Effect of the 3D Artificial Nichoid on the Morphology and Mechanobiological Response of Mesenchymal Stem Cells Cultured In Vitro.

Stem cell fate and behavior are affected by the bidirectional communication of cells and their local microenvironment (the stem cell niche), which includes biochemical cues, as well as physical and mechanical factors. Stem cells are normally cultured in conventional two-dimensional monolayer, with a mechanical environment very different from the physiological one. Here, we compare culture of rat mesenchymal stem cells on flat culture supports and in the "Nichoid", an innovative three-dimensional substrate micro-engineered to recapitulate the architecture of the physiological niche in vitro. Two versions of the culture substrates Nichoid (single-layered or "2D Nichoid" and multi-layered or "3D Nichoid") were fabricated via two-photon laser polymerization in a biocompatible hybrid organic-inorganic photoresist (SZ2080). Mesenchymal stem cells, isolated from rat bone marrow, were seeded on flat substrates and on 2D and 3D Nichoid substrates and maintained in culture up to 2 weeks. During cell culture, we evaluated cell morphology, proliferation, cell motility and the expression of a panel of 89 mesenchymal stem cells' specific genes, as well as intracellular structures organization. Our results show that mesenchymal stem cells adhered and grew in the 3D Nichoid with a comparable proliferation rate as compared to flat substrates. After seeding on flat substrates, cells displayed large and spread nucleus and cytoplasm, while cells cultured in the 3D Nichoid were spatially organized in three dimensions, with smaller and spherical nuclei. Gene expression analysis revealed the upregulation of genes related to stemness and to mesenchymal stem cells' features in Nichoid-cultured cells, as compared to flat substrates. The observed changes in cytoskeletal organization of cells cultured on 3D Nichoids were also responsible for a different localization of the mechanotransducer transcription factor YAP, with an increase of the cytoplasmic retention in cells cultured in the 3D Nichoid. This difference could be explained by alterations in the import of transcription factors inside the nucleus due to the observed decrease of mean nuclear pore diameter, by transmission electron microscopy. Our data show that 3D distribution of cell volume has a profound effect on mesenchymal stem cells structure and on their mechanobiological response, and highlight the potential use of the 3D Nichoid substrate to strengthen the potential effects of MSC in vitro and in vivo.

Protective Effect of Human Mesenchymal Stem Cells on the Survival of Pancreatic Islets.

BACKGROUND AND OBJECTIVES: Transplantation of pancreatic islets is an intriguing new therapeutic option to face the worldwide spread problem of Type-I diabetes. Currently, its clinical use is limited by several problems, mainly based on the high number of islets required to restore normoglycaemia and by the low survival of the transplanted tissue. A promising attempt to overcome the limits to such an approach was represented by the use of Mesenchymal Stem Cells (MSC). Despite the encouraging results obtained with murine-derived MSC, little is still known about their protective mechanisms. The aim of the present study was to verify the effectiveness, (besides murine MSC), of clinically relevant human-derived MSC (hMSC) on protecting pancreatic islets, thus also shedding light on the putative differences between MSC of different origin. METHODS AND RESULTS: Threefold kinds of co-cultures were therefore in vitro set up (direct, indirect and mixed), to analyze the hMSC effect on pancreatic islet survival and function and to study the putative mechanisms involved. Although in a different way with respect to murine MSC, also human derived cells demonstrated to be effective on protecting pancreatic islet survival. This effect could be due to the release of some trophic factors, such as VEGF and Il-6, and by the reduction of inflammatory cytokine TNF-α. CONCLUSIONS: Therefore, hMSC confirmed their great clinical potential to improve the feasibility of pancreatic islet transplantation therapy against diabetes.

Engineering the vasculature of decellularized rat kidney scaffolds using human induced pluripotent stem cell-derived endothelial cells.

