Angiogenic potential of mesenchymal stem cells derived from patients with diabetes seeded on decellularized micro fragments.

Mesenchymal stem cells (MSCs) are a potential source of angiogenic factors which may promote wound healing in poorly vascularized diabetic foot ulcers. We demonstrate that MSCs of patients with diabetic foot ulcers seeded on decellularized micro-fragments transcribe and secrete angiogenic factors in amounts comparable to MSCs derived from healthy individuals.

Paracrine Signaling from a Three-Dimensional Model of Bladder Carcinoma and from Normal Bladder Switch the Phenotype of Stromal Fibroblasts.

We present a three-dimensional model based on acellular scaffolds to recreate bladder carcinoma in vitro that closely describes the in vivo behavior of carcinoma cells. The integrity of the basement membrane and protein composition of the bladder scaffolds were examined by Laminin immunostaining and LC-MS/MS. Human primary bladder carcinoma cells were then grown on standard monolayer cultures and also seeded on the bladder scaffolds. Apparently, carcinoma cells adhered to the scaffold basement membrane and created a contiguous one-layer epithelium (engineered micro-carcinomas (EMCs)). Surprisingly, the gene expression pattern displayed by EMCs was similar to the profile expressed by the carcinoma cells cultured on plastic. However, the pattern of secreted growth factors was significantly different, as VEGF, FGF, and PIGF were secreted at higher levels by EMCs. We found that only the combination of factors secreted by EMCs, but not the carcinoma cells grown on plastic dishes, was able to induce either the pro-inflammatory phenotype or the myofibroblast phenotype depending on the concentration of the secreted factors. We found that the pro-inflammatory phenotype could be reversed. We propose a unique platform that allows one to decipher the paracrine signaling of bladder carcinoma and how this molecular signaling can switch the phenotypes of fibroblasts.

Creation of a vascular inducing device using mesenchymal stem cells to induce angiogenesis.

Conventional treatments of peripheral vascular disease and coronary artery disease have partial success but are still limited. Methods to deliver angiogenic factors into ischemic areas using gene, protein and cell therapies are faced with difficult issues such a delivery, effective concentration and duration of action. Tissue engineering offers the possibility of creating a functional self-contained three-dimensional (3D) unit that works as a coordinated biological pump that can secrete a whole range of angiogenic factors. We report a tissue engineering approach using decellularized micro-fragments and mesenchymal stem cells (MSCs) to create a vascular inducing device (VID). Proteomic analysis of the decellularized micro-fragments and of the VIDs reveals a large number of extracellular-matrix (ECM) proteins. Moreover, the VIDs were found to transcribe and secrete a whole repertoire of angiogenic factors in a sustained manner. Furthermore, preliminary results of implantation VIDs into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice indicate formation of vascular network at the site within a week. We propose that those VIDs could serve as a safe, localized, simple and powerful method for the treatment of certain types of vascular diseases.

An in vivo Like Micro-Carcinoma Model.

We here present a novel micro-system which allows to reconstitute an in vivo lung carcinoma where the various constituting epithelial and/or stromal structural and/or cellular components can be incorporated at will. In contrast to various "organs on a chip" the model is based on the observation that in nature, epithelial cells are always supported by a connective tissue or stroma. The model is based on acellular micro-scaffolds of microscopic dimensions which enable seeded cells to obtain gases and nutrients through diffusion thus avoiding the need for vascularization. As a proof of concept, we show that in this model, Calu-3 cells can form a well-organized, continuous, polarized, one-layer epithelium lining the stromal derived alveolar cavities, and express a different pattern of tumor-related genes than when grown as standard monolayer cultures on plastic culture dishes. To our knowledge, this model, introduces for the first time a system where the function of carcinogenic cells can be tested in vitro in an environment that closely mimics the natural in vivo situation.

Lung-Derived Microscaffolds Facilitate Diabetes Reversal after Mouse and Human Intraperitoneal Islet Transplantation.

