Basu J., Jayo M.J., Ilagan R.M., Guthrie K.I., Sangha N., Genheimer C.W., Quinlan S.F., Payne R., Knight T., Rivera E., Jain D., Bertram T.A., Ludlow J.W. (May 2012). Regeneration of Native-Like Neo-Urinary Tissue from Nonbladder Cell Sources. Tissue Engineering: Part A. 2012 May; 18(9-10):1025-34. 2012 Jan 16. [Epub ahead of print]
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.
Justewicz D.M., Burnette T.B., Shokes J.E., Spencer T., Jain D. (2011, December) Genetic Stability of Autologous Human Smooth Muscle Cells. Oral Presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 11-14, 2010, Houston, Texas.
Smooth muscle cells (SMC) are a key component in autologous cell / biomaterial combination products that target regeneration of diseased tissue. Human adipose tissue represents an abundant source of adult precursor and progenitor SMC. Stable expansion of healthy SMC during manufacturing scale-up is a central requirement of product safety. Assessment of genetic stability during successive passages in culture provides assurance of manufactured product stability. The present study investigated the genetic stability of SMC derived from human adipose tissue. Comparison was made to the behavior of adult SMC obtained from human bladder tissue.
Results demonstrate genetic stability of SMC in culture. Though the functional significance of any alteration remains to be determined, the overall mutational load is markedly lower than reported for precursor cells in the recent literature. This can be attributed to minimal manipulation of the SMC cultures. Taken together, SMC derived from adipose tissue possess reproducible DNA ploidy and chromosomal stability and are comparable to bladder tissue derived SMC.
Shokes J.E., Burnette T.B., McMahan J., Ramachandrannair S., Spencer T., Jain D. , Justewicz D.J. (2011, December) Characterization of Chemotherapy / Radiation Treated Human Smooth Muscle Cells for Use in Regenerative Medicine. Poster presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 11-14, 2010, Houston, Texas.
The repertoire of molecules secreted by smooth muscle cells (i.e., SMC secretome) is a resource for understanding how autologous cell/biomaterial combination products could potentially contribute to regeneration via paracrine signaling mechanisms, in addition to serving as a tool in determining similarities/differences between sources of SMC. We are developing autologous regenerative products for urologic applications including a Neo-Urinary Conduit (NUC) for bladder cancer patients requiring cystectomy, as an alternative to the use of bowel tissue that has many complications (e.g., GI and metabolic disturbances). Bladder cancer patients are typically treated with neoadjuvant cancer drug therapies and/or radiation prior to bladder removal. In this study, we investigated the secretome of three dimensional SMC cultures (3D SMC) initiated from bladder tissue obtained from patients that had undergone cancer treatment. Comparison was made to 3D SMC initiated from non-bladder tissue (SMC derived from subcutaneous adipose tissue) treated a priori in vitro with a chemotherapy drug combination, and to normal bladder SMC (control).
The secretome from 3D SMC generated with treated and untreated SMC were largely homogeneous suggesting that SMC derived from a diseased source produced a similar repertoire of factors. The data presented here further supports our earlier findings that SMC derived from bladder cancer patients who had undergone cancer treatment prior to biopsy are suitable for producing our NUC product for implantation.
Shokes J.E., Burnette T.B, Shrestha S., Justewicz D.M., Spencer T., and Jain D. Characterization of Autologous Smooth Muscle Cells Following Treatment with Bladder Cancer Drugs. Oral Presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 5 - 8, 2010.
Cystectomy, or surgical removal of the bladder, requires reconstructing a channel to eliminate urine from the body. While several surgical options for a urinary diversion exist, they all use a segment from the gastrointestinal (GI) tract. Unfortunately, exposing GI mucosa to urine can result in multiple acute and chronic complications, including GI and metabolic abnormalities. Tengion is developing autologous regenerative products such as the Neo-Urinary Conduit™ (NUC) for urinary diversion and other urologic applications as alternative to the use of gastrointestinal tract segments. The NUC is produced by seeding autologous smooth muscle cells (SMC), expanded ex vivo from subcutaneous adipose tissue, on a biodegradable scaffold; however, bladder cancer patients requiring cystectomy are typically treated with neoadjuvant cancer therapy drugs prior to bladder removal. This study investigated the genetic and phenotypic stability of SMC during ex vivo expansion after in vitro exposure to two cancer drug combinations commonly used to treat bladder cancer patients.
Halberstadt C.R., Fish J., Robbins N., McCoy D., Campbell J., Boyd S., Payne R., Spencer T., Jain D. Development of a Bioreactor System for Preparation of Tubular Organs. Poster Presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 5 - 8, 2010.
A treatment modality for bladder cancer patients is a cystectomy or surgical removal of the bladder, requiring the formation of a channel to remove urine from the body by using a gastrointestinal segment. However, complications arise when using this tissue, including gastrointestinal and metabolic abnormalities. Tengion is developing autologous regenerative products such as the Neo-Urinary Conduit™ (NUC) for urinary diversion and other urologic applications as alternative to the use of gastrointestinal tract segments that consists of adipose-derived smooth muscle cells (SMC) seeded onto a tubular scaffold1. To manufacture this product efficiently, we have designed a multi-functional bioreactor system that provides: (i) placement and positioning of the NUC scaffold; (ii) a closed system for sterilizing the NUC scaffold; (iii) a closed system for cell seeding, medium exchanges, and in-process sample collection; (iv) a container for shipping the NUC to the surgical site; (v) user-friendly handling of the NUC in the surgical suite.
Jain D., Halberstadt C.R., Ludlow J.W., Payne R., Spencer T., Justewicz D., Jayo M.J., Wagner B.J., Bertram T.A. A Regenerative Urinary Diversion Tissue-Engineered from Autologous Smooth Muscle Cells and a Biodegradable Scaffold. Poster Presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 5 - 8, 2010.
