Decellularized Rat Lung Research Articles

A novel ex vivo lung cancer model based on bioengineered rat lungs.

Introduction: Two-dimensional cell cultures have contributed substantially to lung cancer research, but 3D cultures are gaining attention as a new, more efficient, and effective research model. A model reproducing the 3D characteristics and tumor microenvironment of the lungs in vivo, including the co-existence of healthy alveolar cells with lung cancer cells, is ideal. Here, we describe the creation of a successful ex vivo lung cancer model based on bioengineered lungs formed by decellularization and recellularization. Methods: Human cancer cells were directly implanted into a bioengineered rat lung, which was created with a decellularized rat lung scaffold reseeded with epithelial cells, endothelial cells and adipose-derived stem cells. Four human lung cancer cell lines (A549, PC-9, H1299, and PC-6) were applied to demonstrate forming cancer nodules on recellularized lungs and histopathological assessment were made among these models. MUC-1 expression analysis, RNA-seq analysis and drug response test were performed to demonstrate the superiority of this cancer model. Results: The morphology and MUC-1 expression of the model were like those of lung cancer in vivo. RNA sequencing revealed an elevated expression of genes related to epithelial-mesenchymal transition, hypoxia, and TNF-α signaling via NF-κB; but suppression of cell cycle-related genes including E2F. Drug response assays showed that gefitinib suppressed PC-9 cell proliferation equally well in the 3D lung cancer model as in 2D culture dishes, albeit over a smaller volume of cells, suggesting that fluctuations in gefitinib resistance genes such as JUN may affect drug sensitivity. Conclusions: A novel ex vivo lung cancer model was closely reproduced the 3D structure and microenvironment of the actual lungs, highlighting its possible use as a platform for lung cancer research and pathophysiological studies.

Tissue engineered cancer metastases as cancer vaccine to improve cancer immunotherapy

Radiotherapy is often used to improve cancer immunotherapy outcomes. While there are both pre-clinical and clinical data supporting this approach, there are also significant challenges. One key challenge is that not all patients have tumors that can be easily treated with radiotherapy due to potential normal tissue toxicity and prior treatment. In addition, it is difficult to control the tumor microenvironment to promote the immune response after radiosurgery. To overcome these challenges, we hypothesize that we can engineer cancer metastasis and utilize irradiated engineered tumor cells as a personalized cancer vaccine to improve cancer immunotherapy. Herein, we report the development of engineered lung metastasis using decellularized rat lung tissue. Using the B16F10 melanoma tumor model, we showed that radiotherapy-treated engineered metastases are highly effective in improving cancer immunotherapy responses and more effective than in vivo metastasis. Our work has demonstrated the potential of applying tissue engineering to cancer immunotherapy. Statement of significanceCombination of radiation and immunotherapy are an effective way to treat metastasis. Despite their success, long term response still remains low. Tumor microenvironment evading the immune response, normal tissue toxicity to radiation and inaccessibility to radiosurgery are some of the limitations. To overcome these challenges, in this paper we present with data supporting the use of high dose radiation treated ex vivo engineered B16F10 metastasis model using decellularized lung scaffolds. These engineered metastases closely mimic the in vivo tumors and when given into tumor bearing mice along with check point inhibitors are highly effective in improving the cancer immunotherapy response.

Mechanical modeling of lung alveoli: From macroscopic behaviour to cell mechano-sensing at microscopic level

