Fgf10 overexpression enhances the formation of tissue-engineered small intestine.

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. ß-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.

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.

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.

A "living bioreactor" for the production of tissue-engineered small intestine.

Here, we describe the use of a mouse model as a living bioreactor for the generation of tissue-engineered small intestine. Small intestine is harvested from donor mice with subsequent isolation of organoid units (a cluster of mesenchymal and epithelial cells). Some of these organoid units contain pluripotent stem cells with a preserved relationship with the mesenchymal stem cell niche. A preparation of organoid units is seeded onto a biodegradable scaffold and implanted intraperitoneally within the omentum of the host animal. The cells are nourished initially via imbibition until neovascularization occurs. This technique allows the growth of fully differentiated epithelium (composed of Paneth cells, goblet cells, enterocytes and enteroendocrine cells), muscle, nerve, and blood vessels of donor origin. Variations of this technique have been used to generate tissue-engineered stomach, large intestine, and esophagus. The variations include harvest technique, length of digestion, and harvest times.

Human tissue-engineered small intestine forms from postnatal progenitor cells.

PURPOSE: Tissue-engineered small intestine (TESI) represents a potential cure for short bowel syndrome (SBS). We previously reported full-thickness intestine formation using an organoid units-on-scaffold approach in rodent and swine models. Transplanted intestinal xenografts have been documented to survive from human fetal tissue but not from postnatal tissue. We now present the first report of human TESI from postnatal tissue. METHODS: Organoid units (OU) were prepared from human small bowel resection specimens, loaded onto biodegradable scaffolds and implanted into NOD/SCID gamma chain-deficient mice. After 4 weeks, TESI was harvested and immunostained for ß2-microglobulin to identify human tissue, villin for enterocytes, lysozyme for Paneth cells, chromogranin-A for enteroendocrine cells, mucin-2 for goblet cells, smooth muscle actin and desmin to demonstrate muscularis, and S-100 for nerves. RESULTS: All TESI was of human origin. Immunofluorescence staining of human TESI reveals the presence of all four differentiated cell types of mature human small intestine, in addition to the muscularis and the supporting intestinal subepithelial myofibroblasts. Nerve tissue is also present. CONCLUSIONS: Our technique demonstrates survival, growth, and differentiation of postnatally derived human small intestinal OU into full thickness TESI in murine hosts. This regenerative medicine strategy may eventually assist in the treatment of SBS.

Tissue engineering of the intestine in a murine model.

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.(1) In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.(2) In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,(3) the relationship between which preserves the intestinal stem cell niche.(4) In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.(5)The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

Human tissue-engineered colon forms from postnatal progenitor cells: an in vivo murine model.

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 10-fibroblast growth factor receptor 2b mediated signaling is not required for adult glandular stomach homeostasis.

The signaling pathways that are essential for gastric organogenesis have been studied in some detail; however, those that regulate the maintenance of the gastric epithelium during adult homeostasis remain unclear. In this study, we investigated the role of Fibroblast growth factor 10 (FGF10) and its main receptor, Fibroblast growth factor receptor 2b (FGFR2b), in adult glandular stomach homeostasis. We first showed that mouse adult glandular stomach expressed Fgf10, its receptors, Fgfr1b and Fgfr2b, and most of the other FGFR2b ligands (Fgf1, Fgf7, Fgf22) except for Fgf3 and Fgf20. Fgf10 expression was mesenchymal whereas FGFR1 and FGFR2 expression were mostly epithelial. Studying double transgenic mice that allow inducible overexpression of Fgf10 in adult mice, we showed that Fgf10 overexpression in normal adult glandular stomach increased epithelial proliferation, drove mucous neck cell differentiation, and reduced parietal and chief cell differentiation. Although a similar phenotype can be associated with the development of metaplasia, we found that Fgf10 overexpression for a short duration does not cause metaplasia. Finally, investigating double transgenic mice that allow the expression of a soluble form of Fgfr2b, FGF10's main receptor, which acts as a dominant negative, we found no significant changes in gastric epithelial proliferation or differentiation in the mutants. Our work provides evidence, for the first time, that the FGF10-FGFR2b signaling pathway is not required for epithelial proliferation and differentiation during adult glandular stomach homeostasis.

