John W. Ludlow

Long-term durability, tissue regeneration and neo-organ growth during skeletal maturation with a neo-bladder augmentation construct

AIMSnTo comparatively evaluate bladder regeneration following 80% cystectomy and augmentation using a synthetic biopolymer with autologous urothelial and smooth muscle cells (autologous neo-bladder augmentation construct [construct]) or autotransplantation of native bladder (reimplanted native urinary bladder [reimplant]) in canines.nnnMATERIALS & METHODSnVoiding function, urodynamic assessment and neo-organ capacity-to-body-weight ratio (C:BW) were assessed longitudinally for a total of 24 months following trigone-sparing augmentation cystoplasty in juvenile canines.nnnRESULTSnWithin 30 days postimplantation, hematology and urinalysis returned to baseline. Constructs and reimplants yielded neo-organs with statistically equivalent urodynamics and histology. Linear regression analysis of C:BW showed that constructs regained baseline slope and continued to adapt with animal growth.nnnCONCLUSIONSnConstructs and reimplants regained and maintained native bladder histology by 3 months, capacity at 3-6 months and compliance by 12-24 months. Furthermore, construct C:BW demonstrated the ability of regenerated bladder to respond to growth regulation.

Platform technologies for tubular organ regeneration

As a result of recent successes in regenerative medicine approaches to engineering multiple disparate tubular organs, methodology commonalities are emerging. Principal themes include the importance of a biodegradable scaffold seeded with a population of smooth muscle cells. 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 genitourinary 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.

Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb

BackgroundThe product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). At mitotic exit, all three PP1 isoforms, α, γ1 and δ, bind to pRb and dephosphorylate its Ser/Thr sites in a sequential and site-specific way. The pRb-C terminal has been reported to be necessary and sufficient for PP1α binding. The present study investigated whether the three PP1 isoforms from mitotic or asynchronous HeLa cells associate differentially with wild-type and pRb mutants, as well as the holoenzyme composition of the pRb-directed PP1.ResultsThe requirement for the entire pRb molecule to achieve optimal PP1-binding was indicated by the fact that full-length pRb displayed the highest affinity for all three PP1 isoforms. Ser/Thr-to-Ala substitution for up to 14 pRb sites did not affect the ability of pRb to bind the PP1 isoforms derived from mitotic or asynchronous HeLa cells, thus suggesting that the phosphate-accepting residues on pRb do not regulate the interaction with PP1. To probe for the presence of PP1 targeting subunits in the pRb-directed PP1 complex, PP1 from mitotic or asynchronous HeLa cells was isolated by affinity chromatography on GST-Rb (either full-length or its deletion mutants Rb-big pocket or Rb-C-terminal). The PP1 was always obtained as free catalytic subunit, displaying all three isoforms, thus suggesting direct interaction between pRb and PP1. The direct association was confirmed by the ability of pRb to pull-down purified PP1 catalytic subunits and by in vitro reconstitution of a complex between PP1 catalytic subunit and the pRb-C-terminal.ConclusionThe work indicated that the full length of the pRb molecule is required for optimal interaction with the PP1 isoforms and that the association between pRb and PP1 isoforms is direct.

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. 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 neovascularization and cellular infiltration by 4 weeks postimplantation. 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 neokidney tissue formation at 1 week postimplantation. To investigate a potential mechanism by which NKA constructs could impact a disease state, the effect of conditioned media on TGF-β signaling pathways related to tubulo-interstitial fibrosis associated with CKD progression was evaluated. Conditioned medium was observed to attenuate TGF-β-induced epithelial–mesenchymal transition (EMT) in vitro in a human proximal tubular cell line (HK2).

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. Using esophagus as an illustrative example in support of this hypothesis, we demonstrate that patch constructs composed of adipose-derived smooth muscle cells seeded on a biodegradable matrix catalyze complete regeneration of the esophageal wall in a rodent model of esophageal injury. By implication, such regenerative constructs may potentially be used to mediate the regeneration of any laminarly organized tubular organ.

Developmental engineering the kidney: leveraging principles of morphogenesis for renal regeneration.

Multiple methodological approaches are currently under active development for application in tissue engineering and regenerative medicine of tubular and solid organs. Most recently, developmental engineering (TE/RM), or the leveraging of embryonic and morphological paradigms to recapitulate aspects of organ development, has been proposed as a strategy for the sequential, iterative de novo assembly of tissues and organs as discrete developmental modules ex vivo, prior to implantation in vivo. In this article, we focus on the kidney to highlight in detail how principles of developmental biology are impacting approaches to TE of this complex solid organ. Ultimately, such methodologies may facilitate the establishment of clinically relevant therapeutic strategies for regeneration of renal structure and function, greatly impacting treatment regimens for chronic kidney disease.

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. 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.

