Acellular Lung Research Articles

Stem cells, cell therapies, and bioengineering in lung biology and diseases 2023.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade, increasing evidence from preclinical models suggests that mesenchymal stromal cells, which are not normally resident in the lung, can be used to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathological remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

Effects of aging on the biomechanical properties of the lung extracellular matrix: dependence on tissular stretch.

Introduction: Aging induces functional and structural changes in the lung, characterized by a decline in elasticity and diminished pulmonary remodeling and regenerative capacity. Emerging evidence suggests that most biomechanical alterations in the lung result from changes in the composition of the lung extracellular matrix (ECM), potentially modulating the behavior of pulmonary cells and increasing the susceptibility to chronic lung diseases. Therefore, it is crucial to investigate the mechanical properties of the aged lung. This study aims to assess the mechanical alterations in the lung ECM due to aging at both residual (RV) and functional (FV) lung volumes and to evaluate their effects on the survival and proliferation of mesenchymal stromal cells (MSCs). Methods: The lungs from young (4-6-month-old) and aged (20-24-month-old) mice were inflated with optimal cutting temperature compound to reach FV or non-inflated (RV). ECM proteins laminin, collagen I and fibronectin were quantified by immunofluorescence and the mechanical properties of the decellularized lung sections were assessed using atomic force microscopy. To investigate whether changes in ECM composition by aging and/or mechanical properties at RV and FV volumes affects MSCs, their viability and proliferation were evaluated after 72h. Results: Laminin presence was significantly reduced in aged mice compared to young mice, while fibronectin and collagen I were significantly increased in aged mice. In RV conditions, the acellular lungs from aged mice were significantly softer than from young mice. By contrast, in FV conditions, the aged lung ECM becomes stiffer than that of in young mice, revealing that strain hardening significantly depends on aging. Results after MSCs recellularization showed similar viability and proliferation rate in all conditions. Discussion: This data strongly suggests that biomechanical measurements, especially in aging models, should be carried out in physiomimetic conditions rather than following the conventional non-inflated lung (RV) approach. The use of decellularized lung scaffolds from aged and/or other lung disease murine/human models at physiomimetic conditions will help to better understand the potential role of mechanotransduction on the susceptibility and progression of chronic lung diseases, lung regeneration and cancer.

Thoracic Endometriosis Syndrome: a case series and review of literature

Background: The presence of endometrial tissue in the tracheobronchial tree, pleural, and lung is normally referred to as thoracic endometriosis. The association of catamenial pneumothorax, catamenial haemothorax, catamenial haemoptysis and pulmonary nodules is referred to as thoracic endometriosis syndrome (TES). TES is rare but not as rare as it has always been thought of.
 Case summaries:
 Case 1: We present EA, a 26-year-old chef/baker who presented to our unit on account of recurrent cyclical right sided chest pain and difficulty in breathing, recurrent haemoptysis and cyclical abdominal pain and swelling with multiple tender umbilical nodules. On general physical examination she was found to be in respiratory distress but not cyanosed with peripheral arterial oxygen saturation 97% on room air. Examination of the chest revealed a right sided chest fullness and reduced movement with breathing. Percussion note was stony dull and absent of breath sound on right hemithorax. Abdominal examination revealed a moderately distended abdomen with positive fluid thrill. Both Pleurocentesis (thoracocentesis) and paracentesis abdominis yielded a haemorrhagic fluid acellular and pleural and lung biopsy demonstrated endometrial stroma and glands. Imaging examination by way of chest X-ray showed a homogenous opacity of the right hemithorax, and pulmonary nodule following drainage. A diagnosis of Thoracic Endometriosis Syndrome was made. She had VAT with lung and pleural biopsy, pleural abrasion and a thoracostomy tube drainage.
 Case 2: We present IAE, a 29year-old Po+0 who presented to our unit on account of recurrent cyclical right sided chest pain and difficulty in breathing, unproductive cough. On general physical examination she was found to be in moderate respiratory distress but not cyanosed with peripheral arterial oxygen saturation of 98% on room air. Chest examination revealed an apical flattering of anterior chest wall with left trachea deviation. Percussion note was stony dull on the right lower third and hyper resonance middle and upper zones of right hemithorax. Pleurocentesis (thoracocentesis) yielded air and haemorrhagic fluid acellular and pleural and lung biopsies demonstrated endometrial stroma and glands. Imaging examination by way of chest X-ray and chest CT-scan showed air fluid level of the right hemithorax. A diagnosis of TES was made. She had Laparoscopy and VAT at the same sitting with pleural and lung biopsies. She was managed medically after closed thoracostomy tube drainage. She had partial collapse of right lower lobe and thoracotomy with right lower lobectomy was planned but patient declined. She also had Stage IV endometriosis by (rASRM)/ENZIAN systems
 Case 3: We present JA a 29-year-old lady, a filling station attendant who presented to our unit with a history of gradual onset of right sided chest pain, progressive difficulty in breathing and non-productive cough, and abdominal pain and swelling. On general physical examination she was found to be in respiratory distress but not cyanosed with SPO2 of 98% on room air. Examination of the chest revealed a right apical flattening, reduced ipsilateral chest movement with breathing. Percussion notes were stony dull and absent air entry on the ipsilateral hemithorax. Abdominal examination showed a mildly distended abdomen with a positive shifting dullness. Both Pleurocentesis (thoracocentesis) and paracentesis abdominis yielded a haemorrhagic fluid acellular at cytology and pleural and lung biopsy demonstrated endometrial stroma and gland. Imaging examination by way of chest X-ray showed a homogenous opacity of the right hemithorax. A diagnosis of Familial Thoracic Endometriosis Syndrome (FTES). Had USS guided lung and pleural biopsy and a thoracostomy tube drainage. She had chemical pleurodesis with tetracycline
 Conclusion: Thoracic endometriosis syndrome is the diagnosis in a lady of reproductive age who present with cyclic or a cyclical recurrent right sided chest pain, cyclical or a cyclical dyspnoea, haemoptysis, cyclical or a cyclical abdominal swelling and peritonitis.

