Correction: Generation and selection of pluripotent stem cells for robust differentiation to insulin-secreting cells capable of reversing diabetes in rodents.

[This corrects the article DOI: 10.1371/journal.pone.0203126.].

Concise Review: Lessons Learned from Islet Transplant Clinical Trials in Developing Stem Cell Therapies for Type 1 Diabetes.

We examined data and patterns in clinical islet transplant studies registered on ClinicalTrials.gov (CTgov) for treatment of type 1 diabetes (T1D), with a goal of extracting insights to apply in the design of a pluripotent stem cell-derived islet therapy. Clinical islet transplantation, as a cell therapy (rather than solid organ transplant) is a unique precedent for stem cell-based islet therapies. Registration activity shows that the field is not growing significantly, and newer registrations suggest that the reasons for stagnation include need for a more optimal site of infusion/transplantation, and especially a need for better immune protective strategies to advance a more effective and durable therapy for T1D. Stem Cells Translational Medicine 2019;8:209&214.

Generation and selection of pluripotent stem cells for robust differentiation to insulin-secreting cells capable of reversing diabetes in rodents.

Induced pluripotent stem cell (iPSC) technology enables the creation and selection of pluripotent cells with specific genetic traits. This report describes a pluripotent cell line created specifically to form replacement pancreatic cells as a therapy for insulin-dependent diabetes. Beginning with primary pancreatic tissue acquired through organ donation, cells were isolated, re-programmed using non-integrating vectors and exposed to a four day differentiation protocol to generate definitive endoderm, a developmental precursor to pancreas. The best performing iPSC lines were then subjected to a 12-day basic differentiation protocol to generate endocrine pancreas precursors. The line that most consistently generated highly pure populations was selected for further development. This approach created an iPSC-variant cell line, SR1423, with a genetic profile correlated with preferential differentiation toward endodermal lineage at the loss of mesodermal potential. This report further describes an improved differentiation protocol that, coupled with SR1423, generated populations of greater than 60% insulin-expressing cells that secrete insulin in response to glucose and are capable of reversing diabetes in rodents. Created and banked following cGMP guidelines, SR1423 is a candidate cell line for the production of insulin-producing cells useful for the treatment of diabetes.

Directed differentiation of human embryonic stem cells into the pancreatic endocrine lineage.

Human embryonic stem (hES) cells represent a potentially unlimited source of transplantable beta-cells for the treatment of diabetes. Here we describe a differentiation strategy that reproducibly directs HES3, an National Institutes of Health (NIH)-registered hES cell line, into cells of the pancreatic endocrine lineage. HES3 cells are removed from their feeder layer and cultured as embryoid bodies in a three-dimensional matrix in the presence of Activin A and Bmp4 to induce definitive endoderm. Next, growth factors known to promote the proliferation and differentiation of pancreatic ductal epithelial cells to glucose-sensing, insulin-secreting beta-cells are added. Pdx1 expression, which identifies pancreatic progenitors, is detected as early as day 12 of differentiation. By day 34, Pdx1+ cells comprise between 5% and 20% of the total cell population and Insulin gene expression is up-regulated, with release of C-peptide into the culture medium. Unlike another recent report of the induction of insulin+ cells in differentiated hES cell populations, we are unable to detect the expression of other pancreatic hormones in insulin+ cells. When transplanted into severe combined immunodeficiency (SCID) mice, differentiated cell populations retain their endocrine identity and synthesize insulin.

Three-dimensional extracellular matrix stimulates gastrulation-like events in human embryoid bodies.

Human embryonic stem (hES) cells cultured in suspension form aggregates called embryoid bodies (EBs), which structurally resemble the pregastrulation-stage embryo. We show that these EBs express genes characteristic of the visceral endoderm (VE) and form an outer boundary of VE-like cells, but only a minority expresses markers of the primitive streak and the definitive endoderm in patterns suggestive of gastrulation-like events. EBs embedded in a Matrigel matrix, however, lack the presumptive VE but up-regulate expression of gastrulation-related genes, which is correlated with the expression of these genes in a greater proportion of individual EBs. Over time, Matrigelembedded EBs form internally organized structures and exhibit higher expression of markers of the definitive endoderm and mesoderm. We hypothesize that a three-dimensional adhesive support provides an organizing influence analogous to the early basal lamina surrounding the epiblast of the pregastrulation embryo. Furthermore, we identify a synthetic three-dimensional scaffold capable of supporting EB formation that partially mimics the effects of Matrigel.

The Promise of Integrins as Effective Targets for Anticancer Agents.

This review will briefly describe integrin function, address why integrins are attractive targets for chemotherapeutic drug design, and discuss some ongoing studies aimed at inhibiting integrin activity. Integrins are cell surface heterodimeric receptors. They modulate many cellular processes including: growth, death (apoptosis), adhesion, migration, and invasion by activating several signaling pathways. Many potential chemotherapeutic agents target integrins directly (eg, polypeptides, monoclonal antibodies, adenovirus vectors). These agents may be clinically useful in controlling the metastatic spread of cancer.

Perillyl Alcohol Inhibits Breast Cell Migration without Affecting Cell Adhesion.

The monoterpene d-limonene exhibits chemotherapeutic and chemopreventive potential in breast cancer patients. D-limonene and its related compounds, perillyl alcohol and perillyl aldehyde, were chosen as candidate drugs for application in a screen for nontoxic inhibitors of cell migration. Using the nontumorigenic human breast cell line MCF-10A, we delineated the toxicity as greatest for the perillyl aldehyde, intermediate for perillyl alcohol, and least for limonene. A noncytotoxic concentration of 0.5 mmol/L perillyl alcohol inhibited the migration, while the same concentration of limonene failed to do so. Adhesion of the MCF-10A cell line and the human breast cancer cell line MDA-MB 435 to fibronectin was unaffected by 1.5 mmol/L perillyl alcohol. 0.4 mmol/L perillyl alcohol inhibited the growth of MDA-MB 435 cells. All migration-inhibiting concentrations of perillyl alcohol for MDA-MB 435 cells proved to be toxic. These results suggest that subtoxic doses of perillyl alcohol may have prophylactic potential in the treatment of breast cancer.