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.

Mesenchymal-specific inhibition of vascular endothelial growth factor (VEGF) attenuates growth in neonatal mice.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis and vasculogenesis. However, the role of VEGF in the regulation of neonatal mouse development is not completely defined. We sought to determine the effect of VEGF inhibition on the development of the neonatal mouse using a transgenic approach. MATERIALS AND METHODS: We generated triple transgenic mice that express the soluble VEGF receptor, (sFlt-1), specifically in the mesenchyme (dermo-1(Cre)- tetracycline reverse transcriptional activator (rtTA)(flox/flox)-tet(0)-sflt-1). Mothers of the pups (transgenic and littermate controls) were fed doxycycline chow at birth for transgene activation via breast milk, and the pups were sacrificed at various time points. To test reversibility of the phenotype, mice from both groups (n = 6) were switched to normal chow at P50 and monthly weights were measured for 9 mo. RESULTS: Dermo-1(Cre)-rtTA(flox/flox)-tet(0)-sflt-1 mice were smaller compared with littermate controls at P21. There was a significant reduction in tissue VEGF levels following sFlt-1 expression. The rate of growth was reduced but did not impact overall survival after 1 y. A significant reduction in organ size as a percentage body weight was seen in the kidney and stomach, whereas the weight of the colon and spleen were relatively increased; however, no gross histologic difference was observed. After 6 mo on normal diet, the dermo-1(Cre)-rtTA(flox/flox)-tet(0)-sflt-1 mouse's weight doubled, indicating reversibility of phenotype. CONCLUSION: Mesenchymal-specific inhibition of VEGF in neonatal mice results in a severe but reversible arrest in somatic growth that does not affect overall survival at 1 yr. This mouse is a useful tool to test the function of VEGF in somatic growth.

VEGF optimizes the formation of tissue-engineered small intestine.

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.

Contrasting expression of canonical Wnt signaling reporters TOPGAL, BATGAL and Axin2(LacZ) during murine lung development and repair.

Canonical WNT signaling plays multiple roles in lung organogenesis and repair by regulating early progenitor cell fates: investigation has been enhanced by canonical Wnt reporter mice, TOPGAL, BATGAL and Axin2(LacZ). Although widely used, it remains unclear whether these reporters convey the same information about canonical Wnt signaling. We therefore compared beta-galactosidase expression patterns in canonical Wnt signaling of these reporter mice in whole embryo versus isolated prenatal lungs. To determine if expression varied further during repair, we analyzed comparative pulmonary expression of beta-galactosidase after naphthalene injury. Our data show important differences between reporter mice. While TOPGAL and BATGAL lines demonstrate Wnt signaling well in early lung epithelium, BATGAL expression is markedly reduced in late embryonic and adult lungs. By contrast, Axin2(LacZ) expression is sustained in embryonic lung mesenchyme as well as epithelium. Three days into repair after naphthalene, BATGAL expression is induced in bronchial epithelium as well as TOPGAL expression (already strongly expressed without injury). Axin2(LacZ) expression is increased in bronchial epithelium of injured lungs. Interestingly, both TOPGAL and Axin2(LacZ) are up regulated in parabronchial smooth muscle cells during repair. Therefore the optimal choice of Wnt reporter line depends on whether up- or down-regulation of canonical Wnt signal reporting in either lung epithelium or mesenchyme is being compared.

Murine tissue-engineered stomach demonstrates epithelial differentiation.