Mesenchymal cells support the early retention of primary alveolar type 2 cells on acellular mouse lung scaffolds

ObjectivesTissue engineering approaches via repopulation of acellular biological grafts provide an exciting opportunity to generate lung grafts for transplantation. Alveolar type 2 (AT2) cells are a promising cell source for re-epithelialization. There are however inherent limitations with respect to their survival and growth, thus impeding their usability for tissue engineering applications. This study investigates the use of mesenchymal stromal cells to support primary AT2 cells for recellularization of mouse lung scaffolds. MethodsAT2 cells and bone marrow-derived mesenchymal cells (BMC) were co-delivered to decellularized mouse lung scaffolds. Recellularized lungs were evaluated for cell surface coverage, viability, and differentiation at 1 and 4 days after cell seeding. Recellularization was evaluated via histological analysis and immunofluorescence. ResultsSimultaneous delivery of AT2 and BMC into acellular lung scaffolds resulted in enhanced cell surface coverage and reduced AT2 cell apoptosis in the recellularized scaffolds at Day 1 but not Day 4. AT2 cell number decreased after 4 days in both of AT2 only and codelivery groups suggesting limited expansion potential in the scaffold. After retention in the scaffold, AT2 cells differentiated into Aqp5-expressing cells. ConclusionsOur results indicate that BMC support AT2 cell survival during the initial attachment and engraftment phase of recellularization. While our findings suggest only a short-term beneficial effect of BMC, our study demonstrates that AT2 cells can be delivered and retained in acellular lung scaffolds; thus with preconditioning and supporting cells, may be used for re-epithelialization. Selection and characterization of appropriate cell sources for use in recellularization, will be critical for ultimate clinical application.

The Complete Score for Comprehensive Evaluation of Donor Lungs in Ex-Vivo Lung Perfusion: An Approach for Optimizing the Outcomes of Transplantation

