Immunogenicity of decellularized porcine liver for bioengineered hepatic tissue.

Liver disease affects millions of patients each year. The field of regenerative medicine promises alternative therapeutic approaches, including the potential to bioengineer replacement hepatic tissue. One approach combines cells with acellular scaffolds derived from animal tissue. The goal of this study was to scale up our rodent liver decellularization method to livers of a clinically relevant size. Porcine livers were cannulated via the hepatic artery, then perfused with PBS, followed by successive Triton X-100 and SDS solutions in saline buffer. After several days of rinsing, decellularized liver samples were histologically analyzed. In addition, biopsy specimens of decellularized scaffolds were seeded with hepatoblastoma cells for cytotoxicity testing or implanted s.c. into rodents to investigate scaffold immunogenicity. Histological staining confirmed cellular clearance from pig livers, with removal of nuclei and cytoskeletal components and widespread preservation of structural extracellular molecules. Scanning electron microscopy confirmed preservation of an intact liver capsule, a porous acellular lattice structure with intact vessels and striated basement membrane. Liver scaffolds supported cells over 21 days, and no increased immune response was seen with either allogeneic (rat-into-rat) or xenogeneic (pig-into-rat) transplants over 28 days, compared with sham-operated on controls. These studies demonstrate that successful decellularization of the porcine liver could be achieved with protocols developed for rat livers, yielding nonimmunogenic scaffolds for future hepatic bioengineering studies.

Genetic modification of primate amniotic fluid-derived stem cells produces pancreatic progenitor cells in vitro.

Insulin therapy for type 1 diabetes does not prevent serious long-term complications including vascular disease, neuropathy, retinopathy and renal failure. Stem cells, including amniotic fluid-derived stem (AFS) cells - highly expansive, multipotent and nontumorigenic cells - could serve as an appropriate stem cell source for ß-cell differentiation. In the current study we tested whether nonhuman primate (nhp)AFS cells ectopically expressing key pancreatic transcription factors were capable of differentiating into a ß-cell-like cell phenotype in vitro. nhpAFS cells were obtained from Cynomolgus monkey amniotic fluid by immunomagnetic selection for a CD117 (c-kit)-positive population. RT-PCR for endodermal and pancreatic lineage-specific markers was performed on AFS cells after adenovirally transduced expression of PDX1, NGN3 and MAFA. Expression of MAFA was sufficient to induce insulin mRNA expression in nhpAFS cell lines, whereas a combination of MAFA, PDX1 and NGN3 further induced insulin expression, and also induced the expression of other important endocrine cell genes such as glucagon, NEUROD1, NKX2.2, ISL1 and PCSK2. Higher induction of these and other important pancreatic genes was achieved by growing the triply infected AFS cells in media supplemented with a combination of B27, betacellulin and nicotinamide, as well as culturing the cells on extracellular matrix-coated plates. The expression of pancreatic genes such as NEUROD1, glucagon and insulin progressively decreased with the decline of adenovirally expressed PDX1, NGN3 and MAFA. Together, these experiments suggest that forced expression of pancreatic transcription factors in primate AFS cells induces them towards the pancreatic lineage.

Production and implantation of renal extracellular matrix scaffolds from porcine kidneys as a platform for renal bioengineering investigations.

BACKGROUND: It is important to identify new sources of transplantable organs because of the critical shortage of donor organs. Tissue engineering holds the potential to address this issue through the implementation of decellularization-recellularization technology. OBJECTIVE: To produce and examine acellular renal extracellular matrix (ECM) scaffolds as a platform for kidney bioengineering. METHODS: Porcine kidneys were decellularized with distilled water and sodium dodecyl sulfate-based solution. After rinsing with buffer solution to remove the sodium dodecyl sulfate, the so-obtained renal ECM scaffolds were processed for vascular imaging, histology, and cell seeding to investigate the vascular patency, degree of decellularization, and scaffold biocompatibility in vitro. Four whole renal scaffolds were implanted in pigs to assess whether these constructs would sustain normal blood pressure and to determine their biocompatibility in vivo. Pigs were sacrificed after 2 weeks and the explanted scaffolds were processed for histology. RESULTS: Renal ECM scaffolds were successfully produced from porcine kidneys. Scaffolds retained their essential ECM architecture and an intact vascular tree and allowed cell growth. On implantation, unseeded scaffolds were easily reperfused, sustained blood pressure, and were tolerated throughout the study period. No blood extravasation occurred. Pathology of explanted scaffolds showed maintenance of renal ultrastructure. Presence of inflammatory cells in the pericapsular region and complete thrombosis of the vascular tree were evident. CONCLUSIONS: Our investigations show that pig kidneys can be successfully decellularized to produce renal ECM scaffolds. These scaffolds maintain their basic components, are biocompatible, and show intact, though thrombosed, vasculature.