Cystectomy or surgical removal of the bladder requires reconstructing a channel to eliminate urine from the body. While several surgical options for a urinary diversion exist, they all use a segment from the gastrointestinal (GI) tract. Unfortunately, exposing GI mucosa to urine can result in multiple acute and chronic complications, including GI and metabolic abnormalities. Tengion is developing autologous regenerative products such as the Neo-Urinary Conduit™ (NUC) for urinary diversion and other urologic applications as alternative to the use of gastrointestinal tract segments. The NUC is produced by seeding autologous smooth muscle cells (SMC) on a biodegradable scaffold to form a NUC Construct. Earlier products for regenerating urinary tissue (e.g., Tengion's Autologous Neo-Bladder Augment™) used SMC isolated from urinary bladder1. For bladder cancer patients, a SMC source other than the bladder is preferred; therefore, sourcing of autologous SMC from an alternate tissue for use in the NUC was evaluated. Adipose tissue was determined to be a suitable source of SMC. In a porcine model, the NUC Construct regenerated an incontinent urinary diversion composed of native-like urinary tissue.
Jayo M.J., Rivera E., Sharp W., Wagner B.J., Ludlow J.W., Jain D., Bertram T.A. Regeneration of Native-Like Mucocutaneous Region at the Skin-Conduit Junction Following Neo-Urinary Conduit™ Implantation. Poster Presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 5 - 8, 2010.
The urinary tract exits the body via the urethra, a distinct structure incorporating features that defend the orifice against local and/or ascending infections. Specifically, the mucocutaneous region is a non-keratinized stratified squamous epithelium composed of glycogen-rich cells that provide substrate for a protective endogenous lactobacteria flora. Epithelia closer to the skin are associated with acid-phosphatase activity and lysozyme-like immunoreactivity indicative of the presence of macrophages that secrete bactericidal compounds1. Regenerative medicine strategies for channeling urine from ureters to skin have been tested in large mammals and one option, Tengion's Neo-Urinary Conduit™ (NUC), is currently in Phase I clinical trials. This study reports on the ability of the NUC to initiate the formation of a native-like transition between urinary mucosa and skin epithelium that has the structural features of mucocutaneous regions observed in native urethras within 3 months post-implantation in cystectomized pigs.
Genheimer C.W., Guthrie K.I., Shokes J.E., Bruce A.T., Quinlan S.F., Sangha N., Ilagan R.M., Basu J., Burnette T., Ludlow J.W. (2010, October 6). Increased Urothelial Cell Detection in the Primary Bladder Smooth Muscle Cell Cultures with Dual MACS/qRT-PCR Approach . Appl Immunohistochem Mol Morphol. 2010 Oct 6. [Epub ahead of print]
Bladder tissue has been regenerated in humans with neurogenic bladder using an implant produced from autologous urothelial (UC) and smooth muscle cells (SMC) expanded from bladder biopsies seeded onto a biodegradable synthetic scaffold. As the majority of bladder cancers are urothelial carcinomas (aka, transitional cell carcinoma), this 2-cell type autologous sourcing strategy presents significant challenges to product development. Entire bladders have been regenerated in cystectomized animals using a single-cell-type sourcing strategy: implants were seeded with bladder-derived SMC-only. Applying the bladder SMC-only sourcing strategy to produce clinical implants for bladder replacement or urinary diversion in bladder cancer patients requires methods for screening SMC cultures for the presence of potentially cancerous UC cells to provide evidence of SMC culture purity before seeding the scaffold. In this report, we show a 10-fold to 100-fold improvement in the sensitivity of qualitative and quantitative reverse-transcription PCR (qRT-PCR)-based assays for detecting UC positive for Cytokeratin 5 (CK5) in mixed SMC/UC cultures when the cell population was first subjected to magnetic activated cell sorting to enrich for cells expressing the epithelial cell adhesion molecule (known as EPCAM or CD326), a marker known to be present in normal UC and upregulated in the cancerous UC.
Jain D., Ludlow J.W., Guthrie K., Sangha N., Genheimer C., Shokes J., Burnette T., Justewicz D., Ilagan R., Wagner B.J., Bertram T.A., (2010, May 24). Smooth Muscle Cells Derived From Human Adipose Tissue For Use In Urologic Tissue Engineering. Podium Presentation at 16th Annual Meeting of The International Society For Cellular Therapy, Philadelphia, PA May 23-26, 2010.
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.
Basu J, Guthrie K, Ilagan R, Sangha N, Genheimer C, Payne R, Rapoport S, Knight T, Wagner BJ, Jayo MJ, Jain D, Bertram TA, Ludlow JW (2009, July 8-11). Functional De Novo Neo-Urinary Conduit Formation From Porcine Peripheral Blood And Adipose Tissue Derived Smooth Muscle Cells. Poster presented at 2009 International Society for Stem Cell Research meeting, SPAIN.
Current surgical treatment options for many urinary disorders caused by congenital conditions, injury, or cancer involve the use of gastrointestinal (GI) tissue to form an orthotopic neobladder or urinary diversion. Use of GI tissue requires GI tissue resection and exposes gut mucosa to urine, leading to multiple acute and chronic complications, including GI and metabolic derangements. First generation regenerative medical technologies used an implant composed of synthetic biocompatible scaffold seeded with autologous urothelial and smooth muscle cells (SMC) isolated from native bladder tissue to eliminate the need for GI tissue in the urinary tract. 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 can require invasive biopsy of a diseased organ and is not ideal.
In this study, alternative cell sources were investigated for the SMC component of Tengion's Neo-Urinary Conduit (NUC). Porcine cells were cultured from bladder tissue, adipose tissue, and peripheral blood samples harvested from 24 animals. SMC were recovered from 100% of bladder and adipose tissue samples and 96% of peripheral blood samples. SMC phenotype of adipose and blood derived cultures was shown to be indistinguishable from bladder-derived smooth muscle cells by Ca2+ dependant contractility and immunocytochemistry using antibodies against four SMC proteins that are expressed in primary cultures of native porcine bladder SMC: smooth muscle alpha-actin, mycocardin, calponin, and smooth muscle myosin heavy chain.
To evaluate the ability of NUC seeded with SMC derived from bladder, adipose, or peripheral blood to regenerate urinary tissue in vivo, NUC implants were retrieved from recipient swine and processed for histological analysis. NUC seeded with smooth muscle cells from bladder, adipose tissue, or blood led to the development of a urinary tissue wall a mucosa lined with urothelial cells and multiple muscle layers composed of SMC by three months post-implantation. These results demonstrate that SMC can be isolated and expanded from porcine adipose and peripheral blood and seeded onto NUC that develop in vivo into a urinary diversion that passively transports urine from the ureters to outside the swine's body following radical cystectomy. The ability to form urinary tissue with SMC derived from non-bladder tissue sources should facilitate the use of this technology in clinical practice.
