Analyses and Utilization of Selectively Tuned Human Adipose-Derived Stromal/Stem Cell Exosomes.

We have developed a hollow fiber bioreactor-based production system for manufacturing large quantities of extracellular vesicles (EVs) containing exosomes from adult human adipose-derived stromal/stem cells (ASCs). By manipulating the cellular bioreactor environment, we have found that we can alter ASC EV production, secretion, and surface protein composition. The aims of this chapter are to describe the methodology for culturing and tuning of adipose ASCs in a bioreactor, along with the collection and isolation of the EVs containing exosomes demonstrating increased HSP70 content.

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.

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. The overall regenerative response index was assessed by quantitative composite expression of CD24, NODAL and LEFTY1 proteins, which were induced within 1 week of cell treatment and peaked at 12 weeks after treatment, reaching statistical significance (p < 0.05) compared to untreated CKD controls. Molecular assays that incorporate the assessment of SOX2 and the regenerative response index may prove to be valuable tools for the detection and monitoring of the tissue response after the delivery of regenerative treatments for CKD, thereby significantly shortening the developmental timelines associated with such therapies.

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.

Enzymatically labeled chromosomal probes for in situ identification of human cells in xenogeneic transplant models.

Analysis of the viability, differentiation, clonogenicity and function of human stem/progenitor cells requires suitable xenograft models. However, the identification of transplanted cells has been generally difficult. Fluorescence in situ hybridization is a tedious method for analyzing tissues, and localization of transplanted cells with X or Y chromosome probes is limited by the sparse signals produced. Therefore, we examined the possibility of generating either pan-nuclear signals with a total human DNA probe or multiple nuclear signals with a pan-centromeric human DNA probe. The probes were labeled with digoxigenin to make reaction products visible by light microscopy and to allow the use of immunohistochemistry methods incorporating various color schemes to demonstrate specific properties of transplanted cells. The ability to localize all types of nucleated human cells with such probes will facilitate studies of stem cell biology and cell and gene therapy, as well as the development of new animal models.

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. 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. RESULTS: We 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. CONCLUSIONS: Taken 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.

Differentiated human adipose-derived stem cells exhibit hepatogenic capability in vitro and in vivo.

The availability of suitable human livers for transplantation falls short of the number of potential patients. In addition, the availability of primary human hepatocytes for cell-therapy and drug development applications is significantly limited; less than 700 livers per year are available for such studies. However, the majority of these organs cannot be utilized due to pathological infections (e.g., HepB, HepC, or HIV) or excessive levels of steatosis. Thus, the number of cells needed for cell therapy applications far exceeds the number of cells available from donated livers. The ability to implant progenitor cell populations that can form liver tissue in situ, or can be differentiated in vitro would be a major advance in current cell-based therapies. In addition, and importantly for this application, the ability to utilize a non-hepatic progenitor cell to mimic hepatocytes in vitro would enable the scale-up production of cells for bioartifical liver assist devices, cell-therapy and drug discovery applications. We demonstrate the feasibility of inducing adipose-derived stromal (ASC) cells to express several features of human hepatocytes such as glycogen storage and expression of liver specific genes. Importantly, we also show that undifferentiated ASCs and ASC-derived hepatic cells engraft robustly into the liver in a mouse model of toxic injury. These data indicate a significant potential for the use of undifferentiated ASCs and ASC-derived hepatic cells as novel and valuable products for cell therapy.

Initial Assessment of Variability of Responses to Toxicants in Donor-Specific Endothelial Colony Forming Cells.