Regenerative medicine as applied to general surgery.

The present review illustrates the state of the art of regenerative medicine (RM) as applied to surgical diseases and demonstrates that this field has the potential to address some of the unmet needs in surgery. RM is a multidisciplinary field whose purpose is to regenerate in vivo or ex vivo human cells, tissues, or organs to restore or establish normal function through exploitation of the potential to regenerate, which is intrinsic to human cells, tissues, and organs. RM uses cells and/or specially designed biomaterials to reach its goals and RM-based therapies are already in use in several clinical trials in most fields of surgery. The main challenges for investigators are threefold: Creation of an appropriate microenvironment ex vivo that is able to sustain cell physiology and function in order to generate the desired cells or body parts; identification and appropriate manipulation of cells that have the potential to generate parenchymal, stromal and vascular components on demand, both in vivo and ex vivo; and production of smart materials that are able to drive cell fate.

A three-dimensional microfluidic approach to scaling up microencapsulation of cells.

Current applications of the microencapsulation technique include the use of encapsulated islet cells to treat Type 1 diabetes, and encapsulated hepatocytes for providing temporary but adequate metabolic support to allow spontaneous liver regeneration, or as a bridge to liver transplantation for patients with chronic liver disease. Also, microcapsules can be used for controlled delivery of therapeutic drugs. The two most widely used devices for microencapsulation are the air-syringe pump droplet generator and the electrostatic bead generator, each of which is fitted with a single needle through which droplets of cells suspended in alginate solution are produced and cross-linked into microbeads. A major drawback in the design of these instruments is that they are incapable of producing sufficient numbers of microcapsules in a short-time period to permit mass production of encapsulated and viable cells for transplantation in large animals and humans. We present in this paper a microfluidic approach to scaling up cell and protein encapsulations. The microfluidic chip consists of a 3D air supply and multi-nozzle outlet for microcapsule generation. It has one alginate inlet and one compressed air intlet. The outlet has 8 nozzles, each having 380 micrometers inner diameter, which produce hydrogel microspheres ranging from 500 to 700 µm in diameter. These nozzles are concentrically surrounded by air nozzles with 2 mm inner diameter. There are two tubes connected at the top to allow the air to escape as the alginate solution fills up the chamber. A variable flow pump 115 V is used to pump alginate solution and Tygon® tubing is used to connect in-house air supply to the air channel and peristaltic/syringe pump to the alginate chamber. A pressure regulator is used to control the flow rate of air. We have encapsulated islets and proteins with this high throughput device, which is expected to improve product quality control in microencapsulation of cells, and hence the outcome of their transplantation.

Derivation of a novel undifferentiated human foetal phenotype in serum-free cultures with BMP-2.

Skeletal stem and progenitor populations provide a platform for cell-based tissue regeneration strategies. Optimized conditions for ex vivo expansion will be critical and use of serum-free culture may allow enhanced modelling of differentiation potential. Maintenance of human foetal femur-derived cells in a chemically defined medium (CDM) with activin A and fibroblast growth factor-2 generated a unique undifferentiated cell population in comparison to basal cultures, with significantly reduced amino acid depletion, appearance and turnover, reduced alkaline phosphatase (ALP) activity and loss of type I and II collagen expression demonstrated by fluorescence immunocytochemistry. Microarray analysis demonstrated up-regulation of CLU, OSR2, POSTN and RABGAP1 and down-regulation of differentiation-associated genes CRYAB, CSRP1, EPAS1, GREM1, MT1X and SRGN as validated by quantitative real-time polymerase chain reaction. Application of osteogenic conditions to CDM cultures demonstrated partial rescue of ALP activity. In contrast, the addition of bone morphogenetic protein-2 (BMP-2) resulted in reduced ALP levels, increased amino acid metabolism and, strikingly, a marked shift to a cobblestone-like cellular morphology, with expression of SOX-2 and SOX-9 but not STRO-1 as shown by immunocytochemistry, and significantly altered expression of metabolic genes (GFPT2, SC4MOL and SQLE), genes involved in morphogenesis (SOX15 and WIF1) and differentiation potential (C1orf19, CHSY-2,DUSP6, HMGCS1 and PPL). These studies demonstrate the use of an intermediary foetal cellular model for differentiation studies in chemically defined conditions and indicate the in vitro reconstruction of the mesenchymal condensation phenotype in the presence of BMP-2, with implications therein for rescue studies, screening assays and skeletal regeneration research.

Porcine pancreas extracellular matrix as a platform for endocrine pancreas bioengineering.

