Jennifer J. Freeman

Transcriptome-wide Analysis Reveals Hallmarks of Human Intestine Development and Maturation In Vitro and In Vivo.

Summary Human intestinal organoids (HIOs) are a tissue culture model in which small intestine-like tissue is generated from pluripotent stem cells. By carrying out unsupervised hierarchical clustering of RNA-sequencing data, we demonstrate that HIOs most closely resemble human fetal intestine. We observed that genes involved in digestive tract development are enriched in both fetal intestine and HIOs compared to adult tissue, whereas genes related to digestive function and Paneth cell host defense are expressed at higher levels in adult intestine. Our study also revealed that the intestinal stem cell marker OLFM4 is expressed at very low levels in fetal intestine and in HIOs, but is robust in adult crypts. We validated our findings using in vivo transplantation to show that HIOs become more adult-like after transplantation. Our study emphasizes important maturation events that occur in the intestine during human development and demonstrates that HIOs can be used to model fetal-to-adult maturation.

Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids.

ABSTRACT Short bowel syndrome (SBS) is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving, such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes, new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), called human intestinal organoids (HIOs), have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However, given that HIOs are small three-dimensional structures grown in vitro, methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA) scaffolds, and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro, the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast, HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine, which need to be explored further to develop them into fully functional tissue. Summary: HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. These scaffolds appear to be suitable for further tissue engineering approaches to develop functional intestine.

TPN-associated intestinal epithelial cell atrophy is modulated by TLR4/EGF signaling pathways

Recent studies suggest a close interaction between epidermal growth factor (EGF) and TLR signaling in the modulation of intestinal epithelial cell (IEC) proliferation; however, how these signaling pathways adjust IEC proliferation is poorly understood. We utilized a model of total parenteral nutrition (TPN), or enteral nutrient deprivation, to study this interaction as TPN results in mucosal atrophy due to decreased IEC proliferation and increased apoptosis. We identified the novel finding of decreased mucosal atrophy in TLR4 knockout (TLR4KO) mice receiving TPN. We hypothesized that EGF signaling is preserved in TLR4KO‐TPN mice and prevents mucosal atrophy. C57B1/6 and strain‐matched TLR4KO mice were provided either enteral feeding or TPN. IEC proliferation and apoptosis were measured. Cytokine and growth factor abundances were detected in both groups. To examine interdependence of these pathways, ErbB1 pharmacologic blockade was used. The marked decline in IEC proliferation with TPN was nearly prevented in TLR4KO mice, and intestinal length was partially preserved. EGF was significantly increased, and TNF‐α decreased in TLR4KO‐TPN versus wild‐type (WT)‐TPN mice. Apoptotic positive crypt cells were 15‐fold higher in WT‐TPN versus TLR4KO‐TPN mice. Bcl‐2 was significantly increased in TLR4KOTPN mice, while Bax decreased 10‐fold. ErbB1 blockade prevented this otherwise protective effect in TLR4KO‐sTPN mice. TLR4 blockade significantly prevented TPN‐associated atrophy by preserving proliferation and preventing apoptosis. This is driven by a reduction in TNF‐α abundance and increased EGF. Potential manipulation of this regulatory pathway may have significant clinical potential to prevent TPN‐associated atrophy.—Freeman, J. J., Feng, Y., Demehri, F. R., Dempsey, P. J., Teitelbaum, D. H. TPN‐associated intestinal epithelial cell atrophy is modulated by TLR4/EGF signaling pathways. FASEB J. 29, 2943‐2958 (2015). www.fasebj.org

Development of an endoluminal intestinal attachment for a clinically applicable distraction enterogenesis device.

