Evaluation of a decellularized bronchial patch transplant in a porcine model.

Biological scaffolds for airway reconstruction are an important clinical need and have been extensively investigated experimentally and clinically, but without uniform success. In this study, we evaluated the use of a decellularized bronchus graft for airway reconstruction. Decellularized left bronchi were procured from decellularized porcine lungs and utilized as grafts for airway patch transplantation. A tracheal window was created and the decellularized bronchus was transplanted into the defect in a porcine model. Animals were euthanized at 7 days, 1 month, and 2 months post-operatively. Histological analysis, immunohistochemistry, scanning electron microscopy, and strength tests were conducted in order to evaluate epithelialization, inflammation, and physical strength of the graft. All pigs recovered from general anesthesia and survived without airway obstruction until the planned euthanasia timepoint. Histological and electron microscopy analyses revealed that the decellularized bronchus graft was well integrated with native tissue and covered by an epithelial layer at 1 month. Immunostaining of the decellularized bronchus graft was positive for CD31 and no difference was observed with immune markers (CD3, CD11b, myeloperoxidase) at two months. Although not significant, tensile strength was decreased after one month, but recovered by two months. Decellularized bronchial grafts show promising results for airway patch reconstruction in a porcine model. Revascularization and re-epithelialization were observed and the immunological reaction was comparable with the autografts. This approach is clinically relevant and could potentially be utilized for future applications for tracheal replacement.

Comparative immunogenicity of decellularized wild type and alpha 1,3 galactosyltransferase knockout pig lungs.

Decellularized pig lungs recellularized with human lung cells offer a novel approach for organ transplantation. However, the potential immunogenicity of decellularized pig lungs following exposure to human tissues has not been assessed. We found that exposure of native lungs from wildtype and transgenic pigs lacking alpha (1,3)-galactosyltransferase (α-gal KO) to sera from normal healthy human volunteers demonstrated similar robust IgM and IgG immunoreactivity, comparably decreased in decellularized lungs. Similar results were observed with sera from patients who had previously undergone transcutaneous porcine aortic valve replacement (TAVR) or from patients with increased circulating anti-α-gal IgE antibodies (α-gal syndrome). Depleting anti-α-gal antibodies from the sera demonstrated both specificity of α-gal immunoreactivity and also residual immunoreactivity similar between wildtype and α-gal KO pig lungs. Exposure of human monocytes and macrophages to native wildtype lungs demonstrated greater induction of M2 phenotype than native α-gal KO pig lungs, which was less marked with decellularized lungs of either type. Overall, these results demonstrate that native wildtype and α-gal KO pig lungs provoke similar immune responses that are comparably decreased following decellularization. This provides a further platform for potential use of decellularized pig lungs in tissue engineering approaches and subsequent transplantation schemes but no obvious overall immunologic advantage of utilizing lungs obtained from α-gal KO pigs.

Evaluation of the host immune response to decellularized lung scaffolds derived from α-Gal knockout pigs in a non-human primate model.

Whole organ tissue engineering is a promising approach to address organ shortages in many applications, including lung transplantation for patients with chronic pulmonary disease. Engineered lungs may be derived from animal sources after removing cellular content, exposing the extracellular matrix to serve as a scaffold for recellularization with human cells. However, the use of xenogeneic tissue sources in human transplantation raises concerns due to the presence of the antigenic Gal epitope. In the present study, lungs from wild type or α-Gal knockout pigs were harvested, decellularized, and implanted subcutaneously in a non-human primate model to evaluate the host immune response. The decellularized porcine implants were compared to a sham surgery control, as well as native porcine and decellularized macaque lung implants. The results demonstrated differential profiles of circulating and infiltrating immune cell subsets and histological outcomes depending on the implanted tissue source. Upon implantation, the decellularized α-Gal knockout lung constructs performed similarly to the decellularized wild type lung constructs. However, upon re-implantation into a chronic exposure model, the decellularized wild type lung constructs resulted in a greater proportion of infiltrating CD45+ cells, including CD3+ and CD8+ cytotoxic T-cells, likely mediated by an increase in production of Gal-specific antibodies. The results suggest that removal of the Gal epitope can potentially reduce adverse inflammatory reactions associated with chronic exposure to engineered organs containing xenogeneic components.

