Proteomic profiling of regenerated urinary bladder tissue in a non-human primate augmentation model.

Urinary bladder dysfunction can be caused by environmental, genetic, and developmental insults. Depending upon insult severity, the bladder may lose its ability to maintain volumetric capacity and intravesical pressure resulting in renal deterioration. Bladder augmentation enterocystoplasty (BAE) is utilized to increase bladder capacity to preserve renal function using autologous bowel tissue as a "patch." To avoid the clinical complications associated with this procedure, we have engineered composite grafts comprised of autologous bone marrow mesenchymal stem cells (MSCs) co-seeded with CD34+ hematopoietic stem/progenitor cells (HSPCs) onto a pliable synthetic scaffold [poly(1,8-octamethylene-citrate-co-octanol)(POCO)] or a biological scaffold (SIS; small intestinal submucosa) to regenerate bladder tissue in our baboon bladder augmentation model. We set out to determine the global protein expression profile of bladder tissue that has undergone regeneration with the aforementioned stem cell seeded scaffolds along with baboons that underwent BAE. Data demonstrate that POCO and SIS grafted animals share high protein homogeneity between native and regenerated tissues while BAE animals displayed heterogeneous protein expression between the tissues following long-term engraftment. We posit that stem cell-seeded scaffolds can recapitulate tissue that is nearly indistinguishable from native tissue at the protein level and may be used in lieu of procedures such as BAE.

Multipotent bone marrow cell-seeded polymeric composites drive long-term, definitive urinary bladder tissue regeneration.

To date, there are no efficacious translational solutions for end-stage urinary bladder dysfunction. Current surgical strategies, including urinary diversion and bladder augmentation enterocystoplasty (BAE), utilize autologous intestinal segments (e.g. ileum) to increase bladder capacity to protect renal function. Considered the standard of care, BAE is fraught with numerous short- and long-term clinical complications. Previous clinical trials employing tissue engineering approaches for bladder tissue regeneration have also been unable to translate bench-top findings into clinical practice. Major obstacles still persist that need to be overcome in order to advance tissue-engineered products into the clinical arena. These include scaffold/bladder incongruencies, the acquisition and utility of appropriate cells for anatomic and physiologic tissue recapitulation, and the choice of an appropriate animal model for testing. In this study, we demonstrate that the elastomeric, bladder biomechanocompatible poly(1,8-octamethylene-citrate-co-octanol) (PRS; synthetic) scaffold coseeded with autologous bone marrow-derived mesenchymal stem cells and CD34+ hematopoietic stem/progenitor cells support robust long-term, functional bladder tissue regeneration within the context of a clinically relevant baboon bladder augmentation model simulating bladder trauma. Partially cystectomized baboons were independently augmented with either autologous ileum or stem-cell-seeded small-intestinal submucosa (SIS; a commercially available biological scaffold) or PRS grafts. Stem-cell synergism promoted functional trilayer bladder tissue regeneration, including whole-graft neurovascularization, in both cell-seeded grafts. However, PRS-augmented animals demonstrated fewer clinical complications and more advantageous tissue characterization metrics compared to ileum and SIS-augmented animals. Two-year study data demonstrate that PRS/stem-cell-seeded grafts drive bladder tissue regeneration and are a suitable alternative to BAE.

Proteomic profiling of regenerated urinary bladder tissue with stem cell seeded scaffold composites in a non-human primate bladder augmentation model.

Urinary bladder insult can be caused by environmental, genetic, and developmental factors. Depending upon insult severity, the bladder may lose its ability to maintain capacity and intravesical pressures resulting in renal deterioration. Bladder augmentation enterocystoplasty (BAE) is employed to increase bladder capacity to preserve renal function using autologous bowel tissue as a "patch." To avoid the clinical complications associated with this procedure, we have engineered composite grafts comprised of autologous bone marrow mesenchymal stem cells (MSCs) with CD34+ hematopoietic stem/progenitor cells (HSPCs) co-seeded onto a pliable synthetic scaffold [POCO; poly(1,8-octamethylene-citrate-co-octanol)] or a biological scaffold (SIS; small intestinal submucosa) to regenerate bladder tissue in a baboon bladder augmentation model. We set out to determine the protein expression profile of bladder tissue that has undergone regeneration with the aforementioned stem cell seeded scaffolds along with baboons that underwent BAE. Data demonstrate that POCO and SIS grafted animals share high protein homogeneity between native and regenerated tissues while BAE animals displayed heterogenous protein expression between the tissues following long-term engraftment. We posit that stem cell seeded scaffolds can recapitulate tissue that is almost indistinguishable from native tissue at the protein level and may be used in lieu of procedures such as BAE.

