conditional-reprogramming-pediatric-human-esophageal-epithelial-cells

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

Connecticut Children's Medical Center

A 3-yr-old boy with posterior urethral valves underwent cadaveric renal transplant. On the ninth day after transplantation the patient developed a urinary leak, with complete ureteral necrosis. There was insufficient length of undamaged ureter to permit ureteroneocystostomy, unavailability of a native ureter to permit ureteroureterostomy, and an inability to mobilize the transplant kidney or bladder sufficiently to permit direct pyelovesicostomy. As the kidney was otherwise functioning perfectly, we decided to create an appendiceal conduit in the hope of salvaging the patient's renal allograft. At present, 7 months post-transplant, the child is clinically well with a serum creatinine of 0.7 mg/dL. Complete ureteral necrosis is an infrequent but devastating complication following renal transplantation. We report a novel method that allowed an otherwise normally functioning cadaveric graft to be salvaged.

Use of appendix for complete transplant ureteral necrosis.

In this paper, we report on engineering 3-D pulmonary tissue constructs in vitro. Primary isolates of murine embryonic day 18 fetal pulmonary cells (FPC) were comprised of a mixed population of epithelial, mesenchymal, and endothelial cells as assessed by immunohistochemistry and RT-PCR of 2-D cultures. The alveolar type II (AE2) cell phenotype in 2-D and 3-D cultures was confirmed by detection of SpC gene expression and presence of the gene product prosurfactant protein C. Three-dimensional constructs of FPC were generated utilizing Matrigel hydrogel and synthetic polymer scaffolds of poly-lactic-co-glycolic acid (PLGA) and poly-L-lactic-acid (PLLA) fabricated into porous foams and nanofibrous matrices, respectively. Three-dimensional Matrigel constructs contained alveolar forming units (AFU) comprised of cells displaying AE2 cellular ultrastructure while expressing the SpC gene and gene product. The addition of tissue-specific growth factors induced formation of branching, sacculated epithelial structures reminiscent of the distal lung architecture. Importantly, 3-D culture was necessary for inducing expression of the morphogenesis-associated distal epithelial gene fibroblast growth factor receptor 2 (FGFr2). PLGA foams and PLLA nanofiber scaffolds facilitated ingrowth of FPC, as evidenced by histology. However, these matrices did not support the survival of distal lung epithelial cells, despite the presence of tissue-specific growth factors. Our results may provide the first step on the long road toward engineering distal pulmonary tissue for augmenting and/or replacing dysfunctional native lung in diseases, such as neonatal pulmonary hypoplasia.

Engineering three-dimensional pulmonary tissue constructs.

Pulmonary hypoplasia (PH) is found in 15% to 20% of all neonatal autopsies, accounting for 2850 deaths yearly. Development of engineered tissue substitutes that could functionally restore damaged tissue remains a unique opportunity for biotechnology. Recently, we isolated and characterized murine fetal pulmonary cells (FPC) and engineered 3-D pulmonary tissue constructs in vitro. Our goal is to devise a reliable and reproducible method for delivering FPC into a live animal model of PH.

Novel methods for delivery of cell-based therapies.

Intrapulmonary engraftment of engineered lung tissues could provide a potential therapeutic approach for the treatment of pediatric and adult pulmonary diseases. In working toward this goal, we report here on in vivo generation of vascularized pulmonary tissue constructs utilizing the subcutaneous Matrigel plug model. Mixed populations of murine fetal pulmonary cells (FPCs) containing epithelial, mesenchymal, and endothelial cells (ECs) were isolated from the lungs of embryonic day 17.5 fetuses. FPCs were admixed to Matrigel and injected subcutaneously into the anterior abdominal wall of adult C57/BL6 mice to facilitate in vivo pulmonary tissue construct formation. Vascularization was enhanced by placing fibroblast growth factor 2 (FGF2)-loaded polyvinyl sponges into the hydrogel. After 1 week, routine histology and immunohistochemical staining for donor-derived epithelial cells and ECs as well as analysis of patent vasculature in the constructs following tail vein injection of fluorescein isothiocyanate-conjugated dextran were performed. In the Matrigel-only controls, some level of host infiltrate, but no measurable vascularization, was detected. In the presence of FPCs, the constructs contained ductal epithelial structures and patent vasculature. In the absence of FPCs, exogenous FGF2 induced the formation of numerous patent blood vessels throughout the entire constructs; in combination with FPCs, it resulted in enhanced capillary density and abundant interfacing between developing epithelial and vascular structures. The significant findings of this study are that distal pulmonary epithelial differentiation (as assessed by the expression of prosurfactant protein C) can be maintained in vivo and that donor-derived ECs contribute to the formation of patent vessels that interface tightly with ductal epithelial structures.

