epithelial-cell-repopulation-preparation-rodent-extracellular-matrix

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

kidney-organoid-generation-at-the-air-liquid-interface

Kidney Organoid Generation at the Air-Liquid Interface

The prevalence of kidney diseases continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four ...

Northwestern University

JESSE BROWN VA MEDICAL CENTER

Northwestern University Feinberg School of Medicine

The bcr/abl fusion in chronic myelogenous leukemia (CML) creates a chimeric tyrosine kinase with dramatically different properties than intact c-abl. In P210 bcr/abl, the bcr portion includes a coiled-coil oligomerization domain (amino acids 1-63) and a grb2-binding site at tyrosine 177 (Tyr177) that are critical for fibroblast transformation, but give variable results in other cell lines. To investigate the role of the coiled-coil domain and Tyr177 in promoting CML, 4 P210 bcr/abl-derived mutants containing different bcr domains fused to abl were constructed. All 4 mutants, Delta(1-63) bcr/abl, (1-63) bcr/abl, Tyr177Phe bcr/abl, and (1-210) bcr/abl exhibited elevated tyrosine kinase activity and conferred factor-independent growth in cell lines. In contrast, differences in the transforming potential of the 4 mutants occurred in our mouse model, in which all mice receiving P210 bcr/abl-expressing bone marrow cells exclusively develop a myeloproliferative disease (MPD) resembling human CML. Of the 4 mutants assayed, only 1-210 bcr/abl, containing both the coiled-coil domain and Tyr177, induced MPD. Unlike full-length P210, this mutant also caused a simultaneous B-cell acute lymphocytic leukemia (ALL). The other 3 mutants, (1-63) bcr/abl, Tyr177Phe bcr/abl, and Delta(1-63) bcr/abl, failed to induce an MPD but instead caused T-cell ALL. These results show that both the bcr coiled-coil domain and Tyr177 are required for MPD induction by bcr/abl and provide the basis for investigating downstream signaling pathways that lead to CML.

The coiled-coil domain and Tyr177 of bcr are required to induce a murine chronic myelogenous leukemia-like disease by bcr/abl.

The t(9;22) chromosomal translocation results in expression of P210(BCR-ABL), a fusion protein necessary for the development of chronic myelogenous leukemia (CML). The constitutive activation of the P210(BCR-ABL) tyrosine kinase results in phosphorylation of multiple signaling pathways leading to the transformed phenotype. Additionally, extracellular interactions between P210(BCR-ABL)-expressing progenitor cells and bone marrow stroma may provide external signals that facilitate CML development. In contrast to the intracellular signaling pathways involved in CML, little is known about how P210(BCR-ABL) expression modifies cell-cell and cell-substratum interactions. To investigate the role of P210(BCR-ABL) in modulating cellular adhesion, we used a highly sensitive and quantitative cell detachment apparatus that measures the strength of association between a population of cells and an adhesive matrix. Our findings show that P210(BCR-ABL) expression increased adhesion nearly 2-fold between the myeloblastic cell line, 32D, and fibronectin compared to a control vector. We then investigated whether abnormal adhesion due to P210(BCR-ABL) expression was caused by its tyrosine kinase activity. A quantitative analysis of cell-fibronectin adhesion found that neither expression of a kinase-inactive P210(BCR-ABL) mutant in 32D cells or attenuation of kinase activity by STI571 (imatinib mesylate) in 32D cells transduced with wild-type P210(BCR-ABL) could correct the nearly 2-fold increase in cell-fibronectin adhesion. Similarly, STI571 treatment of Meg-01 cells, a P210(BCR-ABL)-expressing cell line derived from a patient in blast crisis, failed to inhibit adhesion to fibronectin. Together, our results indicate that changes in adhesion induced by P210(BCR-ABL) are independent of its tyrosine kinase activity.

BCR-ABL-induced adhesion defects are tyrosine kinase-independent.