The mechanical signals sensed by the alveolar cells through the changes in the local matrix stiffness of the extracellular matrix (ECM) are determinant for regulating cellular functions. Therefore, the study of the mechanical response of lung tissue becomes a fundamental aspect in order to further understand the mechanosensing signals perceived by the cells in the alveoli. This study is focused on the development of a finite element (FE) model of a decellularized rat lung tissue strip, which reproduces accurately the mechanical behaviour observed in the experiments by means of a tensile test. For simulating the complex structure of the lung parenchyma, which consists of a heterogeneous and non-uniform network of thin-walled alveoli, a 3D model based on a Voronoi tessellation is developed. This Voronoi-based model is considered very suitable for recreating the geometry of cellular materials with randomly distributed polygons like in the lung tissue. The material model used in the mechanical simulations of the lung tissue was characterized experimentally by means of AFM tests in order to evaluate the lung tissue stiffness on the micro scale. Thus, in this study, the micro (AFM test) and the macro scale (tensile test) mechanical behaviour are linked through the mechanical simulation with the 3D FE model based on Voronoi tessellation. Finally, a micro-mechanical FE-based model is generated from the Voronoi diagram for studying the stiffness sensed by the alveolar cells in function of two independent factors: the stretch level of the lung tissue and the geometrical position of the cells on the extracellular matrix (ECM), distinguishing between pneumocyte type I and type II. We conclude that the position of the cells within the alveolus has a great influence on the local stiffness perceived by the cells. Alveolar cells located at the corners of the alveolus, mainly type II pneumocytes, perceive a much higher stiffness than those located in the flat areas of the alveoli, which correspond to type I pneumocytes. However, the high stiffness, due to the macroscopic lung tissue stretch, affects both cells in a very similar form, thus no significant differences between them have been observed.

307.3: Bioengineering of an iPSC Derived Vascularized Endocrine Pancreas (iVEP) for T1D

Introduction: Intrahepatic islet transplantation in patients with type 1 diabetes is limited by donor availability and lack of engraftment. To overcome these limitations, new sources of β cells and alternative sites are needed. Organ decellularization is an emerging strategy in organ regeneration. Based on our experience with decellularized rat lung as scaffold for the generation of Vascularized Islet Organ (lung scaffold repopulated by murine islets and HUVEC cells), we used the same platform to engineer an iPSC-based version named iVEP (iPSC-derived Vascularized Endocrine Pancreas). Methods: iPSC-derived β (iβ) and endothelial (iEC) cells were characterized in flow cytometry. Rat lung was decellularized by vascular perfusion with 1% SDS and 0.1% Triton and seeded with commercially available iβ and iEC. The recellularized scaffold matured in vitro for 7 days in a customized perfusion bioreactor specifically designed to allow cell/compartment integration. The iβ death was estimated during ex vivo organ maturation compared to 7 days of iβ in vitro culture evaluating miR-375 expression by droplet digital PCR. At day 7, fluorangiography assay was performed to evaluate the vascular compartment structure and function while for the endocrine compartment, insulin production was measured by dynamic glucose perifusion and insulin quantification (ELISA/IF). Matured iVEPs were then transplanted subcutaneously into NSG diabetic mice followed for 30 days, compared to the deviceless (DL) implantation site and explanted iVEPs were assessed for IF evaluation. Results: iEC/iβ maintained for 7 days in vitro their phenotype expressing endothelial (>95% CD31+/CD105+/CD73+/CD90- cells) and β-cell (>60% PDX1+/insulin+ cells) markers respectively. Matured iVEPs showed a regenerated vascular network (CD31+) able to sustain the direct distribution of a perfusate with iβ cells (insulin+) integrated. In mature iVEP, iβ preserved insulin expression. miR-375, a marker of β cell death, was expressed in iβ but not in iECs. iVEP was able to significantly reduce β cell death: the amount of lost iβ was ≤18% during organ maturation, while >70% during in vitro culture (p<0.05). In iVEPs, lung ECM was able to sustain iβ engraftment and insulin secretion during the maturation process (AUC first phase iVEPs: 3.9465±1.23 Vs 1.44±0.83 p<0.05). Preliminary preclinical results demonstrated the ability of iVEP to engraft and restore normoglycemia in diabetic recipient mice compared to the iβ implanted in the DL site. Conclusion: To our knowledge, we assembled the first human entirely iPSC-derived Vascularized Endocrine Organ able to provide both in vitro and in vivo insulin production and regenerated vascular network.

