Synthetic pre-metastatic niches for detection and analysis of early metastatic cells

Systems and strategies for promoting tissue growth provide enabling technologies for either enhancing regeneration for diseased or injured tissues, or to investigate abnormal tissue formation such as cancer. Breast cancer is known to metastasize to specific sites (e.g., lung, liver), and we have developed implants that mimic key aspects of the pre-metastatic niche, with the objective of capturing metastatic cells for the early detection of metastatic disease. These implants reduce the burden of disease in solid organs and provide a survival advantage when combined with early interventions. Additionally, we have applied systems biology approaches to analyze cancer cell and immune cell phenotypes at primary and metastatic sites, and as a tool to investigate the development of resistance to therapies that are unfortunately all too common for many patients. The analysis of a metastatic site complements existing approaches based on a liquid biopsy, which provides distinct information about disease progression and may ultimately enable a molecular staging of metastatic diseas

Incorporation of Polyethylene Glycol into Self-Assembled Monolayers Enhances Substrate-Mediated Gene Delivery by Nonspecifically- Bound Complexes

Developing systems capable of controlled and efficient gene transfer is a fundamental goal of biotechnology, with applications including functional genomics, gene therapy, and tissue engineering. Substrate-mediated delivery, also termed solid phase delivery, describes the immobilization of DNA, complexed with nonviral vectors, to a biomaterial or substrate through specific or nonspecific interactions. Cells cultured on the substrate are exposed to elevated DNA concentrations within the local microenvironment, which enhances transfection. We investigated transfection resulting from DNA complexes immobilized to a substrate through specific interactions introduced through complementary functional groups on the vector and surface or through nonspecific interactions. Self-assembled monolayers (SAMs) of alkanethiols on gold were used to provide a controlled surface to investigate transfection following specific and non-specific immobilization. DNA, complexed with polyethylenimine (PEI), was immobilized to SAMs through nonspecific mechanisms or covalently linked to SAMs presenting appropriate functional groups through a fraction of the functional groups available on the PEI present in the complex. Nonspecific immobilization of DNA complexes and subsequent transfection was mediated by the hydrophilicity and ionization of the substrate, while covalent tethering resulted in immobilized quantities similar to nonspecific conditions, but provided no transfection. Subsequent studies incorporated polyethylene glycol (PEG)-terminated alkanethiols into the SAMs to reduce nonspecific complex adsorption. Covalent tethering of complexes to PEG/carboxylic acid monolayers resulted in statistically less complex immobilization and no transfection. Nonspecific immobilization to monolayers containing 40% PEG resulted in statistically less DNA complexes immobilized, but substantially greater transfection. Cell adhesion was not affected at this percentage of PEG. Similarly, the addition of Pluronic block copolymers (of polyethylene oxide and polypropylene oxide) to surfaces also enhanced transfection. We hypothesize that the presence of PEG in the monolayer may better preserve complex conformation upon binding to substrates, thereby enhancing the activity of substrate-mediated delivery of DNA complexes

Controlled Release Systems for DNA Delivery

Adapting controlled release technologies to the delivery of DNA has the potential to overcome extracellular barriers that limit gene therapy. Controlled release systems can enhance gene delivery and increase the extent and duration of transgene expression relative to more traditional delivery methods (e.g., injection). These systems typically deliver vectors locally, which can avoid distribution to distant tissues, decrease toxicity to nontarget cells, and reduce the immune response to the vector. Delivery vehicles for controlled release are fabricated from natural and synthetic polymers, which function either by releasing the vector into the local tissue environment or by maintaining the vector at the polymer surface. Vector release or binding is regulated by the effective affinity of the vector for the polymer, which depends upon the strength of molecular interactions. These interactions occur through nonspecific binding based on vector and polymer composition or through the incorporation of complementary binding sites (e.g., biotin–avidin). This review examines the delivery of nonviral and viral vectors from natural and synthetic polymers and presents opportunities for continuing developments to increase their applicability

Mold‐casted non‐degradable, islet macro‐encapsulating hydrogel devices for restoration of normoglycemia in diabetic mice