Selected renal cells modulate disease progression in rodent models of chronic kidney disease via NF-κB and TGF-ß1 pathways.

AIM: Identification of mechanistic pathways for selected renal cell (SRC) therapeutic bioactivity in rodent models of chronic kidney disease. MATERIALS & METHODS: In vivo and in vitro functional bioassays applied to investigate regenerative outcomes associated with delivery of SRC to diseased rodent kidney. RESULTS: In vivo, SRC reduces chronic infiltration by monocytes/macrophages. SRC attenuates NF-κB and PAI-1 responses while simultaneously promoting host tubular cell expansion through trophic cues. In vitro, SRC-derived conditioned media attenuates TNF-α-induced NF-κB response, TGF-ß-mediated PAI-1 response and increases expression of transcripts associated with cell cycle regulation. Observed bioactive responses were from vesicle and nonvesicle-associated factors, including specific miRNAs. CONCLUSION: We identify a paracrine mechanism for SRC immunomodulatory and trophic cues on host renal tissues, catalyzing long-term functional benefits in vivo.

Potency evaluation of tissue engineered and regenerative medicine products.

Methodologies for the rigorous and quantitative evaluation of biological activity or potency are an essential aspect of the developmental pathway for all biologic product candidates. Such assays typically leverage key mechanistic pathways demonstrated to mediate observed therapeutic outcomes. Tissue engineered/regenerative medicine (TE/RM) therapeutics include cell based therapies as well as engineered tissues and neo-organs for which clarity regarding the mechanism or mechanisms of action may not be forthcoming. Here, we discuss how strategies for the development of potency assays for TE/RM product candidates may harness potential mechanisms of action or other therapeutically relevant bioactivity along with cell number and viability. As the pipeline for TE/RM product candidates expands through 2014 and beyond, the establishment of a defined framework for potency assays will facilitate successful translational outcomes.

Isolation of pulsatile cell bodies from esophageal tissue.

Pulsatile cell bodies, three-dimensional cell clusters with satellite streaming cells, can be isolated from -esophageal tissue. One of the key features of these clusters is that they pulsate at rhythmic rates and demonstrate contractility under several in vitro conditions. Their ability to pulsate appears to be due to the presence of interstitial cells of Cajal (ICC), which mediate signal transmission from nerve to muscle cells. As predicted, the cells comprising these clusters express phenotypic and genotypic markers characteristic of smooth and skeletal muscle, neuronal, and epithelial cells. Because of the critical role of ICC in gastrointestinal tract motility, loss of function in these cells can result in a variety of pathologies. Cultures of pulsatile cell bodies may have utility as an in vitro model to study tissue engineering and regenerative medicine approaches to treating defects in gastrointestinal rhythmicity due to disease or injury.

Ex vivo culture and separation of functional renal cells.

The following methods outline the procedures for isolating primary renal cells from kidney tissue via enzymatic digestion, followed by their culture, harvest, and then fractionation of renal subpopulations from primary culture. The current methods describe procedures to sub-fractionate biologically active cells that have been used to treat and stabilize renal function in models of chronic kidney disease (Kelley et al. Am J Physiol Renal Physiol 299(5):F1026-F1039, 2010).

Migration assay to evaluate cellular interactions with biomaterials for tissue engineering/regenerative medicine applications.

