Ziyad Jabaji

Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium.

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth.

Intestinal Subepithelial Myofibroblasts Support the Growth of Intestinal Epithelial Stem Cells

Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.

Type I Collagen as an Extracellular Matrix for the In Vitro Growth of Human Small Intestinal Epithelium

Background We previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells. Methods Human small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry. Results Collagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages. Conclusion Type I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet-like growth, and they eliminate the need for Matrigel for in vitro human intestinal epithelial growth. Future research is required to further develop this cell culture system for tissue engineering applications.

Regeneration of enteric ganglia in mechanically lengthened jejunum after restoration into intestinal continuity

PURPOSE We previously demonstrated that it is feasible to lengthen intestinal segments with mechanical force and to restore them back into intestinal continuity. The changes in the enteric ganglia in the lengthened intestinal segments have not been described. METHODS A 1-cm segment of rodent jejunum was isolated from intestinal continuity and was lengthened using a spring. After lengthening, jejunal segments were either retrieved (n=4) or restored into intestinal continuity (n=4). Rats with restored segments were euthanized 2 to 3 weeks later. Ganglia were identified and quantified by immunostaining of histological sections. RESULTS The normal jejunum had 51 ± 5 myenteric and 31 ± 2 submucosal ganglia per circumferential tissue section. Lengthened segments had 21 ± 7 myenteric and 2 ± 2 submucosal ganglia. Restored segments had 46 ± 14 myenteric and 10 ± 10 submucosal ganglia. Circumferential density of ganglia followed a similar pattern. CONCLUSION Mechanical lengthening led to a paucity of submucosal and myenteric ganglia. After restoration into continuity, the number increased toward normal, indicating regeneration of the enteric ganglia. The function of regenerated ganglia needs to be assessed in the future.

Long-term renewable human intestinal epithelial stem cells as monolayers: A potential for clinical use.

PURPOSE Current culture schema for human intestinal stem cells (hISCs) frequently rely on a 3D culture system using Matrigel™, a laminin-rich matrix derived from murine sarcoma that is not suitable for clinical use. We have developed a novel 2D culture system for the in vitro expansion of hISCs as an intestinal epithelial monolayer without the use of Matrigel. METHODS Cadaveric duodenal samples were processed to isolate intestinal crypts from the mucosa. Crypts were cultured on a thin coat of type I collagen or laminin. Intestinal epithelial monolayers were supported with growth factors to promote self-renewal or differentiation of the hISCs. Proliferating monolayers were sub-cultured every 4-5days. RESULTS Intestinal epithelial monolayers were capable of long-term cell renewal. Less differentiated monolayers expressed high levels of gene marker LGR5, while more differentiated monolayers had higher expressions of CDX2, MUC2, LYZ, DEF5, and CHGA. Furthermore, monolayers were capable of passaging into a 3D culture system to generate spheroids and enteroids. CONCLUSION This 2D system is an important step to expand hISCs for further experimental studies and for clinical cell transplantation. LEVEL OF EVIDENCE 1 Experimental.

Changing relationship of pediatric surgical workforce to patient demographics

BACKGROUND Physician workforce studies indicate that more specialists contribute to higher average costs. The closely monitored pediatric surgery specialty may reflect what is occurring in other specialties. METHODS This report reviews the number of complex operations performed on infants and children in 1970, with <225 trained US pediatric surgeons, and in 2010, when there were 1,130. The number of births remained consistent during this 40-year period. RESULTS In 2010, approximately 10,710 complex index operations were performed on children in the United States by certified pediatric surgeons, resulting in 9.5 per surgeon annually. Data from the University of California, Los Angeles, and the Vanderbilt Childrens Hospital confirm these observations. CONCLUSIONS The progressive disparity in the number of pediatric surgeons trained and the number of complex index operations performed annually may increase costs and calls into question the ability of individual pediatric surgeons to maintain optimal competence. Consideration might be given to performing index operations at centers of excellence.

Basic fibroblast growth factor eluting microspheres enhance distraction enterogenesis.

PURPOSE The purpose of this study was to determine if distraction enterogenesis using self-expanding polycaprolactone (PCL) springs is a potential therapy for short bowel syndrome. Sustained release basic fibroblast growth factor (bFGF) microspheres have been shown to induce angiogenesis and intestinal regeneration in tissue engineered scaffolds. We hypothesized that the provision of bFGF-loaded microspheres would increase angiogenesis and thereby enhance the process of enterogenesis. METHODS A 10-mm segment of rodent jejunum was isolated and an encapsulated PCL spring inserted. Blank or bFGF-loaded microspheres were delivered to the segment. After 4weeks, jejunal segments were assessed for lengthening, morphology, quantification of blood vessels, and ganglia. RESULTS Lengthened intestinal segments receiving bFGF microspheres demonstrated significantly increased microvascular density compared to those with blank microspheres. There were also significantly more submucosal and myenteric ganglia in the segments that received bFGF microspheres. Segments achieved similar lengthening and final muscular thickness in both blank and bFGF groups, but the bFGF microsphere caused a significant increase in luminal diameter of the jejunal segment. CONCLUSION Sustained release bFGF microspheres enhanced distraction enterogenesis through improved vascularity. The synergy of growth factors such as bFGF with distraction enterogenesis may yield improved results for the future treatment of patients with short bowel syndrome.

Newborn with Bilious Emesis

A term newborn female presents with bilious vomiting 12 h after an uneventful delivery. A prenatal ultrasound showed polyhydramnios, but the mother was lost to follow up. The infant passed meconium soon after birth. All vital signs are normal, and on physical examination the infant is well appearing. Her abdomen is soft and nontender with epigastric distension. She has a single palmar crease in both hands. An abdominal radiograph showed a “double-bubble.”

Cold, Painful Right Lower Extremity

A 65-year-old female presents to the emergency department with a 4 h history of sudden onset of right leg pain, coolness, weakness, and numbness. She has no prior similar history. The pain began while she was sitting watching television. She has a history of hypertension and hypercholesterolemia and takes medication for both. She denies smoking. On review of systems, she has no prior history of pain in her right leg with walking. On physical examination, her lungs are clear to auscultation. Her heart rate is irregularly irregular without murmurs, rubs, or gallops. Her abdomen is soft and non-tender without a pulsatile mass. On the left side, the femoral, popliteal, dorsalis pedis, and posterior tibial pulses are 2+, with biphasic handheld Doppler signals. On the right side, femoral, popliteal, dorsalis pedis and posterior tibial pulses are absent, with faint monophasic Doppler signals. Her right calf is tender to palpation but is not edematous. She has normal appearing, supple skin in her legs with normal hair pattern. The left foot is pink and warm with 2 s capillary refill and normal motor and sensory function. Her right foot is pale and feels much colder than the left; capillary refill time is 6 s. The toes have decreased sensation to touch. Her strength is diminished in both plantar flexion and dorsiflexion of the ankle at 4+/5.

Infant with Nonbilious Emesis

A 6-week-old full-term male is brought into the emergency room with vomiting. The mother reports that he began regurgitating breast milk one week ago. Although intermittent at first, vomiting now occurs after every feeding and has become progressively more forceful with increased volume. His appetite remains vigorous, even immediately after vomiting. He is the mother’s first child and his delivery was uneventful. His blood pressure is normal and he is afebrile but tachycardic. On physical examination, he is irritable and has a sunken fontanelle. A small, firm mass is palpated in the right upper quadrant.