Genotypic and phenotypic analysis of in vivo tissue regeneration in an animal model.

Determining the in vivo response to cellular therapies is important in evaluating the effectiveness of regenerative medicine therapies. Such treatment modalities leverage the treated individual's ability to elicit the body's innate healing response to repair/regenerate damaged tissues or organs. Detailed within this chapter is the process of evaluating the host tissue response to a candidate cell therapy through analysis of key transcript and protein targets.

Tissue engineering of esophagus and small intestine in rodent injury models.

Regenerative constructs composed of synthetically sourced, biodegradable biomaterials seeded with smooth muscle-like cells have been leveraged to mediate regeneration of bladder and bladder-like neo-organs. Here, we describe how such constructs may be applied to catalyze regeneration of esophagus and small intestine in preclinical rodent models.

Migration assay to evaluate cellular interactions with biomaterials for tissue engineering/regenerative medicine applications.

Regenerative medicine and tissue engineering approaches for solving current medical dilemmas such as organ failure, congenital defect, or reconstruction following disease or trauma typically require specific considerations regarding biomaterial selection, identification of key cell types, and applicable surgical techniques (Lanza et al. Principles of tissue engineering, Academic, 2007; Kikuchi, Kanama., Quart Rev 24:51-67, 2007).

Isolation of pulsatile cell bodies from esophageal tissue.

Pulsatile cell bodies, three-dimensional cell clusters with satellite streaming cells, can be isolated from -esophageal tissue. One of the key features of these clusters is that they pulsate at rhythmic rates and demonstrate contractility under several in vitro conditions. Their ability to pulsate appears to be due to the presence of interstitial cells of Cajal (ICC), which mediate signal transmission from nerve to muscle cells.

Molecular characterization of the regenerative response induced by intrarenal transplantation of selected renal cells in a rodent model of chronic kidney disease.

Dedifferentiation and proliferation of resident tubular epithelial cells is a mechanism of action potentially contributing to repair and regeneration in kidneys presenting with ischemic or chronic disease. To more efficiently develop cell and tissue engineering technologies for the kidney, we have developed molecular assays to evaluate the acquisition of a pluripotent state associated with stem/progenitor cell phenotype during induction of a regenerative response within the kidneys of rats with chronic kidney disease (CKD) following therapeutic intervention. Intrarenal delivery of selected bioactive renal cells leads to significant upregulation of pluripotency-associated SOX2 mRNA within the diseased kidney tissue from 1 to 24 weeks after treatment.

Regeneration of native-like neo-urinary tissue from nonbladder cell sources.

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.

Extension of bladder-based organ regeneration platform for tissue engineering of esophagus.

Recent successes in regenerative medicine and tissue engineering of bladder and bladder-like neo-organs have leveraged regenerative constructs composed of a biodegradable scaffold seeded with a population of smooth muscle cells. We have shown that such smooth muscle cells are isolatable from adipose and other sources alternate to the primary organ. We hypothesize that this regenerative platform is not limited to regeneration of bladder and bladder-like neo-organs, but rather represents a foundational technology platform broadly applicable for regeneration of laminarly organized hollow organs.

Regeneration of rodent small intestine tissue following implantation of scaffolds seeded with a novel source of smooth muscle cells.

Aims: To apply an organ regeneration platform technology of autologous smooth muscle cell/biomaterial combination products, previously demonstrated to be successful for urinary tissue regeneration, to the regeneration of the small intestine.

Materials & Methods: Patch and tubular constructs were implanted in rodent small intestines and histologically evaluated over a time course for evidence of regeneration of the laminarly organized neo-mucosa and muscle layers.

Results: Laminarly organized neo-mucosa and muscle layer bundles are demonstrated as early as 8 weeks postimplantation.

Organ specific regenerative markers in peri-organ adipose: kidney.

Background: Therapeutically bioactive cell populations are currently understood to promote regenerative outcomes in vivo by leveraging mechanisms of action including secretion of growth factors, site specific engraftment and directed differentiation. Constitutive cellular populations undoubtedly participate in the regenerative process. Adipose tissue represents a source of therapeutically bioactive cell populations.

Smooth muscle phenotypic diversity is mediated through alterations in myocardin gene splicing.

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.

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.

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.

Functional evaluation of primary renal cell/biomaterial neo-kidney augment prototypes for renal tissue engineering.

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 neokidney tissue development in response to NKA constructs delivered to healthy rodent renal parenchyma.

Isolation, characterization, and expansion methods for defined primary renal cell populations from rodent, canine, and human normal and diseased kidneys.

Chronic kidney disease (CKD) is a global health problem; the growing gap between the number of patients awaiting transplant and organs actually transplanted highlights the need for new treatments to restore renal function. Regenerative medicine is a promising approach from which treatments for organ-level disorders (e.g.

Diversity of moxifloxacin resistance during a nosocomial outbreak of a predominantly ribotype ARU 027 Clostridium difficile diarrhea.

To characterize the extent and diversity of moxifloxacin resistance among Clostridium difficile isolates recovered during a predominantly Anaerobe Reference Unit (ARU) ribotype 027-associated nosocomial outbreak of antibiotic associated diarrhea we measured the susceptibility of 34 field isolates and 6 laboratory strains of C. difficile to moxifloxacin. We ribotyped the isolates as well as assaying them by PCR for the metabolic gene, gdh, and the virulence genes, tcdA, tcdB, tcdC, cdtA and cdtB.

Clostridium perfringens toxin genotypes in the feces of healthy North Americans.

We investigated the frequency of Clostridium perfringens in the normal fecal flora of healthy North Americans. About half of 43 subjects were colonized with C. perfringens at levels of approximately 10(6)cfu/g feces.

Molecular characterization and antimicrobial susceptibilities of extra-intestinal Clostridium difficile isolates.

Amongst 25 extra-intestinal clinical isolates of Clostridium difficile, A(+)B(+) (72%) and A(-)B(+) (4%) toxigenic phenotypes, as well as the non-toxigenic phenotype (A(-)B(-)) (24%), were identified. The A(-)B(-) isolates did not express toxin, yet carried part of the tcdA and tcdB gene and are of a previously unreported toxinotype. Six A(+)B(+) isolates also carried binary toxin genes.

Binary toxin-producing, large clostridial toxin-negative Clostridium difficile strains are enterotoxic but do not cause disease in hamsters.

Binary toxin CDT or its genes have been identified in some strains of Clostridium difficile that also produce the large clostridial toxins, toxins A and B (A+B+CDT+), including a newly recognized epidemic strain in the United States and Canada. To study the effects of binary toxin alone, we characterized 4 binary toxin CDT-positive only (A-B-CDT+) C. difficile strains.