P.168: Biofabrication of a Functional Vascularized Endocrine Pancreas (VEP) for Type 1 Diabetes

Background: Intrahepatic islet transplantation in patients with type 1 diabetes is limited by donor availability and lack of engraftment. To overcome these limitations, based on our experience with decellularized rat lung as scaffold for the generation of Vascularized Islet Organ (VIO, lung scaffold repopulated by murine islets and HUVEC cells), we prototyped our platform to engineer an upgrade based on human blood-derived endothelial cells (BOECs - Blood Outgrowing Endothelial Cells) and immature neonatal porcine islet clusters (NPIs). Material and Methods: NPIs and BOECs phenotype profile was assessed by flow cytometry, insulin secretion test and tubule formation assay. Rat lung was decellularized by vascular perfusion with 1% SDS and 0.1% Triton and seeded with NPIs and BOECs, generating a Vascularized Endocrine Pancreas (VEP). For ex vivo maturation VEP was cultured for 7 days in a customized bioreactor specifically designed to allow cell integration. The NPIs β cell death in mature VEPs was estimated during ex vivo organ maturation evaluating miR-375 expression by droplet digital PCR compared to batch matched NPIs in standard condition. Matured VEPs and control NPIs function were measured by dynamic glucose perifusion and insulin quantification (by ELISA/IF). Once validated in vitro, VEPs were subcutaneously transplanted in diabetic immunodeficient NSG recipients and compared for in vivo function with matched NPIs transplanted in different implantation sites: kidney capsule (KC-NPIs), deviceless (DL-NPIs) and liver (LV-NPIs). Results: Matured VEPs showed regenerated vascular network (CD31+) with NPIs (insulin+) integrated. miR-375, a marker of β cell death, was expressed in NPIs but not in BOECs, as expected. VEP was able to significantly reduce β cell death: the amount of lost NPIs were ≤ 5% during VEPs maturation, while >71% during in vitro culture (p<0.05). Matured VEPs were able to sustain NPIs engraftment, survival and significantly improve insulin secretion during the maturation process compared to batch matched NPIs cultured in standard conditions (AUC VEPs first phase: 3.765±0.90; NPIs 1.60±0.25 p<0.01). In long-term transplants in diabetic mice, VEPs demonstrated a significant NPIs engraftment with a prompt VEPs function after implantation and the reversal of the glycemia within 2 days until 60 days after implantation. Additionally, VEPs showed a significant superior function compared to all the internal controls (KC-NPIs, DL-and LV-NPIs). Conclusion: We conclude that ex vivo bio-fabrication of VEPs enables NPIs engraftment, maturation and vascularization before transplantation. VEP is the first organ to our knowledge assembled with relevant source of endocrine and endothelial cells suitable for future clinical translation. Given recent progress in gene editing, this technology may enable assembly of functional personalized endocrine organs to escape immunoreaction against the graft. This study was supported by a grant from Juvenile Diabetes Research Foundation (JDRF 1-SRA-2019-771-S-B).

Histological study of an experimentally constructed decellularized rat lung as a model of whole organ three-dimensional natural scaffold

Abstract Introduction With the increase of end stage lung diseases and the great problems facing lung transplantation tissue engineering become a promising solution. The first step in lung engineering is to obtain a 3D Extracellular matrix lung scaffold via decellularization. Decellularization aims to remove cells from tissue ultrastructure while preserving the mechanical and biological properties of the tissue. Intact ECM provides critical cues for differentiation and migration of cells that are seeded onto the organ scaffold. Objectives This study aimed to obtain an intact and well-preserved ECM lung scaffold by decellularization of rat lungs. Methods Decellularization of lungs of ten Wistar rats was achieved by perfusing detergents through the pulmonary artery. The resultant scaffolds were fixed and analyzed histologically. Results It was found that the decellularization process effectively removed the cellular and nuclear material while retaining native the 3D ECM of lung tissue. The architecture of the collagen and elastic fibers networks were preserved as comparable to the native lungs. Furthermore, the basement membranes of the bronchiolar and interalveolar septa were intact. Conclusions This methodology is expected to allow decellularization of human lung tissues and permits future scientific exploration in tissue engineering.

