The heterogeneous cell population comprising the stromal vascular fraction (SVF) of human adipose includes endothelial, smooth muscle cell (SMC), and mesenchymal stem cell (MSC)-like cells (as defined by the ISCT criteria). We investigated the cellular composition of the primary “passage zero” (P0) adherent human SVF-derived cell population using quantitative real-time PCR (TaqMan). Although the SVF-derived P0 population expressed genetic markers associated with endothelial, SMC, and adipocyte cells; expansion of SVF-derived P0 cells under defined media conditions that select against the growth of MSC yielded a cell population with markedly distinctive biological properties when compared to MSC cultures. The differentiation potential and marker expression profile of this expanded SVF-derived cell population (X-SVF) partially overlapped that historically associated with MSC; however, X-SVF cells have a more pronounced smooth muscle cell phenotype relative to MSC based on FACS and RT-PCR (reverse transcription PCR) analysis of key nuclear and cell surface marker expression. X-SVF cells also expressed noticeably fewer endothelial-specific genes relative to MSC. These observations suggested that the predominant phenotype of the X-SVF cells was that of SMC. Manifestation of SMC phenotype was independent of passage number or individual adipose donor (n=4) and directed differentiation of X-SVF with recombinant cytokines and growth factors was not required. Additionally, X-SVF cells expressed a distinctive SMC-like proteomic signature that unambiguously distinguished it from MSC. Finally, X-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 conclusion that X-SVF cells are more accurately described as adipose-derived smooth muscle cells (Ad-SMC), and represent a separate and distinctive cellular species compared to other classes of adipose-derived cells; including adipocytes, endothelial cells, and MSC.

The term tissue engineering was coined at a National Science Foundation workshop in 1987 to mean "the application of principles and methods of engineering and life sciences toward fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and tissue function" (Viola et al., 2003). Tissue engineering draws on specialized expertise from two traditional disciplines: engineering and the life sciences. The combination of these technologies forms a foundation upon which the commercial development of neo-organs is possible.

Preclinical development of neo-organs faces complex scientific questions and regulatory hurdles. The term neo-organ product will be used to indicate any product composed of synthetic or natural biodegradable materials, with or without living cells and/or cellular products, implanted in the body to incorporate, replace, and/or regenerate a damaged tissue or organ. Today ex vivo development of partial and complete neo-organs by the emerging regenerative medical industry fulfills a significant unmet medical need for patients who have partial or complete organ or tissue loss. In this chapter we will consider development challenges and available solution strategies for bringing these transformational technologies to patients in need.

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.

In the early 1930s Charles Lindbergh, who was better known for his aerial activities, went to Rockefeller University and began to study the culture of organs. After the publication of his book about the culturing of organs ex vivo in order to repair or replace damaged or diseased organs, the field lay dormant for many years. Indeed, delivering respite to failing organs with devices or total replacement (transplant) became far more fashionable. Transplantation medicine has been a dramatic success. But in the late 1980s scientists, engineers, and clinicians began to conceptualize how de novo tissue generation might be used to address the tragic shortage of donated organs. The approach they proposed was as simple as it was dramatic. Biodegradable materials would be seeded with cells and cultured outside the body for a period of time before exchanging this artifi cial bioreactor for a natural bioreactor by implanting the seeded material into a patient. These early pioneers believed that the cells would degrade the material, and after implantation the cell-material construct would become a vascularized native tissue. Tissue engineering, as this approach came to be known, can be accomplished once we understand which materials and cells to use, how to culture these together ex vivo, and how to integrate the resulting construct into the body.