Evolving Experimental Platforms to Evaluate Ulcerative Colitis.

Ulcerative colitis (UC) is a multifactorial disease defined by chronic intestinal inflammation with idiopathic origins. It has a predilection to affect the mucosal lining of the large intestines and rectum. Management of UC depends upon numerous factors that include disease pathogenesis and severity that are maintained via medical or surgical means. Chronic inflammation that is left untreated or managed poorly from a clinical stance can result in intestinal ulceration accompanied by resulting physiological dysfunction. End-stage UC is mediated by surgical intervention with the resection of diseased tissue. This can lead to numerous health-related quality of life issues but is considered a curative approach. Regimens to treat UC are ever evolving and find their basis within various platforms to evaluate and treat UC. Numerous modeling systems have been examined to delineate potential mechanisms of action. However, UC is a heterogenous disease spanning unknown genetic origins coupled with environmental factors that can influence disease outcomes and related treatment procedures. Unfortunately, there is no one-size-fits-all model to fully assess all facets of UC. Within the context of this review article, the utility of various approaches that have been employed to gain insight into different aspects of UC will be investigated.

Caveolin-1 scaffold domain interacts with TRPC1 and IP3R3 to regulate Ca2+ store release-induced Ca2+ entry in endothelial cells.

Caveolin-1 (Cav-1) regulates agonist-induced Ca(2+) entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (NH(2)-terminal residues 82-101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 (IP(3)R3) to regulate Ca(2+) entry. We have shown previously that the TRPC1 COOH-terminal residues 781-789 bind to CSD. In the present study, we show that the TRPC1 COOH-terminal residues 781-789 truncated (TRPC1-CDelta781-789) mutant expression abolished Ca(2+) store release-induced Ca(2+) influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of Ca(2+) influx, we determined TRPC1 binding to IP(3)R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-CDelta781-789 effectively interacted with IP(3)R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-CDelta781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed that the WT-Cav-1 but not the Cav-1DeltaCSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1DeltaCSD was reduced, we measured Ca(2+) store release-induced Ca(2+) influx in Cav-1DeltaCSD-transfected cells. Surprisingly, Cav-1DeltaCSD expression showed a gain-of-function in Ca(2+) entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in Ca(2+) entry when Cav-1DeltaCSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1DeltaCSD interacted with IP(3)R3. Furthermore, we observed using confocal imaging the colocalization of IP(3)R3 with WT-Cav-1 but not with Cav-1DeltaCSD on Ca(2+) store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and IP(3)R3 and thereby regulates Ca(2+) store release-induced Ca(2+) entry in endothelial cells.

Novel transcription factors in human CD34 antigen-positive hematopoietic cells.

Transcription factors (TFs) and the regulatory proteins that control them play key roles in hematopoiesis, controlling basic processes of cell growth and differentiation; disruption of these processes may lead to leukemogenesis. Here we attempt to identify functionally novel and partially characterized TFs/regulatory proteins that are expressed in undifferentiated hematopoietic tissue. We surveyed our database of 15 970 genes/expressed sequence tags (ESTs) representing the normal human CD34(+) cells transcriptosome (http://westsun.hema.uic.edu/cd34.html), using the UniGene annotation text descriptor, to identify genes with motifs consistent with transcriptional regulators; 285 genes were identified. We also extracted the human homologues of the TFs reported in the murine stem cell database (SCdb; http://stemcell.princeton.edu/), selecting an additional 45 genes/ESTs. An exhaustive literature search of each of these 330 unique genes was performed to determine if any had been previously reported and to obtain additional characterizing information. Of the resulting gene list, 106 were considered to be potential TFs. Overall, the transcriptional regulator dataset consists of 165 novel or poorly characterized genes, including 25 that appeared to be TFs. Among these novel and poorly characterized genes are a cell growth regulatory with ring finger domain protein (CGR19, Hs.59106), an RB-associated CRAB repressor (RBAK, Hs.7222), a death-associated transcription factor 1 (DATF1, Hs.155313), and a p38-interacting protein (P38IP, Hs. 171185). The identification of these novel and partially characterized potential transcriptional regulators adds a wealth of information to understanding the molecular aspects of hematopoiesis and hematopoietic disorders.