Generating new kidneys using tissue engineering technologies is an innovative strategy for overcoming the shortage of donor organs for transplantation. Here we report how to efficiently engineer the kidney vasculature of decellularized rat kidney scaffolds by using human induced pluripotent stem cell (hiPSCs)-derived endothelial cells (hiPSC-ECs). In vitro, hiPSC-ECs responded to flow stress by acquiring an alignment orientation, and attached to and proliferated on the acellular kidney sections, maintaining their phenotype. The hiPSC-ECs were able to self-organize into chimeric kidney organoids to form vessel-like structures. Ex vivo infusion of hiPSC-ECs through the renal artery and vein of acellular kidneys resulted in the uniform distribution of the cells in all the vasculature compartments, from glomerular capillaries to peritubular capillaries and small vessels. Ultrastructural analysis of repopulated scaffolds through transmission and scanning electron microscopy demonstrated the presence of continuously distributed cells along the vessel wall, which was also confirmed by 3D reconstruction of z-stack images showing the continuity of endothelial cell coverage inside the vessels. Notably, the detection of fenestrae in the endothelium of glomerular capillaries but not in the vascular capillaries was clear evidence of site-specific endothelial cell specialisation.

A novel hybrid silk-fibroin/polyurethane three-layered vascular graft: towards in situ tissue-engineered vascular accesses for haemodialysis.

Clinically available alternatives of vascular access for long-term haemodialysis-currently limited to native arteriovenous fistulae and synthetic grafts-suffer from several drawbacks and are associated to high failure rates. Bioprosthetic grafts and tissue-engineered blood vessels are costly alternatives without clearly demonstrated increased performance. In situ tissue engineering could be the ideal approach to provide a vascular access that profits from the advantages of vascular grafts in the short-term (e.g. early cannulation) and of fistulae in the long-term (e.g. high success rates driven by biointegration). Hence, in this study a three-layered silk fibroin/polyurethane vascular graft was developed by electrospinning to be applied as long-term haemodialysis vascular access pursuing a 'hybrid' in situ engineering approach (i.e. based on a semi-degradable scaffold). This Silkothane® graft was characterized concerning morphology, mechanics, physical properties, blood contact and vascular cell adhesion/viability. The full three-layered graft structure, influenced by the polyurethane presence, ensured mechanical properties that are a determinant factor for the success of a vascular access (e.g. vein-graft compliance matching). The Silkothane® graft demonstrated early cannulation potential in line with self-sealing commercial synthetic arteriovenous grafts, and a degradability driven by enzymatic activity. Moreover, the fibroin-only layers and extracellular matrix-like morphology, presented by the graft, revealed to be crucial in providing a non-haemolytic character, long clotting time, and favourable adhesion of human umbilical vein endothelial cells with increasing viability after 3 and 7 d. Accordingly, the proposed approach may represent a step forward towards an in situ engineered hybrid vascular access with potentialities for vein-graft anastomosis stability, early cannulation, and biointegration.

Bioengineering Organs for Blood Detoxification.

For patients with severe kidney or liver failure the best solution is currently organ transplantation. However, not all patients are eligible for transplantation and due to limited organ availability, most patients are currently treated with therapies using artificial kidney and artificial liver devices. These therapies, despite their relative success in preserving the patients' life, have important limitations since they can only replace part of the natural kidney or liver functions. As blood detoxification (and other functions) in these highly perfused organs is achieved by specialized cells, it seems relevant to review the approaches leading to bioengineered organs fulfilling most of the native organ functions. There, the culture of cells of specific phenotypes on adapted scaffolds that can be perfused takes place. In this review paper, first the functions of kidney and liver organs are briefly described. Then artificial kidney/liver devices, bioartificial kidney devices, and bioartificial liver devices are focused on, as well as biohybrid constructs obtained by decellularization and recellularization of animal organs. For all organs, a thorough overview of the literature is given and the perspectives for their application in the clinic are discussed.

Decellularized kidney matrix as functional material for whole organ tissue engineering.