Approximately 10,000 cystectomies (surgical removal of the bladder) are performed annually in the US with bladder cancer being the leading indication. Once the bladder is removed, a mechanism for urinary diversion must be constructed. While there are several surgical options for a urinary diversion they all involve the use of a gastrointestinal segment. However, use of GI tissue exposes gut mucosa to urine leading to multiple complications including GI and metabolic abnormalities. Tengion is developing autologous regenerative products such as the Neo-Urinary Conduit™ (NUC) for urologic tissue engineering applications. Tengion’s NUC is engineered to reconstruct the urinary tract as an alternative to using gastrointestinal tract segments. The NUC is produced by seeding smooth muscle cells (SMC) on a biodegradable scaffold to form a NUC Construct. In a porcine model, the NUC regenerated an incontinent urinary diversion composed of native-like urinary tissue. Early products for regenerating urinary tissue used SMC isolated from urinary bladder (e.g., Tengion’s autologous Neo-Bladder Augment™). For NUC development, since most patients will have a cancerous bladder, the feasibility of sourcing autologous SMC from adipose tissue was evaluated. Canine and porcine adipose tissue biopsies were digested with collagenase and the dissociated cells recovered by centrifugation. Cells were cultured and characterized using RT-PCR, FACS, ELISA and immunocytochemistry. Isolated cells showed typical SMC morphology and growth characteristics. Phenotypic characterization revealed a robust population expressing multiple SMC markers involved in muscle contraction. Endothelial, epithelial, and myofibroblast marker expression was not observed. Adipose-derived cells expressed MCP-1 protein indicative of functional SMC. Seeding adipose-derived SMC on the NUC scaffold resulted in a NUC Construct suitable for implantation in preclinical studies. These results support the hypothesis that adipose tissue can be used as a source of SMC for autologous products to regenerate urinary tissue in patients requiring reconstruction of the lower urinary tract.

Background/Objective: In the U.S., bladder cancer is the 4th and 8th most common cancer in men and women, respectively. Cystectomy is an effective therapy for invasive disease (Konety et al., 2007). Management of post-cystectomy urine elimination typically involves the use of gastrointestinal (GI) tissue to form an incontinent urinary diversion (Konety et al., 2007). However, use of GI tissue exposes gut mucosa to urine leading to multiple complications including GI and metabolic maladies (Davidsson et al., 1994). The feasibility of using autologous SMC-seeded PLGA-based biodegradable scaffolds, or Neo-Bladder Conduits, to establish an incontinent urinary diversion was evaluated.

Methods: Scaffold-only controls and Neo-Bladder Conduits seeded with autologous SMC isolated from blood, fat, or urinary bladders were evaluated in a porcine model of percutaneous diversion for 3 months.

Results: At 3 months, scaffold-only implants developed into conduits with walls of fibrous connective tissue that were partially lined with urothelium. In contrast, Neo-Bladder Conduits developed into diversions composed of regenerated urinary tissues (urothelial cell lining and vascular smooth muscle wall) regardless of autologous SMC source.

Conclusions: A Neo-Bladder Conduit seeded with autologous SMC sourced from blood, fat, or bladder is capable of establishing a patent incontinent urinary diversion for post-cystectomy management of urine elimination. Neo-Bladder Conduit may represent an alternative to GI tissue for post-cystectomy management by incontinent urinary diversion.

Current surgical treatment options for urinary disorders caused by congenital conditions, injury, or cancer involve the use of gastrointestinal (GI) tissue to form an orthotopic neobladder or urinary diversion; however, use of GI tissue exposes gut mucosa to urine leading to multiple acute and chronic complications, including GI and metabolic maladies. First generation regenerative medical technologies to avoid using GI tissue in the urinary tract utilize an implant composed of synthetic biocompatible scaffold seeded with autologous urothelial and smooth muscle cells (SMC) isolated from native bladder tissue. Although autologous cells avoid the risk of rejection and the need for lifelong immunosuppression, the use of native bladder tissue as a source of autologous cells to seed implants for bladder cancer patients is not ideal. In this study, skeletal muscle was investigated as an alternative cell source for the SMC component of Tengion’s Neo-Urinary Conduit. Cells were cultured from canine and porcine skeletal muscle and SMC phenotype confirmed by immunocytochemistry using antibodies against six SMC proteins that are expressed in primary cultures of native human bladder SMC: smooth muscle alpha-actin, vimentin, myocardin, myosin, calponin, and desmin. No expression of epithelial marker cytokeratin AE1/AE3 or myofibbroblast marker proteins were detected. These data support the notion that skeletal muscle may be an alternate source of SMCs, thus potentially solving a cell sourcing challenge to developing autologous regenerative medical therapies for patients with cancer of the bladder.