Jain D., Ludlow J.W., Halberstadt C., Payne R., Wagner B.J., Jayo M.J., Bertram T.A. (2009, March 6). An Autologous Smooth Muscle Cell (Smc) Seeded Plga-Based Scaffold (Neo-Bladder Conduit) for Establishing an Incontinent Urinary Diversion. Poster presented at Regenerative Medicine: Advancing Next Generation Therapies meeting, March 5-8, 2009 at Hilton Head Island, SC.
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.
Jain D., Ludlow J.W., Guthrie K., Johnson K., Bruce A., Bertram T. (2009, May 3). Skeletal Muscle Tissue As An Alternate Source Of Smooth Muscle Cells For Tissue-Engineered Neo-Urinary Implants. Poster presented at International Society for Cell Therapy (ISCT) 15th Annual Meeting, San Diego, CA, May 3-6, 2009.
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.
Spencer T., Kelley R., Werdin E., Bruce A., Jain D. (2011, December) Multivariate Analysis of Preclinical Data from the Tengion Neo-Kidney Augment.. Podium presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 11-14, 2010, Houston, Texas.
Chronic Kidney Disease is a public health issue with a substantial un-met clinical need. Tengion is developing a novel Regenerative Medicine treatment, the Neo-Kidney Augment, for patients with renal impairment. In the current study, data from a diabetic rat model will be presented showing the impact of treatment on disease progression. Classical statistical analysis, such as animal survival and ANOVA of serum and urine chemistry data, are compared with novel multivariate techniques. Composite measures are generated based on related physiological functions. The composite measures strongly predict the time to death for the animals that expired during the study period.
Jain D., Basu J., Halberstadt C., Rivera E., Knight T., Payne R., Robbins N., McCoy D., Guthrie K., Sangha N., Genheimer C., Ludlow J., and Bertram T. (2011, December) Selected Regenerative Renal Cells combined with Bioresorbable Natural Biomaterials catalyze in vivo Kidney Tissue Regeneration. Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 11-14, 2010, Houston, Texas.
Chronic kidney disease is a global public health concern involving progressive loss in renal function. Disease progression typically leads to dialysis and eventually a kidney transplant. An urgent need exists for new treatments to restore renal function thereby delaying or eliminating dialysis and transplant. Tengion’s unique integrated regenerative medicine technology platform has generated products that catalyze regeneration of tissues and organs. We report on the development of Neo-Kidney Augment (NKA) prototypes composed of selected regenerative renal cells (SRC) and a biomaterial for catalyzing kidney tissue regeneration.
Neo-Kidney Augment prototype implantation into healthy rat kidneys was well-tolerated and elicited neo-kidney tissue regeneration. Taken together, these data provide evidence that implantable regenerative medicine products developed using tissue engineering principles can be used in reconstructing solid organs (e.g., kidney).
Basu J., Payne R., Rivera E., Robbins N., McCoy D., Knight T., Sangha N., Guthrie K., Spencer T., Genheimer C., Jain D., Ludlow J.W. and Halberstadt C. (October 2011). Bio-Response of a Rodent Hemi-Nephrectomy Model to Implantation of Neo- Kidney Augment Prototypes Composed of Selected Renal Cells and Biomaterials. Poster presentation at Joint Congress 2011 Cell Transplant Society / International Xenotransplantation Association, October 23 – 26, Miami, Florida.
Continual loss of renal function over a time span of months or years is the operational definition of chronic kidney disease. Current renal function replacement therapy includes dialysis and eventual kidney transplant. An unmet need exists for new treatments to restore renal function thereby delaying or eliminating dialysis and transplant. Towards addressing this need, Tengion has developed a unique integrated regenerative medicine technology platform capable of catalyzing regeneration of tissues and organs. In the current study, we report on the development of a Neo-Kidney Augment (NKA) product prototype, comprised of biomaterials and selected regenerative renal cells (SRC), which facilitate regeneration of kidney tissue. SRC are obtained from enzymatic digestion of a kidney biopsy and density gradient separation of cells. Gelatin based hydrogels were used as biomaterial.
Bio-response of mammalian kidney towards implantation of NKA prototypes has previously been evaluated in healthy adult rodents (Basu et al., 2011, Cell Transplantation). However, removal of single kidney from rodents (hemi-nephrectomy) increases sensitivity of the model, permitting detection of systemically acting toxicological effects. In this study, 20 hemi-nephrectomized rodents were injected with NKA prototypes within the renal parenchyma of the remnant kidney. Physiological indices derived from whole blood, serum and urine chemistries were evaluated at 2 and 4 week time points post-implantation. Animals were sacrificed at 4 weeks post-injection and remnant kidney examined histologically for evidence of inflammatory or fibrotic bio-response. Implantation of NKA prototypes did not significantly affect key renal physiological indices, and presented minimal evidence of inflammatory, necrotic or fibrotic bio-response. Therefore, NKA prototypes based on SRC in gelatin based hydrogels are well tolerated by remnant kidney in the rodent hemi-nephrectomy model.
Kelley R., Spencer T., Werdin E., Bruce A., Ilagan R., Wallace S., Watts B., Choudhury S., Cox B., Guthrie K., Jayo M., Bertram T., Presnell S. (June 2011). Intra-renal Transplantation of Bioactive Renal Cells Preserves Renal Functions and Extends Survival in the ZSF1 model of Progressive Diabetic Nephropathy. Poster presentation at 71st Scientific Sessions of American Diabetes Association, June 24 – 27, 2011 in San Diego, CA.