Chronic myelogenous leukemia (CML) is a biphasic neoplasm of the bone marrow that is precipitated by the Philadelphia chromosome, a t(9;22) balanced translocation that encodes a constitutively activated nonreceptor tyrosine kinase termed P210(BCR-ABL). This oncoprotein has several intracellular functions; however, the most important effect of P210(BCR-ABL) leading to cell transformation is phosphorylation of signaling molecules through a constitutively active tyrosine kinase domain. Despite extensive knowledge of the structure and functional domains of BCR-ABL, its precise function in transformation is not known. Progress has been hampered, in part, by the lack of relevant CML models, as cell culture and in vitro assays do not mimic the pathogenesis of CML. Recently, there has been significant progress toward improving murine models that closely resemble human CML. This has allowed researchers to evaluate critical functions of BCR-ABL and has provided a model to test the efficacy of therapeutic medications that block these pathways. Our laboratory has developed two intersecting research programs to better understand the functioning of P210(BCR-ABL) in leukemogenesis. In one approach, we have developed a murine CML model by transferring HSCs that express BCR-ABL from a retroviral vector. All recipients develop a rapidly fatal MPD that shares several important features with CML. This model has been extremely useful for studying the function of BCR-ABL in the pathogenesis of CML. A second approach utilizes a quantitative cell detachment apparatus capable of measuring small changes in cell adhesion to investigate the mechanism by which P210(BCR-ABL) causes abnormal cell binding. Altered cell adhesion may contribute to the imbalance between proliferation and self-renewal in the hematopoietic progenitor compartment. To better understand the role abnormal adhesion may play in the development of leukemia, we have attempted to correlate the effects of functional P210(BCR-ABL) mutants in regulating adhesion and oncogenicity.

The biology of chronic myelogenous leukemia:mouse models and cell adhesion.

We have previously found that P210BCR-ABL increases the adhesion of hematopoietic cell lines to fibronectin by a mechanism that is independent of tyrosine kinase activity. To investigate the pathway(s) by which P210BCR-ABL influences cell adhesion, we used a quantitative cell adhesion device that can discern small changes in cell adhesion to assay P210BCR-ABL with mutations in several critical domains. We expressed P210BCR-ABL mutants in 32D myeloblast cells and found that binding to fibronectin is mediated primarily by the alpha5beta1 integrin. We performed a structure/function analysis to map domains important for cell adhesion. Increased adhesion was mediated by 3 domains: (1) the N-terminal coiled-coil domain that facilitates oligomerization and F-actin localization; (2) bcr sequences between aa 163 to 210; and (3) F-actin localization through the C-terminal actin-binding domain of c-abl. We compared our adhesion results with the ability of these mutants to cause a chronic myelogenous leukemia (CML)-like disease in a murine bone marrow transplantation assay and found that adhesion to fibronectin did not correlate with the ability of these mutants to cause CML. Together, our results suggest that F-actin localization may play a pivotal role in modulating adhesion but that it is dispensable for the development of CML.

Localization of BCR-ABL to F-actin regulates cell adhesion but does not attenuate CML development.

The success of liver transplantation has increased over the past 20 years due to improved immunosuppressive medications, surgical technique and donor-recipient selection. To date, the number of patients waiting for a liver transplant exceeds the number of transplants performed yearly by over a 2 : 1 ratio. Despite efforts to expand the donor pool, mortality of patients waiting for a liver remains high due to the shortage of donor organs. Herein, we discuss options for liver replacement that are currently under development.

Cellular therapy and bioartificial approaches to liver replacement.

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Il y a une demande croissante d&#39;organes transplantables remplacer les tissus aigu&euml;s et chronique endommag&eacute;s. Cette demande ne peut &ecirc;tre satisfaite par les organes de donneurs disponibles actuellement. Efforts pour fournir une source alternative ont conduit au d&eacute;veloppement de l&#39;orgue d&#39;ing&eacute;nierie, une discipline qui combine la biologie cellulaire, g&eacute;nie tissulaire et cellulaire/transplantation d&#39;organes. Au cours des derni&egrave;res ann&eacute;es, des organes ont &eacute;t&eacute; implant&eacute;s dans les rongeurs b&eacute;n&eacute;ficiaires et ont montr&eacute; modeste fonction. Dans cet article, nous r&eacute;sumer les avanc&eacute;es les plus r&eacute;centes dans ce domaine et fournir une perspective sur les d&eacute;fis de traduire cette nouvelle technologie prometteuse dans une th&eacute;rapie r&eacute;g&eacute;n&eacute;ratrice &eacute;prouv&eacute;e.

Perspectives sur l&#39;ensemble d&#39;organes l&#39;Assembl&eacute;e : vers la transplantation sur demande.

Es gibt eine wachsende Nachfrage nach &uuml;bertragbare Organe, akut und chronisch besch&auml;digten Gewebe zu ersetzen. Diese Forderung kann nicht durch die derzeit verf&uuml;gbaren Spenderorganen gedeckt werden. Bem&uuml;hungen um eine alternative Bezugsquelle f&uuml;hrten zur Entwicklung der Orgel engineering, eine Disziplin, die Zellen-Biologie, Tissue-Engineering und Zelle/Organtransplantation kombiniert. In den letzten Jahren ver&auml;nderte Organe haben in Nagetier-Empf&auml;nger implantiert worden und haben bescheidene Funktion gezeigt. In diesem Artikel Wir fassen die j&uuml;ngsten Fortschritte in diesem Bereich und bieten eine Perspektive auf die Herausforderungen dieser vielversprechende neue Technologie in eine bew&auml;hrte regenerative Therapie zu &uuml;bersetzen.