There is increased interest in using high throughput in vitro assays to characterize human population variability in response to toxicants and drugs. Utilizing primary human endothelial colony-forming cells (ECFCs) isolated from blood would be highly useful for this purpose because these cells are involved in neonatal and adult vasculogenesis. We characterized the cytotoxicity of four known toxic chemicals (NaAsO2, CdCl2, tributyltin [TBT], and menadione) and their four relatively nontoxic counterparts (Na2HAsO4, ZnCl2, SnCl2, and phytonadione, respectively) in eight ECFC clones representing four neonatal donors (2 male and 2 female donors, 2 clones per donor). ECFCs were exposed to 9 concentrations of each chemical in duplicate; cell viability was evaluated 48 h later using the fluorescent vital dye fluorescent dye 5-Carboxyfluorescein Diacetate (CFDA), yielding concentration-effect curves from each experiment. Technical (day-to-day) variability of the assay, assessed from three independent experiments, was low: p-values for the differences of results were 0.74 and 0.64 for the comparison of day 2 vs. day 1 and day 3 vs. day 1, respectively. The statistical analysis used to compare the entire concentration-effect curves has revealed significant differences in levels of cytotoxicity induced by the toxic and relatively nontoxic chemical counterparts, demonstrating that donor-specific ECFCs can clearly differentiate between these two groups of chemicals. Partitioning of the total variance in the nested design assessed the contributions of between-clone and between-donor variability for different levels of cytotoxicity. Individual ECFC clones demonstrated highly reproducible responses to the chemicals. The most toxic chemical was TBT, followed by NaAsO2, CdCl2, and Menadione. Nontoxic counterparts exhibited low cytotoxicity at the higher end of concentration ranges tested. Low variability was observed between ECFC clones obtained from the same donor or different donors for CdCl2, NaAsO2, and TBT, but for menadione, the between-donor variability was much greater than the between-clone variability. The low between-clone variability indicates that an ECFC clone may represent an individual donor in cell-based assays, although this finding must be confirmed using a larger number of donors. Such confirmation would demonstrate that an in vitro ECFC-based testing platform can be used to characterize the inter-individual variability of neonatal ECFCs exposed to drugs and/or environmental toxicants.

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

BACKGROUND: The product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). At mitotic exit, all three PP1 isoforms, alpha, gamma1 and delta, 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 PP1alpha 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. RESULTS: The 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. CONCLUSION: The 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.

Exosomes for repair, regeneration and rejuvenation.

INTRODUCTION: Application of regenerative medicine strategies for repair of organs/tissue impacted by chronic disease is an active subject for product development. Such methodologies emphasize the role of stem cells as the active biological ingredient. However, recent developments in elucidating mechanisms of action of these therapies have focused on the role of paracrine, 'action-at-a-distance' modus operandi in mediating the ability to catalyze regenerative outcomes without significant site-specific engraftment. A salient component of this secreted regenerative milieu are exosomes: 40-100 nm intraluminal vesicles that mediate transfer of proteins and nucleic acids across cellular boundaries. AREAS COVERED: Here, we synthesize recent studies from PubMed and Google Scholar highlighting how cell-based therapeutics and cosmeceutics are transitioning towards the secretome generally and exosomes specifically as a principal modulator of regenerative outcomes. EXPERT OPINION: Exosomes contribute to organ development and mediate regenerative outcomes in injury and disease that recapitulate observed bioactivity of stem cell populations. Encapsulation of the active biological ingredients of regeneration within non-living exosome carriers may offer process, manufacturing and regulatory advantages over stem cell-based therapies.

Allogeneic cell therapy for the treatment of liver disease.

The scarcity of human organs available for transplantation is clearly evident. Efforts to maximize the use of available organs and to increase the number of donors have increased the number of transplantations performed, but at a rate that remains far behind the rate of growth of the waiting list. Thus, the likelihood of a patient with severe liver disease receiving a liver replacement is decreasing. In order to offer treatment to most patients with liver disease, alternatives to whole-organ replacement must be found. Cell-based treatments, in which suspensions of liver cells are injected into patients with liver failure and reconstitute the patient's liver functions, may be that alternative. Here, we report on a regulatory-compliant process for the production of a cryopreserved cell therapy product that yields viable, metabolically active hepatocytes that can be infused directly into patients with the goal of reconstituting liver function.

Isolation, culturing, and characterization of cardiac muscle cells from nonhuman primate heart tissue.