<h3>Purpose</h3> The imperfection of the currently utilized tools for assessing graft function in acellular ex-vivo lung perfusion (EVLP) has led our team to develop a comprehensive evaluation scoring system. This study aimed to validate this scoring system in our clinical EVLP program. <h3>Methods</h3> One hundred twelve EVLP cases were performed in our program since 2018. Data from the first 73 cases (derivation cohort) were retrospectively analyzed to identify the included evaluation parameters and to inform the cutoff values in the scoring system using Youden index. The scoring system was then prospectively applied in the remaining 39 cases (validation cohort). The comprehensive lung evaluation EVLP (COMPLETE) score included ultrasound evaluation, lung weight measurement, compliance, airway pressure, deflation, oxygenation, lactate level, bronchoscopy, and x-ray. <h3>Results</h3> Out of 112 EVLP cases, 75 (67%) were transplanted with 98.7% 30-day survival. The incidence of primary graft dysfunction (PGD) grades 0, 1, 2, and 3 at 72 hours post-transplant were 21, 63, 8, and 8 %, respectively. The COMPLETE score cutoff value for transplant suitability identified by the derivation cohort was 12 (AUC 1, 100% sensitivity & specificity) which was then applied in the validation cohort. Only two recipients in the validation cohort had PGD3 (which appeared to be related to intraoperative factors, not donor factors). The COMPLETE score had a significant correlation with IL-1β perfusate levels (r = 0.44, <i>p</i> < .001). Nine cases did not meet the standard criteria (FDA approved) for transplant suitability on EVLP but were transplanted based on their COMPLETE scores with 100% 30-day survival. Of those, 1 had PGD0, 7 had PGD1, and 1 had PGD3. On the other hand, 10 cases met the standard criteria for transplant suitability but were deemed non-suitable based on their COMPLETE scores and the post-EVLP pathological examination showed significant edema in 4 cases, acute lung injury in 4 cases, and pulmonary embolism in 2 cases. <h3>Conclusion</h3> Our novel COMPLETE scoring system provides a protocolized comprehensive evaluation of donor lungs on EVLP. It can be used to optimize the outcomes of lung transplantation by increasing utilization rates after EVLP and by identifying donor factors that might lead to unfavorable outcomes following transplantation.

Novel Decellularization Method for Tissue Slices.

Decellularization procedures have been developed and optimized for the entire organ or tissue blocks, by either perfusion of decellularizing agents through the tissue’s vasculature or submerging large sections in decellularizing solutions. However, some research aims require the analysis of native as well as decellularized tissue slices side by side, but an optimal protocol has not yet been established to address this need. Thus, the main goal of this work was to develop a fast and efficient decellularization method for tissue slices—with an emphasis on lung—while attached to a glass slide. To this end, different decellularizing agents were compared for their effectiveness in cellular removal while preserving the extracellular matrix. The intensity of DNA staining was taken as an indicator of remaining cells and compared to untreated sections. The presence of collagen, elastin and laminin were quantified using immunostaining and signal quantification. Scaffolds resulting from the optimized protocol were mechanically characterized using atomic force microscopy. Lung scaffolds were recellularized with mesenchymal stromal cells to assess their biocompatibility. Some decellularization agents (CHAPS, triton, and ammonia hydroxide) did not achieve sufficient cell removal. Sodium dodecyl sulfate (SDS) was effective in cell removal (1% remaining DNA signal), but its sharp reduction of elastin signal (only 6% remained) plus lower attachment ratio (32%) singled out sodium deoxycholate (SD) as the optimal treatment for this application (6.5% remaining DNA signal), due to its higher elastin retention (34%) and higher attachment ratio (60%). Laminin and collagen were fully preserved in all treatments. The SD decellularization protocol was also successful for porcine and murine (mice and rat) lungs as well as for other tissues such as the heart, kidney, and bladder. No significant mechanical differences were found before and after sample decellularization. The resulting acellular lung scaffolds were shown to be biocompatible (98% cell survival after 72 h of culture). This novel method to decellularize tissue slices opens up new methodological possibilities to better understand the role of the extracellular matrix in the context of several diseases as well as tissue engineering research and can be easily adapted for scarce samples like clinical biopsies.

Decellularized human-sized pulmonary scaffolds for lung tissue engineering: a comprehensive review.

The ultimate goal of lung bioengineering is to produce transplantable lungs for human beings. Therefore, large-scale studies are of high importance. In this paper, we review the investigations on decellularization and recellularization of human-sized lung scaffolds. First, studies that introduce new ways to enhance the decellularization of large-scale lungs are reviewed, followed by the investigations on the xenogeneic sources of lung scaffolds. Then, decellularization and recellularization of diseased lung scaffolds are discussed to assess their usefulness for tissue engineering applications. Next, the use of stem cells in recellularizing acellular lung scaffolds is reviewed, followed by the case studies on the transplantation of bioengineered lungs. Finally, the remaining challenges are discussed, and future directions are highlighted.