Differentiation of human induced pluripotent stem cells to megakaryocyte lineage by using 3D bioreactor, microfluidic system and acellular rat lung

• Acellular rat lung as a natural scaffold can improve cell differentiation. • Polydimethylsiloxane made bioreactor provides a natural microenvironment for cells. • Microfluidic culture system can reduce the cost of cell culture & differentiation. Induced pluripotent stem cells (hiPSCs) are reprogrammed cells that can develop into all human cell types, including megakaryocytes. Extracellular matrix plays a crucial role in the differentiation of hiPSCs into megakaryocytes. Therefore, we aimed to prepare a suitable natural acellular scaffold, and 3D bioreactor for in-vitro proliferation, and differentiation of hiPSCs into megakaryocytes. The rat lung was extracted, and the decellularization process was performed in order to eradicate cellular and nuclear materials, and lung three-dimensional (3D) structure with the protein contents remained intact. Scanning electron microscopy (SEM), hematoxylin and eosin (H&E), and 4′, 6-diamidino-2-phenylindole (DAPI) staining were used to verify tissue decellularization, and to ensure the integrity of the tissue structure. The 3D polydimethylsiloxane (PDMS) based bioreactor was designed, and the recellularization of the acellular lung was performed by hiPSCs. Decellularized rat lung vessels were used to deliver culture media as a microfluidic system. Differentiation of hiPSCs to megakaryocytes was assessed by RT-PCR and flow cytometry. H&E, DAPI staining, and SEM analysis confirmed the integrity of the 3D lung structure. Flow cytometry and RT-PCR analysis revealed the presence of megakaryocyte markers in differentiated cells. It seems that natural acellular scaffold and microfluidic 3D bioreactor provides a suitable natural cost-benefit microenvironment for hiPSCs differentiation into megakaryocytes.

Abstract 6512: Tissue engineered metastases to improve cancer immunotherapy

Abstract Background: Radiotherapy can induce the abscopal effect when combined with immune checkpoint inhibition. The main limitations to this treatment are the lack of control over the tumor microenvironment and the tumor is not amendable for radiosurgery. To overcome this, we hypothesize that using ex-vivo engineered cancer metastases treated with radiotherapy can improve the impact of the abscopal effect, as the immune cascade reaction is radiotherapy dose dependent and tumors can be exposed to higher amounts of radiation ex-vivo without the risk of radiation toxicity using our tissue engineered metastases model to boost immune response. Methods: Using B16F10 as a model, we first engineered B16F10 lung metastasis ex-vivo using decellularized rat lung biomatrix scaffolds. The engineered lung metastases were then lethally irradiated with 100 Gy of radiotherapy. The cells were then injected subcutaneously into tumor bearing C57BL6 mice in combination with anti PD-1 checkpoint inhibitor. Experimental groups included: no treatment, anti PD-1 only, cell lysate with and without anti PD-1, and irradiated engineered metastases with and without anti PD-1. All the mice were inoculated with 50,000 plastic grown B16F10 cells. On days 3, 6, and 9 the mice received immunotherapy and radiotherapy combinations based on their respective groups. The tumors were then analyzed by flow cytometry and IHC staining for different T cell populations. Results: We successfully engineered B16F10 lung metastasis ex vivo. We then demonstrated that mice treated irradiated engineered lung metastasis had significantly better survival than any other treatment arms with median survival twice that of irradiated cells (P &amp;lt;0.01). Correlative studies showed that mice that received irradiated engineered metastasis had increased CD8+ cells and lower Treg cells when compared to other experimental groups. This indicates that engineered metastasis increased immune activation and response to tumor. Conclusions: Our data demonstrates that engineered metastases can indeed improve cancer immunotherapy and induce a robust abscopal/vaccine effect. Citation Format: Nikhila Reddy Sultanpuram. Tissue engineered metastases to improve cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6512.