There are >200,000 diabetic patients in the US with End-Stage Renal Disease. Hemodialysis and pharmacological intervention insufficiently support diminishing kidney function long term, culminating in whole kidney transplantation or death. New treatment paradigms that slow or reverse progression of chronic kidney disease (CKD) are needed to relieve patient and healthcare burdens. Recent work in our laboratory demonstrated that a selected population of bioactive renal cells (BRCs), established from autologous diseased kidney tissue, regenerated in situ functional kidney mass, stabilized filtration function, and prolonged survival following intra-renal delivery in a 5/6 nephrectomy model of terminal CKD1. In the present study, the in vivo function of these BRCs was evaluated in the ZSF1 rodent model of progressive nephropathy secondary to a metabolic syndrome of diabetes, obesity, dyslipidemia, and hypertension. Injection of syngeneic BRCs into the ZSF1 renal parenchyma elicited a regenerative response that significantly improved renal functions, including filtration (BUN, sCre, eGFR), protein handling (albumin), electrolyte balance (K, Na, Phos) and the ability to concentrate urine (osmolarity) confirming similar results observed in a severe mass reduction model of CKD1. Multivariable linear regression analysis showed that each of the renal functions affected by treatment significantly predicted ZSF1 survival beyond the one year timeline for follow up. The characteristic hypertension in the ZSF1 model was attenuated at 50 weeks of age by the cell treatment; these data were further supported by statistically significant modulation of physiological regulators of blood pressure, including the pressor hormones, ACTH, cortisol and renin. Also consistent with previous results, implantation of the BRCs in the ZSF1 model resulted in significant reduction of circulating plasminogen activator inhibitor (PAI), a master regulator of tissue fibrosis. These results collectively form the basis for justifying the clinical use for intra-renal delivery of an adult autologous and regenerative renal cell population for slowing disease progression in CKD patients secondary to metabolic syndrome.
Basu J., Genheimer C., Sangha N., Quinlan S., Guthrie K., Kelley R., Ilagan R., Jain D., Bertram T.A. and Ludlow J.W. (June 2011). Organ Specific Regenerative Markers in Peri-Organ Adipose: Kidney. Poster presentation to the 9th Annual meeting of International Society of Stem Cell Research (ISSCR), Toronto, Ontario Canada,June 15 - 18, 2011.
Therapeutically bioactive cell populations may promote regenerative outcomes in vivo by leveraging mechanisms of action including secretion of growth factors, site specific engraftment and directed differentiation. Constitutive cellular populations also participate in regenerative processes. Adipose tissue is a source of therapeutically bioactive cell populations. The potential of these cells to participate in regenerative processes is broadly demonstrated1. However, organ association of regenerative markers to specific peri-organ adipose depots has not been investigated. To characterize this topographical association, we explored the potential of cells isolated from the stromal vascular fraction (SVF) of kidney and non-kidney sourced adipose to express key renal associated factors. We report that adipose tissue is a novel reservoir for EPO expressing cells. Kidney adipose-derived SVF cells show hypoxia regulated expression of EPO transcript/protein and VEGF transcripts. Using iso-electric focusing, we demonstrate that kidney and non-kidney adipose derived cells present uniquely different patterns of EPO post-translational modification, consistent with the idea that kidney and non-kidney sources are functionally distinct adipose depots. In addition, kidney adipose-SVF cells specifically express the key kidney developmental transcription factor WT1 while non-kidney adipose does not express WT1. Taken together, these data are consistent with the notion that kidney adipose sourced stromal cells could be used to recreate a regenerative microenvironment within kidney. These findings open the possibility of isolating solid organ associated adipose cell populations for therapeutic application in organ-specific regenerative medicine products.
Bruce A., Watts B., Cox B., Genheimer C., Werdin E., Guthrie K., Ilagan R., Kelley R., Presnell S., Wallace S., Choudhury S. (April 2011). Hypoxic Exposure of Cultured Human Renal Cells Induces Mediators of Cell Migration and Attachment and Facilitates the Repair of Tubular Cell Monolayers in vitro. Podium presentation given at Experimental Biology Meeting in Washington, DC, April 10, 2011.
Jain D., Payne R. et al (March 2011). Catalyzing Kidney Regeneration using Selected Renal Cells and Biomaterials in a Neo-Kidney Augment Product. Oral Presentation given at Tissue Engineering meeting, Hilton Head, NC March 2011.
Basu J., Genheimer C.W., Rivera E.A., Payne R., Mihalko K., Guthrie K., Bruce A.T., Robbins N., McCoy D., Sangha N., Ilagan R., Knight T., Spencer T., Wagner B.J., Jayo M.J., Jain D., Ludlow J.W., Halberstadt C. (2011, March 24) Functional evaluation of primary renal cell/biomaterial Neo-Kidney Augment prototypes for renal tissue engineering Cell Transplant. 2011 Mar 24. doi: 10.3727/096368911X566172. [Epub ahead of print]
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 neo-kidney 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 neo-vascularization and cellular infiltration by 4 weeks post-implantation. 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 neo-kidney tissue formation at 1 week post-implantation. To investigate a potential mechanism by which NKA Constructs could impact a disease state, the effect of conditioned media on TGF-Î² signaling pathways related to tubulo-interstitial fibrosis associated with CKD progression was evaluated. Conditioned medium was observed to attenuate TGF-Î²-induced epithelial-mesenchymal transition (EMT) in vitro in a human proximal tubular cell line (HK2).
Payne R., Knight T., Basu J., Rivera E., Robbins N., McCoy D., Jayo M., Halberstadt C., Jain D. (2011, April). Bioresponse of Mammalian Kidney to Implantation of Polymeric Materials Poster presented at 2011 Society for Biomaterials Annual Meeting & Exposition, April 13-16, 2011 in Orlando, Florida.
Biomaterials can modulate regenerative outcomes in tissue engineering or regenerative medicine applications by facilitating cell attachment and delivery and by providing a physical substrate for tissue infiltration1. This study investigated host tissue responses to intra-renal injection of biomaterials in rodent kidneys to identify candidate biomaterials for forming cell/biomaterial composites with selected regenerative cell populations2. Natural and synthetic biomaterials were evaluated in both spherical bead and irregular particle forms. The ultimate goal of this research is to develop Neo-Kidney Augment prototypes that delay the need for dialysis and improve renal function in patients with chronic kidney disease.