Regenerative medicine and tissue engineering approaches for solving current medical dilemmas such as organ failure, congenital defect, or reconstruction following disease or trauma typically require specific considerations regarding biomaterial selection, identification of key cell types, and applicable surgical techniques (Lanza et al. Principles of tissue engineering, Academic, 2007; Kikuchi, Kanama., Quart Rev 24:51-67, 2007). The ability to evaluate these components in vitro under conditions which simulate relevant in vivo environments can reduce development risks including time and money costs associated with early-stage product development. Similarly, such methods can be useful in making progress in researching features of natural and synthetic biomaterial such as porosity, strength, surface topography, and functionalization, and their singular or collective effects on cell behavior (Kikuchi and Kanama., Quart Rev 24:51-67, 2007; Furth et al. Biomaterials 28:5068-5073, 2007; Mieszawska and Kaplan., BMC Biol 8:59, 2010).Adhesion, migration, and gene and protein expression are all cell behaviors that can be affected by properties of a chosen biomaterial and vary based upon organ system (Cornwell et al. J Biomater Res 71A:55-62, 2004; David et al. Tissue Eng 8(5):787-798, 2002). Understanding of these properties and their role in combination with biomaterial in remodeling is sought in order to fully harness and direct regeneration (Lanza et al. Principles of tissue engineering, Academic Press, 2007; Mieszawska and Kaplan. BMC Biol 8:59, 2010; Matragotri and Lahann J. Nat Mater 8:15-23, 2009).

Formulation of selected renal cells for implantation into a kidney.

Delivery of cells to organs has primarily relied on formulating the cells in a nonviscous liquid carrier. We have developed a methodology to isolate selected renal cells (SRC) that have provided functional stability to damaged kidneys in preclinical models (Kelley et al. Poster presentation at 71st scientific sessions of American diabetes association , 2011; Kelley et al. Oral presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS)-North America annual conference, 2010; Presnell et al. Tissue Eng Part C Methods 17:261-273, 2011; Kelley et al. Am J Physiol Renal Physiol 299:F1026-F1039, 2010). In order to facilitate SRC injection into the kidney of patients who have chronic kidney disease, we have developed a strategy to immobilize the cells in a hydrogel matrix. This hydrogel (gelatin) supports cells by maintaining them in a three-dimensional state during storage and shipment (both at cold temperatures) while facilitating the delivery of cells by liquefying when engrafting into the kidney. This chapter will define a method for the formulation of the kidney epithelial cells within a hydrogel.

Tissue engineering of esophagus and small intestine in rodent injury models.

Regenerative constructs composed of synthetically sourced, biodegradable biomaterials seeded with smooth muscle-like cells have been leveraged to mediate regeneration of bladder and bladder-like neo-organs. Here, we describe how such constructs may be applied to catalyze regeneration of esophagus and small intestine in preclinical rodent models.

A population of selected renal cells augments renal function and extends survival in the ZSF1 model of progressive diabetic nephropathy.

New treatment paradigms that slow or reverse progression of chronic kidney disease (CKD) are needed to relieve significant patient and healthcare burdens. We have shown that a population of selected renal cells (SRCs) stabilized disease progression in a mass reduction model of CKD. Here, we further define the cellular composition of SRCs and apply this novel therapeutic approach to the ZSF1 rat, a model of severe progressive nephropathy secondary to diabetes, obesity, dyslipidemia, and hypertension. Injection of syngeneic SRCs into the ZSF1 renal cortex elicited a regenerative response that significantly improved survival and stabilized disease progression to renal structure and function beyond 1 year posttreatment. Functional improvements included normalization of multiple nephron structures and functions including glomerular filtration, tubular protein handling, electrolyte balance, and the ability to concentrate urine. Improvements to blood pressure, including reduced levels of circulating renin, were also observed. These functional improvements following SRC treatment were accompanied by significant reductions in glomerular sclerosis, tubular degeneration, and interstitial inflammation and fibrosis. Collectively, these data support the utility of a novel renal cell-based approach for slowing renal disease progression associated with diabetic nephropathy in the setting of metabolic syndrome, one of the most common causes of end-stage renal disease.

Extension of bladder-based organ regeneration platform for tissue engineering of esophagus.

