Rajesh Pareta

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.

Skeletal myogenic differentiation of urine-derived stem cells and angiogenesis using microbeads loaded with growth factors

To provide site-specific delivery and targeted release of growth factors to implanted urine-derived stem cells (USCs), we prepared microbeads of alginate containing growth factors. The growth factors included VEGF, IGF-1, FGF-1, PDGF, HGF and NGF. Radiolabeled growth factors were loaded separately and used to access the in vitro release from the microbeads with a gamma counter over 4 weeks. In vitro endothelial differentiation of USCs by the released VEGF from the microbeads in a separate experiment confirmed that the released growth factors from the microbeads were bioactive. USCs and microbeads were mixed with the collagen gel type 1 (2 mg/ml) and used for in vivo studies through subcutaneous injection into nude mice. Four weeks after subcutaneous injection, we found that grafted cell survival was improved and more cells expressed myogenic and endothelial cell transcripts and markers compared to controls. More vessel formation and innervations were observed in USCs combined with six growth factors cocktail incorporated in microbeads compared to controls. In conclusion, a combination of growth factors released locally from the alginate microbeads induced USCs to differentiate into a myogenic lineage, enhanced revascularization and innervation, and stimulated resident cell growth in vivo. This approach could potentially be used for cell therapy in the treatment of stress urinary incontinence.

Long-term function of islets encapsulated in a redesigned alginate microcapsule construct in omentum pouches of immune-competent diabetic rats.

Objective Our study aim was to determine encapsulated islet graft viability in an omentum pouch and the effect of fibroblast growth factor 1 (FGF-1) released from our redesigned alginate microcapsules on the function of the graft. Methods Isolated rat islets were encapsulated in an inner core made with 1.5% low-viscosity–high-mannuronic-acid alginate followed by an external layer made with 1.25% low-viscosity high-guluronic acid alginate with or without FGF-1, in microcapsules measuring 300 to 400 µm in diameter. The 2 alginate layers were separated by a perm-selective membrane made with 0.1% poly-l-ornithine, and the inner low-viscosity–high-mannuronic-acid core was partially chelated using 55 mM sodium citrate for 2 minutes. Results A marginal mass of encapsulated islet allografts (∼2000 islets/kg) in streptozotocin-diabetic Lewis rats caused significant reduction in blood glucose levels similar to the effect observed with encapsulated islet isografts. Transplantation of alloislets coencapsulated with FGF-1 did not result in better glycemic control, but induced greater body weight maintenance in transplant recipients compared with those that received only alloislets. Histological examination of the retrieved tissue demonstrated morphologically and functionally intact islets in the microcapsules, with no signs of fibrosis. Conclusions We conclude that the omentum is a viable site for encapsulated islet transplantation.

Design of a bioartificial pancreas.

Islet transplantation has been shown to be a viable treatment option for patients afflicted with type 1 diabetes. However, the lack of availablity of human pancreases and the need to use risky immunosuppressive drugs to prevent transplant rejection remain two major obstacles to the routine use of islet transplantation in diabetic patients. Successful development of a bioartificial pancreas using the approach of microencapsulation with perm-selective coating of islets in hydrogels for graft immunoisolation holds tremendous promise for diabetic patients because it has great potential to overcome these two barriers. In this review article, we will discuss the need for a bioartificial pancreas, provide a detailed description of the microencapsulation process, and review the status of the technology in clinical development. We will also critically review the various factors that will need to be taken into consideration in order to achieve the ultimate goal of routine clinical application.

Immunoisolation: where regenerative medicine meets solid organ transplantation

Immunoisolation refers to an immunological strategy in which nonself antigens present on an allograft or xenograft are not allowed to come in contact with the host immune system, and it is implemented to prevent allorecognition and avoid immunosuppression. In this setting, the two most promising technologies, encapsulation of pancreatic islets (EPI) and immunocloaking (IC), are used. In the case of EPI, islets are inserted in capsules that, allow exchange of oxygen, nutrients and other molecules. In the case of IC, a natural nanofilm is injected prior to renal transplantation within the vasculature of the graft with the intent to pave the inner surface of the vascular lumen and camouflage the antigens located on the membrane of endothelia cells. Significant progress achieved in experimental models is leading EPI and IC to clinical translation.