Emergent technologies of regenerative medicine have the potential to overcome the limitations of organ transplantation by supplying tissues and organs bioengineered in the laboratory. Pancreas bioengineering requires a scaffold that approximates the biochemical, spatial and vascular relationships of the native extracellular matrix (ECM). We describe the generation of a whole organ, three-dimensional pancreas scaffold using acellular porcine pancreas. Imaging studies confirm that our protocol effectively removes cellular material while preserving ECM proteins and the native vascular tree. The scaffold was seeded with human stem cells and porcine pancreatic islets, demonstrating that the decellularized pancreas can support cellular adhesion and maintenance of cell functions. These findings advance the field of regenerative medicine towards the development of a fully functional, bioengineered pancreas capable of establishing and sustaining euglycemia and may be used for transplantation to cure diabetes mellitus.

Decellularization methods of porcine kidneys for whole organ engineering using a high-throughput system.

End-stage renal failure is a devastating disease, with donor organ transplantation as the only functional restorative treatment. The current number of donor organs meets less than one-fifth of demand, so regenerative medicine approaches have been proposed as potential therapeutic alternatives. One such approach for whole large-organ bioengineering is to combine functional renal cells with a decellularized porcine kidney scaffold. The efficacy of cellular removal and biocompatibility of the preserved porcine matrices, as well as scaffold reproducibility, are critical to the success of this approach. We evaluated the effectiveness of 0.25 and 0.5% sodium dodecyl sulfate (SDS) and 1% Triton X-100 in the decellularization of adult porcine kidneys. To perform the decellularization, a high-throughput system was designed and constructed. In this study all three methods examined showed significant cellular removal, but 0.5% SDS was the most effective detergent (<50 ng DNA/mg dry tissue). Decellularized organs retained intact microarchitecture including the renal vasculature and essential extracellular matrix components. The SDS-treated decellularized scaffolds were non-cytotoxic to primary human renal cells. This method ensures clearance of porcine cellular material (which directly impacts immunoreactivity during transplantation) and preserves the extracellular matrix and cellular compatibility of these renal scaffolds. Thus, we have developed a rapid decellularization method that can be scaled up for use in other large organs, and this represents a step toward development of a transplantable organ using tissue engineering techniques.

Design of a bioartificial pancreas(+).

INTRODUCTION: In type 1 diabetes, the ß-cells that secrete insulin have been destroyed such that daily exogenous insulin administration is required for the control of blood glucose in individuals with the disease. After the development of reliable techniques for the isolation of islets from the human pancreas, islet transplantation has emerged as a therapeutic option, albeit for only a few selected patients largely because there are not enough islets for the millions of patients requiring the treatment, and there is also the need to use immunosuppressive drugs to prevent transplant rejection. In 1980, the concept of islet immunoisolation by microencapsulation was introduced as a technique to overcome these 2 major barriers to islet transplantation. Microencapsulation of islets and transplantation in the peritoneal cavity was then described as a bioartificial pancreas. However, it is difficult to retrieve encapsulated islets transplanted in the peritoneal cavity, thus making it difficult to meet all the criteria for a bioartificial pancreas. A new design of a bioartificial pancreas comprising islets co-encapsulated with angiogenic protein in permselective multilayer alginate-poly-L-ornithine-alginate microcapsules and transplanted in an omentum pouch is described in this paper. MATERIALS AND METHODS: The multilayer alginate-poly-L-ornithine-alginate microcapsules are made with ultrapure alginate using poly-L-ornithine as a semipermeable membrane separating the 2 alginate layers. The inner alginate layer is used to encapsulate the islets, and the outer layer is used to encapsulate angiogenic protein, which would induce neovascularization around the graft within the omentum pouch. RESULTS: In in vitro studies, we found that both the wild-type and the heparin-binding growth-associated molecule (HBGAM)-fibroblast growth factor-1 chimera can be encapsulated and released in a controlled and sustained manner from the outer alginate layer with a mean diameter in the range of 113 to 164 µm when 1.25% high guluronic acid alginate is used to formulate this outer layer. DISCUSSION: We are currently performing in vivo experiments to determine the ability of angiogenic proteins released from this outer layer to induce neovascularization around the grafts in the omentum pouch. We will subsequently examine the effect of co-encapsulation of islets with angiogenic protein on blood glucose control in diabetic animals. It is hoped that addition of tissue engineering to encapsulated islet transplantation will result in long-term survival of the islets and their ability to control blood glucose in type 1 diabetes without the necessity to use risky immunosuppressive drugs to prevent transplant rejection.

Whole organ decellularization - a tool for bioscaffold fabrication and organ bioengineering.