Introduction: The ability to use an autologous cell-seeded biodegradable scaffold for bladder augmentation has been demonstrated in patients with neurogenic bladder (NB) due to spina bifida (SB) at Children's Hospital Boston (CHB) [1]. We conducted a confirmatory, prospective multicenter Phase 2 study of the Tengion NBC in a similar population.

Methods: Male or female subjects, 3 to 21 years, with NB due to SB, were eligible if they required augmentation cystoplasty for bladder pressure >40cmH20 and/or new onset of upper tract changes. Eligibility was confirmed by a Steering Committee. Bladder neck sling was the only concomitant surgical procedure permitted. Since biomechanical stimulation (cycling) promotes tissue regeneration, patients were required to bladder cycle postoperatively. Following an open bladder biopsy, urothelial and smooth muscle cells were grown ex vivo for 5 - 7 weeks, then seeded onto a biodegradable scaffold (the NBC).  The implanted NBC served as a template for bladder tissue regeneration. The primary endpoint was urodynamic (UDS) compliance 1 year post implantation.  Evaluations included cystograms, renal ultrasounds, physicals and labs.

Results: Four centers enrolled 11 subjects; 10 (6 females) were implanted. Mean age was 8.2 [3-16] years. The procedure was generally well tolerated. Six patients able to bladder cycle showed clinical improvement. Overall, UDS changes were consistent with those from CHB. Hydronephrosis and/or reflux improved/resolved in 4/5 patients. Patients unable to cycle (3 concomitant bladder neck slings, 1 low pressure high grade reflux) showed no UDS improvement at 12 months.

Discussion: The study supports the potential of regenerative medicine in bladder augmentation. Long term follow-up is ongoing. Additional studies are needed to confirm the benefits of this promising technology.

1. Atala A, Bauer SB, Soker S, Yoo JJ, & Retik AB. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet. 2006; Vol. 367:  1241-1246.

Background: Homeostatic regulation that maintains organ size and structure is a complex relationship between the specific organ, tissues, and body weight or size.  Regulative development, or restoration of organ size and structure after cell or tissue loss, has only been observed during tissue regeneration or organogenesis. Goals for regenerative therapies include both restoration of structure and function and establishment of adaptive regulation specific for the recipient. Adaptive regulation in cystectomized animals implanted with cell-seeded PLGA-based scaffolds was compared with adaptive regulation based on early results from a Phase II clinical trial of the Tengion Autologous Neo-Bladder Augment™ (NBA) in children with neurogenic bladder due to spina bifida.

Methods: Neo-bladder capacity and body weight were measured in cystectomized animals implanted with cell-seeded PLGA-based scaffolds for 6 months post-implantation (p.i.). Cystometric capacity and voiding intervals (VI) were measured and formula predicted bladder capacity (FPBC) was calculated at baseline and 12 months after NBA implant in two age- and weight-matched Phase II NBA clinical trial subjects (PT1 and PT2).

Results: Implanted animals remained healthy and continent for study duration and achieved neo-bladder capacities consistent with body weight as early as 6 months p.i. Histology and immunohistochemistry of neo-bladder tissue revealed a native bladder-like structure and function, indicative of bladder regeneration. PT1's baseline capacity was 33% of FPBC. At 12 months p.i., PT1's capacity had increased 84% from baseline and achieved 60% of FPBC. PT2 had capacities of 100% of FPBC at baseline and 12 months p.i. VI increased for both PT1 and PT2.

Conclusions: These results demonstrate that autologous neo-bladders regenerated and grew appropriately to the recipient's body size in animals and humans. These data support the conclusion that neo-bladders elicited by Tengion Autologous Neo-Bladder Augment™ implantation are bioresponsive to the needs of the recipient.