Kelley R.W., Werdin E.S., Bruce A.T., Wallace S.M., Jayo M.J., Bertram T.A. and Presnell S.C. Bioactive Renal Cells Augment Renal Function in the ZSF1 model of Diabetic Nephropathy. Oral Presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 5 - 8, 2010.
Patients with chronic kidney disease (CKD) face years of dialysis and a complex drug regimen. While kidney transplant can eliminate dialysis and prevent CKD-related death, the ~16,000 donor kidneys available for transplant annually in the US cannot service the >500,000 patients with end-stage renal disease. New treatments that slow CKD progression and reduce dependency on dialysis and transplantation are needed. CKD develops from a progressive imbalance between tissue damage and the kidney's intrinsic repair and regeneration processes. Recent work in our laboratory demonstrated that selected bioactive renal cells, established from primary cultures1,2, regenerated functional kidney mass, stabilized filtration function, and prolonged survival following delivery to the remnant kidney in a 5/6 nephrectomy model of terminal CKD3. In the present study, the selected bioactive renal cells were delivered to ZSF1 rodents to evaluate this candidate treatment for CKD in a model of progressive nephropathy secondary to multiple related disorders of metabolic syndrome, including obesity, diabetes, severe dyslipidemia, and hypertension.
Choudhury S., Bruce A.T., Kelley R.W., Cox B.R., Werdin E.S., Watts B., Presnell S.C., and Ilagan R.M. Paracrine Factors Derived from Bioactive Kidney Cells Provide Anti-fibrotic Signals in vitro and may Mediate Regenerative Outcomes in vivo. Poster Presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 5 - 8, 2010.
The regenerative potential of a particular tissue or treatment modality is often defined as its inherent capacity to re-establish appropriate function in vivo by direct replacement of lost or damaged cells. Of equal importance, however, is the ability of a cell to stimulate regeneration and attenuate the progression of disease through indirect mechanisms. In previous studies, we have shown that intra-renal delivery of bioactive kidney cells into a Lewis rat model of chronic kidney disease preserves kidney functions, resulting in significant reductions in glomerular and tubulointerstitial fibrosis and attenuation of pro-fibrotic pathways when compared to untreated controls1. In the present study, we employed in vitro cell-based assays to investigate potential paracrine mechanism(s) by which bioactive kidney cells could modulate fibrosis through mediators such as Plasminogen Activator Inhibitor-1 (PAI-1).
Ilagan R.M., Guthrie K.I., Cox B.R., Choudhury S., Bruce A.T., Watts B., O'Reilly D.D., Tang C., Genheimer C.W., Werdin E.S., Kelley R.W., and Presnell, S.C. Secreted Factors from Bioactive Kidney Cells Attenuate NF-kappa-B. Poster Presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 5 - 8, 2010.
Nuclear Factor-kappa-B (NF-kappa-B) activation is thought to participate in acute and chronic diseases by eliciting robust innate immune responses in injured or diseased tissues; however, recent work suggests that NF-kappa-B pathways might also participate in the resolution of inflammation in a model of kidney disease1. The bifunctional nature of this pathway suggests that NF-kappa-B may modulate a balance between pro-inflammatory, fibrotic mechanisms of tissue repair and restorative or regenerative outcomes in vivo. We have previously demonstrated that intra-renal delivery of bioactive kidney cells, a Neo Kidney Augment™ (NKA) prototype, can preserve kidney function and tissue integrity in a Lewis rat model of chronic kidney disease2. In this study, we investigated the role of NF-kappa-B pathways in the NKA-initiated attenuation of disease progression in the 5/6 nephrectomy model and to identify properties of the bioactive kidney cells that may contribute to regenerative outcomes through direct modulation of NF-kappa-B activation.
Basu J., Payne R., Rivera E., Robbins N., McCoy D., Knight T., Jayo M.J., Jain D., Halberstadt C.R. In vivo Evaluation of Biomaterials in Mammalian Kidney. Poster Presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 5 - 8, 2010.
Biomaterials can contribute to tissue and organ regeneration by providing a substrate for cell attachment and delivery and a physical template for tissue growth1-3. This study reports a systematic evaluation of the inflammatory and fibrotic responses of mammalian kidney to in vivo microinjection of natural and synthetic biomaterials. The objective was to identify biomaterial candidates that can be combined with therapeutically-relevant renal cell populations4 to form cell/biomaterial composites capable of inducing a regenerative response in chronic kidney disease models.
Basu J., Rivera E., Genheimer C.W., Robbins N., Payne R., McCoy D., Guthrie K.I., Sangha N., Knight T., Jayo M., Ludlow J.W., Jain D., and Halberstadt C.R.. Mammalian Kidney Nephrogenic Response to Primary Renal Cell/Biomaterial-based Neo-Kidney Augment™ Prototype. Poster Presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS) - North America Annual Conference, December 5 - 8, 2010.
Development of a tissue engineered Neo-Kidney Augment (NKA) requires evaluation of defined, therapeutically relevant cell and cell/biomaterial composites for regenerative potential in mammalian kidney. Previous work evaluated in vivo responses to cell-free hydrogel-based biomaterial NKA prototypes1 and the bioactivity of defined renal cell populations2. This report provides evidence that intra-parenchymal delivery of a cell/hydrogel NKA prototype triggered induction of neo-kidney tissue in healthy Lewis rat kidneys within 4 weeks post-implant suggesting that this NKA prototype could potentially modulate renal regeneration in a mammalian model of chronic kidney disease. To the best of our knowledge, the current study and Basu et al.1 are the first in vivo and intra-renal investigations of the biological response of mammalian kidney to implantation of a therapeutically-relevant primary renal cell/biomaterial composite.
Cell Transplant Approaches to Kidney Regeneration. Podium presentation at American Society of Transplantation-Annual Scientific Exchange (AST-ASE), Orlando, FL October 21-24, 2010.
Stem and Progenitor Marker Expression as a Predictor of Renal Regeneration in 5/6 Nephrectomized Rats Treated with Therapeutically Bio-Active Primary Renal Cell Sub-Populations. Poster presented at World Stem Cell Summit, held October 4-6, 2010 in Detroit, MI.