Islet transplantation is a potential cure for diabetic patients, however this procedure is not widely adopted due to the high rate of graft failure. Islet encapsulation within hydrogels is employed to provide a three‐dimensional microenvironment conducive to survival of transplanted islets to extend graft function. Herein, we present a novel macroencapsulation device, composed of PEG hydrogel, that combines encapsulation with lithography techniques to generate polydimethylsiloxane (PDMS) molds. PEG solutions are mixed with islets, which are then cast into PDMS molds for subsequent crosslinking. The molds can also be employed to provide complex architectures, such as microchannels that may allow vascular ingrowth through pre‐defined regions of the hydrogel. PDMS molds allowed for the formation of stable gels with encapsulation of islets, and in complex architectures. Hydrogel devices with a thickness of 600 μm containing 500 islets promoted normoglycemia within 12 days following transplantation into the epididymal fat pad, which was sustained over the two‐month period of study until removal of the device. The inclusion of microchannels, which had a similar minimum distance between islets and the hydrogel surface, similarly promoted normoglycemia. A glucose challenge test indicated hydrogel devices achieved normoglycemia 90 min post‐dextrose injections, similar to control mice with native pancreata. Histochemical staining revealed that transplanted islets, identified as insulin positive, were viable and isolated from host tissue at 8 weeks post‐transplantation, yet devices with microchannels had tissue and vascular ingrowth within the channels. Taken together, these results demonstrate a system for creating non‐degradable hydrogels with complex geometries for encapsulating islets capable of restoring normoglycemia, which may expand islet transplantation as a treatment option for diabetic patients. Biotechnol. Bioeng. 2016;113: 2485–2495. © 2016 Wiley Periodicals, Inc.Macroencapsulating PEG hydrogel devices were created without microchannels (A) or cast in PDMS molds with column‐like features (B) to create hydrogels with microchannels (C, D). Islets were successfully encapsulated in these hydrogel devices (E, F), remained viable post‐encapsulation, and transplanted into the fat pad to restore normoglycemia in diabetic mice.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134144/1/bit26005_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134144/2/bit26005.pd

Spinal Progenitor-Laden Bridges Support Earlier Axon Regeneration Following Spinal Cord Injury.

Impact statementSpinal cord injury (SCI) results in loss of tissue innervation below the injury. Spinal progenitors have a greater ability to repair the damage and can be injected into the injury, but their regenerative potential is hampered by their poor survival after transplantation. Biomaterials can create a cell delivery platform and generate a more hospitable microenvironment for the progenitors within the injury. In this work, polymeric bridges are used to deliver embryonic spinal progenitors to the injury, resulting in increased progenitor survival and subsequent regeneration and functional recovery, thus demonstrating the importance of combined therapeutic approaches for SCI

Cellular Arrays (US Patent Application)

The present invention relates to characterizing transcription within cells. In particular, the present invention provides transfected cell arrays (e.g., two-dimensional and/or three-dimensional arrays) and systems, kits and methods utilizing the same (e.g., for transcriptional activity characterization). Compositions and methods of the present invention find use in, among other things, research, drug discovery and clinical (e.g., diagnostic, preventative and therapeutic) applications

Polycistronic Delivery of IL-10 and NT-3 Promotes Oligodendrocyte Myelination and Functional Recovery in a Mouse Spinal Cord Injury Model.

