Svitlana Vasylovska

Differentiating neural crest stem cells induce proliferation of cultured rodent islet beta cells

Aims/hypothesisEfficient stimulation of cycling activity in cultured beta cells would allow the design of new strategies for cell therapy in diabetes. Neural crest stem cells (NCSCs) play a role in beta cell development and maturation and increase the beta cell number in co-transplants. The mechanism behind NCSC-induced beta cell proliferation and the functional capacity of the new beta cells is not known.MethodsWe developed a new in vitro co-culture system that enables the dissection of the elements that control the cellular interactions that lead to NCSC-dependent increase in islet beta cells.ResultsMouse NCSCs were cultured in vitro, first in medium that stimulated their proliferation, then under conditions that supported their differentiation. When mouse islet cells were cultured together with the NCSCs, more than 35% of the beta cells showed cycle activity. This labelling index is more than tenfold higher than control islets cultured without NCSCs. Beta cells that proliferated under these culture conditions were fully glucose responsive in terms of insulin secretion. NCSCs also induced beta cell proliferation in islets isolated from 1-year-old mice, but not in dissociated islet cells isolated from human donor pancreas tissue. To stimulate beta cell proliferation, NCSCs need to be in intimate contact with the beta cells.Conclusions/interpretationCulture of islet cells in contact with NCSCs induces highly efficient beta cell proliferation. The reported culture system is an excellent platform for further dissection of the minimal set of factors needed to drive this process and explore its potential for translation to diabetes therapy.

Co-transplantation of Human Pancreatic Islets With Post-migratory Neural Crest Stem Cells Increases β-Cell Proliferation and Vascular And Neural Regrowth

CONTEXT Neural crest stem cells (NCSCs) are capable of substantially improving murine islet function by promoting β-cell proliferation. OBJECTIVE The present study aimed to investigate the potential of NCSCs to stimulate human β-cell proliferation, and improve neural and vascular engraftment of human islets. DESIGN, SETTING, AND SUBJECTS Human pancreatic islets from 18 brain-dead cadaveric donors (age range, 19-78 y) were obtained through the Nordic Network for Clinical Islet Transplantation. β-cell proliferation and graft function was investigated at our experimental laboratory. INTERVENTION AND MAIN OUTCOME MEASURES Human islets were transplanted, either alone or together with spheres of NCSCs. β-cell proliferation, as well as islet neural and vascular densities, were assessed by immunohistochemistry. Graft blood perfusion and oxygen tension were measured using laser-Doppler flowmetry and Clark microelectrodes, respectively. RESULTS Two days posttransplantation, the number of Ki67-positive β-cells was doubled in human islets that had been exposed to NCSCs. Similar findings were obtained in vitro, as well as with EdU as proliferation marker. Four weeks posttransplantation, NCSC-exposed human islet grafts had much higher neural and vascular densities. The newly formed blood vessels were also functional, given that these human islets had a substantially higher blood perfusion and oxygen tension when compared with control transplants. CONCLUSION We conclude that exposure to NCSCs stimulates human β-cell proliferation, and that these cells improve both the neural and vascular engraftment of transplanted human islets. NCSCs are a promising cellular therapy for translation into clinical use.

Surface coating of pancreatic islets with neural crest stem cells improves engraftment and function after intraportal transplantation.

The present study aimed to develop techniques for surface coating of islets with neural crest stem cells (NCSCs) in order to enable cotransplantation to the clinically used liver site and then investigate engraftment and function intraportally of such bioengineered islets. Mouse islets were coated during incubation with enhanced green fluorescent protein (EGFP)-expressing mouse NCSCs and transplanted into the portal vein to cure diabetic mice. An intravenous glucose tolerance test was performed at 1 month posttransplantation. Islet grafts were retrieved and evaluated for vascular density, nerves, and glial cells. NCSCs expressed a vast number of key angiogenic and neurotrophic factors. Mice transplanted with NCSC-bioengineered islets responded better to the glucose load than recipient mice with control islets. NCSCs remained present in the vicinity or had often migrated into the NCSC-coated islets, and an improved islet graft reinnervation and revascularization was observed. Transplanted NCSCs differentiated into both glial and neural cells in the islet grafts. We conclude that bioengineering of islets with NCSCs for intraportal transplantation provides a possibility to improve islet engraftment and function. Pending successful establishment of protocols for expansion of NCSCs from, for example, human skin or bone marrow, this strategy may be applied to clinical islet transplantation.