Recent successes in regenerative medicine and tissue engineering of bladder and bladder-like neo-organs have leveraged regenerative constructs composed of a biodegradable scaffold seeded with a population of smooth muscle cells. We have shown that such smooth muscle cells are isolatable from adipose and other sources alternate to the primary organ. We hypothesize that this regenerative platform is not limited to regeneration of bladder and bladder-like neo-organs, but rather represents a foundational technology platform broadly applicable for regeneration of laminarly organized hollow organs. Using esophagus as an illustrative example in support of this hypothesis, we demonstrate that patch constructs composed of adipose-derived smooth muscle cells seeded on a biodegradable matrix catalyze complete regeneration of the esophageal wall in a rodent model of esophageal injury. By implication, such regenerative constructs may potentially be used to mediate the regeneration of any laminarly organized tubular organ.

Regeneration of native-like neo-urinary tissue from nonbladder cell sources.

Urinary pathology requiring urinary diversion, partial or full bladder replacement, is a significant clinical problem affecting ~14,000 individuals annually in the United States alone. The use of gastrointestinal tissue for urinary diversion or bladder reconstruction/replacement surgeries is frequently associated with complications. To try and alleviate or reduce the frequency of these complications, tissue engineering and regenerative medicine strategies have been developed using bio-absorbable materials seeded with cells derived from the bladder. However, bladder-sourced cells may not always be suitable for such applications, especially in patients with bladder cancer. In this study, we describe the isolation and characterization of smooth muscle cells (SMCs) from porcine adipose and peripheral blood that are phenotypically and functionally indistinguishable from bladder-derived SMCs. In a preclinical Good Laboratory Practice study, we demonstrate that autologous adipose- and peripheral blood-derived SMCs may be used to seed synthetic, biodegradable tubular scaffold structures and that implantation of these seeded scaffolds into a porcine cystectomy model leads to successful de novo regeneration of a tubular neo-organ composed of urinary-like neo-tissue that is histologically identical to native bladder. The ability to create urologic structures de novo from scaffolds seeded by autologous adipose- or peripheral blood-derived SMCs will greatly facilitate the translation of urologic tissue engineering technologies into clinical practice.

Regeneration of rodent small intestine tissue following implantation of scaffolds seeded with a novel source of smooth muscle cells.

AIMS: To apply an organ regeneration platform technology of autologous smooth muscle cell/biomaterial combination products, previously demonstrated to be successful for urinary tissue regeneration, to the regeneration of the small intestine. MATERIALS & METHODS: Patch and tubular constructs were implanted in rodent small intestines and histologically evaluated over a time course for evidence of regeneration of the laminarly organized neo-mucosa and muscle layers. RESULTS: Laminarly organized neo-mucosa and muscle layer bundles are demonstrated as early as 8 weeks postimplantation. CONCLUSION: An organ regeneration technology platform of autologous smooth muscle cell/biomaterial combination products can be extended to the regeneration of the small intestine.

Organ specific regenerative markers in peri-organ adipose: kidney.

BACKGROUND: Therapeutically bioactive cell populations are currently understood to promote regenerative outcomes in vivo by leveraging mechanisms of action including secretion of growth factors, site specific engraftment and directed differentiation. Constitutive cellular populations undoubtedly participate in the regenerative process. Adipose tissue represents a source of therapeutically bioactive cell populations. The potential of these cells to participate in various aspects of the regenerative process has been demonstrated broadly. However, organ association of secretory and developmental markers to specific peri-organ adipose depots has not been investigated. To characterize this topographical association, we explored the potential of cells isolated from the stromal vascular fraction (SVF) of kidney sourced adipose to express key renal associated factors. RESULTS: We report that renal adipose tissue is a novel reservoir for EPO expressing cells. Kidney sourced adipose stromal cells demonstrate hypoxia regulated expression of EPO and VEGF transcripts. Using iso-electric focusing, we demonstrate that kidney and non-kidney sourced adipose stromal cells present unique patterns of EPO post-translational modification, consistent with the idea that renal and non-renal sources are functionally distinct adipose depots. In addition, kidney sourced adipose stromal cells specifically express the key renal developmental transcription factor WT1. CONCLUSIONS: Taken together, these data are consistent with the notion that kidney sourced adipose stromal (KiSAS) cells may be primed to recreate a regenerative micro-environment within the kidney. These findings open the possibility of isolating solid-organ associated adipose derived cell populations for therapeutic applications in organ-specific regenerative medicine products.