Submitted November 14, 2008 to the American Urology Association 2009 Annual Meeting - Chicago, IL - April 25-30, 2009 (http://www.aua2009.org/)

Background: Biomechanical stimulation is a process known to promote tissue regeneration and optimal healing. Bladder regeneration is biomechanically stimulated by cycling (filling, storage and evacuation), a process initiated in utero that contributes to the development of a functional bladder in humans. Interruption of cycling in patients with neurogenic bladder from either congenital (e.g., spina bifida) or acquired (e.g., spinal cord injury) impairment leads to significant functional and structural alterations.

Methods: Cycling impacts on bladder tissue regeneration in cystectomized animals implanted with cell-seeded PLGA-based scaffolds were evaluated and learnings applied to outcomes of a Phase II clinical trial of the Tengion Autologous Neo-Bladder Augment™ (NBA) in patients with neurogenic bladder due to spina bifida.

Results: Post-implantation (p.i.) neo-bladder cycling was initiated in animals at 2 weeks p.i. for 3 days/week. Three cycling parameters were collected: total weeks, hr/day, and total hrs. Urodynamic assessments from three cycling cohorts based on mean parameters were evaluated: HIGH (10 weeks, >3.75 hr/day, >60 hrs), LOW (10 weeks, <2.25 hr/day, <25 hrs), and NO cycling. The HIGH cohort developed neo-bladders with improved compliance and capacities that were on average 3-fold higher than the LOW cohort (p < 0.0001). The HIGH cohort achieved 90% of native baseline capacity by 6 mo p.i., while the LOW cohort regained only 40%. Animals not cycled (incontinent) developed tubularized urinary tract diversions. Histology of the neo-bladder wall revealed native-like tissue structure and extracellular matrix composition (e.g., elastin) in cycled bladders. Early Phase II data studying the NBA suggest that patients with challenges in postoperative cycling (e.g., open bladder necks and low pressure high grade reflux) had inferior clinical and urodynamic outcomes to patients without those challenges.

Conclusions: Early post-implantation cycling is essential for promoting regenerative healing following implantation of autologous cell-seeded PLGA-based scaffolds in animals and humans. Insights from Preclinical studies are consistent with early insights from Phase II NBA trial and confirm the importance of cycling in bladder regeneration.

Submitted November 12, 2008 to the American Urology Association 2009 Annual Meeting - Chicago, IL - April 25-30, 2009 (http://www.aua2009.org/)

Purpose: Trigone-sparing cystectomy was used to study the structural and functional aspects of bladder regeneration in a canine model of an augmentation cystoplasty.

Methods: An autologous neo-bladder augmentation construct composed of a PLGA-based biodegradable mesh scaffold and autologous urothelial and smooth muscle cells (Construct) (n=32) was compared to re-implanted native bladder (Reimplant) (n=32), or PLGA-based biodegradable mesh scaffold alone (Scaffold) (n=8) at 1, 3, 6 and 9 months (mo) post-implantation.

Results:Within 14 days, all 72 dogs were continent. Within 1 mo, acute phase responses, hematological and urinalysis parameters returned to baseline. Treatment-related morbidity was only observed in Scaffod and Reimplant dogs. Only Construct dogs achieved functional recovery (i.e., urodynamics) and a regenerative tissue response. Construct dogs regained baseline bladder capacity by 4 mo and compliance by 6 mo and were sustained throughout the study. Urodynamic parameters in Reimplant animals were initially comparable to Construct dogs but were unstable and significantly lower than baseline by 9 mo (60-75% decrease from baseline). Histologically, decreased compliance in Reimplant and Scaffold groups at 9 mo correlated with limited healing and incomplete bladder wall regeneration.

Conclusions: Construct implants were safe and able to restore urodynamic, continence, and voiding functions by 6 mo and retained these functions to study termination. Bladder wall regeneration was obtained only in the Construct implanted animals. Scaffolds lacking cells (Scaffold) elicit repair of bladder wall with incompletely developed components reduced organ capacity and restricted compliance.

Neo-bladder histology was investigated in 21 canines at 30-79 days (n=9) and 80-180 days (n=9) after radical cystectomy and implantation of a Neo-Bladder Replacement Construct (Construct). Pharmacological responses of neo-bladder tissue strips from 9 retrieved neo-bladders (n=1, 30-79 days; n=8, 80-180 days) were compared to age-matched native bladder tissue (n=17).