Co-Culture of Neural Crest Stem Cells (NCSC) and Insulin Producing Beta-TC6 Cells Results in Cadherin Junctions and Protection against Cytokine-Induced Beta-Cell Death

Purpose Transplantation of pancreatic islets to Type 1 diabetes patients is hampered by inflammatory reactions at the transplantation site leading to dysfunction and death of insulin producing beta-cells. Recently we have shown that co-transplantation of neural crest stem cells (NCSCs) together with the islet cells improves transplantation outcome. The aim of the present investigation was to describe in vitro interactions between NCSCs and insulin producing beta-TC6 cells that may mediate protection against cytokine-induced beta-cell death. Procedures Beta-TC6 and NCSC cells were cultured either alone or together, and either with or without cell culture inserts. The cultures were then exposed to the pro-inflammatory cytokines IL-1β and IFN-γ for 48 hours followed by analysis of cell death rates (flow cytometry), nitrite production (Griess reagent), protein localization (immunofluorescence) and protein phosphorylation (flow cytometry). Results We observed that beta-TC6 cells co-cultured with NCSCs were protected against cytokine-induced cell death, but not when separated by cell culture inserts. This occurred in parallel with (i) augmented production of nitrite from beta-TC6 cells, indicating that increased cell survival allows a sustained production of nitric oxide; (ii) NCSC-derived laminin production; (iii) decreased phospho-FAK staining in beta-TC6 cell focal adhesions, and (iv) decreased beta-TC6 cell phosphorylation of ERK(T202/Y204), FAK(Y397) and FAK(Y576). Furthermore, co-culture also resulted in cadherin and beta-catenin accumulations at the NCSC/beta-TC6 cell junctions. Finally, the gap junction inhibitor carbenoxolone did not affect cytokine-induced beta-cell death during co-culture with NCSCs. Conclusion In summary, direct contacts, but not soluble factors, promote improved beta-TC6 viability when co-cultured with NCSCs. We hypothesize that cadherin junctions between NCSC and beta-TC6 cells promote powerful signals that maintain beta-cell survival even though ERK and FAK signaling are suppressed. It may be that future strategies to improve islet transplantation outcome may benefit from attempts to increase beta-cell cadherin junctions to neighboring cells.

Boundary cap neural crest stem cells homotopically implanted to the injured dorsal root transitional zone give rise to different types of neurons and glia in adult rodents

BackgroundThe boundary cap is a transient group of neural crest-derived cells located at the presumptive dorsal root transitional zone (DRTZ) when sensory axons enter the spinal cord during development. Later, these cells migrate to dorsal root ganglia and differentiate into subtypes of sensory neurons and glia. After birth when the DRTZ is established, sensory axons are no longer able to enter the spinal cord. Here we explored the fate of mouse boundary cap neural crest stem cells (bNCSCs) implanted to the injured DRTZ after dorsal root avulsion for their potential to assist sensory axon regeneration.ResultsGrafted cells showed extensive survival and differentiation after transplantation to the avulsed DRTZ. Transplanted cells located outside the spinal cord organized elongated tubes of Sox2/GFAP expressing cells closely associated with regenerating sensory axons or appeared as small clusters on the surface of the spinal cord. Other cells, migrating into the host spinal cord as single cells, differentiated to spinal cord neurons with different neurotransmitter characteristics, extensive fiber organization, and in some cases surrounded by glutamatergic terminal-like profiles.ConclusionsThese findings demonstrate that bNCSCs implanted at the site of dorsal root avulsion injury display remarkable differentiation plasticity inside the spinal cord and in the peripheral compartment where they organize tubes associated with regenerating sensory fibers. These properties offer a basis for exploring the ability of bNCSCs to assist regeneration of sensory axons into the spinal cord and replace lost neurons in the injured spinal cord.

Forced Runx1 expression in human neural stem/progenitor cells transplanted to the rat dorsal root ganglion cavity results in extensive axonal growth specifically from spinal cord-derived neurospheres.

Cell replacement therapy holds great promise for treating a wide range of human disorders. However, ensuring the predictable differentiation of transplanted stem cells, eliminating their risk of tumor formation, and generating fully functional cells after transplantation remain major challenges in regenerative medicine. Here, we explore the potential of human neural stem/progenitor cells isolated from the embryonic forebrain (hfNSPCs) or the spinal cord (hscNSPCs) to differentiate to projection neurons when transplanted into the dorsal root ganglion cavity of adult recipient rats. To stimulate axonal growth, we transfected hfNSPC- and hscNSPC-derived neurospheres, prior to their transplantation, with a Tet-Off Runx1-overexpressing plasmid to maintain Runx1 expression in vivo after transplantation. Although pronounced cell differentiation was found in the Runx1-expressing transplants from both cell sources, we observed extensive, long-distance growth of axons exclusively from hscNSPC-derived transplants. These axons ultimately reached the dorsal root transitional zone, the boundary separating peripheral and central nervous systems. Our data show that hscNSPCs have the potential to differentiate to projection neurons with long-distance axonal outgrowth and that Runx1 overexpression is a useful approach to induce such outgrowth in specific sources of NSPCs.