Chronic kidney disease (CKD) is a global public health concern involving progressive loss in renal function. Preservation of renal function is key to reducing morbidity in CKD patients. The disparity between the number of patients on dialysis awaiting kidney transplant and the number of organs actually transplanted highlights the need for new treatments to preserve renal function. Regenerative medicine approaches may offer hope to those CKD patients waiting for a transplant. Using a rat 5/6 nephrectomy model for CKD, we have developed molecular assays to evaluate the mobilization of resident stem and progenitor cells within the rat 5/6 nephrectomized kidney in response to intra-renal injection of defined, therapeutically bio-active primary renal cell populations. We show that treatment was associated with up-regulation of the key stem cell markers CD24, CD133, UTF1, SOX2, LEFTY1, and NODAL at both transcript and protein levels. Up-regulation was detected by 1 week post-injection and peaked by 12 weeks post-injection. Activation of stem and progenitor cell markers was associated with increased survival relative to untreated nephrectomized controls, consistent with the increased survival and preserved renal function observed in multiple, previous studies.
Presnell S.C., Jayo M.J., Jain D., & Bertram T.A. Discovery and Development of Regenerative Medicine Products Comprised of Autologous Cells and Biomaterials. Podium presentation at International Society for Cellular Therapy (ISCT) 2nd Annual Symposium on Stem Cell Translation, San Francisco, CA on September 27-28, 2010.
Clinical translation of regenerative medicine products requires focused, but adaptable, strategies during discovery and early development phases to increase the probability of success. Iterative in vitro / in vivo testing of early-stage prototypes in parallel with strategic optimization of bioprocess accelerates development timelines. Such a process facilitates bringing forward cutting edge, commercially-viable solutions that address unmet medical needs for evaluation in clinical trials. Applying this strategic approach simplified Urothelial Cell (UC) + Smooth Muscle Cell (SMC) + scaffold production (Tengion's Neo-Bladder Augment) to SMC + scaffold production (Tengion's Neo-Urinary Conduit) for regeneration of autologous urinary tissue. An additional advancement achieved during preclinical development of Tengion's Neo-Urinary Conduit (NUC) was sourcing SMC from autologous adipose tissue instead of a bladder biopsy, thus eliminating the need to source SMC from bladder tissue in patients with bladder cancer. The Neo-Kidney Augment (NKA) represents the first extension of Tengion's platform to solid organ regeneration. An in vivo combinatorial screening strategy enabled rapid identification of specific renal-derived cells as the core Active Biologic Ingredient (ABI) and identified lead biomaterial candidates for formulation and prototype optimization efforts. In vivo proof of concept has been established for the NKA ABI in two animal models of chronic kidney disease (CKD); (i) the surgically-induced remnant kidney model of renal insufficiency and (ii) the ZSF1 model of nephropathy secondary to morbid obesity and Type-2 diabetes. Intra-renal parenchymal delivery of the NKA ABI provided a significant regenerative stimulus in both models of CKD, resulting in delayed disease progression, preservation of functional renal mass, and reduced disease-related mortality. Translational research and development have demonstrated that the NKA ABI can be isolated from biopsy-sized kidney samples in several animal species including human patients with end-stage CKD. Bioprocess development is focused on NKA cell/biomaterial formulations to provide targeted delivery of the ABI, optimize product stability, and potentially expand treatment to subjects with severe fibrosis and end-stage renal disease. Further definition of NKA prototypes will help to establish clinically-relevant delivery methods for future GLP studies.
Presnell S.C., Bruce A.T., Wallace S.M., Choudhury S., Genheimer C., Cox B., Guthrie K.I., Werdin E.S., Tatsumi-Ficht P., Ilagan R., Kelley R.W., Rivera E.A., Ludlow J.W., Wagner B.J., Jayo M.J., Bertram T. (2010, September 16) Isolation, Characterization, and Expansion (ICE) Methods for Defined Primary Renal Cell Populations from Rodent, Canine, and Human Normal and Diseased Kidneys. Tissue Eng Part C Methods. 2010 Sep 16.
Chronic kidney disease (CKD) is a global health problem; the growing gap between the number of patients awaiting transplant and organs actually transplanted highlight 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 following 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, then to rodent and human tissues with advanced chronic kidney disease (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.
Kelley R., Werdin E.S., Bruce A.T., Choudhury S., Wallace S.M., Ilagan R.M., Cox B.R., Tatsumi-Ficht P., Rivera E.A., Spencer T., Rapoport H.S., Wagner B.J., Guthrie K., Jayo M.J., Bertram T.A., Presnell S.C. (2010, September 8). A tubular cell-enriched subpopulation of primary renal cells improves survival and augments kidney function in a rodent model of chronic kidney disease. Am J Physiol Renal Physiol (September 8, 2010). doi:10.1152/ajprenal.00221.2010
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 developing 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 weeks after CKD was established by 2-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 months in duration) with B2 confirmed that B2 significantly extended survival and improved renal filtration (serum creatinine and BUN). 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 six months post-treatment confirmed that B2 treatment reduced severity of kidney tissue pathology. Treatment-associated reduction of transforming growth factor β1 (TGFβ1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin (FN) provided evidence that B2 cells attenuated canonical pathways of profibrotic extracellular matrix production.
Kelley R., Werdin E.S., Bruce A.T., Choudhury S., Wallace S.M., Tatsumi-Ficht P., Rivera E.A., Spencer T., Rapoport H.S., Ilagan R.M., Wagner B.J., Jayo M.J., Bertram T.A., Presnell S.C. (2010, May 24). Bioactive Renal Cells Augment Kidney Function In A Rodent Model Of Chronic Kidney Disease. Podium Presentation at 16th Annual Meeting of The International Society For Cellular Therapy, Philadelphia, PA May 23-26, 2010.
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.
Wallace S.M., Bruce A.T., Choudhury S., Tatsumi-Ficht P., Cox B.R., Wagner B.J., Kelley R., Presnell S.C. (2010, May 26). Quantitative Ex Vivo Characterization Of Human Renal Cell Population Dynamics Via High-Content Image-Based Analysis (HCA). Podium Presentation at 16th Annual Meeting of The International Society For Cellular Therapy, Philadelphia, PA May 23-26, 2010.
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.
