Erik R. Barthel

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.

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.

Mechanisms of the ultrafast production and recombination of solvated electrons in weakly polar fluids: Comparison of multiphoton ionization and detachment via the charge-transfer-to-solvent transition of Na− in THF

The processes by which solvated electrons are generated and undergo recombination are of great interest in condensed phase physical chemistry because of their relevance to both electron transfer reactions and radiation chemistry. Although most of the work in this area has focused on aqueous systems, many outstanding questions remain, especially concerning the nature of these processes in low polarity solvents where the solvated electron has a fundamentally different structure. In this paper, we use femtosecond spectroscopic techniques to explore the dynamics of solvated electrons in tetrahydrofuran (THF) that are produced in two different ways: ejection by multiphoton ionization of the neat solvent, and detachment via the charge-transfer-to-solvent (CTTS) transition of sodide (Na−). Following multiphoton ionization of the solvent, the recombination of solvated electrons can be well described by a simple model that assumes electrons are first ejected to a given thermalization distance and then move diffusi...

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.

Direct observation of charge-transfer-to-solvent (CTTS) reactions: Ultrafast dynamics of the photoexcited alkali metal anion sodide (Na−)

Charge-transfer-to-solvent (CTTS) transitions have been the subject of a great deal of interest recently because they represent the simplest possible charge transfer reaction: The CTTS electron transfer from an atomic ion to a cavity in the surrounding solvent involves only electronic degrees of freedom. Most of the work in this area, both experimental and theoretical, has focused on aqueous halides. Experimentally, however, halides make a challenging choice for studying the CTTS phenomenon because the relevant spectroscopic transitions are deep in the UV and because the charge-transfer dynamics can be monitored only indirectly through the appearance of the solvated electron. In this paper, we show that these difficulties can be overcome by taking advantage of the CTTS transitions in solutions of alkali metal anions, in particular, the near-IR CTTS band of sodide (Na−) in tetrahydrofuran (THF). Using femtosecond pump–probe techniques, we have been able to spectroscopically separate and identify transient ...

Solvent effects on the ultrafast dynamics and spectroscopy of the charge-transfer-to-solvent reaction of sodide

In “outer sphere” electron transfer reactions, motions of the solvent molecules surrounding the donor and acceptor govern the dynamics of charge flow. Are the relevant solvent motions determined simply by bulk solvent properties such as dielectric constant or viscosity? Or are molecular details, such as the local solvent structure around the donor and acceptor, necessary to understand how solvent motions control charge transfer? In this paper, we address these questions by using ultrafast spectroscopy to study a photoinduced electron transfer reaction with only electronic degrees of freedom: the charge-transfer-to-solvent (CTTS) reaction of Na− (sodide). Photoexcitation of Na− places the excited CTTS electron into a solvent-bound excited state; motions of the surrounding solvent molecules in response to this excitation ultimately lead to detachment of the electron. The detached electron can then localize either in an “immediate” contact pair (in the same cavity as the Na atom), which undergoes back electr...

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.

Mapping out the conduction band under CTTS transitions: the photodetachment quantum yield of sodide (Na-) in tetrahydrofuran

Upon photoexcitation of the charge-transfer-to-solvent absorption band of Nain tetrahydrofuran (THF), electrons detach into immediate contact pairs, solvent-separated contact pairs, or as free electrons.In this Letter, we analyze the recombination dynamics of Naat multiple excitation wavelengths to determine the action spectrum for production of each type of electron.The action spectra match well with the four Gaussian sub-bands (corresponding to three p-like CTTS states and the continuum) needed to describe the polarized bleach recovery.We also find that the Nafree- electron action spectrum is linearly related to the Rb � /THF photocurrent action spectrum measured by Levanon and co-workers.

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.