Smooth muscle phenotypic diversity is mediated through alterations in myocardin gene splicing.

Myocardin (MYOCD) is a smooth and cardiac muscle-specific transcriptional coactivator that is required for the proper expression of contraction-related genes. Through its function to transactivate effector genes, MYOCD plays an essential role in mediating the switch between contractile and non-contractile phenotypes, particularly in smooth muscle cells (SMC). There are at least two known transcript variants of MYOCD that are expressed in SMC, differing only by the presence (+) or absence (Δ) of Exon 11. To date, no functional role has been assigned to the domain encoded by Exon 11, nor have any notable differences between the ability of each isoform to activate contraction-related genes been observed. In this study we compared sequences for Exon 11 among several mammalian species and identified a highly conserved, putative target sequence for glycogen synthase kinase 3 (GSK3) phosphorylation, suggesting a regulatory role for Exon 11 that can be modulated by alternative splicing. The function of Exon 11 was investigated by altering MYOCD splice selection in cultured porcine SMC with small interfering RNAs (siRNA) and specific chemical inhibitors, resulting in a relative increase in expression of ΔExon 11 variants in the endogenous pool of MYOCD mRNA. The relative increase in ΔExon 11 mRNAs correlated with a reduction of contractile phenotype in the porcine SMC as evidenced by morphological assessment and molecular analysis of effector genes. Together, these data suggest that MYOCD ΔExon 11 may participate in modulating SMC phenotype, potentially acting as a dominant-negative repressor of contraction-related genes.

Expansion of the human adipose-derived stromal vascular cell fraction yields a population of smooth muscle-like cells with markedly distinct phenotypic and functional properties relative to mesenchymal stem cells.

Adipose tissue contains a heterogeneous cell population composed of endothelial cells, adipocytes, smooth muscle cells (SMC), and mesenchymal progenitors and stromal cells that meet the criteria put forth by the International Society for Cellular Therapy as defining mesenchymal stem cells (MSC). In this study, we expanded the stromal vascular fraction (SVF) of human adipose tissue and characterized the resulting adherent primary cell cultures by quantitative reverse transcription-polymerase chain reaction, antigen expression, protein fingerprinting, growth kinetics, in vitro tri-lineage differentiation bioactivity, and functional responses to small molecules modulating SMC-related developmental pathways and compared the results to those obtained with functionally validated MSC cultures. SVF-derived initial cultures (P0) were expanded in a defined medium that was not optimized for MSC growth conditions, neither were recombinant cytokines or growth factors added to the media to direct differentiation. The adherent cell cultures derived from SVF expansion under these conditions had markedly distinct phenotypic and biological properties relative to functionally validated MSC cultures. SVF-derived adherent cell cultures retained characteristics consistent with the SMC subpopulation within adipose tissue--phenotype, gene, and protein expression--that were independent of passage number and source of SVF (n=4 independent donors). SVF-derived cells presented significantly less robust in vitro tri-lineage differentiation bioactivity relative to validated MSC. Expanded SVF cells and MSC had opposite responses to the thromboxane A2 mimetic U46619, demonstrating an unambiguous functional distinction between the two cell types. Taken together, these data support the conclusions that SVF cells expanded under the conditions described in these studies are accurately described as adipose-derived SMC and represent a cellular subpopulation of adipose SVF that is separate and distinct from other classes of adipose-derived cells.

Functional evaluation of primary renal cell/biomaterial neo-kidney augment prototypes for renal tissue engineering.