Kelley, R., Bruce, A., Wallace, S., Choudhury, S., Tatsumi, P., Das, A., Riveras, E., & Presnell, S. (2008, September 17-19). Enhanced renal cell function in dynamic 3D culture system. Poster presented at the KIDSTEM International Conference, Liverpool, England.
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.
Ilagan RM, Guthrie K, Sangha N, Quinlan S, Delo O'Reilly D, Jain D, Bertram TA, Presnell SC, and Ludlow JW (2009, July 29-31). Characterization of primary adult canine renal cells (CRC) in a three-dimensional (3D) culture system permissive for ex vivo nephrogenesis. Poster presented at the 2009 KIDSTEM International Conference, Edinburgh, Scotland.
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.
Kelley RW, Werdin ES, Bruce AT, Choudhury S, Wallace SM, Tatsumi-Ficht P, Rivera EA, Jayo MJ, Bertram TA, and Presnell SC (2009, July 29-31). Isolation and propagation of therapeutically-relevant cells, including Epo-producing cells, from severely diseased kidneys translates across rodents, large mammals and humans. Poster presented at the 2009 KIDSTEM International Conference, 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.
Presnell S.C., Bruce A., Wallace S.M., Choudhury S., Kelley R., Tatsumi P., Werdin E., Rivera E., Merricks E., Nichols T.C., Jennette J.C., Jayo M.J., Bertram T.A. (2009, April 18). Isolation and characterization of bioresponsive renal cells from human and large mammal with chronic renal failure. Poster presented at the Experimental Biology Meeting, held in New Orleans, LA, April 18-22, 2009.
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.
Ilagan R.M., Genheimer C.W., Quinlan S.F., Guthrie K.I., Sangha N., Ramachandrannair S., Kelley R.W., Presnell S.C., Basu J., Ludlow J.W. (October 2011). Smooth muscle phenotypic diversity is mediated through alterations in Myocardin gene splicing. J Cell Physiol. 2011 Oct;226(10):2702-11. doi: 10.1002/jcp.22622.
Myocardin (MYOCD) is a smooth and cardiac muscle-specific transcriptional coactivator that is required for the proper expression of contraction-related genes. Through its function to transactivate effector genes, MYOCD plays an essential role in mediating the switch between contractile and non-contractile phenotypes, particularly in smooth muscle cells (SMC). There are at least two known transcript variants of MYOCD that are expressed in SMC, differing only by the presence (+) or absence (Δ) of Exon 11. To date, no functional role has been assigned to the domain encoded by Exon 11, nor have any notable differences between the ability of each isoform to activate contraction-related genes been observed. In this study we compared sequences for Exon 11 among several mammalian species and identified a highly conserved, putative target sequence for glycogen synthase kinase 3 (GSK3) phosphorylation, suggesting a regulatory role for Exon 11 that can be modulated by alternative splicing. The function of Exon 11 was investigated by altering MYOCD splice selection in cultured porcine SMC with small interfering RNAs (siRNA) and specific chemical inhibitors, resulting in a relative increase in expression of ΔExon 11 variants in the endogenous pool of MYOCD mRNA. The relative increase in ΔExon 11 mRNAs correlated with a reduction of contractile phenotype in the porcine SMC as evidenced by morphological assessment and molecular analysis of effector genes. Together, these data suggest that MYOCD ΔExon 11 may participate in modulating SMC phenotype, potentially acting as a dominant-negative repressor of contraction-related genes. J. Cell. Physiol. 226: 2702-2711, 2011. © 2010 Wiley-Liss, Inc.
Jain D., Jayo M.J., Bertram T.A. (May 2011). Development of Tissue Engineered Regenerative Medicine Products Using Selected Autologous Cells and Biomaterials. Poster presentation to 17th Annual Meeting of International Society for Cellular Therapy (ISCT) in Rotterdam, The Netherlands, May 18 -21, 2011.
Regenerative medicine holds the promise of fulfilling unmet medical needs, especially in the area of restoration of organ function and organ replacement (transplantation). Development of tissue engineered regenerative medicine products requires a multi-disciplinary effort involving cell biology, biomaterials, bioprocess development, engineering, preclinical development, clinical translation, and manufacturing. Strategies should be evaluated early in development with input from Clinical, Regulatory, and Marketing to improve the probability of success. For example, use of autologous cells can provide early entry into the clinic but may present challenges in manufacturing scale-up and not be commercially acceptable. Biomaterials used to formulate biologically active components may involve a more complicated regulatory approval pathway. Tengion has created a unique integrated technology platform to develop regenerative medicine products from research to commercialization.
Basu J., Genheimer C.W., Guthrie K.I., Sangha N., Quinlan S.F., Bruce A.T., Reavis B., Halberstadt C., Ilagan R.M., Ludlow J.W. (2011 May 19). Expansion of the Human Adipose-Derived Stromal Vascular Cell Fraction Yields a Population of Smooth Muscle-Like Cells with Markedly Distinct Phenotypic and Functional Properties Relative to Mesenchymal Stem Cells. Tissue Eng Part C Methods. 2011 May 19. [Epub ahead of print].
Adipose tissue contains a heterogeneous cell population composed of endothelial cells, adipocytes, smooth muscle cells (SMC), and mesenchymal progenitors and stromal cells that meet the criteria put forth by the International Society for Cellular Therapy as defining mesenchymal stem cells (MSC). In this study, we expanded the stromal vascular fraction (SVF) of human adipose tissue and characterized the resulting adherent primary cell cultures by quantitative reverse transcription-polymerase chain reaction, antigen expression, protein fingerprinting, growth kinetics, in vitro tri-lineage differentiation bioactivity, and functional responses to small molecules modulating SMC-related developmental pathways and compared the results to those obtained with functionally validated MSC cultures. SVF-derived initial cultures (P0) were expanded in a defined medium that was not optimized for MSC growth conditions, neither were recombinant cytokines or growth factors added to the media to direct differentiation. The adherent cell cultures derived from SVF expansion under these conditions had markedly distinct phenotypic and biological properties relative to functionally validated MSC cultures. SVF-derived adherent cell cultures retained characteristics consistent with the SMC subpopulation within adipose tissue-phenotype, gene, and protein expression-that were independent of passage number and source of SVF (n=4 independent donors). SVF-derived cells presented significantly less robust in vitro tri-lineage differentiation bioactivity relative to validated MSC. Expanded 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 conclusions that SVF cells expanded under the conditions described in these studies are accurately described as adipose-derived SMC and represent a cellular subpopulation of adipose SVF that is separate and distinct from other classes of adipose-derived cells.