Renal transplantation is currently the most effective treatment for end-stage renal disease, which represents one of the major current public health problems. However, the number of available donor kidneys is drastically insufficient to meet the demand, causing prolonged waiting lists. For this reason, tissue engineering offers great potential to increase the pool of donated organs for kidney transplantation, by way of seeding cells on supporting scaffolding material. Biological scaffolds are prepared by removing cellular components from the donor organs using a decellularization process with detergents, enzymes or other cell lysing solutions. Extracellular matrix which makes up the scaffold is critical to directing the cell attachment and to creating a suitable environment for cell survival, proliferation and differentiation. Researchers are now studying whole intact scaffolds produced from the kidneys of animals or humans without adversely affecting extracellular matrix, biological activity and mechanical integrity. The process of recellularization includes cell seeding strategies and the choice of the cell source to repopulate the scaffold. This is the most difficult phase, due to the complexity of the kidney. Indeed, no studies have provided sufficient results of complete renal scaffold repopulation and differentiation. This review summarizes the research that has been conducted to obtain decellularized kidney scaffolds and to repopulate the scaffolds, evaluating the best cell sources, the cell seeding methods and the cell differentiation in kidney scaffolds.

Experimental Evaluation of Kidney Regeneration by Organ Scaffold Recellularization.

The rising number of patients needing renal replacement therapy, alongside the significant clinical and economic limitations of current therapies, creates an imperative need for new strategies to treat kidney diseases. Kidney bioengineering through the production of acellular scaffolds and recellularization with stem cells is one potential strategy. While protocols for obtaining organ scaffolds have been developed successfully, scaffold recellularization is more challenging. We evaluated the potential of in vivo and in vitro kidney scaffold recellularization procedures. Our results show that acellular scaffolds implanted in rats cannot be repopulated with host cells, and in vitro recellularization is necessary. However, we obtained very limited and inconsistent cell seeding when using different infusion protocols, regardless of injection site. We also obtained experimental and theoretical data indicating that uniform cell delivery into the kidney scaffolds cannot be obtained using these infusion protocols, due to the permeability of the extracellular matrix of the scaffold. Our results highlight the major physical barriers that limit in vitro recellularization of acellular kidney scaffolds and the obstacles that must be investigated to effectively advance this strategy for regenerative medicine.

Therapeutic potential of Mesenchymal Stem Cells for the treatment of diabetic peripheral neuropathy.

Type-1 Diabetes is generally treated with exogenous insulin administration. Despite treatment, a very common long term consequence of diabetes is the development of a disabling and painful peripheral neuropathy. The transplantation of pancreatic islets is an advanced alternative therapeutic approach, but its clinical application is still very limited, mainly because of the great number of islets required to complete the procedure and of their short-term survival. An intriguing method to improve the performance of pancreatic islets transplantation is the co-transplantation of Mesenchymal Stem Cells (MSCs), adult stem cells already known to support the survival of different cellular populations. In this proof-of-concept study, we demonstrated using an in vivo model of diabetes, the ability of allogenic MSCs to reduce the number of pancreatic islets necessary to achieve glycemic control in diabetic rats, and overall their positive effect on diabetic neuropathy, with the reduction of all the neuropathic signs showed after disease induction. The cutback of the pancreatic islet number required to control glycemia and the regression of the painful neuropathy make MSC co-transplantation a very promising tool to improve the clinical feasibility of pancreatic islet transplantation for diabetes treatment.

Two-photon polymerized "nichoid" substrates maintain function of pluripotent stem cells when expanded under feeder-free conditions.