Perspektiven f&uuml;r ganz-Orgel-Assembly: Bewegung in Richtung Transplantation bei Bedarf.

C&#39;&egrave; una crescente domanda di organi trapiantabili sostituire tessuti acuti e cronicamente danneggiati. Questa domanda non pu&ograve; essere soddisfatte dagli organi dei donatori attualmente disponibili. Gli sforzi per fornire una fonte alternativa hanno portato allo sviluppo di ingegneria, una disciplina che unisce biologia cellulare, ingegneria dei tessuti e delle cellule/trapianto di organo. Negli ultimi anni, ingegnerizzati organi sono stati impiantati nel roditori destinatari e hanno dimostrato la funzione modesta. In questo articolo, abbiamo riassumere i pi&ugrave; recenti progressi in questo campo e fornire una prospettiva sulle sfide di tradurre questa nuova promettente tecnologia comprovata terapia rigenerativa.

Prospettive sull&#39;intero organo assieme: lo spostamento verso il trapianto su richiesta.

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There is an ever-growing demand for transplantable organs to replace acute and chronically damaged tissues. This demand cannot be met by the currently available donor organs. Efforts to provide an alternative source have led to the development of organ engineering, a discipline that combines cell biology, tissue engineering, and cell/organ transplantation. Over the last several years, engineered organs have been implanted into rodent recipients and have shown modest function. In this article, we summarize the most recent advances in this field and provide a perspective on the challenges of translating this promising new technology into a proven regenerative therapy.

Perspectives on whole-organ assembly: moving toward transplantation on demand.

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Hay una demanda creciente de &oacute;rganos transplantables reemplazar tejidos agudos y cr&oacute;nico da&ntilde;ados. Esta demanda no puede cumplirse por los &oacute;rganos de donantes actualmente disponibles. Esfuerzos para proporcionar una fuente alternativa han llevado al desarrollo de la ingenier&iacute;a, una disciplina que combina la biolog&iacute;a celular, ingenier&iacute;a de tejidos y trasplante de &oacute;rgano o c&eacute;lula del &oacute;rgano. En los &uacute;ltimos a&ntilde;os, ingenier&iacute;a de &oacute;rganos se han implantado en los roedores receptores y han mostrado modesta funci&oacute;n. En este art&iacute;culo, resumimos los avances m&aacute;s recientes en este campo y proporcionar una perspectiva sobre los retos de la traducci&oacute;n de esta nueva tecnolog&iacute;a prometedora a una probada terapia regenerativa.

Perspectivas en la Asamblea General-&oacute;rgano: hacia el trasplante en la demanda.

An accurate clinical assessment of hepatic steatosis before transplantation is critical for successful outcomes after liver transplantation, especially if a pathologist is not available at the time of procurement. This prospective study investigated the surgeon's accuracy in predicting hepatic steatosis and organ quality in 201 adult donor livers. A steatosis assessment by a blinded expert pathologist served as the reference gold standard. The surgeon's steatosis estimate correlated more strongly with large-droplet macrovesicular steatosis [ld-MaS; nonparametric Spearman correlation coefficient (rS ) = 0.504] versus small-droplet macrovesicular steatosis (sd-MaS; rS  = 0.398). True microvesicular steatosis was present in only 2 donors (1%). Liver texture criteria (yellowness, absence of scratch marks, and round edges) were mainly associated with ld-MaS (variance = 0.619) and were less associated with sd-MaS (variance = 0.264). The prediction of ≥30% ld-MaS versus <30% ld-MaS was excellent when liver texture criteria were used (accuracy = 86.2%), but it was less accurate when the surgeon's direct estimation of the steatosis percentage was used (accuracy = 75.5%). The surgeon's quality grading correlated with the degree of ld-MaS and the surgeon's steatosis estimate as well as the incidence of poor initial function and primary nonfunction. In conclusion, the precise estimation of steatosis remains challenging even in experienced hands. Liver texture characteristics are more helpful in identifying macrosteatotic organs than the surgeon's actual perception of steatosis. These findings are especially important when histological assessment is not available at the donor's hospital.

Assessment of hepatic steatosis by transplant surgeon and expert pathologist: a prospective, double-blind evaluation of 201 donor livers.