Effects of Allogeneic Bone Marrow Derived Mesenchymal Stromal Cell Therapy on Voiding Function in a Rat Model of Parkinson Disease

PURPOSE Cellular therapy induced transient urodynamic improvement in a rat model of Parkinson disease in which bladder dysfunction was noted after unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. We sought to prolong the effect by injecting allogeneic rat bone marrow mesenchymal stromal cells before and after microencapsulation into the substantia nigra pars compacta. MATERIALS AND METHODS Female rats underwent unilateral stereotactic injection of 6-hydroxydopamine in the medial forebrain bundle. Injection was performed in the ipsilateral substantia nigra pars compacta using vehicle alone or vehicle with nonmicroencapsulated or microencapsulated rat bone marrow derived mesenchymal stromal cells. Rats were evaluated by cystometry 7, 14, 28 and 42 days after treatment. Brains were extracted for immunostaining. RESULTS At 42 days the nonmicroencapsulated group had lower threshold and intermicturition pressure, spontaneous activity and AUC than vehicle treated animals. Rats that received microencapsulated cells had lower threshold pressure at 28 days and lower spontaneous activity at 42 days than vehicle treated rats. Microencapsulated and nonmicroencapsulated rat bone marrow derived mesenchymal stromal cells were noted in the substantia nigra pars compacta up to 42 days after transplantation. At 42 days tyrosine hydroxylase positive neurons were more numerous in the substantia nigra pars compacta of the nonmicroencapsulated group, followed by the microencapsulated and vehicle treated groups. CONCLUSIONS Urodynamic effects of the 6-hydroxydopamine lesion persisted up to 42 days after vehicle injection. Transplantation of nonmicroencapsulated rat bone marrow derived mesenchymal stromal cells improved urodynamic pressure by 42 days after treatment more markedly than microencapsulated cells. This was associated with more tyrosine hydroxylase positive neurons in the treated substantia nigra pars compacta of the nonmicroencapsulated group, suggesting that functional improvement requires a juxtacrine effect.

Bioartificial Pancreas: Evaluation of Crucial Barriers to Clinical Application

The pancreas is a dual-function organ featuring both endocrine and exocrine tissue. Endocrine functionality is provided by approximately one million cell clusters called the islets of Langerhans. Islets consist of four main cell types, 1) ┙ cells: secrete glucagon (increases glucose in blood); ┚ cells: secrete insulin (decreases glucose in blood); ├ cells: secrete somatostatin (regulates ┙ and ┚ cells) and PP cells: secrete pancreatic polypeptide. Thus, the islet plays a diverse role in glucose metabolism and blood glucose homeostasis.

Chapter 43 – Microencapsulation Technology

The bioartificial pancreas (BAP) can be defined as a drug delivery device consisting of biopolymeric materials and functional islets that are responsive to changes in glucose concentrations. This should be distinguished from the artificial pancreas also called the mechanical artificial pancreas consisting of a glucose sensor, an insulin pump, and a computer which determines the rate of insulin delivery. The desirable attributes of a BAP include little or no use of immunosuppressive drugs, sustained graft function, technical ease of grafting, and retrievability for biopsy and posttransplant evaluation. Based on these criteria, the use of microencapsulation technology that incorporates permselectivity in the construct has been the most researched approach to develop a BAP. Also, macroencapsulation of functional islets has been used as an approach to develop a BAP, but this chapter will focus on the use of the microencapsulation technology. Various modifications of the original microencapsulated islet technology have been described including the incorporation of encapsulated islets into other devices designed to enhance the survival and function of the BAP. Additional technologies are emerging such as the use of the body’s tissue scaffolds to enhance the viability of islets, which could impact the development of the BAP. This chapter will discuss the roles that techniques in islet microencapsulation and extracellular matrix technologies may play either separately or in combination to produce a viable BAP for clinical use in diabetic patients.