Cryopreservation of Stem Cell-Derived ß-Like Cells Enriches for Insulin-Producing Cells With Improved Function.

The generation of stem cell-derived ß-like cells (sBCs) holds promise as not only an abundant insulin-producing cell source for replacement therapy of type 1 diabetes (T1D) but also as an invaluable model system for investigating human ß-cell development, immunogenicity, and function. Several groups have developed methodology to direct differentiate human pluripotent stem cells into pancreatic cell populations that include glucose-responsive sBCs. Nevertheless, the process of generating sBCs poses substantial experimental challenges. It involves lengthy differentiation periods, there is substantial variability in efficiency, and there are inconsistencies in obtaining functional sBCs. Here, we describe a simple and effective cryopreservation approach for sBC cultures that yields homogeneous sBC clusters that are enriched for insulin-expressing cells while simultaneously depleting proliferative progenitors. Thawed sBCs have enhanced glucose-stimulated insulin release compared with controls in vitro and can effectively engraft and function in vivo. Collectively, this approach alleviates current challenges with inefficient and variable sBC generation while improving their functional state. We anticipate that these findings can inform ongoing clinical application of sBCs for the treatment of patients with T1D and serve as an important resource for the wider diabetes field that will allow for accelerated research discoveries.

Peptide-Coated Polycaprolactone-Benzalkonium Chloride Nanocapsules for Targeted Drug Delivery to the Pancreatic ß-Cell.

Targeting current therapies to treat or prevent the loss of pancreatic islet ß-cells in Type 1 Diabetes (T1D) may provide improved efficacy and reduce off-target effects. Current efforts to target the ß-cell are limited by a lack of ß-cell-specific targets and the inability to test multiple targeting moieties with the same delivery vehicle. Here, we fabricate a tailorable polycaprolactone nanocapsule (NC) in which multiple different targeting peptides can be interchangeably attached for ß-cell-specific delivery. Incorporation of a cationic surfactant in the NC shell allows for the attachment of Exendin-4 and an antibody for ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) for ß-cell-specific targeting. The average NC size ranges from 250 to 300 nm with a polydispersity index under 0.2. The NCs are nontoxic, stable in media culture, and can be lyophilized and reconstituted. NCs coated with a targeting peptide were taken up by human cadaveric islet ß-cells and human stem cell-derived ß-like cells (sBC) in vitro with a high level of specificity. Furthermore, NCs successfully delivered both hydrophobic and hydrophilic cargo to human ß-cells. Additionally, Exendin-4-coated NCs were stable and targeted the mouse pancreatic islet ß-cell in vivo. Overall, our tailorable NCs have the potential to improve cell-targeted drug delivery and can be utilized as a screening platform to test the efficacy of cell-targeting peptides.

Peptide Coated Polycaprolactone-Benzalkonium Chloride Nanocapsules for Targeted Drug Delivery to the Pancreatic ß-Cell.

Targeting of current therapies to treat or prevent loss of pancreatic islet ß-cells in Type 1 Diabetes (T1D) may provide improved efficacy and reduce off target effects. Current efforts to target the ß-cell are limited by a lack of ß-cell specific targets and the inability to test multiple targeting moieties with the same delivery vehicle. Here we fabricate a novel tailorable polycaprolactone nanocapsule (NC) where multiple different targeting peptides can be interchangeably attached for ß-cell specific delivery. Incorporation of a cationic surfactant in the NC shell allows for the attachment of Exendin-4 and an antibody for ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) for ß-cell specific targeting. The average NC size ranges from 250-300nm with a polydispersity index under 0.2. The NCs are non-toxic, stable in media culture, and can be lyophilized and reconstituted. NCs coated with targeting peptide were taken up by human cadaveric islet ß-cells and human stem cell-derived ß-like cells (sBC) in vitro with a high level of specificity. Furthermore, NCs successfully delivered both hydrophobic and hydrophilic cargo to human ß-cells. Finally, Exendin-4 coated NCs were stable and targeted the mouse pancreatic islet ß-cell in vivo . Our unique NC design allows for the interchangeable coating of targeting peptides for future screening of targets with improved cell specificity. The ability to target and deliver thera-peutics to human pancreatic ß-cells opens avenues for improved therapies and treatments to help the delay onset, prevent, or reverse T1D.

Introducing Wound Healing Assays in the Undergraduate Biology Laboratory Using Ibidi Plates.

The wound healing assay is a simple and inexpensive method that allows researchers to experimentally mimic cell growth and migration leading to wound healing. In this assay, a wound is created on a monolayer of cultured mammalian cells and cell migration is monitored. Micrographs are captured at regular intervals during the duration of the experiment. These microscopy images are analyzed to compare cell migration and wound closure under different conditions. Introduction of different cytotoxic treatments into a wound healing assay can provide information as to whether a particular drug or compound of interest has the ability to affect cell migration. This type of analysis is important when assessing the ability of a particular cancer cell line to display invasive and metastatic behaviors. One of the challenges of this assay is to create the original wound in a way that is consistent across plates or treatments, facilitating comparisons across experimental groups. This is a particular challenge when using the wound healing assay in the context of an undergraduate biology class to expose students to a distinct form of mammalian cell culture and help them apply scientific knowledge and research skills. We found an easy way to overcome this obstacle by using ibidi plates. In this article, we provide a simple protocol to use ibidi plates and HeLa cells to set up wound healing assays. This laboratory exercise allows undergraduate students to utilize different skills developed through cell culture experience, such as growing, treating, and imaging mammalian cells.

Using The Cancer Genome Atlas as an Inquiry Tool in the Undergraduate Classroom.

Undergraduate students in the biomedical sciences are often interested in future health-focused careers. This presents opportunities for instructors in genetics, molecular biology, and cancer biology to capture their attention using lab experiences built around clinically relevant data. As biomedical science in general becomes increasingly dependent on high-throughput data, well-established scientific databases such as The Cancer Genome Atlas (TCGA) have become publicly available tools for medically relevant inquiry. The best feature of this database is that it bridges the molecular features of cancer to human clinical outcomes-allowing students to see a direct connection between the molecular sciences and their future professions. We have developed and tested a learning module that leverages the power of TCGA datasets to engage students to use the data to generate and test hypotheses and to apply statistical tests to evaluate significance.