In vivo pulmonary tissue engineering: contribution of donor-derived endothelial cells to construct vascularization.

In the present study, mouse embryonic stem cells (ESCs) were differentiated into alveolar epithelial type II (AEII) cells for endotracheal injection. These enriched lung-like populations expressed lung epithelial markers SP-A, SP-B, SP-C, and CC10. First we show that rapid differentiation of ESCs requires a dissociated seeding method instead of an embryoid body culture method. We then investigated a two-step differentiation of ESCs into definitive endoderm by activin or A549-conditioned medium as a precursor to lung epithelial cells. When conditioned medium from A549 cells was used to derive endoderm, yield was increased above that of activin alone. Further studies showed that Wnt3a may be one of the secreted factors produced by A549 cells and promotes definitive endoderm differentiation, in part, through suppression of primitive endoderm. Activin and Wnt3a together at appropriate doses with dissociated cell seeding promoted greater endoderm yield than activin alone. Next, fibroblast growth factor 2 was shown to induce a dose-dependent expression of SPC, and these cells contained lamellar bodies characteristic of mature AEII cells from ESC-derived endoderm. Finally, ES-derived lung cells were endotracheally injected into preterm mice with evidence of AEII distribution within the lung parenchyma. This study concludes that a recapitulation of development may enhance derivation of an enriched population of lung-like cells for use in cell-based therapy.

Efficient derivation of alveolar type II cells from embryonic stem cells for in vivo application.

Cell-based therapy is a promising treatment option for lung disease, but no studies have demonstrated its benefit in promoting perinatal lung growth. Embryonic day 18 (E18) fetal lungs treated with vascular inhibitors were grown as explant organ cultures to inhibit endothelial growth in the explant cultures. Disruption of pulmonary vasculature decreased explant mean cord length and viability, whereas coculture with fetal pulmonary or predifferentiated embryonic stem cells rescued both parameters. These results demonstrate in a model of perinatal lung growth, exogenous addition of fetal pulmonary cells or differentiated embryonic stem (ES) cells promotes survival and alveolar morphogenesis. These experiments represent the first evidence of the benefits of cell-based therapy for perinatal lung growth.

Cell-based therapy improves cell viability and increases airway size in an explant model.

Congenital hernias arising in the subcostal region are rare. We describe a case of a former preterm infant, born with congenitally fused right 11th and 12th ribs and a protuberant mass in the right subcostal region. This mass was associated with a small fascial defect and herniation of abdominal contents. At operation, the mass was determined to be a hernia with an incarcerated ovarian remnant and fallopian tube.

Congenital subcostal hernia with unusual contents.

Use of a defined massive transfusion (MT) protocol for severe intraoperative bleeding in a pediatric patient has never been described. Herein we present a case whereby use of hemostatic resuscitation delineated in an MT protocol optimally treated hemorrhage resulting from a large tumor during right hepatectomy. The MT protocol principles, benefits, and postoperative course of the patient are described.

Use of a massive transfusion protocol with hemostatic resuscitation for severe intraoperative bleeding in a child.

Primary renal synovial sarcoma is a rare entity with fewer than 40 cases reported in the literature. Its clinical presentation and radiographic features, namely, its often complex cystic appearance, make it difficult to differentiate from other benign or malignant renal lesions. Although there are certain consistent morphological and immunohistochemical features, diagnosis ultimately depends on molecular studies. Prognosis is poor, and there currently exists no defined treatment protocol. Herein, we describe the youngest reported case of primary renal synovial sarcoma in the literature.

Primary renal synovial sarcoma in a 13-year-old boy.

