Preclinical biosafety evaluation of cell-based therapies: emerging global paradigms.

Cell-based therapies have the potential to treat a diversity of disease conditions, many representing significant and long-standing unmet medical needs. Certain properties of cell-based therapies, such as differentiation potential and proliferative potential, present safety concerns uniquely distinct from those of small molecule drugs and other macromolecule biologics. These cellular products carry risks associated with localized host tissue response, long-term persistence, ectopic tissue formation, differentiation to undesirable cell and tissue types, uncontrollable biodistribution, tumorigenicity, and immunogenicity. Such risks are generally evaluated in preclinical animal model studies as part of a comprehensive safety program prior to administration in humans. However, safety assessment for these products can be challenging because of inconsistent approaches to product characterization, inadequately defined product parameters that anticipate adverse events, and the lack of standardized approaches in evaluating in vivo host responses. In this symposium, we introduced cell-based therapies as an emerging product class to the Society of Toxicologic Pathology (STP) and highlighted key challenges for consideration during product biosafety evaluation.

Regenerative medicine of the gastrointestinal tract.

Regenerative biology/tissue engineering offers potential solutions for the repair and augmentation of diseased tissues and organs. Tissue engineering technology platforms currently under development for organ regeneration may function in part by recapitulating key mechanistic and signaling pathways associated with embryonic organogenesis. Temporal observations of observed morphological outcomes from the regeneration of tubular organs provide insights into the mechanisms of action associated with the activation of regenerative pathways in preclinical animal models and humans. These include induction of a neo-blastema, regeneration of laminarily organized mural elements (i.e., lamina propria, sub-mucosa, and muscularis), and formation of context appropriate transitional junctions at the point of anastomosis with other tissue elements. These results provide the foundation for a regenerative technology applicable to hollow organs of the gastrointestinal (GI) tract including esophagus and small intestine. Factors affecting the efficacy of observed regenerative outcomes within the GI tract include the roles of vascularization, innervations, and mesenchymal signaling. These will be discussed in the context of an overall mechanism of adult regeneration potentially applicable by the tissue engineering and regenerative medicine industry for continued development of hollow neo-organ products.

Cross-linked gelatin microspheres with continuously tunable degradation profiles for renal tissue regeneration.

Collagen and gelatin-based biomaterials are widely used in tissue engineering applications. Various methods have been reported for the cross-linking of these macromolecules for the purpose of delaying their biodegradation to prolong their in vivo residence (in tissue engineering applications) or tailoring their drug releasing capacity (when used as drug carriers). In this study, a carbodiimide-based cross-linking method, also used in the production of United States Food and Drug Administration-approved products, was employed to obtain differentially cross-linked gelatin beads. The colorimetric determination of the in vitro enzymatic susceptibility of the beads indicated that the resistance to degradation linearly correlated with the concentration of carbodiimide used for the cross-linking reaction. This result was also confirmed in vivo by the histological evaluation of the residence time of orthotopically injected cell-seeded beads. These data would indicate that the production of gelatin-based microbeads with tunable degradation profiles might be applicable toward the development of products that catalyze regeneration of kidney and other solid organs.

Selected renal cells harbor nephrogenic potential.

Selected renal cells (SRCs), a renal epithelial cell-enriched platform, are being advanced as an autologous cell-based therapy for the treatment of chronic kidney disease. However, the mechanism underlying its renal reparative and restorative effects remains to be fully elucidated. In this study, we coupled knowledgebase data with empirical findings to demonstrate that genes differentially expressed by SRCs form interactomes within tubules and glomeruli and mediate a suite of renal developmental activities including epithelial cell differentiation, renal vasculature development, and glomerular and nephron development. In culture, SRCs form organoids which self-assemble into tubules in the presence of a scaffold. Implanted into the kidneys of subtotally nephrectomized rats, SRCs are associated with comma- and S-shaped body cell formation and glomerular development, and improvement in renal filtration indices and renal microarchitecture. These data suggest that SRCs harbor nephrogenic potential, which may explain, at least in part, their therapeutic activity.

