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.

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.

Evidence of pluripotent human prostate stem cells in a human prostate primary xenograft model.

INTRODUCTION: The phenotypic plasticity of the human prostate stem cell within human prostate tissue was examined to determine the response of the stem cell to changes in the androgenic environment. METHODS: Prostate xenografts were transplanted into athymic nu/nu mice implanted with testosterone pellets, allowed to establish for 1 month time point, the hosts were castrated and pellets removed, and following 1 month of androgen deprivation, the hosts were stimulated with androgen for 2 days to induce proliferation of the residual population of stem cells (2-month time point). RESULTS: Glands in benign xenografts harvested at the 1- and 2-month time points contained basal cell layers that expressed p63 and high molecular weight cytokeratin, and in which essentially all of the cellular proliferation was localized, consistent with the proposed localization of the prostate stem cell. Benign glandular structures in the xenografts were populated by basal, secretory epithelial, neuroendocrine (NE), or squamous cells overlaying the basal cell layer, whereas, adenocarcinoma glands in the xenografts resembled the original prostate cancer (CaP) tissue. CONCLUSIONS: In this human prostate primary xenograft model, the residual stem cell population that survives transplantation, or androgen deprivation, maintains significant pluripotentiality as demonstrated by the capacity to generate progeny that differentiate along multiple lineages in response to microenvironmental signals, particularly along the secretory epithelial lineage in response to androgen, and along the NE cell lineage in response to androgen deprivation.

Short-term human prostate primary xenografts: an in vivo model of human prostate cancer vasculature and angiogenesis.

Transgenic spontaneously occurring and transplantable xenograft models of adenocarcinoma of the prostate (CaP) are established tools for the study of CaP progression and metastasis. However, no animal model of CaP has been characterized that recapitulates the response of the human prostate vascular compartment to the evolving tumor microenvironment during CaP progression. We report that primary xenografts of human CaP and of noninvolved areas of the human prostate peripheral zone transplanted to athymic nude mice provide a unique model of human angiogenesis occurring in an intact human prostate tissue microenvironment. Angiogenesis in human kidney primary xenografts established from human renal cell carcinoma and noninvolved kidney tissue, a highly vascular organ and cancer, was compared with angiogenesis in xenografts from the relatively less vascularized prostate. Immunohistochemical identification of the human versus mouse host origin of the endothelial cells and of human endothelial cell proliferation in the human prostate and human kidney xenografts demonstrated that: (a) the majority of the vessels in primary xenografts of benign and malignant tissue of both organs were lined with human endothelial cells through the 30-day study period; (b) the mean vessel density was increased in both the CaP and benign prostate xenografts relative to the initial tissue, whereas there was no significant difference in mean vessel density in the renal cell carcinoma and benign kidney xenografts compared with the initial tissue; and (c) the number of vessels with proliferating endothelial cells in primary xenografts of CaP and benign prostate increased compared with their respective initial tissue specimens, whereas the number of vessels with proliferating endothelial cells decreased in the benign kidney xenografts. Short-term primary human prostate xenografts, therefore, represent a valuable in vivo model for the study of human angiogenesis within a human tissue microenvironment and for comparison of angiogenesis in CaP versus benign prostate.