BACKGROUND: Gastric cancer remains the second largest cause of cancer-related mortality worldwide. Postgastrectomy morbidity is considerable and quality of life is poor. Tissue-engineered stomach is a potential replacement solution to restore adequate food reservoir and gastric physiology. In this study, we performed a detailed investigation of the development of tissue-engineered stomach in a mouse model, specifically evaluating epithelial differentiation, proliferation, and the presence of putative stem cell markers. MATERIALS AND METHODS: Organoid units were isolated from <3 wk-old mouse glandular stomach and seeded onto biodegradable scaffolds. The constructs were implanted into the omentum of adult mice. Implants were harvested at designated time points and analyzed with histology and immunohistochemistry. RESULTS: Tissue-engineered stomach grows as an expanding sphere with a simple columnar epithelium organized into gastric glands and an adjacent muscularis. The regenerated gastric epithelium demonstrates differentiation of all four cell types: mucous, enteroendocrine, chief, and parietal cells. Tissue-engineered stomach epithelium proliferates at a rate comparable to native glandular stomach and expresses two putative stem cell markers: DCAMKL-1 and Lgr5. CONCLUSIONS: This study demonstrates the successful generation of tissue-engineered stomach in a mouse model for the first time. Regenerated gastric epithelium is able to appropriately proliferate and differentiate. The generation of murine tissue-engineered stomach is a necessary advance as it provides the transgenic tools required to investigate the molecular and cellular mechanisms of this regenerative process. Delineating the mechanism of how tissue-engineered stomach develops in vivo is an important precursor to its use as a human stomach replacement therapy.

A multicellular approach forms a significant amount of tissue-engineered small intestine in the mouse.

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.

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.

Tissue-engineered small intestine and stomach form from autologous tissue in a preclinical large animal model.

BACKGROUND: Tissue-engineered small intestine, stomach, large intestine, esophagus, and gastroesophageal (GE) junction have been successfully formed from syngeneic cells, and employed as a rescue therapy in a small animal model. The purpose of this study is to determine if engineered intestine and stomach could be generated in an autologous, preclinical large animal model, and to identify if the tissue-engineered intestine retained features of an intact stem cell niche. METHODS: A short segment of jejunum or stomach was resected from 6-wk-old Yorkshire swine. Organoid units, multicellular clusters with predominantly epithelial content, were generated and loaded onto biodegradable scaffold tubes. The constructs were then implanted intraperitoneally in the autologous host. Seven wk later, all implants were harvested and analyzed using histology and immunohistochemistry techniques. RESULTS: Autologous engineered small intestine and stomach formed. Tissue-engineered intestinal architecture replicated that of native intestine. Histology revealed tissue-engineered small intestinal mucosa composed of a columnar epithelium with all differentiated intestinal cell types adjacent to an innervated muscularis mucosae. Intestinal subepithelial myofibroblasts, specialized cells that participate in the stem cell niche formation, were identified. Moreover, cells positive for the putative intestinal stem cell marker, doublecortin and CaM kinase-like-1 (DCAMKL-1) expression were identified at the base of the crypts. Finally, tissue-engineered stomach also formed with antral-type mucosa (mucus cells and surface foveolar cells) and a muscularis. CONCLUSION: We successfully generated tissue-engineered intestine with correct architecture, including features of an intact stem cell niche, in the pig model. To our knowledge, this is the first demonstration in which tissue-engineered intestine was successfully generated in an autologous manner in an animal model, which may better emulate a human host and the intended therapeutic pathway for humans.

Wnt5a knock-out mouse as a new model of anorectal malformation.

BACKGROUND: Anorectal malformations (ARM) represent a variety of congenital disorders that involve abnormal termination of the anorectum. Mutations in Shh signaling and Fgf10 produce a variety of ARM phenotypes. Wnt signaling has been shown to be crucial during gastrointestinal development. We therefore hypothesized that Wnt5a may play a role in anorectal development. METHODS: Wild type (WT), Wnt5a(+/-) and Wnt5a(-/-) embryos were harvested from timed pregnant mice from E15.5 to E18.5, and analyzed for anorectal phenotype. Tissues were processed for whole-mount in situ hybridization and histology. RESULTS: Wnt5a is expressed in the embryonic WT colon and rectum. Wnt5a(-/-) mutants exhibit multiple deformities including anorectal malformation. A fistula between the urinary and intestinal tracts can be identified as early as E15.5. By E18.5, the majority of the Wnt5a(-/-) mutants display a blind-ending pouch of the distal gut. CONCLUSIONS: The expression pattern of Wnt5a and the ARM phenotype seen in Wnt5a(-/-) mutants demonstrate the critical role of Wnt5a during anorectal development. This study establishes a new model of ARM involving the Wnt5a pathway.