One million estimated cases of spinal cord injury (SCI) have been reported in the United States and repairing an injury has constituted a difficult clinical challenge. The complex, dynamic, inhibitory microenvironment postinjury, which is characterized by proinflammatory signaling from invading leukocytes and lack of sufficient factors that promote axonal survival and elongation, limits regeneration. Herein, we investigated the delivery of polycistronic vectors, which have the potential to coexpress factors that target distinct barriers to regeneration, from a multiple channel poly(lactide-co-glycolide) (PLG) bridge to enhance spinal cord regeneration. In this study, we investigated polycistronic delivery of IL-10 that targets proinflammatory signaling, and NT-3 that targets axonal survival and elongation. A significant increase was observed in the density of regenerative macrophages for IL-10+NT-3 condition relative to conditions without IL-10. Furthermore, combined delivery of IL-10+NT-3 produced a significant increase of axonal density and notably myelinated axons compared with all other conditions. A significant increase in functional recovery was observed for IL-10+NT-3 delivery at 12 weeks postinjury that was positively correlated to oligodendrocyte myelinated axon density, suggesting oligodendrocyte-mediated myelination as an important target to improve functional recovery. These results further support the use of multiple channel PLG bridges as a growth supportive substrate and platform to deliver bioactive agents to modulate the SCI microenvironment and promote regeneration and functional recovery. Impact statement Spinal cord injury (SCI) results in a complex microenvironment that contains multiple barriers to regeneration and functional recovery. Multiple factors are necessary to address these barriers to regeneration, and polycistronic lentiviral gene therapy represents a strategy to locally express multiple factors simultaneously. A bicistronic vector encoding IL-10 and NT-3 was delivered from a poly(lactide-co-glycolide) bridge, which provides structural support that guides regeneration, resulting in increased axonal growth, myelination, and subsequent functional recovery. These results demonstrate the opportunity of targeting multiple barriers to SCI regeneration for additive effects

PLG Bridge Implantation in Chronic SCI Promotes Axonal Elongation and Myelination.

Spinal cord injury (SCI) is a devastating condition that may cause permanent functional loss below the level of injury, including paralysis and loss of bladder, bowel, and sexual function. Patients are rarely treated immediately, and this delay is associated with tissue loss and scar formation that can make regeneration at chronic time points more challenging. Herein, we investigated regeneration using a poly(lactide-co-glycolide) multichannel bridge implanted into a chronic SCI following surgical resection of necrotic tissue. We characterized the dynamic injury response and noted that scar formation decreased at 4 and 8 weeks postinjury (wpi), yet macrophage infiltration increased between 4 and 8 wpi. Subsequently, the scar tissue was resected and bridges were implanted at 4 and 8 wpi. We observed robust axon growth into the bridge and remyelination at 6 months after initial injury. Axon densities were increased for 8 week bridge implantation relative to 4 week bridge implantation, whereas greater myelination, particularly by Schwann cells, was observed with 4 week bridge implantation. The process of bridge implantation did not significantly decrease the postinjury function. Collectively, this chronic model follows the pathophysiology of human SCI, and bridge implantation allows for clear demarcation of the regenerated tissue. These data demonstrate that bridge implantation into chronic SCI supports regeneration and provides a platform to investigate strategies to buttress and expand regeneration of neural tissue at chronic time points

Fibrin-Mediated Delivery of an Ovarian Follicle Pool in a Mouse Model of Infertility

The cryopreservation and autotransplantation of ovarian tissue is emerging as a powerful approach for preserving fertility. However, for cancer patients, it may not be possible to transplant ovarian tissue due to the risk of re-seeding disease. We investigated strategies for transplantation of individually isolated follicles to minimize the risk of re-introducing cancer cells present within the vasculature of ovarian stroma. Procedures for large-scale isolation of early-stage follicles and their encapsulation into fibrin hydrogels were developed. For in vivo validation studies, mice were ovariectomized and transplanted with encapsulated follicles into the ovarian bursa. A substantial increase in the number of secondary follicles was observed in the graft at 9 days after transplantation, and antral follicles by day 21, demonstrating primordial follicle recruitment into the growing pool. Initially, elevated follicle-stimulating hormone levels declined substantially by day 21, indicating feedback from the graft; presence of corpora lutea showed the graft's capability of restoring hormone cyclicity. Taken together, the transplanted follicles were able to engraft, mature, and restore ovarian function in an infertile mouse. This biomaterial may, thus, provide a platform for follicle transplantation with a low risk of cancer contamination and for developing strategies that preserve fertility for women facing a cancer diagnosis.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140230/1/ten.tea.2013.0675.pd