BACKGROUND: The use of pluripotent cells in stem cell therapy has major limitations, mainly related to the high costs and risks of exogenous conditioning and the use of feeder layers during cell expansion passages. METHODS: We developed an innovative three-dimensional culture substrate made of "nichoid" microstructures, nanoengineered via two-photon laser polymerization. The nichoids limit the dimension of the adhering embryoid bodies during expansion, by counteracting cell migration between adjacent units of the substrate by its microarchitecture. We expanded mouse embryonic stem cells on the nichoid for 2 weeks. We compared the expression of pluripotency and differentiation markers induced in cells with that induced by flat substrates and by a culture layer made of kidney-derived extracellular matrix. RESULTS: The nichoid was found to be the only substrate, among those tested, that maintained the expression of the OCT4 pluripotency marker switched on and, simultaneously, the expression of the differentiation markers GATA4 and α-SMA switched off. The nichoid promotes pluripotency maintenance of embryonic stem cells during expansion, in the absence of a feeder layer and exogenous conditioning factors, such as the leukocyte inhibitory factor. CONCLUSIONS: We hypothesized that the nichoid microstructures induce a genetic reprogramming of cells by controlling their cytoskeletal tension. Further studies are necessary to understand the exact mechanism by which the physical constraint provided by the nichoid architecture is responsible for cell reprogramming. The nichoid may help elucidate mechanisms of pluripotency maintenance, while potentially cutting the costs and risks of both feed-conditioning and exogenous conditioning for industrial-scale expansion of stem cells.

Regression of Renal Disease by Angiotensin II Antagonism Is Caused by Regeneration of Kidney Vasculature.

Chronic renal insufficiency inexorably progresses in patients, such as it does after partial renal ablation in rats. However, the progression of renal diseases can be delayed by angiotensin II blockers that stabilize renal function or increase GFR, even in advanced phases of the disease. Regression of glomerulosclerosis can be induced by angiotensin II antagonism, but the effect of these treatments on the entire vascular tree is unclear. Here, using microcomputed tomography and scanning electron microscopy, we compared the size and extension of kidney blood vessels in untreated Wistar rats with those in untreated and angiotensin II antagonist-treated Munich Wistar Frömter (MWF) rats that spontaneously develop kidney disease with age. The kidney vasculature underwent progressive rarefaction in untreated MWF rats, substantially affecting intermediate and small vessels. Microarray analysis showed increased Tgf-ß and endothelin-1 gene expression with age. Notably, 10-week inhibition of the renin-angiotensin system regenerated kidney vasculature and normalized Tgf-ß and endothelin-1 gene expression in aged MWF rats. These changes were associated with reduced apoptosis, increased endothelial cell proliferation, and restoration of Nrf2 expression, suggesting mechanisms by which angiotensin II antagonism mediates regeneration of capillary segments. These results have important implications in the clinical setting of chronic renal insufficiency.

Mesenchymal stem cells help pancreatic islet transplantation to control type 1 diabetes.

Islet cell transplantation has therapeutic potential to treat type 1 diabetes, which is characterized by autoimmune destruction of insulin-producing pancreatic islet ß cells. It represents a minimal invasive approach for ß cell replacement, but long-term blood control is still largely unachievable. This phenomenon can be attributed to the lack of islet vasculature and hypoxic environment in the immediate post-transplantation period that contributes to the acute loss of islets by ischemia. Moreover, graft failures continue to occur because of immunological rejection, despite the use of potent immunosuppressive agents. Mesenchymal stem cells (MSCs) have the potential to enhance islet transplantation by suppressing inflammatory damage and immune mediated rejection. In this review we discuss the impact of MSCs on islet transplantation and focus on the potential role of MSCs in protecting islet grafts from early graft failure and from autoimmune attack.

A double mechanism for the mesenchymal stem cells' positive effect on pancreatic islets.