Conversion of human and mouse fibroblasts into lung-like epithelial cells

Cell lineage conversion of fibroblasts to specialized cell types through transdifferentiation may provide a fast and alternative cell source for regenerative medicine. Here we show that transient transduction of fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) in addition to the early lung transcription factor Nkx2-1 (also known as Ttf1), followed by directed differentiation of the cells, can convert mouse embryonic and human adult dermal fibroblasts into induced lung-like epithelial cells (iLEC). These iLEC differentiate into multiple lung cell types in air liquid interface cultures, repopulate decellularized rat lung scaffolds, and form lung epithelia composed of Ciliated, Goblet, Basal, and Club cells after transplantation into immune-compromised mice. As proof-of-concept, differentiated human iLEC harboring the Cystic Fibrosis mutation dF508 demonstrated pharmacological rescue of CFTR function using the combination of lumacaftor and ivacaftor. Overall, this is a promising alternative approach for generation of patient-specific lung-like progenitors to study lung function, disease and future regeneration strategies.

Nonlinear elasticity of the lung extracellular microenvironment is regulated by macroscale tissue strain

The extracellular matrix (ECM) of the lung provides physical support and key mechanical signals to pulmonary cells. Although lung ECM is continuously subjected to different stretch levels, detailed mechanics of the ECM at the scale of the cell is poorly understood. Here, we developed a new polydimethylsiloxane (PDMS) chip to probe nonlinear mechanics of tissue samples with atomic force microscopy (AFM). Using this chip, we performed AFM measurements in decellularized rat lung slices at controlled stretch levels. The AFM revealed highly nonlinear ECM elasticity with the microscale stiffness increasing with tissue strain. To correlate micro- and macroscale ECM mechanics, we also assessed macromechanics of decellularized rat lung strips under uniaxial tensile testing. The lung strips exhibited exponential macromechanical behavior but with stiffness values one order of magnitude lower than at the microscale. To interpret the relationship between micro- and macromechanical properties, we carried out a finite element (FE) analysis which revealed that the stiffness of the alveolar cell microenvironment is regulated by the global strain of the lung scaffold. The FE modeling also indicates that the scale dependence of stiffness is mainly due to the porous architecture of the lung parenchyma. We conclude that changes in tissue strain during breathing result in marked changes in the ECM stiffness sensed by alveolar cells providing tissue-specific mechanical signals to the cells. Statement of SignificanceThe micromechanical properties of the extracellular matrix (ECM) are a major determinant of cell behavior. The ECM is exposed to mechanical stretching in the lung and other organs during physiological function. Therefore, a thorough knowledge of the nonlinear micromechanical properties of the ECM at the length scale that cells probe is required to advance our understanding of cell-matrix interplay. We designed a novel PDMS chip to perform atomic force microscopy measurements of ECM micromechanics on decellularized rat lung slices at different macroscopic strain levels. For the first time, our results reveal that the microscale stiffness of lung ECM markedly increases with macroscopic tissue strain. Therefore, changes in tissue strain during breathing result in variations in ECM stiffness providing tissue-specific mechanical signals to lung cells.

A Fully Automated High-Throughput Bioreactor System for Lung Regeneration.

This work presents methods for ex vivo lung recellularization and biomimetic culture in a high-throughput and consistent manner. These methods allow for the testing of multiple variables, all of which are simultaneously controlled and monitored on a single fully automated pump system, and subsequent assessment of both epithelial and endothelial repair and tissue regeneration. This system provides a controlled environment for tissue repair, wherein key variables can be modified, monitored, reproduced, and optimized to advance the goal of ex vivo tissue regeneration based on native organ scaffolds.

Decellularized Rat Lung Scaffolds Using Sodium Lauryl Ether Sulfate for Tissue Engineering.

Perfusion decellularization with detergents is effective to maintain the architecture and proteins of extracellular matrix (ECM) for use in the field of lung tissue engineering (LTE). However, it is unclear which detergent is ideal to produce an acellular lung scaffold. In this study, we obtained two decellularized rat lung scaffolds using a novel detergent sodium lauryl ether sulfate (SLES) and a conventional detergent sodium dodecyl sulfate (SDS). Both decellularized lung scaffolds were assessed by histology, immunohistochemistry, scanning electron microscopy, DNA quantification, sulfated glycosaminoglycans (GAGs) quantification and western blot. Subsequently, the scaffolds were implanted subcutaneously in rats for 6 weeks and were evaluated via hematoxylin and eosin staining and Masson staining. Results indicated that SLES was effective to remove cells; moreover, lungs decellularized with SLES showed better preservation of sulfated GAGs, lung architecture, and ECM proteins than SDS. After 6 weeks, SLES scaffolds demonstrated a significantly greater potential for cell infiltration and blood vessel formation compared with SDS scaffolds. Taken together, we conclude that SLES is a promising detergent to produce an acellular scaffold using LTE for eventual transplantation.