There is a need to develop three-dimensional structures that mimic the natural islet tissue microenvironment. Endocrine micro-pancreata (EMPs) made up of acellular organ-derived micro-scaffolds seeded with human islets have been shown to express high levels of key beta-cell specific genes and secrete quantities of insulin per cell similar to freshly isolated human islets in a glucose-regulated manner for more than three months in vitro. The aim of this study was to investigate the capacity of EMPs to restore euglycemia in vivo after transplantation of mouse or human islets in chemically diabetic mice. We proposed that the organ-derived EMPs would restore the extracellular components of the islet microenvironment, generating favorable conditions for islet function and survival. EMPs seeded with 500 mouse islets were implanted intraperitoneally into streptozotocin-induced diabetic mice and reverted diabetes in 67% of mice compared to 13% of controls (p = 0.018, n = 9 per group). Histological analysis of the explanted grafts 60 days post-transplantation stained positive for insulin and exhibited increased vascular density in a collagen-rich background. EMPs were also seeded with human islets and transplanted into the peritoneal cavity of immune-deficient diabetic mice at 250 islet equivalents (IEQ), 500 IEQ and 1000 IEQ. Escalating islet dose increased rates of normoglycemia (50% of the 500 IEQ group and 75% of the 1000 IEQ group, n = 3 per group). Human c-peptide levels were detected 90 days post-transplantation in a dose-response relationship. Herein, we report reversal of diabetes in mice by intraperitoneal transplantation of human islet seeded on EMPs with a human islet dose as low as 500 IEQ.

Frog skin cultures secrete anti-yellow fever compounds.

There is an urgent need to develop novel antimicrobial substances. Antimicrobial peptides (AMPs) are considered as promising candidates for future therapeutic use. Because of the re-emergence of the Flavivirus infection, and particularly the yellow fever virus (YFV), we have compared the antiviral activities from skin secretions of seven different frog species against YFV (strain 17D). Secretions from Sphaenorhynchus lacteus, Cryptobatrachus boulongeri and Leptodactylus fuscus displayed the more powerful activities. S. lacteus was found to inhibit viral lysis of Vero E6 cells even at the highest viral concentration evaluated of 10 LD50. We also report the identification of a novel frenatin-related peptide from S. lacteus and found that this peptide-on its own-can lead to 35% protection against YVF, while displaying no cytotoxicity against somatic cells even at fivefold higher concentrations. These results are attractive and support the need for continued exploration of new sources of AMPs from frog skin secretions such as those described here in the development of new compounds for the treatment of infectious diseases in general and specific viral infections in particular.

Beta Cells Secrete Significant and Regulated Levels of Insulin for Long Periods when Seeded onto Acellular Micro-Scaffolds.

The aim of this work is to obtain significant and regulated insulin secretion from human beta cells ex vivo. Long-term culture of human pancreatic islets and attempts at expanding human islet cells normally result in loss of beta-cell phenotype. We propose that to obtain proper ex vivo beta cell function, there is a need to develop three-dimensional structures that mimic the natural islet tissue microenvironment. We here describe the preparation of endocrine micro-pancreata (EMPs) that are made up of acellular organ-derived micro-scaffolds seeded with human intact or enzymatically dissociated islets. We show that EMPs constructed by seeding whole islets, freshly enzymatically-dissociated islets or even dissociated islets grown first in standard monolayer cultures express high levels of key beta-cell specific genes and secrete quantities of insulin per cell similar to freshly isolated human islets in a glucose-regulated manner for more than 3 months in vitro.

Kidney-specific microscaffolds and kidney-derived serum-free conditioned media support in vitro expansion, differentiation, and organization of human embryonic stem cells.

We report a novel method for culturing human embryonic stem cells (HES) using 300-µm-thick acellular kidney-derived microscaffolds (KMSs) that allow cells to obtain nutrients and gasses simply by diffusion, enabling the KMSs to be used readily ex vivo under defined culture conditions, without the need for vascularization/perfusion or transplantation. Standard histology and scanning electron microscopy show that HES grow and expand according to the complex structure dictated by the scaffold. We further show that the expression levels of NPHS-1, REN, AQP-1, SLC2A2, and ANPEP were 7.6-, 5.1-, 128-, 4.3-, and 3.9-fold higher, respectively, when the HES were grown on KMS compared with the HES grown on collagen. Similarly, the levels of these genes were 10-, 30-, 4.6-, 7.5-, and 3-fold higher, respectively, when the HES were grown on KMS compared with the HES grown on Matrigel. We have also shown that culturing HES in 5% kidney-derived serum-free conditioned media can lead to the upregulation of NPHS-1, REN, and EPO by 3-, 18-, and 15-fold, respectively. This article demonstrates a novel way of growing HES in vitro whereby the beneficial biophysical as well as biochemical surroundings of the seeded cells provide a more in vivo-like environment, thereby assisting in differentiation of the HES toward a renal lineage. The approach presented here may provide a powerful tool for in vitro study of HES differentiation toward kidney-specific cell lineages under controlled conditions.

Kidney derived micro-scaffolds enable HK-2 cells to develop more in-vivo like properties.

