Xi Ren

Perfusion decellularization of human and porcine lungs: Bringing the matrix to clinical scale

BACKGROUND Organ engineering is a theoretical alternative to allotransplantation for end-stage organ failure. Whole-organ scaffolds can be created by detergent perfusion via the native vasculature, generating an acellular matrix suitable for recellularization with selected cell types. We aimed to up-scale this process, generating biocompatible scaffolds of a clinically relevant scale. METHODS Rat, porcine, and human lungs were decellularized by detergent perfusion at constant pressures. Collagen, elastin, and glycosaminoglycan content of scaffolds were quantified by colorimetric assays. Proteomic analysis was performed by microcapillary liquid chromatography tandem mass spectrometry. Extracellular matrix (ECM) slices were cultured with human umbilical vein endothelial cells (HUVEC), small airway epithelial cells (SAEC), or pulmonary alveolar epithelial cells (PAECs) and evaluated by time-lapse live cell microscopy and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Whole-organ culture was maintained under constant-pressure media perfusion after seeding with PAECs. RESULTS Rat lungs were decellularized using: (1) sodium dodecyl sulfate (SDS), (2) sodium deoxycholate (SDC), or (3) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Resulting scaffolds showed comparable loss of DNA but greatest preservation of ECM components in SDS-decellularized lungs. Porcine (n = 10) and human (n = 7) lungs required increased SDS concentration, perfusion pressures, and time to achieve decellularization as determined by loss of DNA, with preservation of intact matrix composition and lung architecture. Proteomic analysis of human decellularized lungs further confirmed ECM preservation. Recellularization experiments confirmed scaffold biocompatibility when cultured with mature cell phenotypes and scaffold integrity for the duration of biomimetic culture. CONCLUSIONS SDS-based perfusion decellularization can be applied to whole porcine and human lungs to generate biocompatible organ scaffolds with preserved ECM composition and architecture.

Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish

We describe an enhancer trap transgenic zebrafish line, ETvmat2:GFP, in which most monoaminergic neurons are labeled by green fluorescent protein (GFP) during embryonic development. The reporter gene of ETvmat2:GFP was inserted into the second intron of vesicular monoamine transporter 2 (vmat2) gene, and the GFP expression pattern recapitulates that of the vmat2 gene. The GFP positive neurons include the large and pear-shaped tyrosine hydroxylase positive neurons (TH populations 2 and 4) in the posterior tuberculum of ventral diencephalon (PT neurons), which are thought to be equivalent to the midbrain dopamine neurons in mammals. We found that these PT neurons and two other GFP labeled non-TH type neuronal groups, one in the paraventricular organ of the posterior tuberculum and the other in the hypothalamus, were significantly reduced after exposure to MPTP, while the rest of GFP-positive neuronal clusters, including those in telencephalon, pretectum, raphe nuclei and locus coeruleus, remain largely unchanged. Furthermore, we showed that the effects of hedgehog signaling pathway inhibition on the development of monoaminergic neurons can be easily visualized in individual living ETvmat2:GFP embryos. This enhancer trap line should be useful for genetic and pharmacological analyses of monoaminergic neuron development and processes underlying Parkinsons disease.

Perfusion decellularization of whole organs

The native extracellular matrix (ECM) outlines the architecture of organs and tissues. It provides a unique niche of composition and form, which serves as a foundational scaffold that supports organ-specific cell types and enables normal organ function. Here we describe a standard process for pressure-controlled perfusion decellularization of whole organs for generating acellular 3D scaffolds with preserved ECM protein content, architecture and perfusable vascular conduits. By applying antegrade perfusion of detergents and subsequent washes to arterial vasculature at low physiological pressures, successful decellularization of complex organs (i.e., hearts, lungs and kidneys) can be performed. By using appropriate modifications, pressure-controlled perfusion decellularization can be achieved in small-animal experimental models (rat organs, 4–5 d) and scaled to clinically relevant models (porcine and human organs, 12–14 d). Combining the unique structural and biochemical properties of native acellular scaffolds with subsequent recellularization techniques offers a novel platform for organ engineering and regeneration, for experimentation ex vivo and potential clinical application in vivo.