In both groups, regenerated bladder was histologically consistent with native bladder including mucosal and serosal linings, detrusor muscle, vascular, and nerve composition. Logistic analysis revealed similar EC50 and slope factor values for contraction of bladder tissue strips derived from both groups induced by carbachol (Car) and electrical field stimulation (EFS). A progressive increase in the mean Emax values occurred in response to both Car and EFS over time. While contractile responses to Car and EFS increased over time in the neo-bladder tissue, they were lower than native bladder responses in the 30-79 day group. Car-induced contractions, but not EFS-induced contractions, became equivalent to native tissue in 80-180 days post-implantation (p.i.).

The Neo-Bladder Replacement Construct is capable of regenerating urinary bladder in vivo with histology similar to native bladder in 30-79 days p.i. and pharmacological responses became similar to native bladder in 80-180 days p.i. with no evidence of abnormal cell growth, immune response, or adverse systemic effects.

Tengion is currently conducting a GLP study to support clinical trial studies in 2009.

Purpose: Internal organ regeneration holds promise for changing medical technology and reducing organ shortages. Current medical treatment for internal organ failure is largely limited to organ transplantation. A synthetic biopolymer with autologous cells (Construct) has exhibited long-term clinical benefit in patients undergoing augmentation cystectomy; however, early cellular and stromal events during bladder regeneration have not been elucidated.

Materials and Methods: In situ cellular responses to two biopolymer implants; one with autologous cells (Construct) and one 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 post-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 neo- tissues. 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 both Construct and Scaffold implantation; however, only Construct had normal stroma, well developed detrusor, and abundant aSMA-staining cells at early time points leading to a structurally and functionally complete, regenerated bladder wall at 9 months. 

Conclusion: 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 regeneration of complete mucosal and muscular components whereas the response to Scaffold implantation is consistent with reparative healing; with mucosal growth but incomplete tissue layer development.

Neo-bladder structure and function was investigated in 23 canines at 30- 79 days (n=9) and 80-188 days (n=14) after radical cystectomy and implantation of a Neo-Bladder Replacement Construct (Construct). Pharmacological responses of neo-bladder tissue strips from 11 retrieved neo-bladders (n=1, 30-79 days; n=10, 80-188 days) were compared to age- matched native bladder tissue (n=17).

Regenerated bladder was histologically consistent with native bladder including mucosal and serosal linings, detrusor muscle, vascular, and nerve composition. Logistic analysis revealed similar EC50 and slope factor values for contraction of bladder tissue strips derived from both groups induced by carbachol (Car) and electrical field stimulation (EFS). A progressive increase in the mean Emax values occurred in response to both Car and EFS over time. While contractile responses to Car and EFS increased over time in the neo-bladder tissue, they were lower than native bladder responses in the 30-79 day group. In contrast, strips from 80-188 day post-implantation neo-bladders reached the 95% confidence interval of native tissue in the linear range of the response.

The Neo-Bladder Replacement Construct is capable of regenerating urinary bladder in vivo with histology similar to native bladder in 30-79 days p.i. and pharmacological and electrical field responses became similar to native bladder in 80-188 days p.i. with no evidence of abnormal cell growth, immune response, or adverse systemic effects.

Tengion is currently conducting a GLP study to support clinical trial studies in 2009.

AIMS: To comparatively evaluate bladder regeneration following 80% cystectomy and augmentation using a synthetic biopolymer with autologous urothelial and smooth muscle cells (autologous neo-bladder augmentation construct [construct]) or autotransplantation of native bladder (reimplanted native urinary bladder [reimplant]) in canines.

MATERIALS & METHODS: Voiding function, urodynamic assessment and neo-organ capacity-to-body-weight ratio (C:BW) were assessed longitudinally for a total of 24 months following trigone-sparing augmentation cystoplasty in juvenile canines.

RESULTS: Within 30 days postimplantation, hematology and urinalysis returned to baseline. Constructs and reimplants yielded neo-organs with statistically equivalent urodynamics and histology. Linear regression analysis of C:BW showed that constructs regained baseline slope and continued to adapt with animal growth.

