Andrew T. Bruce

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 kidneys 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)-β1, plasminogen activator inhibitor (PAI)-1, and fibronectin (FN) provided evidence that B2 cells attenuated canonical pathways of profibrotic extracellular matrix production.

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-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).

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.

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.

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).

Phenotypic analysis of bioactive cells for application in regenerative medicine.

The following chapter outlines methodologies to phenotypically characterize primary cells for the use in tissue-engineered and regenerative medicine applications. Methods covered include analyzing cells using immunocytochemistry, fluorescence-activated cell sorting, and confocal microscopy of adherent and suspended cells, as well as combinations of formulated cell-biomaterial constructs.

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.