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.

Cardiomyocyte enrichment from human embryonic stem cell cultures by selection of ALCAM surface expression.

AIMS: The production of a homogenous population of human cardiomyocytes that can be expanded in vitro may facilitate development of replacement tissue lost as a result of cardiac disease and injury. MATERIALS AND METHODS: We evaluated the utility of activated leukocyte cell-adhesion molecule, CD166 (ALCAM) expression as a marker for isolating cardiomyocytes from differentiating cultures of human embryonic stem cells (hESCs). Using RT-qPCR, immunohistochemistry and DNA methylation studies, we evaluated the developmental age of hESC-derived cardiomyocytes. RESULTS AND CONCLUSIONS: We demonstrate that cardiomyocytes derived from hESC cultures express ALCAM and that this surface antigen can be used to select a population of differentiated cells that are enriched for cardiomyocytes. Expression of contractile proteins and ion channels, and DNA methylation patterns, suggest that ALCAM-enriched cardiomyocytes have an embryonic phenotype. Selected cardiomyocyte populations survive sorting, adhere to collagen-coated tissue culture plastic and proliferate in short-term culture. Long-term in vitro survival of cardiomyocytes was achieved by culturing cells in 3D aggregates.

Attachment and growth of human embryonic stem cells on microcarriers.

The use of human embryonic stem cells (hESCs) for cell-based therapies will require large quantities of genetically stable pluripotent cells and their differentiated progeny. Traditional hESC propagation entails adherent culture and is sensitive to enzymatic dissociation. These constraints hamper modifying method from 2-dimensional flat-bed culture, which is expensive and impractical for bulk cell production. Large-scale culture for clinical use will require innovations such as suspension culture for bioprocessing. Here we describe the attachment and growth kinetics of both murine embryonic stem cells (mESCs) and hESCs on trimethyl ammonium-coated polystyrene microcarriers for feeder-free, 3-dimensional suspension culture. mESCs adhered and expanded according to standard growth kinetics. For hESC studies, we tested aggregate (collagenase-dissociated) and single-cell (TrypLE-dissociated) culture. Cells attached rapidly to beads followed by proliferation. Single-cell cultures expanded 3-fold over approximately 5 days, slightly exceeding that of hESC aggregates. Importantly, single-cell cultures were maintained through 6 passages with a 14-fold increase in cell number while still expressing the undifferentiated markers Oct-4 and Tra 1-81. Finally, hESCs retained their capacity to differentiate towards pancreatic, neuronal, and cardiomyocyte lineages. Our studies provide proof-of-principle of suspension-based expansion of hESCs on microcarriers, as a novel, economical and practical feeder-free means of bulk hESC production.

Efficient expansion of clinical-grade human fibroblasts on microcarriers: cells suitable for ex vivo expansion of clinical-grade hESCs.

Human embryonic stem cells hold considerable potential for cell-based treatments of a variety of degenerative diseases, including diabetes, ischemic heart failure, and Parkinson's disease. However, advancing research to provide clinical-grade product requires scale-up to therapeutic quantities of stem cells and their differentiated progeny. Most human embryonic stem cell culture platforms require direct support by a fibroblast feeder layer or indirect support using fibroblast conditioned medium. Accordingly, large numbers of clinically compliant fibroblasts will be requisite for stem cell production. Published platforms for feeder production are insufficient for stem cell scale-up, being costly to operate and requiring considerable effort to prepare, maintain and harvest. Here we describe the expansion of cGMP-grade, FDA-approved human foreskin fibroblasts using cGMP-grade reagents and polystyrene-based cationic trimethyl ammonium-coated microcarriers in spinner flasks. Fibroblasts attach rapidly to the microcarriers (T(1/2)=75 min), and expand with a maximum doubling time of 22.5h. Importantly, microcarrier-expanded fibroblasts and their conditioned medium support pluripotent stem cell growth through >5 passages, enabling extended self-renewal and expansion while retaining full differentiation potential. In summary, the method described is an economical and cGMP-compliant means of producing human fibroblast cells in support of cGMP human embryonic stem cell culture.

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.