In vitro reconstitution of human kidney structures for renal cell therapy

Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston Salem, NC, USA.
Nephrology Dialysis Transplantation (Impact Factor: 3.49). 01/2012; 27(8):3082-90. DOI: 10.1093/ndt/gfr785
Source: PubMed

ABSTRACT Recent advances in cell therapies have provided potential opportunities for the treatment of chronic kidney diseases (CKDs). We investigated whether human kidney structures could be preformed in vitro for subsequent implantation in vivo to maximize tissue-forming efficiency.
Human renal cells were isolated from unused donor kidneys. Human renal cells were cultured and expanded. Migration was analyzed using growth factors. To form structures, cells were placed in a three-dimensional culture system. Cells were characterized by immunofluorescence, western blots and fluorescence-activated cell sorting using renal cell-specific markers for podocin, proximal and distal tubules and collecting ducts. An albumin uptake assay was used to analyze function. Three-dimensional cultures were implanted into athymic rat kidneys to evaluate survival.
Human renal cells were effectively expanded in culture and retained their phenotype, migration ability and albumin uptake functions. Human renal cell in three-dimensional culture-formed tubules, which stained positively for proximal, distal tubule and collecting duct markers, and this was confirmed by western blot. Polarity of the tubular cells was determined by the presence of E-cadherin, N-cadherin and Na-K ATPase. Colocalization of labeled albumin and proximal tubule markers proved functionality and specificity of the newly formed tubules. An in vivo study showed that cells survived in the kidney for up to 6 weeks.
These findings demonstrate that human renal cell grown in three-dimensional culture are able to generate kidney structures in vitro. This system may ultimately be developed into an efficient cell-based therapy for patients with CKD.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Renal transplantation is currently the only definitive treatment for end-stage renal disease; however, this treatment is severely limited by the shortage of implantable kidneys. To address this shortcoming, development of an engineered, transplantable kidney has been proposed. Although current advances in engineering kidneys based on decellularization and recellularization techniques have offered great promises for the generation of functional kidney constructs, most studies have been conducted using rodent kidney constructs and short-term in-vivo evaluation. Toward clinical translations of this technique, several limitations need to be addressed. Human-sized renal scaffolds are desirable for clinical application, and the fabrication is currently feasible using native porcine and discarded human kidneys. Current progress in stem cell biology and cell culture methods have demonstrated feasibility of the use of embryonic stem cells, induced pluripotent stem cells, and primary renal cells as clinically relevant cell sources for the recellularization of renal scaffolds. Finally, approaches to long-term implantation of engineered kidneys are under investigation using antithrombogenic strategies such as functional reendothelialization of acellular kidney matrices. In the field of bioengineering, whole kidneys have taken a number of important initial steps toward clinical translations, but many challenges must be addressed to achieve a successful treatment for the patient with end-stage renal disease.
    Current Opinion in Organ Transplantation 04/2015; 20(2). DOI:10.1097/MOT.0000000000000173 · 2.38 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: It is thought that the universe began as a singularity of enormous potential (negative) energy of a magnitude equal to the total sum of its rest energy (mass) and kinetic energy, originated from the quantum fluctuations of the vacuum; so, it has no other way than to fall irreversibly down the hill, like the skier. However, for some reason that still seems supremely mysterious to me, while falling beautiful forms of increasing complexity are created.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Potency is a critical quality attribute of biological products, defined by the US FDA as the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result. Ideally, a potency assay will leverage the product's mechanism of action. Alternatively, the assay may focus on a therapeutically relevant biological activity. The absence of rigorous mechanistic data for the majority of cell-based therapeutics currently in the process research pipeline has impeded efforts to design and validate indices of product potency. Development of a systematic battery of parallel functional assays that, taken together, can address all potential mechanisms of action believed to be relevant for the product platform is recommended. Such an approach is especially important during preclinical development. Here, we summarize the principal and unique challenges facing the development of functionally relevant and rigorous potency assays for cell-based therapeutics. We present perspectives regarding potency assay development for these products as illustrated by our experiences in process R&D of cryopreserved hepatocytes (Incara Pharmaceuticals) and selected renal cells (Tengion).
    Regenerative Medicine 07/2014; 9(4):497-512. DOI:10.2217/rme.14.25 · 3.50 Impact Factor