Induction of fibroblast growth factor 10 (FGF10) in the ileal crypt epithelium after massive small bowel resection suggests a role for FGF10 in gut adaptation.

We have previously reported that fibroblast growth factor 10 (FGF10) is crucial for the survival and proliferation of progenitor cells during embryonic gastrointestinal development. We sought to characterize the potential role of FGF10 signaling in the adaptive response following small bowel resection. Adult wild-type and Fgf10(LacZ) mice underwent 50% small bowel resection (SBR) or sham operation. Tissues were harvested 24 or 48 hr after surgery for histology, immunohistochemistry, and in situ hybridization. After SBR, Fgf10 expression was demonstrated in the epithelium at the base of the crypts. Moreover, there was a statistically significant increase in proliferating cells and goblet cells after SBR. In vitro studies using rat intestinal epithelial crypt (IEC-6) cells exposed to medium with or without recombinant FGF10 showed increased proliferation and phosphorylation of Raf and AKT with the addition of FGF10. Our results suggest that FGF10 may play a therapeutic role in diseases involving intestinal failure.

Tissue-engineered spleen protects against overwhelming pneumococcal sepsis in a rodent model.

BACKGROUND/PURPOSE: Solid organs production is an ultimate goal of tissue engineering. After refining a technique for intestinal engineering, we applied it to a solid organ, the spleen. Overwhelming postsplenectomy sepsis results in death in nearly half of all cases. This risk is pronounced in children. Necrosis of autotransplanted spleen slices occurs prior to regeneration. We postulate that tissue engineering techniques might be superior. METHODS: Four groups of Lewis rats were compared: sham laparotomy, tissue-engineered spleen (TES), traditional spleen slices, and splenectomy. TES was generated from splenic units, multicellular components of juvenile spleen implanted on a biodegradable polymer scaffold, and spleen slices were derived from age-matched juveniles. Pneumococcal sepsis was induced at wk 16, and survival curves were constructed. RESULTS: Tissue-engineered spleen protected against pneumococcal septicemia with a survival proportion of 85.7% compared with 41.17% of splenectomized animals. Spleen slice was also protective with 71.43% survival. Compared with splenectomy, control and TES groups were statistically significant (P = 0.0002, P = 0.0087; hazard ratio of splenectomy = 5.493) and the Slice group was nearly statistically significant (P = 0.0642, hazard ratio of splenectomy = 2.673). CONCLUSIONS: TES is a novel application of tissue engineering to splenic regeneration and creates a functional spleen. This approach could be advantageous in severe pediatric trauma.

Tracheal occlusion increases the rate of epithelial branching of embryonic mouse lung via the FGF10-FGFR2b-Sprouty2 pathway.

Tracheal occlusion during lung development accelerates growth in response to increased intraluminal pressure. In order to investigate the role of internal pressure on murine early lung development, we cauterized the tip of the trachea, to occlude it, and thus to increase internal pressure. This method allowed us to evaluate the effect of tracheal occlusion on the first few branch generations and on gene expression. We observed that the elevation of internal pressure induced more than a doubling in branching, associated with increased proliferation, while branch elongation speed increased 3-fold. Analysis by RT-PCR showed that Fgf10, Vegf, Sprouty2 and Shh mRNA expressions were affected by the change of intraluminal pressure after 48h of culture, suggesting mechanotransduction via internal pressure of these key developmental genes. Tracheal occlusion did not increase the number of branches of Fgfr2b-/- mice lungs nor of wild type lungs cultured with Fgfr2b antisense RNA. Tracheal occlusion of Fgf10(LacZ/-) hypomorphic lungs led to the formation of fewer branches than in wild type. We conclude that internal pressure regulates the FGF10-FGFR2b-Sprouty2 pathway and thus the speed of the branching process. Therefore pressure levels, fixed both by epithelial secretion and boundary conditions, can control or modulate the branching process via FGF10-FGFR2b-Sprouty2.