Tubular cell-enriched subpopulation of primary renal cells improves survival and augments kidney function in rodent model of chronic kidney disease.

Established chronic kidney disease (CKD) may be identified by severely impaired renal filtration that ultimately leads to the need for dialysis or kidney transplant. Dialysis addresses only some of the sequelae of CKD, and a significant gap persists between patients needing transplant and available organs, providing impetus for development of new CKD treatment modalities. Some postulate that CKD develops from a progressive imbalance between tissue damage and the kidney's intrinsic repair and regeneration processes. In this study we evaluated the effect of kidney cells, delivered orthotopically by intraparenchymal injection to rodents 4-7 wk after CKD was established by two-step 5/6 renal mass reduction (NX), on the regeneration of kidney function and architecture as assessed by physiological, tissue, and molecular markers. A proof of concept for the model, cell delivery, and systemic effect was demonstrated with a heterogeneous population of renal cells (UNFX) that contained cells from all major compartments of the kidney. Tubular cells are known contributors to kidney regeneration in situ following acute injury. Initially tested as a control, a tubular cell-enriched subpopulation of UNFX (B2) surprisingly outperformed UNFX. Two independent studies (3 and 6 mo in duration) with B2 confirmed that B2 significantly extended survival and improved renal filtration (serum creatinine and blood urea nitrogen). The specificity of B2 effects was verified by direct comparison to cell-free vehicle controls and an equivalent dose of non-B2 cells. Quantitative histological evaluation of kidneys at 6 mo after treatment confirmed that B2 treatment reduced severity of kidney tissue pathology. Treatment-associated reduction of transforming growth factor (TGF)-ß1, plasminogen activator inhibitor (PAI)-1, and fibronectin (FN) provided evidence that B2 cells attenuated canonical pathways of profibrotic extracellular matrix production.

Early cellular and stromal responses in regeneration versus repair of a mammalian bladder using autologous cell and biodegradable scaffold technologies.

PURPOSE: Internal organ regeneration holds promise for changing medical technology and decreasing organ shortages. Current medical treatment for internal organ failure is largely limited to organ transplantation. A construct composed of synthetic biopolymer with autologous cells has shown long-term clinical benefit in patients undergoing augmentation cystoplasty. However, to our knowledge early cellular and stromal events during bladder regeneration have not been elucidated. MATERIALS AND METHODS: In situ cellular responses to 2 biopolymer implants, including a poly(lactic-co-glycolic acid) (Sigma-Aldrich) based biodegradable mesh scaffold with autologous urothelial and smooth muscle cells (construct) and a poly(lactic-co-glycolic acid) based biodegradable mesh scaffold alone without cells (scaffold), were compared in a canine model of augmentation cystoplasty. Healing events were correlated with urodynamic assessments. RESULTS: Construct implants regenerated baseline urodynamics as early as 4 months after implantation. Urodynamics following scaffold implantation failed to return to baseline by study termination at 9 months. Functional differences elicited by construct and scaffold implants correlated with structural differences in the neotissues. Construct stroma had greater vascularization with gently folded, interwoven connective tissue elements. Scaffold stroma was dense, haphazardly organized connective tissue. Urothelium regenerated in response to construct and scaffold implantation. However, only construct had normal stroma, well developed detrusor and abundant alpha-smooth muscle actin (Vector Laboratories, Burlingame, California) cell staining at early time points, leading to a structurally and functionally complete regenerated bladder wall at 9 months. CONCLUSIONS: Early cellular and stromal events distinguish healing processes that lead to bladder wall regeneration or repair. Construct implants containing cells elicit early healing processes that culminate with the regeneration of complete mucosal and muscular components, whereas the response to scaffold implantation is consistent with reparative healing, that is with mucosal growth but incomplete tissue layer development.

Tissue engineering and regenerative medicine: role of toxicologic pathologists for an emerging medical technology.

