Allison L. Speer

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.

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.

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 β2-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.

Solid pseudopapillary tumor of the pancreas: a single-institution 20-year series of pediatric patients

PURPOSE Solid pseudopapillary tumor (SPT) of the pancreas is a rare neoplasm. The objective of this study was to review our institutions 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.

Contemporary management of lipoblastoma

PURPOSE Lipoblastoma is a rare, benign, adipose tissue tumor. We report the largest single institution experience managing these uncommon neoplasms. METHODS We retrospectively reviewed 32 cases of lipoblastoma entered in the pathology database at our institution between January 1991 and August 2005. We conducted a comprehensive literature review of lipoblastoma and summarized the results of the largest series published. RESULTS Most patients presented with an enlarging, palpable, firm, nontender, mobile mass. The male-to-female ratio was 1.9:1. The anatomical distribution was trunk (n = 12), extremity (n = 12), groin (n = 5), and neck (n = 3). Average age at resection was 2.8 years (range, 2.6 months to 12 years). Thirty-one cases were completely excised, although 1 patient underwent staged partial excision to preserve nerve function. Chromosomal analysis performed in selected patients revealed characteristic aberrations in chromosome 8. Complications included keloid formation (n = 3), wound infection/dehiscence (n = 2), wound seroma (n = 1), and transient brachial plexus neurapraxia (n = 1). Average follow-up was 7.4 months (range, 1 day to 6.5 years); 2 patients were lost to follow-up. There were no recurrences. CONCLUSIONS A staged approach with meticulous sparing of the neurovascular bundle provides excellent functional outcome for patients with large tumors. Nonmutilating surgical excision is the treatment of choice.

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.

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.

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 Rosa26rtTA/+, tet(o)Fgf10/– or Fgf10Mlc‐nlacZ‐v24 (hereafter called Fgf10lacZ ) 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 Fgf10LacZ duodenum. However, this was not the case with TESI generated from jejunum and ileum. We conclude that Fgf10 enhances the formation of TESI. Copyright

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.