Platforms to test the temporospatial capabilities of carrier systems in delivering growth factors to benefit vascular bioengineering

In this study we produced a set of in vitro culture platforms to model vascular cell responses to growth factors and factor delivery vehicles. Two of the systems (whole vessel and whole lung vascular development) were supported by microfluidic systems facilitating media circulation and waste removal. We assessed vascular endothelial growth factor (VEGF) delivery by Pluronic F-127 hydrogel, 30 nm pore-sized microparticles (MPs), 60 nm pore-sized MP or a 50/50 mixture of 30 and 60 nm pore-sized MP. VEGF was delivered to porcine acellular lung vascular scaffolds (2.5 cm2 square pieces or whole 3D segments of acellular blood vessels) as well as whole acellular lung scaffolds. Scaffold-cell attachment was examined as was vascular tissue formation. We showed that a 50/50 mixture of 30 and 60 nm pore-sized silicon wafer MPs allowed for long-term release of VEGF within the scaffold vasculature and supported vascular endothelial tissue development during in vitro culture.

Photodynamic Therapy in the Extracellular Matrix of Mouse Lungs: Preliminary Results of an Alternative Tissue Sterilization Process

Lung transplantation is one of the most difficult and delicate procedures among organ transplants. For the success of the procedure and survival of the new organ, the sterilization step for acellular lungs prior to recellularization is important to ensure that they are free of any risk of transmitting infections from the donor to the recipient subject. However, there are no available information concerning the lung mechanical parameters after sterilizing photodynamic therapy. The aim of this study was to evaluate the extracellular matrix (ECM) and lung mechanical parameters of decellularized lungs undergoing sterilizing photodynamic therapy (PDT). Besides, we also analyzed the lung after controlled infection with C. albicans in order to evaluate the effectiveness of PDT. The lung mechanical evaluation parameters, resistance ( R L ) and elastance ( E L ), exhibited no significant differences between groups. In addition, there were no PDT-induced changes in lung properties, with maintenance of the viscoelastic behavior of the lung scaffold after 1 h exposure to PDT. The ECM components remained virtually unchanged in the acellular lungs of both groups. We also showed that there was a reduction in fungal infection population after 45 minutes of PDT. However, more studies should be performed to establish and verify the effectiveness of PDT as a possible means for sterilizing lung scaffolds. This manuscript was presented as a master thesis of Nadua Apostólico at the postgraduate program in rehabilitation sciences, University Nove de Julho—UNINOVE.

Harnessing the ECM Microenvironment to Ameliorate Mesenchymal Stromal Cell-Based Therapy in Chronic Lung Diseases.

It is known that the cell environment such as biomechanical properties and extracellular matrix (ECM) composition dictate cell behaviour including migration, proliferation, and differentiation. Important constituents of the microenvironment, including ECM molecules such as proteoglycans and glycosaminoglycans (GAGs), determine events in both embryogenesis and repair of the adult lung. Mesenchymal stromal/stem cells (MSC) have been shown to have immunomodulatory properties and may be potent actors regulating tissue remodelling and regenerative cell responses upon lung injury. Using MSC in cell-based therapy holds promise for treatment of chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). However, so far clinical trials with MSCs in COPD have not had a significant impact on disease amelioration nor on IPF, where low cell survival rate and pulmonary retention time are major hurdles to overcome. Research shows that the microenvironment has a profound impact on transplanted MSCs. In our studies on acellular lung tissue slices (lung scaffolds) from IPF patients versus healthy individuals, we see a profound effect on cellular activity, where healthy cells cultured in diseased lung scaffolds adapt and produce proteins further promoting a diseased environment, whereas cells on healthy scaffolds sustain a healthy proteomic profile. Therefore, modulating the environmental context for cell-based therapy may be a potent way to improve treatment using MSCs. In this review, we will describe the importance of the microenvironment for cell-based therapy in chronic lung diseases, how MSC-ECM interactions can affect therapeutic output and describe current progress in the field of cell-based therapy.