PURPOSE Previous methods of distraction enterogenesis have relied upon blind-ending intestinal segments or transmural device fixation, requiring multiple operations and potential bowel injury. We hypothesized that using a novel attachment would allow reversible device coupling to the luminal bowel surface, achieving effective endoluminal distraction. METHODS A telescopic hydraulic device was designed with latex balloon attachments covered with high-friction mesh and a dilating fenestrated elastic mask (DFM attachment), allowing mesh-to-mucosa contact only with inflation. Yorkshire pigs underwent jejunal Roux-en-Y limb creation and device placement via jejunostomy. Devices underwent 3 cycles of balloon inflation and hydraulic extension/retraction per day for 7 days and then explanted and studied for efficacy. RESULTS DFM attachment allowed reversible, high-strength endoluminal coupling without tissue injury or reduction in bowel perfusion. After 7 day implant, distracted bowel achieved a 44 ± 2% increase in length vs. fed, nondistracted bowel, corresponding to a gain of 7.1 ± 0.3 cm. Distracted bowel demonstrated increased epithelial cell proliferation vs. control bowel. Attachment sites demonstrated villus flattening, increased crypt depth, thicker muscularis mucosa, and unchanged muscularis propria thickness vs. CONCLUSION Novel high-strength, reversible attachments enabled fully endoluminal distraction enterogenesis, achieving length gains comparable to open surgical techniques. This approach may allow development of clinically applicable technology for SBS treatment.

Redo pullthrough for Hirschsprung disease: A single surgical group's experience

INTRODUCTION This study presents our surgical experience for redo-pullthrough (RedoPT) for Hirschsprung disease (HD). It reviews the patients clinical outcomes and assesses stooling patterns after RedoPT. METHODS A retrospective review of our institutions RedoPTs as well as one authors overseas cases was performed. Stooling scores were tabulated using an established survey tool and compared to primary PT matched patients. RESULTS Between 1974 and 2012, 46 individuals (52% males) underwent RedoPT, representing 3 percent of all HD pullthroughs. Median age at primary PT and RedoPT was 1year (range 1week-18years) and 3.5years (range 8weeks-41years), respectively. Indications for RedoPT were predominately for aganglionosis/transition zone pathology (71%); followed by stricture or an obstructing Duhamel pouch (19%), tight cuff (8%) and a twisted PT (4%). None were performed for an isolated clinical diagnosis of repeated bouts of enterocolitis. RedoPT surgical approach depended upon the initial pullthrough technique and any previous complications. Stooling scores were significantly (P<0.05) worse in the RedoPT patients compared to the historically-matched group of children undergoing a primary PT for HD (5.5±1.2 vs. 12.2±1.4, primary PT versus RedoPT, respectively). When breaking down this total score into individual parameters, stooling pattern scores (1.0±0.2 vs. 4.1±0.4, P=0.001) and enterocolitis scores (2.0±0.4 vs. 4.2±0.4, P=0.001) were statistically worse in the RedoPT group. Patients in both groups had similar overall continence rates. CONCLUSION Appropriately selected children undergoing a RedoPT can achieve good results, with comparable continence rates to those undergoing a primary PT.

Improving central line infection rates in the neonatal intensive care unit: Effect of hospital location, site of insertion, and implementation of catheter-associated bloodstream infection protocols

INTRODUCTION Catheter associated blood stream infections (CABSIs) are morbid and expensive for all ages, including neonates. Thus far, the impact of CABSI prevention protocols, such as insertion and maintenance bundles, in the neonatal intensive care unit (NICU) is largely unknown. We hypothesized that lines placed in the operating room (OR) would have a lower infection rate due to established insertion protocols and a more sterile environment. METHODS A retrospective chart review of NICU patients who received a percutaneous or tunneled central venous catheter between 2005 and 2012 was performed. ECMO cannulas, PICC and umbilical catheters were excluded. Variables of interest included demographics, anatomical site, hospital location, line days, and line infection. Line infection was defined as a positive blood culture drawn through the catheter. RESULTS A total of 368 catheters were placed in 285 NICU patients. Majority of catheters (65.5%) were placed in OR. Saphenous and femoral veins were most common anatomical sites (50.8%). Twenty-eight catheters were infected (7.6%). After adjusting for preoperative antibiotics, anatomical site, and SNAPPE-II scores, lines placed in OR were three times less likely to become infected (Odds Ratio=0.32, p=0.038). Although implementation of CABSI prevention protocols resulted in statistically significant reductions in infection (Odds Ratio=0.4, p=0.043), lines placed in the OR remained less likely to become infected. CONCLUSIONS NICU line infection rates decreased with implementation of CABSI prevention protocols. Despite this implementation, catheters placed in the NICU continued to have higher infection rates. As a result, when patient status allows it, we recommend that central lines in newborns be placed in the operating room.