252 - Interactions between induced pluripotent stem cell (iPSC)-derived smooth muscle and endothelial cells enhance angiogenic gene expression improving vascular repaving of decellularized rat lung scaffolds

252 - Interactions between induced pluripotent stem cell (iPSC)-derived smooth muscle and endothelial cells enhance angiogenic gene expression improving vascular repaving of decellularized rat lung scaffolds

Bioengineered lungs generated from human iPSCs-derived epithelial cells on native extracellular matrix.

The development of an alternative source for donor lungs would change the paradigm of lung transplantation. Recent studies have demonstrated the potential feasibility of using decellularized lungs as scaffolds for lung tissue regeneration and subsequent implantation. However, finding a reliable cell source and the ability to scale up for recellularization of the lung scaffold still remain significant challenges. To explore the possibility of regeneration of human lung tissue from stem cells in vitro, populations of lung progenitor cells were generated from human iPSCs. To explore the feasibility of producing engineered lungs from stem cells, we repopulated decellularized human lung and rat lungs with iPSC-derived epithelial progenitor cells. The iPSCs-derived epithelial progenitor cells lined the decellularized human lung and expressed most of the epithelial markers when were cultured in a lung bioreactor system. In decellularized rat lungs, these human-derived cells attach and proliferate in a manner similar to what was observed in the decellularized human lung. Our results suggest that repopulation of lung matrix with iPSC-derived lung epithelial cells may be a viable strategy for human lung regeneration and represents an important early step toward translation of this technology.

Transplantation of bioengineered rat lungs recellularized with endothelial and adipose-derived stromal cells

Bioengineered lungs consisting of a decellularized lung scaffold that is repopulated with a patient’s own cells could provide desperately needed donor organs in the future. This approach has been tested in rats, and has been partially explored in porcine and human lungs. However, existing bioengineered lungs are fragile, in part because of their immature vascular structure. Herein, we report the application of adipose-derived stem/stromal cells (ASCs) for engineering the pulmonary vasculature in a decellularized rat lung scaffold. We found that pre-seeded ASCs differentiated into pericytes and stabilized the endothelial cell (EC) monolayer in nascent pulmonary vessels, thereby contributing to EC survival in the regenerated lungs. The ASC-mediated stabilization of the ECs clearly reduced vascular permeability and suppressed alveolar hemorrhage in an orthotopic transplant model for up to 3 h after extubation. Fibroblast growth factor 9, a mesenchyme-targeting growth factor, enhanced ASC differentiation into pericytes but overstimulated their proliferation, causing a partial obstruction of the vasculature in the regenerated lung. ASCs may therefore provide a promising cell source for vascular regeneration in bioengineered lungs, though additional work is needed to optimize the growth factor or hormone milieu for organ culture.

Efficient and Functional Endothelial Repopulation of Whole Lung Organ Scaffolds.