The Complete Score for Assessment of Donor Lungs: A Comprehensive Evaluation System in Clinical Ex-Vivo Lung Perfusion

Purpose Evaluating marginal donor lungs for transplant suitability in acellular ex-vivo lung perfusion (EVLP) can be challenging. Relying on a single parameter for evaluation can be misleading and may result in unfavorable outcomes following lung transplantation (LTx). This study aimed to establish a comprehensive scoring system incorporating important evaluation parameters in a simple reproducible way. Methods The comp rehensive l ung e valua t ion E VLP ( COMPLETE ) score is the composite result of ten evaluation parameters including: lung weight measurement, ultrasound evaluation, airway pressure, lung compliance, P/F ratio, lactate level, deflation, palpation, bronchoscopy, and x-ray. Each parameter is scored from 0 to 3, 3 being the worst, then the COMPLETE score is calculated by the summation of the individual parameter scores. Data was collected prospectively, then analyzed retrospectively utilizing receiver operating characteristic (ROC) analysis to inform the cut off values for each parameter scoring. Results Seventy-three EVLP cases were performed resulting in 48 lung transplants with 100% 30-day survival and 96% 1-year survival. Median time to extubation was 1 day and median ICU stay was 7 days. The incidence of primary graft dysfunction (PGD) grades 0, 1, 2, and 3 at 72 hours were 23%, 61%, 9%, and 7%, respectively. The COMPLETE score was significantly higher in lungs non-suitable for LTx compared to transplanted lungs (17 [15-20] vs 7 [4-8], P Conclusion The COMPLETE scoring system is a comprehensive system that can facilitate decision making in EVLP in a protocolized manner. The COMPLETE score was significantly higher in EVLP lungs deemed non-suitable for transplant and, in those transplanted after EVLP, the COMPLETE score had a significant correlation with time to extubation, ICU stay, and incidence of PGD3. Consideration should be given to the routine use of the COMPLETE score during EVLP evaluation.

TP63 basal cells are indispensable during endoderm differentiation into proximal airway cells on acellular lung scaffolds

The use of decellularized whole-organ scaffolds for bioengineering of organs is a promising avenue to circumvent the shortage of donor organs for transplantation. However, recellularization of acellular scaffolds from multicellular organs like the lung with a variety of different cell types remains a challenge. Multipotent cells could be an ideal cell source for recellularization. Here we investigated the hierarchical differentiation process of multipotent ES-derived endoderm cells into proximal airway epithelial cells on acellular lung scaffolds. The first cells to emerge on the scaffolds were TP63+ cells, followed by TP63+/KRT5+ basal cells, and finally multi-ciliated and secretory airway epithelial cells. TP63+/KRT5+ basal cells on the scaffolds simultaneously expressed KRT14, like basal cells involved in airway repair after injury. Removal of TP63 by CRISPR/Cas9 in the ES cells halted basal and airway cell differentiation on the scaffolds. These findings suggest that differentiation of ES-derived endoderm cells into airway cells on decellularized lung scaffolds proceeds via TP63+ basal cell progenitors and tracks a regenerative repair pathway. Understanding the process of differentiation is key for choosing the cell source for repopulation of a decellularized organ scaffold. Our data support the use of airway basal cells for repopulating the airway side of an acellular lung scaffold.

Cross-linked acellular lung for application in tissue engineering: Effects on biocompatibility, mechanical properties and immunological responses

The concept of providing tissue engineering scaffolds with natural physical properties and minimal immunogenicity has not been systematically approached for the lungs yet. Here, the rat acellular lung tissue (ALT) was cross-linked to provide either EDC/NHS cross-linked tissue (EDC/NHS-CLT) or tannic acid cross-linked tissue (TA-CLT). Young's modulus revealed that EDC/NHS-CLT had mechanical properties similar to the native lung and culture of lung mesenchymal cells showed a higher potential of cell proliferation on EDC/NHS-CLT versus TA-CLT and ALT. The in vitro immunogenicity tests showed a strong induction of T-cell proliferation by TA-CLT and an attenuated macrophage induction by TA-CLT. Processed rat lungs were implanted xenogenically into the mouse peritoneal cavity and the host-implant interactions showed that tannic acid is not released from TA-CLT in a physiologically effective dose. The profile of peritoneal fluid proinflammatory (TNFα, IL-1β, IL-12p70 and IL-17) and anti-inflammatory (IL-10 and TGFβ1) cytokines, and CD3+ T-lymphocytes and CD11b+ macrophages revealed that apart from induction of high levels of IL-17 during the first week and IL-10 during the second to third weeks after implantation by TA-CLT, other indicators of immune reactions to cross-linked tissues were not significantly different from ALT. Also, a high fibrotic reaction to TA-CLT was observed on the weeks 2–3, but alveolar structures were preserved in EDC/NHS-CLT. Our findings show that by controlled EDC/NHS cross-linking, an acellular lung scaffold could be provided with mechanical properties similar to native lung, which promotes mesenchymal lung cells proliferation and does not stimulate recipient's immune system more than a non-cross-linked tissue.