Serban M.A., Knight T., Payne R., Basu J., Rivera E.A., Robbins N., McCoy D., Halberstadt C., Jain D., Bertram T.A. (2011, April). Fabrication of Chemically Crosslinked Gelatin Microspheres with Tunable Degradation Profiles Poster presented at 2011 Society for Biomaterials Annual Meeting & Exposition, April 13-16, 2011 in Orlando, Florida.
The goal of this work was to investigate the tunability of the enzymatic degradation of gelatin-based biomaterials through the control of the extent of crosslinking. For this, we chose to use a well characterized and widely used reagent, N-(3-Dimethylaminopropyl)-Nâ€²-ethylcarbodiimide hydrochloride (EDC). This zero-length crosslinker promotes the formation of an amide bond between spatially adjacent carboxyl and primary amine functionalities located either intra- or intermolecularly. As indicated by our results, the extent of crosslinking directly correlates to the rate of collagenase-mediated degradation of gelatin beads. Commercially available counterparts provide versatility relative to the size of the beads and possess the porosity desired for cell attachment but offer limited control over the enzymatic degradation rates. For applications where the material biodegradability needs to be finely modulated, the process described herein offers a simple, time and cost efficient alternative.
Presnell S.C., Jayo M.J., Jain D., & Bertram T.A. Discovery and Development of Regenerative Medicine Products Comprised of Autologous Cells and Biomaterials. Podium presentation at International Society for Cellular Therapy (ISCT) 2nd Annual Symposium on Stem Cell Translation, San Francisco, CA on September 27-28, 2010.
Clinical translation of regenerative medicine products requires focused, but adaptable, strategies during discovery and early development phases to increase the probability of success. Iterative in vitro / in vivo testing of early-stage prototypes in parallel with strategic optimization of bioprocess accelerates development timelines. Such a process facilitates bringing forward cutting edge, commercially-viable solutions that address unmet medical needs for evaluation in clinical trials. Applying this strategic approach simplified Urothelial Cell (UC) + Smooth Muscle Cell (SMC) + scaffold production (Tengion's Neo-Bladder Augment) to SMC + scaffold production (Tengion's Neo-Urinary Conduit) for regeneration of autologous urinary tissue. An additional advancement achieved during preclinical development of Tengion's Neo-Urinary Conduit (NUC) was sourcing SMC from autologous adipose tissue instead of a bladder biopsy, thus eliminating the need to source SMC from bladder tissue in patients with bladder cancer. The Neo-Kidney Augment (NKA) represents the first extension of TengionÐ¿Ñ—Ð…s platform to solid organ regeneration. An in vivo combinatorial screening strategy enabled rapid identification of specific renal-derived cells as the core Active Biologic Ingredient (ABI) and identified lead biomaterial candidates for formulation and prototype optimization efforts. In vivo proof of concept has been established for the NKA ABI in two animal models of chronic kidney disease (CKD); (i) the surgically-induced remnant kidney model of renal insufficiency and (ii) the ZSF1 model of nephropathy secondary to morbid obesity and Type-2 diabetes. Intra-renal parenchymal delivery of the NKA ABI provided a significant regenerative stimulus in both models of CKD, resulting in delayed disease progression, preservation of functional renal mass, and reduced disease-related mortality. Translational research and development have demonstrated that the NKA ABI can be isolated from biopsy-sized kidney samples in several animal species including human patients with end-stage CKD. Bioprocess development is focused on NKA cell/biomaterial formulations to provide targeted delivery of the ABI, optimize product stability, and potentially expand treatment to subjects with severe fibrosis and end-stage renal disease. Further definition of NKA prototypes will help to establish clinically-relevant delivery methods for future GLP studies.
Basu J., Ludlow J.W. (2010, August 25). Platform technologies for tubular organ regeneration. Trends Biotechnol. 2010 Oct;28(10):526-33. Epub 2010 Aug 25.
As a result of recent successes in regenerative medicine approaches to engineering multiple, disparate tubular organs, commonalities of methodology are emerging. Principal themes include the importance of a biodegradable scaffold nucleated with a population of smooth muscle cells (SMCs). Such composites trigger a regenerative response following in vivo implantation, resulting in de novo organogenesis. In this review, we examine bladder regeneration as a foundational platform technology to highlight key principles applicable to the regeneration of any tubular organ, and illustrate how these general concepts underlie current strategies to regenerate components of gastrointestinal, vascular, pulmonary and genito-urinary systems. We focus on identifying the elements of this platform that have facilitated the transition of tubular organ regeneration from academic proof-of-concept to commercial viability.
Ludlow J.W., Basu J., Genheimer C., Guthrie K., Sangha N., Quinlan S., Bruce A.T., Ilagan R.M., Bertram T.A., Jain D. (2010, June 16). Phenotypic and Functional Distinction Between Mesenchymal Stem Cells and the Stromal Vascular Cell Fraction of Human Adipose Tissue. Poster presented at 8th Annual Meeting of the International Society for Stem Cell Research, held June 16-19, 2010 in San Francisco, California.
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.
Bertram, T. A., & Jayo, M. J. (2008). Tissue engineered products: Preclinical development of neo-organs. In J. Cavagnero (Ed.), Preclinical safety evaluation of biopharmaceuticals: A science-based approach to facilitating clinical trials (pp. 799-826). New York: John Wiley & Sons.
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.
Jayo, M. J., Watson, D. D., Wagner, B. J., & Bertram, T. A. (2008). Tissue engineering and regenerative medicine: role of toxicologic pathologists for an emerging medical technology. Toxicol Pathol, 36(1), 92-96.
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.
Russell, A. J., & Bertram, T. A. (2007). Moving into the clinic. In R. Lanza, R. Langer & J. P. Vacanti (Eds.), Principles of tissue engineering (3rd ed., pp. 15-32). Burlington, MA: Elsevier Academic Press.
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.