Many cell lines, despite the fact that they are easy to culture, tend to lose some of their in vivo characteristics in vitro, we therefore decided to investigate whether culturing HK-2 cells on kidney derived micro-scaffolds (KMS) could improve proximal tubule functionality to these cells. Kidney derived micro-scaffolds (KMS) have been prepared that, due to the fact that they are only 300 µm in depth, allow for transfer of gases and nutrients via diffusion whilst maintaining the kidney's intricate microstructure. Culturing HK-2 on KMS shows significant increase in expression of AQP-1, ATP1B1, SLC23A1 and SLC5A2 after 1, 2 and 3 weeks compared with HK-2 grown under standard tissue culture conditions. Additionally, very high levels of expression of CCL-2 (15-30 fold increase) and LRP-2 (25-200 fold increase) were observed when the HK-2 were grown on KMS compared with HK-2 grown under standard tissue culture conditions. Furthermore, HK-2 cells grown under standard conditions released higher levels of Il-6 and Il-8 compared with primary tubule cells (Asterand AS-9-2) and secreted no MCP-1 or RANTES as opposed to primary cells that released MCP-1 and RANTES following stimulation. However, HK-2 grown on KMS showed both a marked decrease in Il-6/Il-8 secretion in line with the primary cells and secreted MCP-1 as well. These results show that the micro-environment of the KMS assists in restoring in vivo like properties to the HK-2 cells.

Genes related to differentiation are correlated with the gene regulatory network structure.

MOTIVATION: Many secondary messengers, receptors and transcription factors are related to cell differentiation. Their role in cell differentiation can be affected by their position in the gene regulatory network. Here, we test whether the properties of the gene regulatory network can highlight which genes and proteins are associated with cell differentiation. We use a previously developed purely theoretical algorithm built to detect nodes that can induce a state change in Boolean gene regulatory networks, and show that most genes predicted to participate in differentiation in the theoretical framework are also experimentally known to be associated with such differentiation. These results show that genes related to differentiation are associated with specific features of the genetic regulatory network. The proposed algorithm produces a better classification than simple network measures such as the nodes degree or centrality. Boolean networks were used in many previous theoretical models. Here, we show a direct application of such networks to the detection of genes and subnetworks related to differentiation. The subnetwork emerging from the genes and edges that are predicted to be associated with differentiation are the most active molecular pathways experimentally described to be involved in cell differentiation. AVAILABILITY AND IMPLEMENTATION: http://peptibase.cs.biu.ac.il/homepage/Boolean_network_conversion_code.zip.

Mathematical conditions for induced cell differentiation and trans-differentiation in adult cells.

We present a theoretical framework for the analysis of the effect of a fully differentiated cell population on a neighboring stem cell population in Multi-Cellular Organisms (MCOs). Such an organism is constituted by a set of different cell populations, each set of which converges to a different cycle from all possible options, of the same Boolean network. Cells communicate via a subset of the nodes called signals. We show that generic dynamic properties of cycles and nodes in random Boolean networks can induce cell differentiation.Specifically we propose algorithms, conditions and methods to examine if a set of signaling nodes enabling these conversions can be found. Surprisingly we find that robust conversions can be obtained even with a very small number of signals. The proposed conversions can occur in multiple spatial organizations and can be used as a model for regeneration in MCOs, where an islet of cells of one type (representing stem cells) is surrounded by cells of another type (representing differentiated cells). The cells at the outer layer of the islet function like progenitor cells (i.e. dividing asymmetrically and differentiating). To the best of our knowledge, this is the first work showing a tissue-like regeneration in MCO simulations based on random Boolean networks.We show that the probability to obtain a conversion decreases with the log of the node number in the network, showing that the model is relevant for large networks as well. We have further checked that the conversions are not trivial, i.e. conversions do not occur due to irregular structures of the Boolean network, and the converting cycle undergoes a respectable change in its behavior.Finally we show that the model can also be applied to a realistic genetic regulatory network, showing that the basic mathematical insight from regular networks holds in more complex experiment-based networks.

Skin micro-organs from several frog species secrete a repertoire of powerful antimicrobials in culture.

This work is an attempt to take advantage of the rich biodiversity that exists in Colombia in order to start a systematic analysis of antimicrobial substances that have emerged through amphibian evolution. For this purpose we have developed a technique to grow intact frog skin derived micro-organs (SMOs) in vitro in the absence of serum. We show that in SMOs, the skin glands remain intact and continue to secrete into the medium substances with potent antibacterial activity, for several days in culture. Our strategy has been to create a bank of substances secreted by amphibian skin from different species. This bank contains at present around 50 species and is of particular importance as some of the species are in danger of disappearing. We show that some of the species tested displayed very strong antibacterial activity without being toxic to somatic cell lines, even at 10-fold higher concentration.