Engineering pulmonary vasculature in decellularized rat and human lungs

Bioengineered lungs produced from patient-derived cells may one day provide an alternative to donor lungs for transplantation therapy. Here we report the regeneration of functional pulmonary vasculature by repopulating the vascular compartment of decellularized rat and human lung scaffolds with human cells, including endothelial and perivascular cells derived from induced pluripotent stem cells. We describe improved methods for delivering cells into the lung scaffold and for maturing newly formed endothelium through co-seeding of endothelial and perivascular cells and a two-phase culture protocol. Using these methods we achieved ∼75% endothelial coverage in the rat lung scaffold relative to that of native lung. The regenerated endothelium showed reduced vascular resistance and improved barrier function over the course of in vitro culture and remained patent for 3 days after orthotopic transplantation in rats. Finally, we scaled our approach to the human lung lobe and achieved efficient cell delivery, maintenance of cell viability and establishment of perfusable vascular lumens.

Scl isoforms act downstream of etsrp to specify angioblasts and definitive hematopoietic stem cells

Recent lineage studies suggest that hematopoietic stem cells (HSCs) may be derived from endothelial cells. However, the genetic hierarchy governing the emergence of HSCs remains elusive. We report here that zebrafish ets1-related protein (etsrp), which is essential for vascular endothelial development, also plays a critical role in the initiation of definitive hematopoiesis by controlling the expression of 2 stem cell leukemia (scl) isoforms (scl-alpha and scl-beta) in angioblasts. In etsrp morphants, which are deficient in endothelial and HSC development, scl-alpha alone partially rescues angioblast specification, arterial-venous differentiation, and the expression of HSC markers, runx1 and c-myb, whereas scl-beta requires angioblast rescue by fli1a to restore runx1 expression. Interestingly, when vascular endothelial growth factor (Vegf) signaling is inhibited, HSC marker expression can still be restored by scl-alpha in etsrp morphants, whereas the rescue of arterial ephrinb2a expression is blocked. Furthermore, both scl isoforms partially rescue runx1 but not ephrinb2a expression in embryos deficient in Vegf signaling. Our data suggest that downstream of etsrp, scl-alpha and fli1a specify the angioblasts, whereas scl-beta further initiates HSC specification from this angioblast population, and that Vegf signaling acts upstream of scl-beta during definitive hematopoiesis.

Interleukin-1β downregulates the L-type Ca2+ channel activity by depressing the expression of channel protein in cortical neurons

Interleukin‐1β (IL‐1β), a proinflammatory cytokine, has been involved in various diseases of the central nervous system (CNS). Due to the diverse, “contradictory” effects of IL‐1β on neurons during insults to the brain, the mechanisms underlying these effects have not been elucidated. Calcium influx through the L‐type Ca2+ channels (LCCs) is believed to play a critical role in the cascade of biochemical events leading to neuron death in these pathophysiological conditions. So far, the mechanism of the interaction of IL‐1β and LCCs in the initiation and progression of these diseases is unclear. In this study, we investigate systemically the effects of IL‐1β on the LCCs current, which are believed to be implicated in the cascade of biochemical events leading to neuron death in neuropathological conditions. Using patch clamp, we observe that IL‐1β treatment (10 ng/ml, 24 h) suppresses LCC currents by ∼38%, which made up half of the whole‐cell Ca2+ current determined by nifedipine. IL‐1β does not alter the characteristics of single LCC including current amplitude, open probability, and conductance, but decreases the number of the functioning channel by 40%. Moreover, immunoblot assay exhibits that IL‐1β reduces the expression of LCC proteins by 38∼42% in both whole neuron and plasma membrane fraction, and demonstrates that IL‐1β downregulates the LCC activity via the reduction of LCC density. According to early research pretreatments longer than 12 h may play a crucial role in the neuroprotective effects of IL‐1β, our findings may establish an explanation for the protective effects of this interleukin on neurons in the late stage of injury, and could raise a new issue to clinical treatment for insults to brain.