The management of lymphangiomas in children is a complex problem with frequent recurrence and infection. Vacuum-assisted closure (VAC) devices have been shown to accelerate the healing of open wounds. We hypothesized that VAC therapy might decrease complications after resection of lymphangiomas.

Vacuum-assisted closure in the treatment of extensive lymphangiomas in children.

Enterourachal fistulas are exceedingly rare in Crohn's patients. We report a case of a presumed enterourachal fistula that led to an infected urachal cyst. Preoperative medical treatment obliterated the fistula and avoided the need to resect bowel at the time of operation. We recommend consideration of this diagnosis in a Crohn's patient with a midline abdominal mass.

Infected urachal cyst secondary to a Crohn's enterourachal fistula.

For patients with end-stage lung diseases, lung transplantation is the only available therapeutic option. However, the number of suitable donor lungs is insufficient and lung transplants are complicated by significant graft failure and complications of immunosuppressive regimens. An alternative to classic organ replacement is desperately needed. Engineering of bioartificial organs using either natural or synthetic scaffolds is an exciting new potential option for generation of functional pulmonary tissue for human clinical application. Natural organ scaffolds can be generated by decellularization of native tissues; these acellular scaffolds retain the native organ ultrastructure and can be seeded with autologous cells towards the goal of regenerating functional tissues. Several decellularization strategies have been employed for lungs; however, there is no consensus on the optimal approach. A variety of cell types have been investigated as potential candidates for effective recellularization of acellular lung scaffolds. Candidate cells that might be best utilized are those which can be easily and reproducibly isolated, expanded in vitro, seeded onto decellularized matrices, induced to differentiate into pulmonary lineage cells, and which survive to functional maturity. Whole lung cell suspensions, endogenous progenitor cells, embryonic and adult stem cells and induced pluripotent stem (iPS) cells have been investigated for their applicability to repopulate acellular lung matrices. Ideally, patient-derived autologous cells would be used for lung recellularization as they have the potential to reduce the need for post-transplant immunosuppression. Several studies have performed transplantation of rudimentary bioengineered lung scaffolds in animal models with limited, short-term functionality but much further study is needed.

Can stem cells be used to generate new lungs? Ex vivo lung bioengineering with decellularized whole lung scaffolds.

Ovarian steroid cell tumors, not otherwise specified (OSCTs), are extremely rare and present a diagnostic challenge when evaluating an ovarian mass. We present a case of such a tumor in a patient with known Congenital Adrenal Hyperplasia (CAH), secondary to 21-hydroxylase deficiency, who was noncompliant with her medications. The workup, diagnosis, and treatment of this rare condition are described.

Ovarian steroid cell tumor, not otherwise specified, associated with congenital adrenal hyperplasia: rare tumors of an endocrine disease.

The optimal method for creating a de-cellularized lung scaffold that is devoid of cells and cell debris, immunologically inert, and retains necessary extracellular matrix (ECM) has yet to be identified. Herein, we compare automated detergent-based de-cellularization approaches utilizing either constant pressure (CP) or constant flow (CF), to previously published protocols utilizing manual pressure (MP) to instill and rinse out the de-cellularization agents. De-cellularized lungs resulting from each method were evaluated for presence of remaining ECM proteins and immunostimulatory material such as nucleic acids and intracellular material. Our results demonstrate that the CP and MP approaches more effectively remove cellular materials but differentially retain ECM proteins. The CP method has the added benefit of being a faster, reproducible de-cellularization process. To assess the functional ability of the de-cellularized scaffolds to maintain epithelial cells, intra-tracheal inoculation with GFP expressing C10 alveolar epithelial cells (AEC) was performed. Notably, the CP de-cellularized lungs were able to support growth and spontaneous differentiation of C10-GFP cells from a type II-like phenotype to a type I-like phenotype.

Automated procedure for biomimetic de-cellularized lung scaffold supporting alveolar epithelial transdifferentiation.

