VEGF-delivering PEG hydrogels promote vascularization in the porcine subcutaneous space.

For cell therapies, the subcutaneous space is an attractive transplant site due to its large surface area and accessibility for implantation, monitoring, biopsy, and retrieval. However, its poor vascularization has catalyzed research to induce blood vessel formation within the site to enhance cell revascularization and survival. Most studies focus on the subcutaneous space of rodents, which does not recapitulate important anatomical features and vascularization responses of humans. Herein, we evaluate biomaterial-driven vascularization in the porcine subcutaneous space. Additionally, we report the first use of cost-effective fluorescent microspheres to quantify perfusion in the porcine subcutaneous space. We investigate the vascularization-inducing efficacy of vascular endothelial growth factor (VEGF)-delivering synthetic hydrogels based on 4-arm poly(ethylene) glycol macromers with terminal maleimides (PEG-4MAL). We compare three groups: a non-degradable hydrogel with a VEGF-releasing PEG-4MAL gel coating (Core+VEGF gel); an uncoated, non-degradable hydrogel (Core-only); and naïve tissue. After 2 weeks, Core+VEGF gel has significantly higher tissue perfusion, blood vessel area, blood vessel density, and number of vessels compared to both Core-only and naïve tissue. Furthermore, healthy vital signs during surgery and post-procedure metrics demonstrate the safety of hydrogel delivery. We demonstrate that VEGF-delivering synthetic hydrogels induce robust vascularization and perfusion in the porcine subcutaneous space.

Hydrolytic hydrogels tune mesenchymal stem cell persistence and immunomodulation for enhanced diabetic cutaneous wound healing.

Diabetes is associated with an altered global inflammatory state with impaired wound healing. Mesenchymal stem/stromal cells (MSC) are being explored for treatment of diabetic cutaneous wounds due to their regenerative properties. These cells are commonly delivered by injection, but the need to prolong the retention of MSC at sites of injury has spurred the development of biomaterial-based MSC delivery vehicles. However, controlling biomaterial degradation rates in vivo remains a therapeutic-limiting challenge. Here, we utilize hydrolytically degradable ester linkages to engineer synthetic hydrogels with tunable in vivo degradation kinetics for temporally controlled delivery of MSC. In vivo hydrogel degradation rate can be controlled by altering the ratio of ester to amide linkages in the hydrogel macromers. These hydrolytic hydrogels degrade at rates that enable unencumbered cutaneous wound healing, while enhancing the local persistence MSC compared to widely used protease-degradable hydrogels. Furthermore, hydrogel-based delivery of MSC modulates local immune responses and enhances cutaneous wound repair in diabetic mice. This study introduces a simple strategy for engineering tunable degradation modalities into synthetic biomaterials, overcoming a key barrier to their use as cell delivery vehicles.

Immunotherapy via PD-L1-presenting biomaterials leads to long-term islet graft survival.

Antibody-mediated immune checkpoint blockade is a transformative immunotherapy for cancer. These same mechanisms can be repurposed for the control of destructive alloreactive immune responses in the transplantation setting. Here, we implement a synthetic biomaterial platform for the local delivery of a chimeric streptavidin/programmed cell death-1 (SA-PD-L1) protein to direct "reprogramming" of local immune responses to transplanted pancreatic islets. Controlled presentation of SA-PD-L1 on the surface of poly(ethylene glycol) microgels improves local retention of the immunomodulatory agent over 3 weeks in vivo. Furthermore, local induction of allograft acceptance is achieved in a murine model of diabetes only when receiving the SA-PD-L1-presenting biomaterial in combination with a brief rapamycin treatment. Immune characterization revealed an increase in T regulatory and anergic cells after SA-PD-L1-microgel delivery, which was distinct from naïve and biomaterial alone microenvironments. Engineering the local microenvironment via biomaterial delivery of checkpoint proteins has the potential to advance cell-based therapies, avoiding the need for systemic chronic immunosuppression.

Hydrodynamic shear-based purification of cancer cells with enhanced tumorigenic potential.

Tumor-initiating cells (TICs), a subpopulation of cancerous cells with high tumorigenic potential and stem-cell-like properties, drive tumor progression and are resistant to conventional therapies. Identification and isolation of TICs are limited by their low frequency and lack of robust markers. Here, we characterize the heterogeneous adhesive properties of a panel of human and murine cancer cells and demonstrate differences in adhesion strength among cells, which exhibit TIC properties and those that do not. These differences in adhesion strength were exploited to rapidly (~10 min) and efficiently isolate cancerous cells with increased tumorigenic potential in a label-free manner by use of a microfluidic technology. Isolated murine and human cancer cells gave rise to larger tumors with increased growth rate and higher frequency in both immunocompetent and immunocompromised mice, respectively. This rapid and label-free TIC isolation technology has the potential to be a valuable tool for facilitating research into TIC biology and the development of more efficient diagnostics and cancer therapies.