Organ specific regenerative markers in peri-organ adipose: kidney

BackgroundTherapeutically 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. The potential of these cells to participate in various aspects of the regenerative process has been demonstrated broadly. However, organ association of secretory and developmental 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 sourced adipose to express key renal associated factors.ResultsWe report that renal adipose tissue is a novel reservoir for EPO expressing cells. Kidney sourced adipose stromal cells demonstrate hypoxia regulated expression of EPO and VEGF transcripts. Using iso-electric focusing, we demonstrate that kidney and non-kidney sourced adipose stromal cells present unique patterns of EPO post-translational modification, consistent with the idea that renal and non-renal sources are functionally distinct adipose depots. In addition, kidney sourced adipose stromal cells specifically express the key renal developmental transcription factor WT1.ConclusionsTaken together, these data are consistent with the notion that kidney sourced adipose stromal (KiSAS) cells may be primed to recreate a regenerative micro-environment within the kidney. These findings open the possibility of isolating solid-organ associated adipose derived cell populations for therapeutic applications in organ-specific regenerative medicine products.

Chapter 56 – Digestive Tract

When considering the potential toxic activity of various agents on the gastrointestinal tract, a number of signalments are possible. Acute effects may result from direct irritants (e.g., strong acids and bases), whereas chronic effects may be observed, for example, as increased muscular layer thickness from bulking agents. Importantly, delayed effects may be expressed years after exposure to ulcerogenic or carcinogenic agents. In addition to the array of tissue responses, interpretation of functional and morphological alterations can be complex. The focus of this chapter is the examination of developmental, structural, and functional components of the gastrointestinal tract that are important in understanding mechanisms involved in the toxicologic pathology of this organ system. A major part of the chapter stresses basic mechanisms of toxicologic damage and how the gastrointestinal tract responds to toxicologic insult. Also discussed are approaches that can be used to study selected pathologic mechanisms of toxicological significance and how microbiology of the gut can affect toxicology. Towards the end of the chapter the regenerative biology of the gastrointestinal tract is covered, with an emphasis on tissue engineering of the small intestine, as a means to overcome the effects of injury or toxicity.

Regenerating Kidney Structure and Function: An Industry Perspective

Summary Recent progress toward the application of tissue engineering methodologies for the regeneration of tubular and solid organs including the kidney is identifying shared methodologies that may underlie the development of foundational platform technologies broadly applicable toward the regeneration of multiple organ systems. Central themes emerging for both tubular and solid neo-organs include the application of a biodegradable scaffold to provide structural support for developing neo-organs and the role of committed or progenitor cell populations in establishing the regenerative micro-environment of key secreted growth factors, constitutive components of the regenerated tissue and extra-cellular matrix critical for catalyzing de novo organogenesis. However, aspects of these strategies currently under active development for tissue engineering of neo-organs may not be relevant for successful commercialization as novel TE/RM products for clinical application. For example, difficulties in large scale sourcing and quality control of biomaterials derived from decellularization of cadaveric organs imply that such biomaterials may be less suitable for incorporation into TE/RM products when compared to biomaterials of synthetic origin. Similarly, TE/RM technologies that attempt to leverage populations of stem and progenitor cells are less likely than platforms focused on committed cell populations or acellular biomaterials to facilitate rapid development of viable products. In this chapter, we present our experience in the development of RegenMedTX LLC’s Neo-Kidney Augment to identify elements of this foundational organ regeneration technology platform that may be broadly applicable toward the design and development of additional solid neo-organ products. We will focus specifically on highlighting aspects of this neo-organ regenerative platform conducive to the commercial viability of this technology.Recent progress toward the application of tissue engineering methodologies for the regeneration of tubular and solid organs including the kidney is identifying shared methodologies that may underlie the development of foundational platform technologies broadly applicable toward the regeneration of multiple organ systems. Central themes emerging for both tubular and solid neo-organs include the application of a biodegradable scaffold to provide structural support for developing neo-organs and the role of committed or progenitor cell populations in establishing the regenerative micro-environment of key secreted growth factors, constitutive components of the regenerated tissue and extra-cellular matrix critical for catalyzing de novo organogenesis. However, aspects of these strategies currently under active development for tissue engineering of neo-organs may not be relevant for successful commercialization as novel TE/RM products for clinical application. For example, difficulties in large scale sourcing and quality control of biomaterials derived from decellularization of cadaveric organs imply that such biomaterials may be less suitable for incorporation into TE/RM products when compared to biomaterials of synthetic origin. Similarly, TE/RM technologies that attempt to leverage populations of stem and progenitor cells are less likely than platforms focused on committed cell populations or acellular biomaterials to facilitate rapid development of viable products. In this chapter, we present our experience in the development of RegenMedTX LLC’s Neo-Kidney Augment to identify elements of this foundational organ regeneration technology platform that may be broadly applicable toward the design and development of additional solid neo-organ products. We will focus specifically on highlighting aspects of this neo-organ regenerative platform conducive to the commercial viability of this technology.