Cardiac safety pharmacology requires in vitro testing of all drug candidates before clinical trials in order to ensure they are screened for cardiotoxic effects which may result in severe arrhythmias and, ultimately, cardiomyopathy (Chi, Nat Rev Drug Discov 12:565-567, 2013). Given the physiological similarities between nonhuman primates and humans, isolated primate cardiac muscle cells are an ideal animal model for such in vitro testing. The aims of this chapter are to describe two methods for isolating and culturing primate cardiac muscle cells. One method uses mechanical dissociation of the tissue followed by placing the small pieces onto a Petri dish and culturing these tissue explants. The other method also uses mechanical dissociation but is then followed by enzymatic digestion and culturing of the cell suspension. Methods are also described for phenotypically characterizing cardiac muscle cells by flow cytometry. Based on the location within the heart tissue chosen for cell isolation, a dividing population of cardiac muscle cells expressing cardiomyocyte cell markers was obtained.

Inducible expression of catalytically active type 1 serine/threonine protein phosphatase in a human carcinoma cell line.

BACKGROUND: One of the major cellular serine/threonine protein phosphatases is protein phosphatase type 1 (PP1). Studies employing many eukaryotic systems all point to a crucial role for PP1 activity in controlling cell cycle progression. One physiological substrate for PP1 appears to be the product of the retinoblastoma susceptibility gene (pRB), a demonstrated tumor suppressor. The growth suppressive activity of pRB is regulated by its phosphorylation state. Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation. As a first step towards addressing this question, we developed an inducible PP1 expression system to investigate the regulation of PP1 activity. RESULTS: We have established a cell line for inducing protein expression of the type 1, alpha-isotype, serine/threonine protein phosphatase (PP1alpha). A plasmid encoding a fusion protein of the catalytic subunit of PP1alpha with a 6-histidine peptide (6His) and a peptide from hemagluttinin (HA) was transfected into the UMUC3 transitional cell carcinoma cell line, previously transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo. A stable cell line designated LLWO2F was established by selection with hygromycin B. 6His-HA-PP1alpha protein appeared in cell lysates within two hours following addition of doxycycline to the culture medium. This protein localizes to the nucleus as does endogenous PP1alpha, and was shown to associate with PNUTS, a PP1-nuclear targeting subunit. Like endogenous PP1alpha, immunocomplexed 6His-HA-PP1alpha is active toward phosphorylase a and the product of the retinoblastoma susceptibility gene, pRB. When forcibly overexpressing 6His-HA-PP1alpha, there is a concomitant decrease in endogenous PP1alpha levels. CONCLUSIONS: These data suggest the existence of an autoregulatory mechanism by which PP1alpha protein levels and activity remain relatively constant. RT-PCR analyses of isolated polysome fractions support the notion that this putative autoregulatory mechanism is exerted, at least in part, at the translational level. Implications of these findings for the study of PP1alpha function in vivo are discussed.

Interaction between the retinoblastoma protein and protein phosphatase 1 during the cell cycle.

The functions of the retinoblastoma protein (pRb) are in part regulated by reversible and cell cycle-dependent phosphorylation. While the regulation of pRb by cyclin-dependent kinases (Cdks) has been studied extensively, the role(s) of protein phosphatase 1 (PP1) in controlling pRb are only partially understood. In this chapter, we will describe experimental approaches to investigate the interactions between pRb and PP1. Methods will be presented to study the cell cycle-dependent dephosphorylation of pRb by various PP1 isozymes, the specificity of PP1 isozymes for distinct pRb phosphorylation sites, the dephosphorylation of pRb associated with apoptosis, and the cell cycle- and pRb-dependent phosphorylation of PP1.

Cell-based therapeutic products: potency assay development and application.