CONCLUSIONS: Constructs and reimplants regained and maintained native bladder histology by 3 months, capacity at 3-6 months and compliance by 12-24 months. Furthermore, construct C:BW demonstrated the ability of regenerated bladder to respond to growth regulation.

Purpose: Neo-bladder pharmacology was evaluated in animals after total cystectomy and implant of Autologous Neo-Bladder Replacement constructs (NBR) made from bladder smooth muscle cells (SMC).

Methods: NBR were seeded with SMC to form three test groups in which 25, 12, or 4x106 SMC were seeded per construct (n= 8/grp). A fourth group (n=8) in which radically cystectomized bladders were immediately reimplanted (R) served as a control. Ex-vivo pharmacological and histological studies were conducted on neobladder tissues at study termination.

Results: At 9 mo post-implantation, neo-bladders of all groups had tissue histology (including mucosal and serosal linings, detrusor muscle, vasculature, and nerve components) consistent with native bladder. Contractile responses to parasympathomimetic (carbachol) and sympathomimetic (phenylephrine) agonists were similar among all groups. Logistic analysis of bladder tissue strips subjected to electrical field stimulation (EFS) revealed similar EC50 and slope factor values for all groups.

Conclusions: An Autologous Neo-bladder Replacement construct is capable of regenerating a complete urinary bladder as a total organ with structural, urodynamic, and pharmacological features similar to native bladder. There was no evidence of abnormal tissue development, immune response, or systemic response to the neo-bladder regeneration in any group.

The rising cost of established chronic kidney disease (CKD) management, the morbidity associated with dialysis, and the limited numbers of donor kidneys suitable for transplant provide impetus for developing new treatment paradigms for CKD. Previous studies demonstrated that ex vivo cell cultures propagated from whole kidney tissue (UNFX) contained renal cells from all major compartments of the kidney and were capable of stimulating regeneration and stabilizing renal function after orthotopic transplantation in the rodent remnant kidney model of renal failure. Also demonstrated previously was that a subpopulation of UNFX (B2), enriched for specific renal epithelial cells, enhanced nearly all aspects of nephron function and outperformed UNFX in all parameters. The present study further segregated UNFX into component cell subpopulations, evaluated in vitro characteristics, and tested in vivo performance of specific subpopulations alone and in combination. The selected subpopulations had unique profiles encompassing phenotypic, genotypic, and functional characteristics and elicited distinct systemic outcomes after orthotopic transplantation. One such subpopulation (B4), selected for its enrichment of glomerular, vascular and endocrine cells, enhanced renal endocrine functions compared to UNFX. Interestingly, a controlled admixture of the B2/B4 cell populations provided the most comprehensive benefit, outperforming B2, B4, UNFX, and four other cell prototypes across most parameters, providing statistically-significant improvements in survival, weight gain, systemic blood pressure, protein retention, and endocrine function when compared to untreated controls. Histological evaluation of kidney and bone marrow six months post-treatment confirmed that two treatments (B2 and the B2/B4 combination) initiated a tissue-level regenerative response in the kidney, reducing glomerulosclerosis and tubulointerstitial fibrosis throughout the renal parenchyma. Taken together, these studies support continued investigation into alternative treatments for progressive CKD that employ specific, biologically-active renal cells in the stimulation of a regenerative response.

Autologous cells can be harnessed and deployed as key components of products that functionally augment or replace degenerating organs and tissues. Thorough ex vivo characterization of cellular component(s) is essential for establishing defined prototypes for in vivo evaluation. High-content image-based analysis (HCA) provides robust characterization of heterogeneous cell populations, yet quantitates phenotypic and functional data on a cell-by-cell basis, yielding insights into the relationships between cellular composition of selected prototypes and in vivo therapeutic outcomes.

The present study used HCA and fluorescence-based assays to assess the albumin-transport functionality of specific tubular cell subpopulations in heterogeneous primary cultures of human renal cells established from 22 donors with (n=5) or without (n=17) chronic kidney disease (CKD) and compare the relative distribution of specific tubular cell phenotypes and functionality between CKD and non-CKD specimens.

