Modified hyaluronan hydrogels support the maintenance of mouse embryonic stem cells and human induced pluripotent stem cells.

These studies provide evidence for the ability of a commercially available, defined, hyaluronan-gelatin hydrogel, HyStem-C™, to maintain both mouse embryonic stem cells (mESCs) and human induced pluripotent stem cells (hiPSCs) in culture while retaining their growth and pluripotent characteristics. Growth curve and doubling time analysis show that mESCs and hiPSCs grow at similar rates on HyStem-C™ hydrogels and mouse embryonic fibroblasts and Matrigel™, respectively. Immunocytochemistry, flow cytometry, gene expression and karyotyping reveal that both human and murine pluripotent cells retain a high level of pluripotency on the hydrogels after multiple passages. The addition of fibronectin to HyStem-C™ enabled the attachment of hiPSCs in a xeno-free, fully defined medium.

The stem cell laboratory: design, equipment, and oversight.

This chapter describes some of the major issues to be considered when setting up a laboratory for the culture of human pluripotent stem cells (hPSCs). The process of establishing a hPSC laboratory can be divided into two equally important parts. One is completely administrative and includes developing protocols, seeking approval, and establishing reporting processes and documentation. The other part of establishing a hPSC laboratory involves the physical plant and includes design, equipment and personnel. Proper planning of laboratory operations and proper design of the physical layout of the stem cell laboratory so that meets the scope of planned operations is a major undertaking, but the time spent upfront will pay long-term returns in operational efficiency and effectiveness. A well-planned, organized, and properly equipped laboratory supports research activities by increasing efficiency and reducing lost time and wasted resources.

The teratoma assay: an in vivo assessment of pluripotency.

A teratoma is a nonmalignant tumor comprised of a disorganized mixture of cells and small foci of tissue comprised of cells from all three of the embryonic germ-layers. By definition, a cell is pluripotent if it can differentiate into cells derived from all three of the embryonic germ-layers: ectoderm, mesoderm, and endoderm. In the teratoma assay, putative pluripotent stem cells (PSCs) are implanted into an immune-compromised mouse where they may proliferate and differentiate to form a teratoma. The PSCs grow at the implantation site supported by a complex mixture of factors from the local milieu, as well as circulating factors that are vital components of normal mammalian physiology. After a predetermined time of 6-12 weeks or when the tumor has reached sufficient size, it is removed and subjected to histopathological analysis. The teratoma may be further processed by immunocytochemistry and gene expression profiling. This chapter describes methods to generate teratomas through the implantation of putative PSC lines in the SCID mouse. Implantation at the following sites is described: (1) intramuscular, (2) subcutaneous, (3) under the testis capsule, and (4) under the kidney capsule.

Cdc37 regulates Ryk signaling by stabilizing the cleaved Ryk intracellular domain.

Ryk is a Wnt receptor that plays an important role in neurogenesis, neurite outgrowth, and axon guidance. We have reported that the Ryk receptor is cleaved by gamma-secretase and that its intracellular domain (ICD) translocates to the nucleus upon Wnt stimulation. Cleavage of Ryk and its ICD is important for the function of Ryk in neurogenesis. However, the question of how the Ryk ICD is stabilized and translocated into the nucleus remains unanswered. Here, we show that the Ryk ICD undergoes ubiquitination and proteasomal degradation. We have identified Cdc37, a subunit of the molecular chaperone Hsp90 complex, as a Ryk ICD-interacting protein that inhibits proteasomal degradation of the Ryk ICD. Overexpression of Cdc37 increases Ryk ICD levels and promotes its nuclear localization, whereas Cdc37 knockdown reduces Ryk ICD stability. Furthermore, we have discovered that the Cdc37-Ryk ICD complex is disrupted during neural differentiation of embryonic stem cells, resulting in Ryk ICD degradation. These results suggest that Cdc37 plays an essential role in regulating Ryk ICD stability and therefore in Ryk-mediated signal transduction.

Generation of embryonic stem cells from mouse insulin I promoter-green fluorescent protein transgenic mice and characterization in a teratoma model.

Insulin-secreting pancreatic beta cells play a key role in the pathogenesis of diabetes mellitus. Potential new treatments for this disease include cell-replacement therapies using embryonic stem cells (ESCs). We have generated ESCs from a transgenic mouse model, mouse insulin 1 promoter (MIP) green fluorescent protein (GFP) mice, in which embryonic and adult beta cells are genetically tagged with GFP. The aim of the present study is to examine the differentiation potential of MIP-GFP ESCs in the microenvironment of the kidney capsule. The ESCs grew rapidly and formed a teratoma with GFP-expressing beta-like cells present in clusters that formed a cord-like structure similar to what is seen in the embryonic pancreas. These structures also included glucagon-expressing alpha cells and amylase-expressing acinar cells. Electron microscopic analysis showed insulin-like granules in columnar epithelium with microvilli adjacent to exocrine-like granule-containing cells. The MIP-GFP ESCs should be a useful research tool to study the differentiation capacity of ESCs toward pancreatic lineages.