per hemivillus compared with non-heat shock-sham, while the heat shock-short bowel syndrome demonstrated decreased Alcian blue(+) cells compared with non-heat shock-short bowel syndrome. In contrast, SU5402 inhibited epithelial proliferation while increasing weight loss. Conclusion Inhibition of fibroblast growth factor-1 signaling in short bowel syndrome decreases epithelial adaptation, increases Bromodeoxyuridine-labeled cells at 2 weeks, and exacerbates weight loss while decreasing epithelial goblet cells.

A case of intraoperative cyanosis in a patient with a common atrioventricular canal palliated with a pulmonary artery (PA) band is presented. The patient's physiology was consistent with cyanosis due to inadequate pulmonary blood flow, and responded quickly to typical interventions used for a hypercyanotic episode in a patient with unrepaired Tetralogy of Fallot. Differences and similarities in the physiology of PA banding compared with Tetralogy of Fallot are presented, including a rationale for treatment options for hemodynamic decompensation occurring in the setting of anesthesia and surgery.

Aim: Loss of colon reservoir function after colectomy can adversely affect patient outcomes. In previous work, human fetal intestinal cells developed epithelium without mesenchyme following implantation in mice. However, for humans, postnatal tissue would be the preferred donor source. We generated tissue-engineered colon (TEC) from postnatal human organoid units. Materials & methods: Organoid units were prepared from human colon waste specimens, loaded onto biodegradable scaffolds and implanted into immunocompromised mice. After 4 weeks, human TEC was harvested. Immunofluorescence staining confirmed human origin, identified differentiated epithelial cell types and verified the presence of supporting mesenchyme. Results: Human TEC demonstrated a simple columnar epithelium. Immunofluorescence staining demonstrated human origin and the three differentiated cell types of mature colon epithelium. Key mesenchymal components (smooth muscle, intestinal subepithelial myofibroblasts and ganglion cells) were seen. Conclusion: Colon can form from human progenitor cells on a scaffold in a mouse host. This proof-of-concept experiment is an important step in transitioning TEC to human therapy.

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.

The purpose of this review is to describe recent advancements in tissue-engineering of the gastrointestinal system. For some patients, a congenital or acquired defect in the alimentary system results in digestive or nutritional deficiencies requiring intervention. Unfortunately, these treatments are associated with morbid complications. Advances in the growth of tissue-engineered esophagus, stomach, small intestine, colon and anus have been made in recent years. The progress reviewed here hopefully will someday benefit patients with gastrointestinal organ loss by providing a tissue replacement with morphology and function similar to native tissue.In native gastrointestinal tissue, epithelial homeostasis is governed largely by the interaction of the stem cell and its surrounding cellular niche. In particular, the small intestinal stem cell populations identified as the crypt base columnar cell (CBCC) and at cell position 4 (cp4) are responsible for mucosal maintenance and response to injury. This work influences efforts to generate bioengineered tissues for both in-vitro mucosal models and full-thickness in-vivo tissue-engineered esophagus, stomach, intestine and colon.Gastrointestinal organ loss is a challenge to manage. Current therapy can be life-saving, but is associated with morbid complications. Tissue-engineering will someday restore normal gastrointestinal function and eliminate the need for nutritional supplementation or transplant.

Purpose Solid pseudopapillary tumor (SPT) of the pancreas is a rare neoplasm. The objective of this study was to review our institution's experience and provide an update on current management in the pediatric population. Methods Our pathology database identified all patients with SPT for a 20-year period (1991-2011). Demographics, clinical characteristics, operative details, pathology, and outcomes data were retrospectively reviewed. Results Eleven patients with SPT were identified. Most were female and Hispanic. Median age at resection was 14 years (9-17 years). Most patients presented with abdominal pain. Diagnostic imaging was most commonly an ultrasound or computed tomography. All tumors were resected en bloc. Median greatest tumor diameter was 5 cm (3.5-12 cm). Median length of stay was 8 days (5-19 days). Complications included pancreatic leak, chyle leak, delayed gastric emptying, fat malabsorption, and incisional keloid. Recurrence developed after 2.5 years in 1 patient with positive surgical margins. There were no metastases or deaths. Median follow-up was 1.4 years (0.6-5.9 years). Conclusion This pediatric series of SPT from a single institution corroborates previous reports in the literature. In our experience, SPT behaves like a low-grade malignancy and has an excellent prognosis. Surgical resection is dictated by tumor location and remains the treatment of choice.

Large type II and III congenital pulmonary airway malformations (CPAMs) can cause pulmonary hypoplasia, non-immune hydrops fetalis and fetal demise. Fetal intervention is indicated if hydrops fetalis develops. In this report, we describe three cases of type II and III CPAMs complicated by hydrops and treated with percutaneous sclerotherapy by ethanolamine injection into the tumor. All 3 cases demonstrated reduction in size of the CPAM and resolution of the hydrops with subsequent delivery at term. We believe that fetal percutaneous sclerotherapy can be used as a minimally invasive palliative strategy to treat CPAM-induced hydrops fetalis. Further studies are needed to delineate the risks of this novel technique.

Aim: To determine the effect of VEGF overexpression on tissue-engineered small intestine (TESI) formation. Materials & methods: Organoid units were isolated from the intestines of 2-week-old transgenic mouse pups capable of inducible, ubiquitous VEGF overexpression (CMV-Cre/rtTA/tet(0)-VEGF) and implanted into nonobese diabetic/severe combined immunodeficiency mice. Resulting TESI were explanted at 2 and 4 weeks, and studied by histology, tissue ELISA and immunofluorescence. Results: At 2 weeks postimplantation, the TESI mucosa from the VEGF overexpression group formed rudimentary villi and more crypts compared with controls, which demonstrated a flat epithelium with few crypts and no villi. At 4 weeks postimplantation, the TESI from the VEGF overexpression group was larger and significantly heavier than controls. Within the mucosa, the villus height and crypt depth was significantly longer, contained a greater percentage of proliferating crypt epithelial cells and consisted of all four terminally differentiated epithelial cell types. There was also a significant increase in the capillary density within the submucosa. Conclusions: Overexpression of VEGF optimizes the formation of TESI by increasing the submucosal capillary density, crypt epithelial proliferation and the rate of mucosa formation. A larger construct with increased villus and crypt height was noted after 4 weeks in vivo.

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection. In this study, we transitioned the technique to a mouse model, allowing investigation of the processes involved during TESI formation through the transgenic tools available in this species. This is a necessary step toward applying the technique to human therapy. Multicellular organoid units were derived from small intestines of transgenic mice and transplanted within the abdomen on biodegradable polymers. Immunofluorescence staining was used to characterize the cellular processes during TESI formation. We demonstrate the preservation of Lgr5- and DcamKl1-positive cells, two putative intestinal stem cell populations, in proximity to their niche mesenchymal cells, the intestinal subepithelial myofibroblasts (ISEMFs), at the time of implantation. Maintenance of the relationship between ISEMF and crypt epithelium is observed during the growth of TESI. The engineered small intestine has an epithelium containing a differentiated epithelium next to an innervated muscularis. Lineage tracing demonstrates that all the essential components, including epithelium, muscularis, nerves, and some of the blood vessels, are of donor origin. This multicellular approach provides the necessary cell population to regenerate large amounts of intestinal tissue that could be used to treat short bowel syndrome.

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.