HCA analysis revealed that the cellular composition of renal cultures established from CKD and non-CKD donors was similar, representing tubular, glomerular, ductular, endocrine, and vascular compartments. Assessment of protein transport function via receptor-mediated albumin uptake illustrated that cells possessing this function comprised a significant and similar proportion of CKD (40-80%) and non-CKD (40-80%) primary cultures. Serial passage of these heterogeneous cultures under standard media and culture conditions resulted in a gradual loss of the protein-transport activity in both CKD and non-CKD cultures, but the transport-positive population was preferentially retained in CKD-derived cultures.

In summary, HCA provided an accurate and meaningful comparative assessment of renal cell phenotype and function, yielding quantitative cellular-level data and revealing cell population dynamics that may be unable to be detected by other population-based assays (i.e., gene/protein expression). The synergy between advancements in image collection automation and evolution of more sophisticated analysis software packages has made application of HCA feasible in an industrial R&D environment, enabling detection and tracking of cell subpopulations throughout various processing steps.

Traditional primary culture of renal cells involves enzymatic dissociation of kidney tissue followed by propagation of the cells on tissue-culture treated plastic with or without extracellular matrix coatings. In the present study, we examined the effects of three-dimensional (3D) architecture and perfusion on metabolism, phenotype, and tubular function of primary kidney cells in a variety of culture configurations. 3D architecture promoted cell-cell interaction and organization, as determined by scanning electron microscopy, histology, and confocal immunochemistry. The addition of perfusion to the culture system resulted in enhanced metabolic activity and a significant and sustained upregulation of genes associated with tubular function. Importantly, the tubular function of renal cells was confirmed in culture systems via the demonstration of megalin/cubilin-mediated uptake of albumin. In summary, dynamic 3D culture systems provide a means to examine tubular cell phenotype and function in an environment that better recapitulates in vivo biology.

Primary renal cells have been isolated and propagated successfully from rodent, swine, and human kidney tissue¹ and therapeutic benefits of the rodent cell populations have been demonstrated in vivo in a rodent model of progressive renal failure². Key therapeutic attributes associated with in vivo application of rodent renal cell populations included positive effects on tubule-associated functions with histologic evidence of tubular regeneration, indicating that the cells were capable of stimulating some degree of nephrogenesis, either through direct or indirect mechanisms.

The goal of this study was to build an in vitro 3D culture system to assess the capability of these cells for autonomous ex vivo nephrogenesis and/or screen for exogenous factor requirements. 3D culture systems that replicate renal organogenesis have been used for mechanistic studies of tubule, collecting duct, and nephron formation and morphogenesis. Most studies used immortalized or embryonic cells and tissues, which may or may not translate to clinical use.

Developmentally-relevant genes were responsive to hypoxia in primary CRC 2D cultures. The ability of CRC to reform mature kidney structures ex vivo was evaluated in 3D cultures made by suspending expanded primary CRC in Matrigel™ Growth Factor Reduced extracellular matrix (M-GFR). Extent of ex vivo nephrogenesis was evaluated by immunofluorescence.

Under atmospheric oxygen conditions, duct-derived cells and nephron tubule-derived cells contained within the population of CRC formed epithelial cysts in the 3D culture system, reminiscent of the ureteric buds and renal vesicles, respectively. However, no advanced morphogenetic processes such as duct branching, epithelial fusion, and nephron formation that rely on mutual induction in vivo were observed. A limited number of exogenous cues (e.g. GDNF, HGF, FGF7, and hypoxia) were investigated for their ability to drive nephrogenesis in the 3D CRC cultures, but structure formation was still restricted to buds and vesicles.

CRC grown in 3D cultures with M-GFR under atmospheric or hypoxic conditions were not capable of autonomous ex vivo nephrogenesis that relies on mutual induction (e.g., tubule outgrowth, branching, and fusion); however, hypoxia may prime CREC to be more responsive to a nephrogenic environment, provided the proper exogenous cues are present. Additional work is required to identify the complex cues necessary to drive CRC to nephrogenesis in vitro.

