Sivanandane Sittadjody

The human pancreas as a source of protolerogenic extracellular matrix scaffold for a new-generation bioartificial endocrine pancreas

Objectives: Our study aims at producing acellular extracellular matrix scaffolds from the human pancreas (hpaECMs) as a first critical step toward the production of a new-generation, fully human-derived bioartificial endocrine pancreas. In this bioartificial endocrine pancreas, the hardware will be represented by hpaECMs, whereas the software will consist in the cellular compartment generated from patients own cells. Background: Extracellular matrix (ECM)-based scaffolds obtained through the decellularization of native organs have become the favored platform in the field of complex organ bioengineering. However, the paradigm is now switching from the porcine to the human model. Methods: To achieve our goal, human pancreata were decellularized with Triton-based solution and thoroughly characterized. Primary endpoints were complete cell and DNA clearance, preservation of ECM components, growth factors and stiffness, ability to induce angiogenesis, conservation of the framework of the innate vasculature, and immunogenicity. Secondary endpoint was hpaECMs’ ability to sustain growth and function of human islet and human primary pancreatic endothelial cells. Results: Results show that hpaECMs can be successfully and consistently produced from human pancreata and maintain their innate molecular and spatial framework and stiffness, and vital growth factors. Importantly, hpaECMs inhibit human naïve CD4+ T-cell expansion in response to polyclonal stimuli by inducing their apoptosis and promoting their conversion into regulatory T cells. hpaECMs are cytocompatible and supportive of representative pancreatic cell types. Discussion: We, therefore, conclude that hpaECMs has the potential to become an ideal platform for investigations aiming at the manufacturing of a regenerative medicine-inspired bioartificial endocrine pancreas.

Novel 3D Co-Culture Model for Epithelial-Stromal Cells Interaction in Prostate Cancer

Paracrine function is a major mechanism of cell-cell communication within tissue microenvironment in normal development and disease. In vitro cell culture models simulating tissue or tumor microenvironment are necessary tools to delineate epithelial-stromal interactions including paracrine function, yet an ideal three-dimensional (3D) tumor model specifically studying paracrine function is currently lacking. In order to fill this void we developed a novel 3D co-culture model in double-layered alginate hydrogel microspheres, incorporating prostate cancer epithelial and stromal cells in separate compartments of the microspheres. The cells remained confined and viable within their respective spheres for over 30 days. As a proof of principle regarding paracrine function of the model, we measured shedded component of E-cadherin (sE-cad) in the conditioned media, a major membrane bound cell adhesive molecule that is highly dysregulated in cancers including prostate cancer. In addition to demonstrating that sE-cad can be reliably quantified in the conditioned media, the time course experiments also demonstrated that the amount of sE-cad is influenced by epithelial-stromal interaction. In conclusion, the study establishes a novel 3D in vitro co-culture model that can be used to study cell-cell paracrine interaction.

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.

Engineered multilayer ovarian tissue that secretes sex steroids and peptide hormones in response to gonadotropins.

Although hormone replacement therapy is an option for the loss of ovarian function, hormone delivery through pharmacological means results in various clinical complications. The present study was designed to deliver sex steroids by a functional construct fabricated using encapsulation techniques. Theca and granulosa cells isolated from ovaries of 21-day old rats were encapsulated in multilayer alginate microcapsules to recapitulate the native follicular structure. Cells encapsulated in two other schemes were used as controls to assess the importance of the multilayer structure. The endocrine functions of the encapsulated cells were assessed in vitro for a period of 30 days. Encapsulated cells showed sustained viability during long-term in vitro culture with those encapsulated in multilayer capsules secreting significantly higher and sustained concentrations of 17 β-estradiol (E(2)) than the two other encapsulation schemes (p < 0.05, n = 6) in response to follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In addition, cells in the multilayer microcapsules also secreted activin and inhibin in vitro. In contrast, when granulosa and theca cells were cultured in 2D culture, progesterone (P(4)) secretion increased while E(2) secretion decreased over a 30-day period. In summary, we have designed a multilayer engineered ovarian tissue that secretes sex steroids and peptide hormones and responds to gonadotropins, thus demonstrating the ability to recapitulate native ovarian structure ex vivo.