Tissue Engineering Regenerative Medical (TERM) products are a new technology currently in human clinical testing for a variety of unmet medical needs involving tissue and organ dysfunction and failure. Safety evaluation of TERM products overlaps 3 established product paradigms: pharmaceuticals (biologically active substances), transplantation (cells or tissue), and devices (biomaterials). As TERM products recapitulate organ or tissue structure and function with unique biological activity and characteristics, they require new preclinical paradigms to bring TERM products through to clinical trials. Establishing TERM-product safety programs requires broad-based knowledge of tissue and organ homeostasis, regenerative biology, and translational medicine to design new preclinical paradigms. Therefore, toxicologic pathologists have a compelling scientific role in evaluating TERM products, characterizing tissue responses, and helping distinguish optimal (regeneration) from deficient or incomplete outcomes indicative of substandard functionality (repair). As new-tissue engineering and regenerative medical technologies develop for tissue and organ regeneration, the toxicologic pathologist will be asked to develop novel testing, reevaluate established toxicologic diagnostic criteria, and reinterpret tissue responses that may extend beyond current standards.

Enhancing Tissue Engineering and Regenerative Medicine Product Commercialization: The Role of Science in Regulatory Decision-Making for the TE/RM Product Development.

TERMIS-AM Industry Committee (TERMIS-AM/IC), in collaboration with the TERMIS-Europe (EU)/IC, conducted a symposium involving the European Medicines Agency and the U.S. Food and Drug Administration (FDA) toward building an understanding of the rational basis for regulatory decision-making and providing a framework for decisions made during the evaluation of safety and efficacy of TE/RM technologies. This symposium was held in August 2012 during the TERMIS-WC in Vienna, Austria. Emerging from this international initiative by the European Union and the United States, representatives from the respective agencies demonstrated that there are ongoing interagency efforts for developing common national practices toward harmonization of regulatory requirements for the TE/RM products. To extend a broad-based understanding of the role of science in regulatory decision-making, TERMIS-AM/IC, in cooperation with the FDA, organized a symposium at the 2014 TERMIS-AM Annual Meeting, which was held in Washington, DC. This event provided insights from leaders in the FDA and TERMIS on the current status of regulatory approaches for the approved TE/RM products, the use of science in making regulatory decisions, and TE/RM technologies that are in the development pipeline to address unmet medical needs. A far-ranging discussion with FDA representatives, industrialists, physicians, regenerative medicine biologists, and tissue engineers considered the gaps in today's scientific and regulatory understanding of TE/RM technologies. The identified gaps represent significant opportunities to advance TE/RM technologies toward commercialization.

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.

Identification of platform-independent gene expression markers of cisplatin nephrotoxicity.

Within the International Life Sciences Institute Committee on Genomics, a working group was formed to focus on the application of microarray technology to preclinical assessments of drug-induced nephrotoxicity. As part of this effort, Sprague-Dawley rats were treated with the nephrotoxicant cisplatin at doses of 0.3-5 mg/kg over a 4- to 144-hr time course. RNA prepared from these animals was run on a variety of microarray formats at multiple sites. A set of 93 differentially expressed genes associated with cisplatin-induced renal injury was identified on the National Institute of Environmental Health Sciences (NIEHS) custom cDNA microarray platform using quadruplicate measurements of pooled animal RNA. The reproducibility of this profile of statistically significant gene changes on other platforms, in pooled and individual animal replicate samples, and in an independent study was investigated. A good correlation in response between platforms was found among the 48 genes in the NIEHS data set that could be matched to probes on the Affymetrix RGU34A array by UniGene identifier or sequence alignment. Similar results were obtained with genes that could be linked between the NIEHS and Incyte or PHASE-1 arrays. The degree of renal damage induced by cisplatin in individual animals was commensurate with the number of differentially expressed genes in this data set. These results suggest that gene profiles linked to specific types of tissue injury or mechanisms of toxicity and identified in well-performed replicated microarray experiments may be extrapolatable across platform technologies, laboratories, and in-life studies.

Overview on the application of transcription profiling using selected nephrotoxicants for toxicology assessment.