The therapeutic role of endothelial progenitor cells in Type 1 diabetes mellitus

Pancreatic β-cells sense and adjust the blood glucose level by secretion of insulin. In Type 1 diabetes mellitus, these insulin-producing cells are destroyed, leaving the patients incapable of regulating blood glucose homeostasis. At the time of diagnosis, most patients still have 20-30% of their original β-cell mass remaining. These residual β-cells are targets for intervention therapies aimed at preventing further autoimmune destruction, in addition to increasing the number of existing β-cells. Such a therapeutic option is highly desirable since it may lead to a full recovery of newly diagnosed patients, with no need for further treatment with immunosuppressant drugs or exogenous insulin administration. In this article, we propose that endothelial progenitor cells, a cell type known to promote and support neovascularization following endothelial injury, may be used as part of a combinational stem cell therapy aimed to improve the vascularization, survival and proliferation of β-cells.

Neural crest stem cells from hair follicles and boundary cap have different effects on pancreatic islets in vitro

Purpose: Neural crest stem cells derived from the boundary cap (bNCSCs), markedly promote survival, proliferation and function of insulin producing β-cells in vitro and in vivo after coculture/transplantation with pancreatic islets [1, 2]. Recently, we have shown that beneficial effects on β-cells require cadherin contacts between bNCSCs and β-cells [3, 4]. Here we investigated whether hair follicle (HF) NCSCs, a potential source for human allogeneic transplantation, exert similar positive effects on β-cells. Materials and Methods: We established cocultures of HF-NCSCs or bNCSCs from mice expressing enhanced green fluorescent protein together with pancreatic islets from DxRed expressing mice or NMRI mice and compared their migration towards islet cells and effect on proliferation of β-cells as well as intracellular relations between NCSCs and islets using qRT-PCR analysis and immunohistochemistry. Results: Whereas both types of NCSCs migrated extensively in the presence of islets, only bNCSCs demonstrated directed migration toward islets, induced β-cell proliferation and increased the presence of cadherin at the junctions between bNCSCs and β-cells. Even in direct contact between β-cells and HF-NCSCs, no cadherin expression was detected. Conclusions: These observations indicate that HF-NCSCs do not confer the same positive effect on β-cells as demonstrated for bNCSCs. Furthermore, these data suggest that induction of cadherin expression by HF-NCSCs may be useful for their ability to support β-cells in coculture and after transplantation.

Co-culture of insulin producing human EndoC-βH1 cells with boundary cap neural crest stem cells protects partially against cytokine-induced cell death

We have recently observed that co-culture of mouse and rat beta-cells with mouse boundary cap neural crest stem cells (bNCSCs) protected against inflammatory cytokine-induced beta-cell death, possibly via direct cadherinmediated cell-to-cell junctions. However, it has not been addressed whether also human beta-cells can be protected via this strategy. If possible it would be an important approach for development of new protocols for improved outcome of islet transplantation to Type-1 diabetes patients. The aim of this investigation was therefore to study the effect of bNCSC co-culture with insulin producing human EndoC-βH1 cells on cytokine-induced cell death. For this purpose GFP-positive bNCSCs were cultured together with GFP-negative human EndoC-βH1 cells in the presence of the cytokines IL-1γ (50 U/ml) and IFN-γ (1000 U/ml). Cells were then stained with propidium iodide and trypsinized for flow cytometry analysis. Analysis of propidium iodide fluorescence in GFP-positive and GFP-negative cells revealed that EndoC-βH1 cells died to a lower extent when co-cultured with bNCSCs than when cultured without bNCSCs. We also observed that EndoC-βH1 cells formed N-cadherin, but not E-cadherin junctions with the bNCSCs. The bNCSC cell population contained a large proportion of beta-tubulin expressing cells indicating neuronal differentiation. A protective function of the N-cadherin junctions was verified by the finding that a neutralizing N-cadherin antibody counteracted the effect of co-culture. We conclude that the interaction between human insulin producing cells and bNCSCs results in a lowered susceptibility of insulin producing cells to pro-inflammatory cytokines in vitro.

Boundary Cap Neural Crest Stem Cells Promote Survival of Mutant SOD1 Motor Neurons.

ALS is a devastating disease resulting in degeneration of motor neurons (MNs) in the brain and spinal cord. The survival of MNs strongly depends on surrounding glial cells and neurotrophic support from muscles. We previously demonstrated that boundary cap neural crest stem cells (bNCSCs) can give rise to neurons and glial cells in vitro and in vivo and have multiple beneficial effects on co-cultured and co-implanted cells, including neural cells. In this paper, we investigate if bNCSCs may improve survival of MNs harboring a mutant form of human SOD1 (SOD1G93A) in vitro under normal conditions and oxidative stress and in vivo after implantation to the spinal cord. We found that survival of SOD1G93A MNs in vitro was increased in the presence of bNCSCs under normal conditions as well as under oxidative stress. In addition, when SOD1G93A MN precursors were implanted to the spinal cord of adult mice, their survival was increased when they were co-implanted with bNCSCs. These findings show that bNCSCs support survival of SOD1G93A MNs in normal conditions and under oxidative stress in vitro and improve their survival in vivo, suggesting that bNCSCs have a potential for the development of novel stem cell-based therapeutic approaches in ALS models.