The clinical usability of pancreatic islet transplantation for the treatment of type I diabetes, despite some encouraging results, is currently hampered by the short lifespan of the transplanted tissue. In vivo studies have demonstrated that co-transplantation of Mesenchymal Stem Cells (MSCs) with transplanted pancreatic islets is more effective with respect to pancreatic islets alone in ensuring glycemia control in diabetic rats, but the molecular mechanisms of this action are still unclear. The aim of this study was to elucidate the molecular mechanisms of the positive effect of MSCs on pancreatic islet functionality by setting up direct, indirect and mixed co-cultures. MSCs were both able to prolong the survival of pancreatic islets, and to directly differentiate into an "insulin-releasing" phenotype. Two distinct mechanisms mediated these effects: i) the survival increase was observed in pancreatic islets indirectly co-cultured with MSCs, probably mediated by the trophic factors released by MSCs; ii) MSCs in direct contact with pancreatic islets started to express Pdx1, a pivotal gene of insulin production, and then differentiated into insulin releasing cells. These results demonstrate that MSCs may be useful for potentiating pancreatic islets' functionality and feasibility.

Recellularization of well-preserved acellular kidney scaffold using embryonic stem cells.

For chronic kidney diseases, there is little chance that the vast majority of world's population will have access to renal replacement therapy with dialysis or transplantation. Tissue engineering would help to address this shortcoming by regeneration of damaged kidney using naturally occurring scaffolds seeded with precursor renal cells. The aims of the present study were to optimize the production of three-dimensional (3D) rat whole-kidney scaffolds by shortening the duration of organ decellularization process using detergents that avoid nonionic compounds, to investigate integrity of extracellular matrix (ECM) structure and to enhance the efficacy of scaffold cellularization using physiological perfusion method. Intact rat kidneys were successfully decellularized after 17 h perfusion with sodium dodecyl sulfate. The whole-kidney scaffolds preserved the 3D architecture of blood vessels, glomeruli, and tubuli as shown by transmission and scanning electron microscopy. Micro-computerized tomography (micro-CT) scan confirmed integrity, patency, and connection of the vascular network. Collagen IV, laminin, and fibronectin staining of decellularized scaffolds were similar to those of native kidney tissues. After infusion of whole-kidney scaffolds with murine embryonic stem (mES) cells through the renal artery, and pressure-controlled perfusion with recirculating cell medium for 24 and 72 h, seeded cells were almost completely retained into the organ and uniformly distributed in the vascular network and glomerular capillaries without major signs of apoptosis. Occasionally, mES cells reached peritubular capillary and tubular compartment. We observed the loss of cell pluripotency and the start of differentiation toward meso-endodermal lineage. Our findings indicate that, with the proposed optimized protocol, rat kidneys can be efficiently decellularized to produce renal ECM scaffolds in a relatively short time, and rapid recellularization of vascular structures and glomeruli. This experimental setup may open the possibility to obtain differentiation of stem cells with long lasting in vitro perfusion.

Effect of inborn pancreatic islet deficit in the Munich Wister Frömter rat.

The total mass of pancreatic islet cells is a critical factor in glucose metabolic control. The aim of the present study was to investigate whether in the Munich Wistar Frömter (MWF) rat, beside a reduction in the number of nephrons, there are also alterations in the number of pancreatic islets and of ß cell mass. We also examined glucose metabolism, both in normal conditions and following intravenous glucose injection. The number of islets per pancreas, estimated by morphometrical analysis, was significantly lower in MWF rats than in Wistar rats (3,501±1,285 vs. 7,259±2,330 islet/rat, respectively). Also the mean number of islets per gram of body weight was significantly lower in MWF rats than in Wistar rats (18±7 in MWF rats vs. 28±10 islets/g bw in Wistar rats). Morphometric analysis of ß cell mass showed an average of 77.1±7% islet cells staining for insulin in MWF rats and 83.9±2.1% in the control Wistar rats. Despite the lower number of islets and ß cells, MWF and Wistar rats had comparable fasting blood glucose levels but significant differences in blood glucose following an intraperitoneal glucose tolerance test. In summary, pancreatic islets of MWF and Wistar rats showed a marked difference in morphometrical characteristics. While this difference is not associated with blood glucose levels, glucose metabolism after IPGTT between MWF and Wistar rats is significantly different. These data suggest that an inborn deficit in ß cell mass of about 60% is responsible for altered glucose metabolism and could favor the development of diabetes.