Regeneration and bioengineering of transplantable abdominal organs: current status and future challenges

Introduction: The most critical issue to organ transplantation is the identification of new sources of organs. The present manuscript illustrates the state-of-the-art regenerative medicine (RM) investigations aiming to manufacturing abdominal organs for transplant purposes. Areas covered: This manuscript focuses on research in the bioengineering and regeneration of kidneys, insulin-producing cells, livers and small bowel. The main technology currently under development exploits the seeding of cells on supporting scaffolding material. Despite favorable preliminary results obtained with relatively simple, hollow organs, when more complex organs are considered, the scenario changes dramatically. Investigations are still in early stages, and clinical translation is not yet foreseeable based on current knowledge and information. Obstacles are numerous but we believe the critical factor hampering success is lack of in-depth understanding of the extracellular matrix (ECM) and cell–ECM interactions, as well as the mechanisms with which organs develop in utero. Expert opinion: The success of RM to generate transplantable abdominal organs relies heavily on progress in (stem) cell therapies, developmental and ECM biology, and in the thorough understanding of the intricate relationship and interplay between cells and the ECM. This will require enormous investments in financial and medical resources, which ideally should be embarked upon by governments, the private sector and academia.

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.

Scientific principles of regenerative medicine and their application in the female reproductive system

The goal of regenerative medicine is to repair, replace, or regenerate diseased tissues/organs in order to restore normal function. In this paper we will first discuss the general principle of regenerative medicine and the various techniques and approaches that have been used to replace or regenerate cells in diseased tissues and organs. Then, we will review different regenerative medicine approaches that have been used to treat specific diseased tissues and organs of the reproductive system in both animal and human experiments. It is clear from this article that regenerative medicine holds significant promise, and we hope that the review will serve as a platform for further development of regenerative medicine technologies for the treatment of inadequacies of the reproductive system.

Retrieval of Microencapsulated Islet Grafts for Post-transplant Evaluation.

Microencapsulation of islets is a procedure used to immunoisolate islets in order to obviate the need for immunosuppression of islet transplant recipients. Although microencapsulated islets have routinely been transplanted in the peritoneal cavity, the ideal site for their engraftment remains to be determined. The omentum, a highly vascularized tissue, has been proposed as an alternative site for microencapsulated islet transplantation. An added benefit to the omentum is that implanted microcapsules can be easily retrieved for post-transplant evaluation. This chapter describes a collagenase-based procedure for the retrieval of microencapsulated islets following the harvest of omentum pouch site of transplantation.

Non-androgen Signaling Pathways in Castration-Resistant Prostate Cancer

Prostate cancer (PCa), while initially androgen-sensitive, the lethal form of the disease inevitably progresses to hormone refractory or castration-resistant state and accounts for vast majority of mortality among PCa patients. During the transition from the hormone naive disease into CRPC, the progression of PCa cells can be driven by alternative (non-androgen) signaling pathways. Upregulation of ligands, receptors and intracellular signaling molecules along with activating mutations of proto-oncogenes and/or suppression of tumor suppressor genes are the major causes of the deregulation of these alternative pathways. In addition, loss of negative feedback mechanism of the signal cascades further amplifies the effects of the pathways and thus contributing to the emergence of CRPC. This chapter covers studies investigating the potential involvement of non-androgen signaling pathways in PCa and the current strategies employed in PCa cell lines, animal models and clinical trials for controlling these aberrant signaling pathways. The understanding of non-androgen signaling pathway target(s) in CRPC could provide novel biomarkers and newer strategies in management of metastatic PCa.

In vivo transplantation of 3D encapsulated ovarian constructs in rats corrects abnormalities of ovarian failure

Safe clinical hormone replacement (HR) will likely become increasingly important in the growing populations of aged women and cancer patients undergoing treatments that ablate the ovaries. Cell-based HRT (cHRT) is an alternative approach that may allow certain physiological outcomes to be achieved with lower circulating hormone levels than pharmacological means due to participation of cells in the hypothalamus-pituitary-ovary feedback control loop. Here we describe the in vivo performance of 3D bioengineered ovarian constructs that recapitulate native cell–cell interactions between ovarian granulosa and theca cells as an approach to cHRT. The constructs are fabricated using either Ca++ or Sr++ to crosslink alginate. Following implantation in ovariectomized (ovx) rats, the Sr++-cross-linked constructs achieve stable secretion of hormones during 90 days of study. Further, we show these constructs with isogeneic cells to be effective in ameliorating adverse effects of hormone deficiency, including bone health, uterine health, and body composition in this rat model.Cell-based hormone replacement therapy (cHRT) may be an alternative therapy to pharmacological (p)HRT. Here, the authors show that implanted 3D bioengineered ovarian constructs of granulosa and theca cells in ovariectomized rats recapitulate native cell interactions and improve efficacy compared to similar doses of pHRT.