Fertility preservation in girls.

Children that undergo treatment for cancer are at risk of suffering from subfertility or hormonal dysfunction due to the detrimental effects of radiotherapy and chemotherapeutic agents on the gonads. Cryopreservation of ovarian tissue prior to treatment offers the possibility of restoring gonadal function after resumption of therapy. Effective counseling and management of pediatric patients is crucial for preserving their future reproductive potential. The purpose of this article is to review recent literature and to revise recommendations we made in a 2007 article. Pediatric hemato-oncology, reproductive endocrinology, surgery, anesthesia and bioethics perspectives are discussed and integrated to propose guidelines for offering ovarian cryopreservation to premenarcheal girls with cancer.

Organ-specific scaffolds for in vitro expansion, differentiation, and organization of primary lung cells.

In light of the increasing need for differentiated primary cells for cell therapy and the rapid dedifferentiation occurring during standard in vitro cultivation techniques, there is an urgent need for developing three-dimensional in vitro systems in which expanded cells display in vivo-like differentiated phenotypes. It is becoming clear that the natural microenvironment provides the optimal conditions for achieving this aim. To this end, we prepared natural decellularized scaffolds of microscopic dimensions that would allow appropriate diffusion of gases and nutrients to all seeded cells. Scaffolds from either the lung or the liver were analyzed for their ability to support growth and differentiation of progenitor alveolar cells and hepatocytes. We observed that progenitor alveolar cells that have been expanded on plastic culture and thus dedifferentiated grew within the lung-derived scaffolds into highly organized structures and regained differentiation markers classical for type I and type II alveolar cells. The cells generated proper alveolar structures, and only 15%-30% of them secreted surfactant proteins in a localized manner for extended periods. Vice versa, liver-derived scaffolds supported the differentiation state of primary hepatocytes. We further demonstrate that the natural scaffolds are organ specific, that is, only cells derived from the same organ become properly differentiated. A proteomic analysis shows significant different composition of lung and liver scaffolds, for example, decorin, thrombospondin 1, vimentin, and various laminin isoforms are especially enriched in the lung. Altogether, our data demonstrate that complex interactions between the seeded cells and a highly organized, organ-specific stroma are required for proper localized cell differentiation. Thus, our novel in vitro culture system can be used for ex vivo differentiation and organization of expanded primary cells.

Micro-organ ovarian transplantation enables pregnancy: a case report.

A 19-year-old thalassemic woman had tissue from one of her ovaries cryopreserved prior to bone marrow transplantation, total body irradiation and sterilizing chemotherapy. As expected, premature ovarian failure resulted from this treatment. Transplantation of her thawed ovarian tissue resulted in return of menstrual cycling and the patient then underwent several IVF cycles. The patient, however, had poor ovarian response to hyperstimulation. We thus considered an alternative approach based on the observation that very thin ovarian fragments that preserve the basic ovarian structure [ovarian micro-organs (MOs)] induce angiogenesis and remained viable after autologous transplantation in animals. We report that preparation of autologous tiny ovarian fragments (MO)s and reimplantation into our patient resulted in IVF pregnancy and delivery of a healthy baby.

Biopump: Autologous skin-derived micro-organ genetically engineered to provide sustained continuous secretion of therapeutic proteins.

A novel approach for sustained production of therapeutic proteins is described, using genetic modification of intact autologous micro-organ tissue explants from the subject's own skin. The skin-derived micro-organ can be maintained viable ex vivo for extended periods and is transduced with a transgene encoding a desired therapeutic protein, resulting in protein-secreting micro-organ (biopump (BP)). The daily protein production from each BP is quantified, enabling drug dosing by subcutaneous implantation of the requisite number of BPs into the patient to provide continuous production to the circulation of a known amount of the therapeutic protein. Each implanted BP remains localized and is accessible, to enable removal or ablation if needed. Examples from preclinical and clinical studies are presented, including use of associated virus vector 1 and helper-dependent adenoviral vectors producing BPs to provide long-term sustained secretion of recombinant interferon-α and erythropoietin.

Skin-derived microorgan autotransplantation as a novel approach for therapeutic angiogenesis.