1. Presnell, S.C. et al. (2009) EXPERIMENTAL BIOLOGY 2009 Meeting, New Orleans, LA, USA
2. Kelley, R.W. et al. (2009) KIDSTEM 2009 Meeting, Edinburgh, SCOTLAND

Chronic Kidney Disease (CKD) often develops in patients with co-morbidities such as obesity, chronic hypertension, and metabolic disorders and is characterized by severely impaired renal filtration (uremia) and impaired erythropoiesis (anemia). The combination of a rising cost burden of dialysis on the healthcare system, a limited number of donor kidneys suitable for transplant, and the side effects associated with Erythropoiesis Stimulating Agents (ESAs) provide impetus for developing new treatment paradigms for CKD.

Aboushwareb et al (2008) showed that ex vivo cell cultures established from mouse whole kidney tissue contain highly-specialized cells that express erythropoietin (Epo) in addition to other kidney cell types1. Similar methods were used to establish cultures from rat kidney. Epo-producing cells, as well as tubular, ductal, vascular, interstitial, and glomerular cells were identified in the rat cultures by immunofluorescence and qRT-PCR. A pilot study was conducted, whereby intrarenal transplantation of the ex vivo-cultured rat cells in an established rodent model of progressive renal failure stabilized renal filtration and tubular functions, restored erythroid homeostasis, and prolonged survival versus untreated rats. These systemic observations were confirmed histologically, with clear demonstration of tubular and glomerular repair and regeneration, reduction of glomerular and tubulointerstitial fibrosis, stabilization of erythroid function, and reduction of bone catabolism. These results demonstrated that this heterogeneous culture contained therapeutically relevant cells.

Cell isolation was extended successfully to swine and human kidney tissue; with starting material isolated from CKD and non-CKD kidneys. All cell compartments identified in the rodent cultures were identified in swine and human cell cultures. Retention of functional proximal tubular cells in cultures propagated from both CKD and non-CKD kidney was demonstrated by receptor-mediated albumin uptake. Epo-expressing cells were also present in both CKD and non-CKD kidney-derived cultures and retained oxygen-responsive, HIF1-α-driven EPO expression during expansion.

Taken together, these results suggest that autologous sourcing of therapeutically-relevant cell populations is feasible in advanced CKD. Experiments are ongoing to identify the bioactive cellular component(s) responsible for the observed therapeutic benefits and establish potential methanism(s) of action.

Aboushwareb T, Egydio F, et al. (2008) World J Urol 26, 295-300.

Chronic kidney disease (CKD) is a global public health problem; U.S. patients on dialysis awaiting organ transplant more than doubled between 1991 and 2004. To avoid an immune response to implanted cells, functional autologous cells would be preferred components of regenerative medicine therapies for CKD; however, evidentiary support for autologous sourcing of therapeutically-relevant cells from CKD patients and large animal models is lacking. Fresh kidney tissue (CKD and non-CKD) was obtained from porcine* and human** subjects. CKD was confirmed by serology and histopathology. Tissue dissociation and cell isolation methods developed with non-CKD tissue were employed to isolate and propagate cells from CKD tissue and yielded tubular cells and erythropoietin (EPO)-expressing cells. Comparative in vitro studies demonstrated expression of megalin:cubilin and receptor-mediated uptake of fluorescein-conjugated albumin in tubular cell cultures from both CKD and non-CKD tissues. Cells expressing erythropoietin (EPO) were present in both CKD and non-CKD tissues and could be isolated and expanded with retention of oxygen-responsive, HIF1-?-driven EPO expression. Taken together, these results suggest that autologous sourcing of at least two therapeutically-relevant cell populations is feasible in advanced CKD.

*porcine: adult male with severe diffuse chronic interstitial fibrosis and crescentic glomerulonephritis with multifocal fibrosis, azotemia, and mild anemia; BUN of 75 and Creatinine of 9.5 at death.
**human: female (48 years old) with a history of hypertension, type II diabetes, and chronic renal failure, on dialysis for 7 years. Cause of death: anoxia, secondary to cardiovascular failure; BUN of 40 and Creatinine of 8.6 at death.