To date, efforts to generate engineered lung tissue capable of long-term function have been limited by incomplete barrier formation between air and blood and by thrombosis of the microvasculature upon exposure of blood to the collagens within the decellularized scaffold. Improved barrier function and resistance to thrombosis both depend upon the recapitulation of a confluent monolayer of functional endothelium throughout the pulmonary vasculature. This manuscript describes novel strategies to increase cell coverage of the vascular surface area, compared to previous reports in our lab and others, and reports robust production of multiple anticoagulant substances that will be key to long-term function in vivo once additional strides are made in improving barrier function. Rat lung microvascular endothelial cells were seeded into decellularized rat lungs by both the pulmonary artery and veins with the use of low-concentration cell suspensions, pulsatile, gravity-driven flow, and supraphysiological vascular pressures. Together, these strategies yielded 72.44 ± 10.52% endothelial cell nuclear coverage of the acellular matrix after 3-4 d of biomimetic bioreactor culture compared to that of the native rat lung. Immunofluorescence, Western blot, and PCR analysis of these lungs indicated robust expression of phenotypic markers such as CD31 and VE-Cadherin after time in culture. Endothelial-seeded lungs had CD31 gene expression of 0.074 ± 0.015 vs 0.021 ± 0.0023 for native lungs, p = 0.025, and VE-Cadherin gene expression of 0.93 ± 0.22 compared to that of the native lung at 0.13 ± 0.02, p = 0.023. Precursors to antithrombotic substances such as tissue plasminogen activator, prostacyclin synthase, and endothelial nitric oxide synthase were expressed at levels equal to or greater than those of the native lung. Engineered lungs reseeded with endothelial cells were implanted orthotopically and contained patent microvascular networks that had gas exchange function during mechanical ventilation on 100% O2 greater than that of decelluarized lungs. Taken together, these data suggest that these engineered constructs could be compatible with long-term function in vivo when utilized in future studies in tandem with improved barrier function.

Enhanced Re-Endothelialization of Decellularized Rat Lungs.

Decellularized lung tissue has been recognized as a potential platform to engineer whole lung organs suitable for transplantation or for modeling a variety of lung diseases. However, many technical hurdles remain before this potential may be fully realized. Inability to efficiently re-endothelialize the pulmonary vasculature with a functional endothelium appears to be the primary cause of failure of recellularized lung scaffolds in early transplant studies. Here, we present an optimized approach for enhanced re-endothelialization of decellularized rodent lung scaffolds with rat lung microvascular endothelial cells (ECs). This was achieved by adjusting the posture of the lung to a supine position during cell seeding through the pulmonary artery. The supine position allowed for significantly more homogeneous seeding and better cell retention in the apex regions of all lobes than the traditional upright position, especially in the right upper and left lobes. Additionally, the supine position allowed for greater cell retention within large diameter vessels (proximal 100-5000 μm) than the upright position, with little to no difference in the small diameter distal vessels. EC adhesion in the proximal regions of the pulmonary vasculature in the decellularized lung was dependent on the binding of EC integrins, specifically α1β1, α2β1, and α5β1 integrins to, respectively, collagen type-I, type-IV, and fibronectin in the residual extracellular matrix. Following in vitro maturation of the seeded constructs under perfusion culture, the seeded ECs spread along the vascular wall, leading to a partial reestablishment of endothelial barrier function as inferred from a custom-designed leakage assay. Our results suggest that attention to cellular distribution within the whole organ is of paramount importance for restoring proper vascular function.

Fate of distal lung epithelium cultured in a decellularized lung extracellular matrix.

Type II cells are the defenders of the alveolus. They produce surfactant to prevent alveolar collapse, they actively transport water to prevent filling of the air sacs that would otherwise prevent gas exchange, and they differentiate to type I epithelial cells. They are an indispensable component of functional lung tissue. To understand the functionality of type II cells in isolation, we sought to track their fate in decellularized matrices and to assess their ability to contribute to barrier function by differentiation to type I alveolar epithelial cells. Rat type II cells were isolated from neonatal rat lungs by labeling with the RTII-70 surface marker and separation using a magnetic column. This produced a population of ∼50% RTII-70-positive cells accompanied by few type I epithelial cells or α-actin-positive mesenchymal cells. This population was seeded into decellularized rat lung matrices and cultured for 1 or 7 days. Culture in Dulbecco's modified Eagle's medium +10% fetal bovine serum (FBS) resulted in reduced expression of epithelial markers and increased expression of mesenchymal markers. By 7 days, no epithelial markers were visible by immunostaining; nearly all cells were α-actin positive. Gene expression for the mesenchymal markers, α-actin, vimentin, and TGF-βR, was significantly upregulated on day 1 (p=0.0005, 0.0005, and 2.342E-5, respectively). Transcript levels of α-actin and TGF-βR remained high at 7 days (p=1.364E-10 and 0.0002). Interestingly, human type II cells cultured under the same conditions showed a similar trend in the loss of epithelial markers, but did not display high expression of mesenchymal markers. Rat cells additionally showed the ability to produce and degrade the basement membrane and extracellular matrix components, such as fibronectin, collagen IV, and collagen I. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) showed significant increases in expression of the fibronectin and matrix metalloprotease-2 (MMP-2) genes after 1 day in culture (p=0.0135 and 0.0128, respectively) and elevated collagen I expression at 7 days (p=0.0016). These data suggest that the original type II-enriched population underwent a transition to increased expression of mesenchymal markers, perhaps as part of a survival or wound-healing program. These results suggest that additional medium components and/or the application of physiologically appropriate stimuli such as ventilation may be required to promote lung-specific epithelial phenotypes.