The use of synthetic and naturally-derived scaffolds for bioengineering of solid organs has been limited due to a lack of an integrated vascular network. Here, we describe fabrication of a bioscaffold system with intact vascular tree. Animal-donor organs and tissues, ranging in size up-to thirty centimeters, were perfused with decellularization solution to selectively remove the cellular component of the tissue and leave an intact extracellular matrix and vascular network. The vascular tree demonstrated sequential fluid flow through a central inlet vessel that branched into an extensive capillary bed and coalesced back into a single outlet vessel. In one example, the liver, we used central inlet vessels to perfuse human and animal liver cells through the bioscaffold to create a functional liver tissue construct in vitro. These results demonstrate a novel yet simple and scalable method to obtain whole organ vascularized bioscaffolds with potential for liver, kidney, pancreas, intestine and other organs' bioengineering. These bioscaffolds can further provide a tool to study cells in their natural three-dimensional environment, which is superior for drug discovery platform compared with cells cultured in two-dimensional petri dishes.

Biocompatibility and osteogenic potential of human fetal femur-derived cells on surface selective laser sintered scaffolds.

For optimal bone regeneration, scaffolds need to fit anatomically into the requisite bone defects and, ideally, augment cell growth and differentiation. In this study we evaluated novel computationally designed surface selective laser sintering (SSLS) scaffolds for their biocompatibility as templates, in vitro and in vivo, for human fetal femur-derived cell viability, growth and osteogenesis. Fetal femur-derived cells were successfully cultured on SSLS-poly(d,l)-lactic acid (SSLS-PLA) scaffolds expressing alkaline phosphatase activity after 7days. Cell proliferation, ingrowth, Alcian blue/Sirius red and type I collagen positive staining of matrix deposition were observed for fetal femur-derived cells cultured on SSLS-PLA scaffolds in vitro and in vivo. SSLS-PLA scaffolds and SSLS-PLA scaffolds seeded with fetal femur-derived cells implanted into a murine critical-sized femur segmental defect model aided the regeneration of the bone defect. SSLS techniques allow fabrication of biocompatible/biodegradable scaffolds, computationally designed to fit any defect, providing a template for cell osteogenesis in vitro and in vivo.

Adipocyte differentiation induced using nonspecific siRNA controls in cultured human mesenchymal stem cells.

RNA interference (RNAi) is gene silencing induced by double-stranded RNA of 21-23 nucleotides in length, termed small interfering RNA, or siRNA. RNAi-based techniques have been widely applied to elucidate gene function, identify drug targets, and used in trials as a promising adjunct to silence disease-causing genes. However, emerging evidence suggests unexpected changes in expression of untargeted genes as a consequence of an off-target effect by RNAi in mammalian cells. To date, our understanding of such effects on stem cells is limited. We transfected human fetal femur-derived mesenchymal stem cells using commercially available nonspecific siRNA controls and examined adipocyte differentiation in the cells using morphology, histochemistry, and quantitative real-time PCR to examine the expression of key genes for adipogenic or osteogenic differentiation. We report here the induction of adipocyte differentiation in human mesenchymal stem cells using nonspecific siRNAs raising concerns as to the specificity of RNAi in stem cells and, critically, a need to understand and delineate the rules governing the specificity of RNAi.

Characterization and multipotentiality of human fetal femur-derived cells: implications for skeletal tissue regeneration.

To date, the plasticity, multipotentiality, and characteristics of progenitor cells from fetal skeletal tissue remain poorly defined. This study has examined cell populations from human fetal femurs in comparison with adult-derived mesenchymal cell populations. Real-time quantitative polymerase chain reaction demonstrated expression of mesenchymal progenitor cell markers by fetal-derived cells in comparison with unselected adult-derived and immunoselected STRO-1-enriched adult populations. Multipotentiality was examined using cells derived from femurs and single-cell clones, culture-expanded from explants, and maintained in basal medium prior to exposure to adipogenic, osteogenic, and chondrogenic conditions. Adipocyte formation was confirmed by Oil Red O lipid staining and aP2 immunocytochemistry, with expression of peroxisome proliferation-activated receptor-gamma detected only in adipogenic conditions. In chondrogenic pellets, chondrocytes lodged within lacunae and embedded within dense proteoglycan matrix were observed using Alcian blue/Sirius red staining and type II collagen immunocytochemistry. Osteogenic differentiation was confirmed by alkaline phosphatase staining and type I collagen immunocytochemistry as well as by gene expression of osteopontin and osteocalcin. Single-cell clonal analysis was used to demonstrate multipotentiality of the fetal-derived populations with the formation of adipogenic, chondrogenic, and osteogenic populations. Mineralization and osteoid formation were observed after culture on biomimetic scaffolds with extensive matrix accumulation both in vitro and in vivo after subcutaneous implantation in severely compromised immunodeficient mice. These studies demonstrate the proliferative and multipotential properties of fetal femur-derived cells in comparison with adult-derived cells. Selective differentiation and immunophenotyping will determine the potential of these fetal cells as a unique alternative model and cell source in the restoration of damaged tissue.