You have accessJournal of UrologyCME1 May 2022MP20-16 CLINICAL TRANSLATION OF ENGINEERED PENILE TISSUE Sita Somara, Teresa Burnette, Namrata Sangha, Lindsey Creahan, Tsghe Abraha, Kathryn Krupp, Tiana Stewart, Torie Westendorf, Brad Damratoski, Lisa Hinshaw, Todd Meinecke, Darren Hickerson, Cynthia Wilkins-Port, Ryan Terlecki, James Yoo, Julie Allickson, Anthony Atala, and John Jackson Sita SomaraSita Somara More articles by this author , Teresa BurnetteTeresa Burnette More articles by this author , Namrata SanghaNamrata Sangha More articles by this author , Lindsey CreahanLindsey Creahan More articles by this author , Tsghe AbrahaTsghe Abraha More articles by this author , Kathryn KruppKathryn Krupp More articles by this author , Tiana StewartTiana Stewart More articles by this author , Torie WestendorfTorie Westendorf More articles by this author , Brad DamratoskiBrad Damratoski More articles by this author , Lisa HinshawLisa Hinshaw More articles by this author , Todd MeineckeTodd Meinecke More articles by this author , Darren HickersonDarren Hickerson More articles by this author , Cynthia Wilkins-PortCynthia Wilkins-Port More articles by this author , Ryan TerleckiRyan Terlecki More articles by this author , James YooJames Yoo More articles by this author , Julie AllicksonJulie Allickson More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and John JacksonJohn Jackson More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002553.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Loss or damage of penile tissue due to trauma and disease are life-defining injuries for those affected. Restoration of sexual function is necessary to support recovering psychologically and reestablish the prospect of leading a full and productive life. Recently, the concept of tissue engineering has been proposed to address the goal of restoring normal anatomical tissue configuration and erectile function. METHODS: A penile construct has been engineered by seeding autologous, ex vivo-expanded smooth muscle and endothelial cells seeded onto a naturally derived acellular corporal tissue matrix that possesses the same architecture as native corpora. The proposed clinical indication for the engineered product was for treatment of damaged penile corpora cavernosa. Autologous endothelial cells (EC) and smooth muscle cells (SMC) were isolated from a biopsy obtained from the patient’s cavernosum. Scaffolding was generated by decellularizing a donor penile body. This acellular scaffold was seeded with cells isolated and expanded from the patient’s biopsy. Once seeded, the engineered penile tissue construct was matured in a bioreactor. RESULTS: A robust process, developed in WFIRM RMCC Process Development, is being translated into GMP Manufacturing for the production of a clinical grade engineered penile tissue construct. In-process and release testing procedures have been established for the utilization of autologous cells and decellularized donor scaffold, to deliver a sterile engineered penile tissue final product. Cells were characterized by studying growth kinetics, identity and purity through immunophenotyping and viability assessments. Decellularized scaffolds are evaluated for acellularity through the absence of DNA and cell nuclei. The final product construct has been characterized for cell viability and distribution on the donor scaffold. CONCLUSIONS: Processes for isolating and expanding the cells, decellularization of a donor penile body and production of a final product construct is currently being been optimized and validated to conform to FDA requirements. An investigational New Drug (IND) application for the project has been approved from the Food and Drug Administration (FDA) for carrying out Phase I safety study for this engineered penile tissue product. Source of Funding: This work was supported by the Army, Navy, NIH, Air Force, VA and Health Affairs to support the AFIRM II effort, under Award No. W81XWH-13-2-0052. The U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick MD 21702-5014 is the awarding and administering acquisition office. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e324 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sita Somara More articles by this author Teresa Burnette More articles by this author Namrata Sangha More articles by this author Lindsey Creahan More articles by this author Tsghe Abraha More articles by this author Kathryn Krupp More articles by this author Tiana Stewart More articles by this author Torie Westendorf More articles by this author Brad Damratoski More articles by this author Lisa Hinshaw More articles by this author Todd Meinecke More articles by this author Darren Hickerson More articles by this author Cynthia Wilkins-Port More articles by this author Ryan Terlecki More articles by this author James Yoo More articles by this author Julie Allickson More articles by this author Anthony Atala More articles by this author John Jackson More articles by this author Expand All Advertisement PDF downloadLoading ...

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-β-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.

Presented below is a methodology for the isolation, expansion, and maintenance of urothelial cells derived from human bladder. Such bladder-derived urothelial cells, taken together with bladder or alternately sourced smooth muscle cells, may be complexed with an appropriately shaped biodegradable scaffold to create regenerative constructs capable of seeding formation of new bladder or bladder-like neo-organs upon implantation in human cystectomy patients.

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.

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.

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.

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).

Surgical reduction of kidney mass (RKM) models have been used to study cardiovascular ramifications of early CKD, however long‐term features of the model have not been well defined. This study characterized a canine RKM model and its use in evaluating efficacy of autologous selected renal cells (SRC) through one‐year post‐nephrectomy by monitoring renal specific, whole animal, and terminal histological parameters. SRC preserve tubular and glomerular functions in rodent nephrectomy models. Dogs underwent 2‐step nephrectomy (RKM) and hemi‐nephrectomized dogs served as controls. Using International Renal Interest Society guidelines, 15 weeks post‐nephrectomy RKM dogs were in late Stage I/early Stage II CKD as defined by sCre (1.6), systolic BP (144–157) and mild proteinuria (UPC 0.2). SRC were then delivered to 4 RKM dogs. All animals demonstrated improving sCre values indicating sufficient nephron mass remained for increased singlular nephron GFR to compensate for RKM and maintain eGFR, thus limiting the sensitivity in detecting filtration related treatment effects. SRC therapy significantly reduced proteinuria and evidence of interstitial tubular disease and juxtamedullary glomerular degeneration. Analysis of urine proteins supports the presence of specific treatment benefit to the tubulointerstitial renal compartment. Since proteinuria is considered to be an early indicator of and a contributor to progression of human and canine CKD, this model may provide a useful approach to evaluate potential therapeutics for CKD and supports continued effort toward developing regenerative medicine products containing SRC.

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.

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.

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.

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.

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.

Urinary pathology requiring urinary diversion, partial or full bladder replacement, is a significant clinical problem affecting similar to 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 neoorgan 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.

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-β signaling pathways related to tubulo-interstitial fibrosis associated with CKD progression was evaluated. Conditioned medium was observed to attenuate TGF-β-induced epithelial–mesenchymal transition (EMT) in vitro in a human proximal tubular cell line (HK2).