Development of a tissue-engineered neo-kidney augment (NKA) requires evaluation of defined, therapeutically relevant cell and cell/biomaterial composites (NKA constructs) for regenerative potential in mammalian kidney. Previous work identified primary renal cell populations that extended survival and improved renal function in a rodent model of chronic kidney disease (CKD). This study extends that work toward the goal of developing NKA by (i) screening in vivo inflammatory and fibrotic responses to acellular biomaterials delivered to healthy rodent renal parenchyma, (ii) evaluating the functionality of renal cell/biomaterial combinations in vitro, (iii) generating NKA constructs by combining therapeutically relevant cell populations with biocompatible biomaterial, and (iv) evaluating in vivo neokidney tissue development in response to NKA constructs delivered to healthy rodent renal parenchyma. Gelatin and hyaluronic acid (HA)-based hydrogels elicited the least inflammatory and fibrotic responses in renal parenchyma relative to polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA) beads or particles and were associated with neovascularization and cellular infiltration by 4 weeks postimplantation. Renal cell populations seeded onto gelatin or HA-based hydrogels were viable and maintained a tubular epithelial functional phenotype during an in vitro maturation of 3 days as measured by transcriptomic, proteomic, secretomic, and confocal immunofluorescence assays. In vivo delivery of cell-seeded NKA constructs (bioactive renal cells + gelatin hydrogels) to healthy rodent renal parenchyma elicited neokidney tissue formation at 1 week postimplantation. To investigate a potential mechanism by which NKA constructs could impact a disease state, the effect of conditioned media on TGF-ß signaling pathways related to tubulo-interstitial fibrosis associated with CKD progression was evaluated. Conditioned medium was observed to attenuate TGF-ß-induced epithelial-mesenchymal transition (EMT) in vitro in a human proximal tubular cell line (HK2).

Increased urothelial cell detection in the primary bladder smooth muscle cell cultures with dual MACS/qRT-PCR approach.

Bladder tissue has been regenerated in humans with neurogenic bladder using an implant produced from autologous urothelial (UC) and smooth muscle cells (SMC) expanded from bladder biopsies seeded onto a biodegradable synthetic scaffold. As the majority of bladder cancers are urothelial carcinomas (aka, transitional cell carcinoma), this 2-cell type autologous sourcing strategy presents significant challenges to product development. Entire bladders have been regenerated in cystectomized animals using a single-cell-type sourcing strategy: implants were seeded with bladder-derived SMC-only. Applying the bladder SMC-only sourcing strategy to produce clinical implants for bladder replacement or urinary diversion in bladder cancer patients requires methods for screening SMC cultures for the presence of potentially cancerous UC cells to provide evidence of SMC culture purity before seeding the scaffold. In this report, we show a 10-fold to 100-fold improvement in the sensitivity of qualitative and quantitative reverse-transcription PCR (qRT-PCR)-based assays for detecting UC positive for Cytokeratin 5 (CK5) in mixed SMC/UC cultures when the cell population was first subjected to magnetic activated cell sorting to enrich for cells expressing the epithelial cell adhesion molecule (known as EPCAM or CD326), a marker known to be present in normal UC and upregulated in the cancerous UC.

Isolation, characterization, and expansion methods for defined primary renal cell populations from rodent, canine, and human normal and diseased kidneys.

Chronic kidney disease (CKD) is a global health problem; the growing gap between the number of patients awaiting transplant and organs actually transplanted highlights the need for new treatments to restore renal function. Regenerative medicine is a promising approach from which treatments for organ-level disorders (e.g., neurogenic bladder) have emerged and translated to clinics. Regenerative templates, composed of biodegradable material and autologous cells, isolated and expanded ex vivo, stimulate native-like organ tissue regeneration after implantation. A critical step for extending this strategy from bladder to kidney is the ability to isolate, characterize, and expand functional renal cells with therapeutic potential from diseased tissue. In this study, we developed methods that yield distinct subpopulations of primary kidney cells that are compatible with process development and scale-up. These methods were translated to rodent, large mammal, and human kidneys, and then to rodent and human tissues with advanced CKD. Comparative in vitro studies demonstrated that phenotype and key functional attributes were retained consistently in ex vivo cultures regardless of species or disease state, suggesting that autologous sourcing of cells that contribute to in situ kidney regeneration after injury is feasible, even with biopsies from patients with advanced CKD.