Bioartificial or laboratory-grown organs is a growing field centered on developing replacement organs and tissues to restore body function and providing a potential solution to the shortage of donor organs for transplantation. With the entry of engineered planar tissues, such as bladder and trachea, into clinical studies, an increasing focus is being given to designing complex, three-dimensional solid organs. As tissues become larger, thicker and more complex, the vascular network becomes crucial for supplying nutrients and maintaining viability and growth of the neo-organ. Perfusion decellularization, the process of removing cells from an entire organ, leaves the matrix of the vascular network intact. Organ engineering requires a delicate process of decellularization, sterilization, reseeding with appropriate cells, and organ maturation and stimulation to ensure optimal development. The design of bioreactors to facilitate this sequence of events has been refined to the extent that some bioartificial organs grown in these systems have been transplanted into recipient animals with sustained, though limited, function. This review focuses on the state-of-art in bioreactor development for perfusion-based bioartificial organs and highlights specific design components in need of further refinement.

Bioreactor design for perfusion-based, highly-vascularized organ regeneration.

We would like to welcome our readers to the regenerated Organogenesis and to share with you the reasoning behind the refocusing of this journal toward the development of tissues and organs as a future means to improve the health of patients.

The regeneration of organogenesis.

The severe shortage of suitable donor kidneys limits organ transplantation to a small fraction of patients suffering from end-stage renal failure. Engineering autologous kidney grafts on-demand would potentially alleviate this shortage, thereby reducing healthcare costs, improving quality of life, and increasing longevity for patients suffering from renal failure.

New strategies in kidney regeneration and tissue engineering.

Organ engineering using decellularized scaffolds is a potential long-term solution to donor organ shortage. However, this technology is severely limited by small vessel thrombosis due to incompletely recellularized vessels, resulting in exposure of extracellular matrix (ECM) components to platelets and clotting factors in flowing blood. To address this limitation, we designed a polymer-ECM composite and demonstrated its potential to reduce thrombosis and facilitate re-endothelialization in a vascular graft model. Rat aortas were decellularized using a sequential combination of weak detergents followed by a nuclease treatment that resulted in 96.5±1.3% DNA removal, while ECM components and mechanical properties were well maintained. A biodegradable and biocompatible elastomer poly(1,8 octanediol citrate) (POC, 1wt.%) was infused throughout the ECM at mild conditions (37°C and 45°C) and was functionalized with heparin using carbodiimide chemistry. The polymer-ECM composite significantly reduced platelet adhesion (67.4±8.2% and 82.7±9.6% reduction relative to untreated ECM using one of two processing temperatures, 37°C or 45°C, respectively); inhibited whole blood clotting (85.9±4.3% and 87.0±11.9% reduction relative to untreated ECM at 37°C or 45°C processing temperature, respectively); and supported endothelial cell-and to a lesser extent smooth muscle cell-adhesion in vitro. Taken together, this novel POC composite may provide a solution for thrombosis of small vessel conduits commonly seen in decellularized scaffolds used in tissue engineering applications.

A polymer-extracellular matrix composite with improved thromboresistance and recellularization properties.

Clinical interventions to preserve fertility and restore hormone levels in female patients with therapy-induced ovarian failure are insufficient, particularly for pediatric cancer patients. Laparoscopic isolation of cortical ovarian tissue followed by cryopreservation with subsequent autotransplantation has temporarily restored fertility in at least 27 women who survived cancer, and aided in pubertal transition for one pediatric patient. However, reintroducing cancer cells through ovarian transplantation has been a major concern. Decellularization is a process of removing cellular material, while maintaining the organ skeleton of extracellular matrices (ECM). The ECM that remains could be stripped of cancer cells and reseeded with healthy ovarian cells. We tested whether a decellularized ovarian scaffold could be created, recellularized and transplanted to initiate puberty in mice. Bovine and human ovaries were decellularized, and the ovarian skeleton microstructures were characterized. Primary ovarian cells seeded onto decellularized scaffolds produced estradiol in vitro. Moreover, the recellularized grafts initiated puberty in mice that had been ovariectomized, providing data that could be used to drive future human transplants and have broader implications on the bioengineering of other organs with endocrine function.

Initiation of puberty in mice following decellularized ovary transplant.

Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of physical and biochemical parameters that measure dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Intricately designed bioreactors that house developing tissue are critical to properly recapitulate the in vivo environment, deliver nutrients within perfused media, and monitor physiological parameters of tissue development. Herein, we provide an in-depth description and analysis of two dual-purpose perfusion bioreactors that improve upon current bioreactor designs and enable comparative analyses of ex vivo scaffold recellularization strategies and cell growth performance during long-term maintenance culture of engineered kidney or liver tissues. Both bioreactors are effective at maximizing cell seeding of small-animal organ scaffolds and maintaining cell survival in extended culture. We further demonstrate non-invasive monitoring capabilities for tracking dynamic changes within scaffolds as the native cellular component is removed during decellularization and model human cells are introduced into the scaffold during recellularization and proliferate in maintenance culture. We found that hydrodynamic pressure drop (∆P) across the retained scaffold vasculature is a non-invasive measurement of scaffold integrity. We further show that ∆P, and thus resistance to fluid flow through the scaffold, decreases with cell loss during decellularization and correspondingly increases to near normal values for whole organs following recellularization of kidney or liver scaffolds. Perfused media may be further sampled in real-time to measure soluble biomarkers (e.g. resazurin, albumin, or kidney injury molecule-1) that indicate degree of cellular metabolic activity, synthetic function, or engraftment into the scaffold. Cell growth within bioreactors is validated for primary and immortalized cells, and the design of each bioreactor is scalable to accommodate any three-dimensional scaffold (e.g., synthetic or naturally-derived matrix) that contains conduits for nutrient perfusion to deliver media to growing cells and monitor non-invasive parameters during scaffold repopulation, broadening the applicability of these bioreactor systems.

Dual-purpose bioreactors to monitor non-invasive physical and biochemical markers of kidney and liver scaffold recellularization.

Guided bone regeneration is a new concept of large bone defect therapy, which employs a barrier membrane to afford a protected room for osteogenesis and prevent the invasion of fibroblasts. In this study, we developed a novel barrier membrane made from lyophilized multilayered acellular human amnion membranes (AHAM). After decellularization, the AHAM preserved the structural and biomechanical integrity of the amnion extracellular matrix (ECM). The AHAM also showed minimal toxic effects when cocultured with mesenchymal stem cells (MSCs), as evidenced by high cell density, good cell viability, and efficient osteogenic differentiation after 21-day culturing. The effectiveness of the multilayered AHAM in guiding bone regeneration was evaluated using an in vivo rat tibia defect model. After 6 weeks of surgery, the multilayered AHAM showed great efficiency in acting as a shield to avoid the invasion of the fibrous tissues, stabilizing the bone grafts and inducing the massive bone growth. We hence concluded that the advantages of the lyophilized multilayered AHAM barrier membrane are as follows: preservation of the structural and mechanical properties of the amnion ECM, easiness for preparation and handling, flexibility in adjusting the thickness and mechanical properties to suit the application, and efficiency in inducing bone growth and avoiding fibrous tissues invasion.

ArticleTotal : 2	Year
Publication title Cited by 17	2015
Publication title

Article	Year
The coiled-coil domain and Tyr177 of bcr are required to induce a murine chronic myelogenous leukemia-like disease by bcr/abl. Blood\| PubMed ID: 11929787	2002
BCR-ABL-induced adhesion defects are tyrosine kinase-independent. Blood\| PubMed ID: 12010816	2002
The biology of chronic myelogenous leukemia:mouse models and cell adhesion. Oncogene\| PubMed ID: 12476308	2002
Localization of BCR-ABL to F-actin regulates cell adhesion but does not attenuate CML development. Blood\| PubMed ID: 12791659	2003
Cellular therapy and bioartificial approaches to liver replacement. Current opinion in organ transplantation\| PubMed ID: 22476224	2012
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Assessment of hepatic steatosis by transplant surgeon and expert pathologist: a prospective, double-blind evaluation of 201 donor livers. Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society\| PubMed ID: 23408461	2013
Bioreactor design for perfusion-based, highly-vascularized organ regeneration. Current opinion in chemical engineering\| PubMed ID: 23542907	2013
The regeneration of organogenesis. Organogenesis\| PubMed ID: 23594929	2013
New strategies in kidney regeneration and tissue engineering. Current opinion in nephrology and hypertension\| PubMed ID: 24848937	2014
A polymer-extracellular matrix composite with improved thromboresistance and recellularization properties. Acta biomaterialia\| PubMed ID: 25712388	2015
Initiation of puberty in mice following decellularized ovary transplant. Biomaterials\| PubMed ID: 25736492	2015
Dual-purpose bioreactors to monitor non-invasive physical and biochemical markers of kidney and liver scaffold recellularization. Tissue engineering. Part C, Methods\| PubMed ID: 25929317	2015
Investigating the Potential of Amnion-Based Scaffolds as a Barrier Membrane for Guided Bone Regeneration. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 26158559	2015

Jason A. Wertheim

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