Induction of morphogenesis by competent lung progenitor cells in a 3D environment is a central goal of pulmonary tissue engineering, yet little is known about the microenvironmental signals required to induce de novo assembly of alveolar-like tissue in vitro. In extending our previous reports of alveolar-like tissue formation by fetal pulmonary cells stimulated by exogenous fibroblast growth factors (FGFs), we identified some of the key endogenous mediators of FGF-driven morphogenesis (organoid assembly), for example, epithelial sacculation, endothelial network assembly, and epithelial-endothelial interfacing. Sequestration of endogenously secreted vascular endothelial growth factor-A (VEGF-A) potently inhibited endothelial network formation, with little or no effect on epithelial morphogenesis. Inhibition of endogenous sonic hedgehog (SHH) partially attenuated FGF-driven endothelial network formation, while the addition of exogenous SHH in the absence of FGFs was able to induce epithelial and endothelial morphogenesis, although with distinct morphological characteristics. Notably, SHH-induced endothelial networks exhibited fewer branch points, reduced sprouting behavior, and a periendothelial extracellular matrix (ECM) virtually devoid of tenascin-C (TN-C). By contrast, focal deposition of endogenous TN-C was observed in the ECM-surrounding endothelial networks of FGF-induced organoids, especially around sprouting tips. In the FGF-induced organoids, TN-C was also observed in the clefts of sacculated epithelium and at the epithelial-endothelial interface. In support of a critical role in the formation of alveolar-like tissue in vitro, TN-C blocking inhibited endothelial network formation and epithelial sacculation. Upon engraftment of in-vitro-generated pulmonary organoids beneath the renal capsule of syngeneic mice, robust neovascularization occurred in 5 days with a large contribution of patent vessels from engrafted organoids, providing proof of principle for exploring intrapulmonary engraftment of prevascularized hydrogel constructs. Expression of proSpC, VEGF-A, and TN-C following 1 week in vivo mirrored the patterns observed in vitro. Taken together, these findings advance our understanding of endogenous growth factor and ECM signals important for de novo formation of pulmonary tissue structures in vitro and demonstrate the potential of an organoid-based approach to lung tissue augmentation.

Article	Year
Use of appendix for complete transplant ureteral necrosis. Pediatric transplantation\| PubMed ID: 12756052	2003
Engineering three-dimensional pulmonary tissue constructs. Tissue engineering\| PubMed ID: 16674286	2006
Novel methods for delivery of cell-based therapies. The Journal of surgical research\| PubMed ID: 17686493	2008
In vivo pulmonary tissue engineering: contribution of donor-derived endothelial cells to construct vascularization. Tissue engineering. Part A\| PubMed ID: 18333788	2008
Efficient derivation of alveolar type II cells from embryonic stem cells for in vivo application. Tissue engineering. Part A\| PubMed ID: 19388834	2009
Cell-based therapy improves cell viability and increases airway size in an explant model. Experimental lung research\| PubMed ID: 19842834	2009
Congenital subcostal hernia with unusual contents. Journal of pediatric surgery\| PubMed ID: 20152370	2010
Use of a massive transfusion protocol with hemostatic resuscitation for severe intraoperative bleeding in a child. Journal of pediatric surgery\| PubMed ID: 20638538	2010
Primary renal synovial sarcoma in a 13-year-old boy. Journal of pediatric surgery\| PubMed ID: 21930002	2011
Vacuum-assisted closure in the treatment of extensive lymphangiomas in children. Journal of pediatric surgery\| PubMed ID: 22325392	2012
Infected urachal cyst secondary to a Crohn's enterourachal fistula. Journal of pediatric surgery\| PubMed ID: 23217916	2012
Can stem cells be used to generate new lungs? Ex vivo lung bioengineering with decellularized whole lung scaffolds. Respirology (Carlton, Vic.)\| PubMed ID: 23614471	2013
Ovarian steroid cell tumor, not otherwise specified, associated with congenital adrenal hyperplasia: rare tumors of an endocrine disease. Journal of pediatric surgery\| PubMed ID: 23845653	2013
Automated procedure for biomimetic de-cellularized lung scaffold supporting alveolar epithelial transdifferentiation. Biomaterials\| PubMed ID: 24095252	2013
Engineering de novo assembly of fetal pulmonary organoids. Tissue engineering. Part A\| PubMed ID: 24825442	2014

Christine M. Finck

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