Multiple clinically relevant immunotherapies prolong the function of microencapsulated porcine islet xenografts in diabetic NOD mice without the use of anti-CD154 mAb.

BACKGROUND: Our goal was to identify clinically relevant immunotherapies that synergize with microencapsulation to protect adult porcine islet (API) xenografts in diabetic NOD mice. We have shown previously that dual costimulatory blockade (CTLA4-Ig plus anti-CD154 mAb) combined with encapsulation protects APIs long-term in NOD mice. Since no anti-CD154 mAbs currently are approved for use in humans, we tested the efficacy of other targeted immunosuppression regimens that might be used for diabetic patients receiving encapsulated islets. METHODS: Microencapsulated APIs were transplanted i.p. in diabetic NOD mice given either no immunosuppression or combinations immunosuppressive reagents. Graft function was monitored by blood glucose levels, i.p. glucose tolerance tests, and histology. Mechanisms of rejection were investigated by phenotyping host peritoneal cells and measuring graft site cytokine and chemokine levels. RESULTS: New immunosuppressive therapies were compared to CTLA4-Ig plus anti-CD154 mAb, used here as a control. The most effective was triple treatment with CTLA4-Ig, anti-CD154 mAb, and intracapsular CXCL12, and the next most effective was a non-depleting anti-CD4 mAb (YTS177.9) plus intracapsular CXCL12. Three additional regimens (CTLA4-Ig plus YTS177.9, YTS177.9 alone, and anti-OX40-Ligand mAb alone) significantly prolonged encapsulated API function. Dual treatment with CTLA4-Ig plus anti-CD40 mAb was as effective as CTLA4-Ig plus anti-CD154 mAb. Five other monotherapies and three combination therapies did not augment encapsulated API survival. Most peritoneal cytokines and chemokines were either absent or minimal. At necropsy, the capsules were intact, not fibrosed, and clean when function was maintained, but were coated with host cells if rejection had occurred. CONCLUSIONS: Multiple different immunotherapies which specifically inhibit CD4+ T cells, modulate T-cell trafficking, or interfere with antigen presentation can substitute for anti-CD154 mAb to prolong encapsulated islet xenograft function in diabetic NOD mice.

Engineered insulin secretion from neuroendocrine cells isolated from human thyroid.

BACKGROUND: Insulin-secreting beta-like cells are vulnerable to diabetic autoimmunity. We hypothesized that human thyroid neuroendocrine (NE) cells could be engineered to secrete human insulin, be glucose-responsive, and avoid autoimmunity. METHODS: Collagenase-digested thyroid tissue was cultured and subjected to size-based fluorescence-activated cell sorting. Insulin secretion and storage in NE cells transduced with viral vectors carrying an insulin sequence was assessed by enzyme-linked immunosorbent assay (ELISA) and immunogold transmission electron microscopy (TEM). Baseline mRNA expression was assessed by Illumina expression array analysis. Transduction with retrovirus expressing transcription factors PDX1, NGN3, MAFA, or HNF6 altered mRNA expression in a custom polymerase chain reaction (PCR) array. Gastrin-releasing peptide (GRP) in conditioned medium and cell lysates was determined by reverse transcription (RT)-PCR, ELISA, and immunohistochemistry. RESULTS: Isolation yielded an average of 2.2 × 10(6) cells/g thyroid tissue, which stained for calcitonin/calcitonin gene-related protein, expressed genes consistent with NE origins, and secreted GRP. Transduced cells secreted 56 % and retained 48 % of total insulin produced. Immunogold TEM revealed insulin in secretory vesicles. PDX1, NGN3, and MAFA overexpression increased expression of genes typical for hepatocytes and beta cells. Overexpression of HNF6 also increased the message of genes critical for glucose sensing. CONCLUSIONS: Human thyroid NE cells can produce human insulin, fractions of which are both secreted and retained in secretory granules. Overexpression of HNF6, PDX1, or NGN3 enhances expression of both hepatocyte and beta cell typical mRNAs, including the message of proteins critical for glucose sensing. These data suggest that reimplantation of engineered autologous NE cells may develop as a viable treatment for diabetes mellitus type 1.