Despite promising preclinical results, transient single-factor-based therapeutic angiogenesis has shown no definitive benefits in clinical trials. The use of skin-derived microorgans (SMOs), capable of sustained expression of angiogenic factors and sustained viability with their cellular and extracellular elements, constitutes an attractive alternative. We sought to evaluate the efficacy of SMO implantation in a porcine model of chronic myocardial ischemia. Eighteen pigs underwent placement of an ameroid constrictor on the proximal circumflex artery. Three weeks later, split-thickness skin biopsies were harvested and pigs were randomized to lateral wall implantation of either 8 or 16 SMOs or blank injections. The procedure was safe and resulted in no adverse events. Three weeks after treatment, SMO implantation resulted in significant improvement of lateral wall perfusion during pacing, assessed by isotope-labeled microspheres [post- vs. pretreatment ratios of lateral/anterior wall blood flow were 1.31 +/- 0.09 (SMOs) and 1.04 +/- 0.06 (controls); P = 0.03]. No significant difference in angiographic scores was observed. Microvascular relaxation in response to VEGF was impaired in the ischemic territory of the control group but returned to normal after SMO implantation, indicating restoration of endothelial function. Molecular studies showed significant increases in VEGF and CD31 expression in the ischemic area of treated animals. Morphometric analysis showed increased neovascularization with SMO treatment. Autotransplantation of SMOs constitutes a novel approach for safe and effective therapeutic angiogenesis with improvement in perfusion, normalization of microvascular reactivity, and increased expression of VEGF and CD31.

Skin-derived micro-organs induce angiogenesis in rabbits.

We have recently reported an alternative cell therapy approach to induce angiogenesis. The approach is based on small organ fragments--micro-organs (MOs)--whose geometry allows preservation of the natural epithelial/mesenchymal interactions and ensures appropriate diffusion of nutrients and gases to all cells. We have shown that lung-derived MOs, when implanted into hosts, transcribe a wide spectrum array of angiogenic factors and can induce an angiogenic response that can rescue experimentally induced ischemic regions in mice. From a clinical perspective, skin-derived MOs are particularly appealing as they could readily be obtained from a skin biopsy taken from the same target patient. In the present work we have investigated the angiogenesis-inducing capacity of rabbit and human skin-derived micro-organs in vitro and in vivo. Rabbit skin MOs were implanted into homologous adult rabbits and human skin MOs were encapsulated and implanted into xenogenic mice. Skin-derived MOs, as lung-derived MOs, were found to secrete a whole array of angiogenic factors and to induce a powerful angiogenic response when implanted back into animals. We believe the approach presented suggests a novel, efficacious and simple approach for therapeutic angiogenesis.

Epithelial-mesenchymal interactions allow for epidermal cells to display an in vivo-like phenotype in vitro.

We here report that preservation of the basic epithelial-mesenchymal interactions allows for highly complex ex vivo function of epidermal cells. The approach taken is based on the preparation of organ fragments that preserve the basic epithelial/mesenchymal interactions but also ensure appropriate diffusion of nutrients and gases to all cells. Human and mice keratinocytes in such organ fragments, remain viable, proliferate and express epidermal-specific gene products when cultured in serum-free medium without added growth factors, for several weeks in vitro. When implanted into syngeneic animals they remain viable, become vascularized and continue to function and transcribe tissue-specific gene products for several months. Such fragments allow primary cells ex vivo to preserve most of the functional attributes of the in vivo system. Clearly, the effect of the extracellular matrix is critical in this system in order for the cells to proliferate and differentiate ex vivo. We are not aware of any other system which allows for localized expression of epidermal-specific genes ex vivo for significant periods in culture in defined serum-free medium.

A cell-based multifactorial approach to angiogenesis.

We here propose an alternative cell therapy approach to induce angiogenesis. We prepared small organ fragments whose geometry allows preservation of the natural epithelial/mesenchymal interactions and ensures appropriate diffusion of nutrients and gases to all cells. Fragments derived from lung are shown to behave as fairly independent units, to undergo a marked upregulation of angiogenic factors and to continue to function for several weeks in vitro in serum-free media. When implanted into hosts, they transcribe a similar array of angiogenic factors that specifically induce the formation of a potent vascular network. The angiogenic induction capacity of these fragments was also tested in a mouse and rat model of limb ischemia. We report that such fragments, when implanted in the vicinity of the ischaemic area, induce an angiogenic response which can rescue the ischaemia-induced damage. The approach presented differs from single factor application, gene therapy and other cell therapy methods in that it exploits the complex behaviour of autologous cells in their near to normal environment in order to achieve secretion of a whole range of angiogenic stimuli continuously and in an apparently coordinated fashion.