The differentiation of rat-induced pluripotent stem cells into alveolar type II epithelial cells with a three-step induction protocol

Induced pluripotent stem (iPS) cells derive from autologous somatic cells, the application prospect of iPS cells for regenerative medicine and tissue engineering is better than embryonic stem cells (ESCs) to some extent. Alveolar type II (AT II) epithelial cells play key role in the injured lung tissue regeneration and function recovery. The differentiation of iPS cells into AT II cells could provide available source for injured lung treatment. In this study, rat iPS (riPS) cells were resuscitated and proliferated for 14 days before differentiation. A modified three-step induction protocol similar to the reported ESCs inducing procedure was used in this study for the differentiation groups. Routine cell culture was done to the riPS cell control group (riPS-con). At stage 3, cells of day 7 (Diff. 7) and day 14 (Diff. 14) were collected for the real-time polymerase chain reaction tests for gene expressions of Oct4, Nanog, SPA, SPB, SPC, SPD, and CC10. Immunofluorescence staining of SPC and SSEA-1 was conducted. At the end of the differentiation, cell morphology became outstretched and epithelium-like. Cells of the Diff. 14 group positively expressed SPC and negatively expressed SSEA-1, which is contrary to the riPS-con group. In the Diff. 7 and the Diff. 14 groups, the expression of Oct4, Nanog, and SPB decreased (P < 0.05), whereas the expression of SPA, SPC, SPD (P < 0.05), and CC10 (P > 0.05) increased. This study indicated that riPS cells can successfully differentiate into AT II epithelial cells with the three-step induction protocol and may be further applied to implanting in decellularized rat lung scaffolds and building a bio-artificial lung.

Second and third trimester amniotic fluid mesenchymal stem cells can repopulate a de-cellularized lung scaffold and express lung markers

Background/PurposeThis study examined the potential of amniotic fluid mesenchymal stem cells (AF-MSCs) to generate lung precursor cells in vitro and on a xenologous three-dimensional de-cellularized lung scaffold. MethodsAF-MSCs were isolated from human amniotic fluid obtained from 17–37 weeks gestation. Lung differentiation was induced on Matrigel or on de-cellularized rat lungs intra-tracheally injected with AF-MSCs by culturing with a modification of small airway growth medium (mSAGM) lacking retinoic acid (RA) and triodothyronine (T3) with addition of fibroblast growth factor-10 (FGF10). Cells and scaffolds were characterized by immunofluorescence and RT-PCR for markers of viability, proliferation, and lung distal airway differentiation (TTF-1+ and SPC+) in the absence of markers of brain (TuJ1−) and thyroid (Pax8−). ResultsAfter culture in mSAGM on either Matrigel or lung scaffolds, there were TTF-1+/TuJ1−/Pax8− cells, indicating a lung precursor phenotype. In addition, SPC+ cells also evolved suggesting a more mature lung phenotype. ConclusionsWe demonstrate that mid- to late-trimester AF-MSCs can be induced to develop into lung precursor cells when cultured on the appropriate extracellular matrix (ECM), making them a viable source for use in cell therapy or development of an ex vivo tissue engineered lung.