Effects of albumin, transferrin and humic-like substances on iron-mediated OH radical formation in human lung fluids

Inhalation of particulate matter is hypothesized to contribute to health effects by overproducing reactive oxygen species (ROS) and inducing oxidative stress. Fe(II) has been shown to contribute to ROS generation in acellular simulated lung fluids. Atmospheric humic-like substances (HULIS) have been shown to chelate Fe(II) and significantly enhance this ROS generation. Here, we investigate Fe(II)-mediated .OH generation from the iron active proteins in lung fluid, albumin and transferrin, and fulvic acid, a surrogate for HULIS, in human bronchoalveolar lavage fluid (BALF). We find that albumin enhances .OH generation from inorganic Fe(II) and that transferrin attenuates this enhancement. We estimate the rate constants for albumin-Fe(II) and fulvic acid-Fe(II) mediated O2.- reduction (1.9 ± 0.3) M−1 s−1 and (2.7 ± 0.3) M−1s−1 (pH = 5.5, T = 37 °C), 17–25 times the rate for free iron, which we measured to be (110 ± 20) × 10−3 M−1s−1, in agreement with the literature. .OH generation measured from fulvic acid-Fe(II) in BALF from 8 individuals with added fulvic acid is successfully predicted rates of .OH generation by mixtures of Fe(II), albumin, transferrin, fulvic acid, and ascorbate in saline solution. This indicates that fulvic acid enhances .OH formation in BALF, and that albumin and transferrin in BALF moderate the effect. We propose that fulvic acid, and thereby HULIS, is capable of mobilizing Fe(II) away from albumin and transferrin and this increases the formation rate of O2.- and ultimately of .OH. Furthermore, we find that albumin and transferrin have significantly different impacts on Fe(II)-mediated .OH than citrate, a common component of simulated lung fluids, a factor that should be considered carefully in the interpretation of results obtained from solutions containing citrate.

Transcriptional activators YAP/TAZ and AXL orchestrate dedifferentiation, cell fate, and metastasis in human osteosarcoma

Osteosarcoma (OS) is a molecularly heterogeneous, aggressive, poorly differentiated pediatric bone cancer that frequently spreads to the lung. Relatively little is known about phenotypic and epigenetic changes that promote lung metastases. To identify key drivers of metastasis, we studied human CCH-OS-D OS cells within a previously described rat acellular lung (ACL) model that preserves the native lung architecture, extracellular matrix, and capillary network. This system identified a subset of cells—termed derived circulating tumor cells (dCTCs)—that can migrate, intravasate, and spread within a bioreactor-perfused capillary network. Remarkably, dCTCs highly expressed epithelial-to-mesenchymal transition (EMT)-associated transcription factors (EMT-TFs), such as ZEB1, TWIST, and SOX9, which suggests that they undergo cellular reprogramming toward a less differentiated state by coopting the same epigenetic machinery used by carcinomas. Since YAP/TAZ and AXL tightly regulate the fate and plasticity of normal mesenchymal cells in response to microenvironmental cues, we explored whether these proteins contributed to OS metastatic potential using an isogenic pair of human OS cell lines that differ in AXL expression. We show that AXL inhibition significantly reduced the number of MG63.2 pulmonary metastases in murine models. Collectively, we present a laboratory-based method to detect and characterize a pure population of dCTCs, which provides a unique opportunity to study how OS cell fate and differentiation contributes to metastatic potential. Though the important step of clinical validation remains, our identification of AXL, ZEB1, and TWIST upregulation raises the tantalizing prospect that EMT-TF-directed therapies might expand the arsenal of therapies used to combat advanced-stage OS.

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.