Bioengineering of functional human induced pluripotent stem cell-derived intestinal grafts

Patients with short bowel syndrome lack sufficient functional intestine to sustain themselves with enteral intake alone. Transplantable vascularized bioengineered intestine could restore nutrient absorption. Here we report the engineering of humanized intestinal grafts by repopulating decellularized rat intestinal matrix with human induced pluripotent stem cell-derived intestinal epithelium and human endothelium. After 28 days of in vitro culture, hiPSC-derived progenitor cells differentiate into a monolayer of polarized intestinal epithelium. Human endothelial cells seeded via native vasculature restore perfusability. Ex vivo isolated perfusion testing confirms transfer of glucose and medium-chain fatty acids from lumen to venous effluent. Four weeks after transplantation to RNU rats, grafts show survival and maturation of regenerated epithelium. Systemic venous sampling and positron emission tomography confirm uptake of glucose and fatty acids in vivo. Bioengineering intestine on vascularized native scaffolds could bridge the gap between cell/tissue-scale regeneration and whole organ-scale technology needed to treat intestinal failure patients.There is a need for humanised grafts to treat patients with intestinal failure. Here, the authors generate intestinal grafts by recellularizing native intestinal matrix with human induced pluripotent stem cell-derived epithelium and human endothelium, and show nutrient absorption after transplantation in rats.

Bioengineering Human Lung Grafts on Porcine Matrix

Objective: Bioengineering of viable, functional, and implantable human lung grafts on porcine matrix. Summary Background Data: Implantable bioartificial organ grafts could revolutionize transplant surgery. To date, several milestones toward that goal have been achieved in rodent models. To make bioengineered organ grafts clinically relevant, scaling to human cells and graft size are the next steps. Methods: We seeded porcine decellularized lung scaffolds with human airway epithelial progenitor cells derived from rejected donor lungs, and banked human umbilical vein endothelial cells. We subsequently enabled tissue formation in whole organ culture. The resulting grafts were then either analyzed in vitro (n = 15) or transplanted into porcine recipients in vivo (n = 3). Results: By repopulating porcine extracellular matrix scaffolds with human endothelial cells, we generated pulmonary vasculature with mature endothelial lining and sufficient anti-thrombotic function to enable blood perfusion. By repopulating the epithelial surface with human epithelial progenitor cells, we created a living, functioning gas exchange graft. After surgical implantation, the bioengineered lung grafts were able to withstand physiological blood flow from the recipients pulmonary circulation, and exchanged gases upon ventilation during the 1-hour observation. Conclusions: Engineering and transplantation of viable lung grafts based on decellularized porcine lung scaffolds and human endothelial and epithelial cells is technically feasible. Further graft maturation will be necessary to enable higher-level functions such as mucociliary clearance, and ventilation-perfusion matching.

On the road to bioartificial organs

Biological organs are highly orchestrated systems with well-coordinated positioning, grouping, and interaction of different cell types within their specialized extracellular environment. Bioartificial organs are intended to be functional replacements of native organs generated through bioengineering techniques and hold the potential to alleviate donor organ shortage for transplantation. The development, production, and evaluation of such bioartificial organs require synergistic efforts of biology, material science, engineering, and medicine. In this review, we highlight the emerging platforms enabling structured assembly of multiple cell types into functional grafts and discuss recent advances and challenges in the development of bioartificial organs, including cell sources, in vitro organ culture, in vivo evaluation, and clinical considerations.

Characterization of two novel small molecules targeting melanocyte development in zebrafish embryogenesis

Melanocytes are pigment cells that are closely associated with many skin disorders, such as vitiligo, piebaldism, Waardenburg syndrome, and the deadliest skin cancer, melanoma. Through studies of model organisms, the genetic regulatory network of melanocyte development during embryogenesis has been well established. This network also seems to be shared with adult melanocyte regeneration and melanoma formation. To identify chemical regulators of melanocyte development and homeostasis, we screened a small‐molecule library of 6000 compounds using zebrafish embryos and identified five novel compounds that inhibited pigmentation. Here we report characterization of two compounds, 12G9 and 36E9, which disrupted melanocyte development. TUNEL assay indicated that these two compounds induced apoptosis of melanocytes. Furthermore, compound 12G9 specifically inhibited the viability of mammalian melanoma cells in vitro. These two compounds should be useful as chemical biology tools to study melanocytes and could serve as drug candidates against melanocyte‐related diseases.