Microarrays allow for the simultaneous measurement of changes in the levels of thousands of messenger RNAs within a single experiment. As such, the potential for the application of transcription profiling to preclinical safety assessment and mechanism-based risk assessment is profound. However, several practical and technical challenges remain. Among these are nomenclature issues, platform-specific data formats, and the lack of uniform analysis methods and tools. Experiments were designed to address biological, technical, and methodological variability, to evaluate different approaches to data analysis, and to understand the application of the technology to other profiling methodologies and to mechanism-based risk assessment. These goals were addressed using experimental information derived from analysis of the biological response to three mechanistically distinct nephrotoxins: cisplatin, gentamicin, and puromycin aminonucleoside. In spite of the technical challenges, the transcription profiling data yielded mechanistically and topographically valuable information. The analyses detailed in the articles from the Nephrotoxicity Working Group of the International Life Sciences Institute Health and Environmental Sciences Institute suggest at least equal sensitivity of microarray technology compared to traditional end points. Additionally, microarray analysis of these prototypical nephrotoxicants provided an opportunity for the development of candidate bridging biomarkers of nephrotoxicity. The potential future extension of these applications for risk assessment is also discussed.

Identification of putative gene based markers of renal toxicity.

This study, designed and conducted as part of the International Life Sciences Institute working group on the Application of Genomics and Proteomics, examined the changes in the expression profile of genes associated with the administration of three different nephrotoxicants--cisplatin, gentamicin, and puromycin--to assess the usefulness of microarrays in the understanding of mechanism(s) of nephrotoxicity. Male Sprague-Dawley rats were treated with daily doses of puromycin (5-20 mg/kg/day for 21 days), gentamicin (2-240 mg/kg/day for 7 days), or a single dose of cisplatin (0.1-5 mg/kg). Groups of rats were sacrificed at various times after administration of these compounds for standard clinical chemistry, urine analysis, and histological evaluation of the kidney. RNA was extracted from the kidney for microarray analysis. Principal component analysis and gene expression-based clustering of compound effects confirmed sample separation based on dose, time, and degree of renal toxicity. In addition, analysis of the profile components revealed some novel changes in the expression of genes that appeared to be associated with injury in specific portions of the nephron and reflected the mechanism of action of these various nephrotoxicants. For example, although puromycin is thought to specifically promote injury of the podocytes in the glomerulus, the changes in gene expression after chronic exposure of this compound suggested a pattern similar to the known proximal tubular nephrotoxicants cisplatin and gentamicin; this prediction was confirmed histologically. We conclude that renal gene expression profiling coupled with analysis of classical end points affords promising opportunities to reveal potential new mechanistic markers of renal toxicity.

The future of regenerative medicine: urinary system.

Regeneration of tissues and organs is now within the technological reach of modern medicine. With such advancements, substantial improvements to existing standards-of-care are very real possibilities. This review will focus on regenerative medicine approaches to treating specific maladies of the bladder and kidney, including the biological basis of regeneration and the history of regenerative medicine in the urinary system. Current clinical management approaches will be presented within the context of future directions including cell-based regenerative therapies.

Hurdles in tissue engineering/regenerative medicine product commercialization: a pilot survey of governmental funding agencies and the financial industry.

The Tissue Engineering and Regenerative Medicine International Society of the Americas (TERMIS-AM) Industry Committee conducted a semiquantitative opinion survey in 2010 to delineate potential hurdles to commercialization perceived by the TERMIS constituency groups that participate in the stream of technology commercialization (academia, start-up companies, development-stage companies, and established companies). A significant hurdle identified consistently by each group was access to capital for advancing potential technologies into development pathways leading to commercialization. A follow-on survey was developed by the TERMIS-AM Industry Committee to evaluate the financial industry's perspectives on investing in regenerative medical technologies. The survey, composed of 15 questions, was developed and provided to 37 investment organizations in one of three sectors (governmental, private, and public investors). The survey was anonymous and confidential with sector designation the only identifying feature of each respondent's organization. Approximately 80% of the survey was composed of respondents from the public (n=14) and private (n=15) sectors. Each respondent represents one investment organization with the potential of multiple participants participating to form the organization's response. The remaining organizations represented governmental agencies (n=8). Results from this survey indicate that a high percentage (<60%) of respondents (governmental, private, and public) were willing to invest >$2MM into regenerative medical companies at the different stages of a company's life cycle. Investors recognized major hurdles to this emerging industry, including regulatory pathway, clinical translation, and reimbursement of these new products. Investments in regenerative technologies have been cyclical over the past 10-15 years, but investors recognized a 1-5-year investment period before the exit via Merger and Acquisition (M&A). Investors considered musculoskeletal products and their top technology choice with companies in the clinical stage of development being the most preferred investment targets. All sectors indicated a limited interest in early-stage start-up companies potentially explaining why start-up companies have struggled to access to capital and investors based their investment on the stage of a company's life cycle, reflecting each sector's risk tolerance, exit strategy, time of holding an investment, and investment strategy priorities. Investors highlighted the limited number of regenerative medical companies that have achieved commercial status as a basis for why public investors have been approached by so few companies. Based on respondents to this survey, regenerative medical sponsors seeking capital from the financial industry must keep the explanation of their technology simple, since all sectors considered regenerative medical technology as difficult to evaluate. This survey's results indicate that under the current financial environment, many regenerative medical companies must consider codevelopment or even M&A as nondilutive means of raising capital. The overall summary for this survey highlights the highly varied goals and motivations for the various sectors of the government and financial industries.