Bioengineered Human Acellular Vessels as Dialysis Access Grafts

Cardiovascular regenerative medicine has taken many avenues. One approach currently in clinical trials does not require any cells from the patient, and is an engineered tissue that is available “off‐the‐shelf”. Our approach to vascular engineering involves seeding allogeneic vascular cells onto a degradable substrate to culture vascular tissues in a biomimetic bioreactor. After a period of 8–10 weeks, engineered tissues are then decellularized to produce an engineered extracellular matrix‐based graft. The advantage of using allogeneic cells for graft production is that no biopsy need be harvested from the patient, and no patient‐specific culture time is required. These grafts are currently being tested in a Phase III clinical trial in Europe and in the US. Early experience supports the potential utility of this novel tissue engineered vascular graft to provide vascular access for hemodialysis.The decellularization approach has also allowed us to generate scaffolds to support whole lung regeneration. Using rat, porcine and human sources of organs, lungs have been subjected to a range of decellularization procedures, with the goal of removing a maximal amount of cellular material while retaining matrix constituents. Next‐generation proteomics approaches have shown that gentle decellularization protocols result in near‐native retention of key matrix molecules involved in cell adhesion, including proteoglycans and glycoproteins. Repopulation of the acellular lung matrix with mixed populations of neonatal lung epithelial cells results in regio‐specific epithelial seeding in correct anatomic locations. Survival and differentiation of lung epithelium is enhanced by culture in a biomimetic bioreactor that is designed to mimic some aspects of the fetal lung environment, including vascular perfusion and liquid ventilation. Current challenges involve the production of a uniformly recellularized scaffold within the vasculature, in order to shield blood elements from the collagenous matrix which can stimulate clot formation. In addition, we have developed methods to quantify barrier function of acellular and repopulated matrix, in order to predict functional gas exchange in vivo.Support or Funding InformationNIH R01 HL127386 (Niklason)

Biofabrication of a vascularized islet organ for type 1 diabetes

Islet transplantation is superior to extrinsic insulin supplementation in the treating severe Type 1 diabetes. However, its efficiency and longevity are limited by substantial islet loss post-transplantation due to lack of engraftment and vascular supply. To overcome these limitations, we developed a novel approach to bio-fabricate functional, vascularized islet organs (VIOs) ex vivo. We endothelialized acellular lung matrixes to provide a biocompatible multicompartment scaffold with an intact hierarchical vascular tree as a backbone for islet engraftment. Over seven days of culture, islets anatomically and functionally integrated into the surrounding bio-engineered vasculature, generating a functional perfusable endocrine organ. When exposed to supra-physiologic arterial glucose levels in vivo and ex vivo, mature VIOs responded with a physiologic insulin release from the vein and provided more efficient reduction of hyperglycemia compared to intraportally transplanted fresh islets. In long-term transplants in diabetic mice, subcutaneously implanted VIOs achieved normoglycemia significantly faster and more efficiently compared to islets that were transplanted in deviceless fashion. We conclude that ex vivo bio-fabrication of VIOs enables islet engraftment and vascularization before transplantation, and thereby helps to overcome limited islet survival and function observed in conventional islet transplantation. Given recent progress in stem cells, this technology may enable assembly of functional personalized endocrine organs.

Immune cells inhibit the tumor metastasis in the 4D cellular lung model by reducing the number of live circulating tumor cells

The immune system and tumor microenvironment play a decisive role in tumor progression. We developed a novel model to better understand tumor progression and interaction with immune cells and the cellular components. We grew 393 P non-metastatic and 344SQ metastatic murine cells in an acellular metastatic lung cancer model, where both cell lines formed circulating tumor cells (CTC) and metastatic lesions. When the CTC from this model were placed in the tail vein of nu/nu mice, both cell lines formed metastatic lesions. However, in syngeneic immune-competent mice, the CTC from the non-metastatic cell line did not metastasize while the CTC from the metastatic cell line metastasized. When we placed the activated immune cells in the cellular lung model, it decreased CTC and metastatic lesion formation for the non-metastatic cell line while it had no impact on metastatic cell line. The metastatic cell line had a significant increase in expression of programmed death-ligand 1 (PDL-1) compared to the non-metastatic cell line in the model. Overall, the immune cells showed an impact on viability of CTC for cell lines with a decreased expression of PDL-1 that leads to decreased metastatic lesion formation. Further studies are needed to understand the subtype of immune cells and mechanism of decreased CTC viability and metastasis inhibition.

Acellular and Cellular Lung Model to Study Tumor Metastasis.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.