Potency is a critical quality attribute of biological products, defined by the US FDA as the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result. Ideally, a potency assay will leverage the product's mechanism of action. Alternatively, the assay may focus on a therapeutically relevant biological activity. The absence of rigorous mechanistic data for the majority of cell-based therapeutics currently in the process research pipeline has impeded efforts to design and validate indices of product potency. Development of a systematic battery of parallel functional assays that, taken together, can address all potential mechanisms of action believed to be relevant for the product platform is recommended. Such an approach is especially important during preclinical development. Here, we summarize the principal and unique challenges facing the development of functionally relevant and rigorous potency assays for cell-based therapeutics. We present perspectives regarding potency assay development for these products as illustrated by our experiences in process R&D of cryopreserved hepatocytes (Incara Pharmaceuticals) and selected renal cells (Tengion).

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. As predicted, the cells comprising these clusters express phenotypic and genotypic markers characteristic of smooth and skeletal muscle, neuronal, and epithelial cells. Because of the critical role of ICC in gastrointestinal tract motility, loss of function in these cells can result in a variety of pathologies. Cultures of pulsatile cell bodies may have utility as an in vitro model to study tissue engineering and regenerative medicine approaches to treating defects in gastrointestinal rhythmicity due to disease or injury.

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). The ability to evaluate these components in vitro under conditions which simulate relevant in vivo environments can reduce development risks including time and money costs associated with early-stage product development. Similarly, such methods can be useful in making progress in researching features of natural and synthetic biomaterial such as porosity, strength, surface topography, and functionalization, and their singular or collective effects on cell behavior (Kikuchi and Kanama., Quart Rev 24:51-67, 2007; Furth et al. Biomaterials 28:5068-5073, 2007; Mieszawska and Kaplan., BMC Biol 8:59, 2010).Adhesion, migration, and gene and protein expression are all cell behaviors that can be affected by properties of a chosen biomaterial and vary based upon organ system (Cornwell et al. J Biomater Res 71A:55-62, 2004; David et al. Tissue Eng 8(5):787-798, 2002). Understanding of these properties and their role in combination with biomaterial in remodeling is sought in order to fully harness and direct regeneration (Lanza et al. Principles of tissue engineering, Academic Press, 2007; Mieszawska and Kaplan. BMC Biol 8:59, 2010; Matragotri and Lahann J. Nat Mater 8:15-23, 2009).

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.

The future of regenerative medicine: urinary system.

Regeneration of tissues and organs is now within the technological reach of modern medicine. With such advancements, substantial improvements to existing standards-of-care are very real possibilities. This review will focus on regenerative medicine approaches to treating specific maladies of the bladder and kidney, including the biological basis of regeneration and the history of regenerative medicine in the urinary system. Current clinical management approaches will be presented within the context of future directions including cell-based regenerative therapies.

Characterization of the human smooth muscle cell secretome for regenerative medicine.

Smooth muscle cells (SMC) play a central role in maintaining the structural and functional integrity of muscle tissue. Little is known about the early in vitro events that guide the assembly of 'bioartificial tissue' (constructs) and recapitulate the key aspects of smooth muscle differentiation and development before surgical implantation. Biomimetic approaches have been proposed that enable the identification of in vitro processes which allow standardized manufacturing, thus improving both product quality and the consistency of patient outcomes. One essential element of this approach is the description of the SMC secretome, that is, the soluble and deposited factors produced within the three-dimensional (3D) extracellular matrix (ECM) microenvironment. In this study, we utilized autologous SMC from multiple tissue types that were expanded ex vivo and generated with a rigorous focus on operational phenotype and genetic stability. The objective of this study was to characterize the spatiotemporal dynamics of the first week of organoid maturation using a well-defined in vitro-like, 3D-engineered scale model of our validated manufacturing process. Functional proteomics was used to identify the topological properties of the networks of interacting proteins that were derived from the SMC secretome, revealing overlapping central nodes related to SMC differentiation and proliferation, actin cytoskeleton regulation, and balanced ECM accumulation. The critical functions defined by the Ingenuity Pathway Analysis included cell signaling, cellular movement and proliferation, and cellular and organismal development. The results confirm the phenotypic and functional similarity of the SMC generated by our platform technology at the molecular level. Furthermore, these data validate the biomimetic approaches that have been established to maintain manufacturing consistency.