Characterization of the human smooth muscle cell secretome for regenerative medicine.

Smooth muscle cells (SMC) play a central role in maintaining the structural and functional integrity of muscle tissue. Little is known about the early in vitro events that guide the assembly of 'bioartificial tissue' (constructs) and recapitulate the key aspects of smooth muscle differentiation and development before surgical implantation. Biomimetic approaches have been proposed that enable the identification of in vitro processes which allow standardized manufacturing, thus improving both product quality and the consistency of patient outcomes. One essential element of this approach is the description of the SMC secretome, that is, the soluble and deposited factors produced within the three-dimensional (3D) extracellular matrix (ECM) microenvironment. In this study, we utilized autologous SMC from multiple tissue types that were expanded ex vivo and generated with a rigorous focus on operational phenotype and genetic stability. The objective of this study was to characterize the spatiotemporal dynamics of the first week of organoid maturation using a well-defined in vitro-like, 3D-engineered scale model of our validated manufacturing process. Functional proteomics was used to identify the topological properties of the networks of interacting proteins that were derived from the SMC secretome, revealing overlapping central nodes related to SMC differentiation and proliferation, actin cytoskeleton regulation, and balanced ECM accumulation. The critical functions defined by the Ingenuity Pathway Analysis included cell signaling, cellular movement and proliferation, and cellular and organismal development. The results confirm the phenotypic and functional similarity of the SMC generated by our platform technology at the molecular level. Furthermore, these data validate the biomimetic approaches that have been established to maintain manufacturing consistency.

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.

Hurdles in tissue engineering/regenerative medicine product commercialization: a survey of North American academia and industry.

The Tissue Engineering and Regenerative Medicine International Society-North America (TERMIS-NA) Industry Committee was formed in February 2009 to address the common roadblocks (i.e., hurdles) in the commercialization of tissue engineering/regenerative medicine products for its members. A semiquantitative online opinion survey instrument that delineated potentially sensitive hurdles to commercialization in each of the TERMIS constituency groups that generally participate in the stream of technology commercialization (academia, startup companies, development-stage companies, and established companies) was developed. The survey was opened to each of the 863 members of TERMIS-NA for a period of 5 weeks from October to November 2009. By its conclusion, 215 members (25%) had responded. Their proportionate numbers were closely representative of TERMIS-NA constituencies. The resulting data delineate what each group considers to be its most difficult and also its easiest hurdles in taking a technology to full product development. In addition, each group ranked its perception of the difficult and easy hurdles for all other groups, enabling an assessment of the degree of understanding between groups. The data depict not only critical hurdles in the path to commercialization at each stage in product development but also a variable understanding of perceptions of hurdles between groups. This assessment has provided the Industry Committee with activity foci needed to assist individual groups in the technology-commercialization stream. Moreover, the analysis suggests that enhanced communication between groups engaged in commercialization will be critical to the successful development of products in the tissue engineering/regenerative medicine sector.

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.

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.