PRODUCTS AND TECHNOLOGY

ADULT STEM CELLS FOR TREATMENT OF DIABETES AND OTHER DISEASES

The Company has generated 29 adult human pancreatic stem cell lines that are distinct from the limited number of embryonic stem cell lines that are presently approved for federally funded research. Some of these lines have been extensively expanded in cell culture and have demonstrated self-renewal properties by growth through 100 to 200 population doublings (PD) while mortal cells typically do not expand beyond 50 population doublings. Also, we have developed methods of differentiation that result in expression of biomarkers characteristic of alpha and beta cells suggesting that the cell lines have at least multipotent differentiation capacity. Some markers of non-pancreatic cell lineage are expressed in these cells raising the possibility of differentiation into other cell types.

Growth of the clonal cell line, VIT4-G9 (black and red symbols) and the effects of cyclopamine (green and blue symbols), an inhibitor of the hedgehog pathway. Growth rate increased in the initial five passages and then was steady (mean t d = 16.9 hrs) through 25 passages (130 PD). Five micromolar cyclopamine slowed growth (blue symbols), 10 micromolar cyclopamine slowed growth further (green symbol) and 20 micromolar cyclopamine blocked growth (not shown). Detailed methods are in patent application, see link below.

The graph above shows expansion of one of our stem cells lines, VIT4-G9, through 130 population doublings and inhibition of growth by cyclopamine in a dose-dependent manner. Since cyclopamine is a specific antagonist of smoothened and thus the hedgehog pathway, these results suggest that the VIT4-G9 cell line exhibits self-renewal through a mechanism dependent on the hedgehog pathway, which is a common characteristic of stem cells. This cell line may also have application to discovery of new smoothened antagonists as potential cancer therapies, since some tumors may proliferate based on hedgehog-driven stem cells e.g., glioblastoma tumor cells, (Nature 444: 687, 2006).

Type I diabetes (juvenile onset or insulin-dependent diabetes mellitus IDDM) results from loss of the insulin-producing beta cells of the pancreas gland due to autoimmune reactions that destroy beta cells. The therapy of Type I diabetes typically involves multiple daily injections of insulin combined with dietary restrictions and frequent glucose monitoring to control blood glucose levels. However, insulin therapy is not a cure and Type I diabetics are at high risk for cardiovascular disease, blindness, diminished wound healing and other complications that diminish life expectancy. There is considerable ongoing research into understanding the mechanisms of autoimmune rejection with the goal of blocking or reversing the autoimmune rejection of beta cells.

Also, through a procedure known as the Edmonton protocol, beta islets are harvested and purified from donated pancreas glands and then transplanted into the liver where implanted islets become functional. This has resulted in long-term insulin independence and reduction of the complications of diabetes. However, to prevent immune rejection, continuous immunosuppressive therapy is necessary and this diminishes function of the transplanted islets and causes other complications as well. Transplant recipients may revert to insulin therapy as function of the transplanted cells diminishes. A further major obstacle to widespread use of the Edmonton protocol is the limited number of available islets for transplantation since these are derived from donated organs.

We are presently engaged in pre-clinical research comparing stem cell-derived beta islet cells and human pancreatic beta cells. We have ongoing in-vitro studies optimizing the process of stem cell differentiation into beta islet cells. Through an approved research protocol with the National Disease Research Interchange we have access to purified human beta islets for direct comparison with stem cell-derived islet cells. We measure expression of various biomarkers and determine insulin secretion profiles, especially glucose-stimulated insulin secretion. Our preliminary results indicate that our stem cell lines may be differentiated into insulin and glucagon-containing cells and that basal insulin secretion may be increased by graded increases in extracellular glucose levels. We plan to extend our efficacy studies to determine if stem cell-derived beta islet cells reverse diabetes in commonly used animal models.

The following link is to a PDF file of our pending patent application, “Generation and Differentiation of Adult Stem Cell Lines”, which provides more detail regarding our technology and stem cell lines.

Generation and Differentiation of Adult Stem Cell Lines (PDF)

PRODUCTS FOR STEM CELL AND DRUG DISCOVERY RESEARCH

We manufacture select human cell lines and related products according to GMP standards for use in stem cell and drug discovery research. Our research products include two human cell lines of pancreatic origin and supporting products including optimized growth and cryopreservation media. These lines are mesenchymal, attachment-dependent cells that exhibit properties of human mesenchymal stem cells, affording researchers several unique opportunities.

Origin of Cell Lines and Properties

The cell lines, VIT1 and LT2 were generated by methods that select mesenchymal (fibroblastic) cells from dispersed cell preparations derived from human fetal pancreatic glands. The LT2 cell line was derived by immunomagnetic purification of CD 90 positive cells, a fibroblast-specific marker, followed by transfection with a plasmid containing the cDNA for large T antigen, while the primary VIT1 cell line was generated by selection of mesenchymal cells through low density culture of dispersed human pancreatic cells. Both cell lines express the mesenchymal markers, vimentin and CD 90 but are negative for epithelial markers, Ep-CAM, E-cadherin and cytokeratin AE1/AE3 and are diploid by flow cytometry cell cycle DNA analysis.

The graphs above show growth characteristics of the VIT1 and LT2 cell lines. Doubling time in hours is shown as a function of passage number for cells of origin (VIT1) or from cryopreserved cells at other passages (legend). VIT1 cells grew at intermediate rates initially and then more rapid growth rates (t d ~ 17 hours) through 26 passages (130 PD), while LT2 cells grew slowly at first and then maintained rapid expansion (t d 15 to 25 hours) through 40 passages (200 PD). Photographs are phase contrast micrographs (100x) of VIT1 cells (origin) at pass 4 and 6 (lines) showing a change in morphology from classic fibroblastic to epithelial.

Both cell lines expand substantially beyond the doubling limit for mortal cells (50 PD) indicating immortality while only the LT2 cells were immortalized by transfection with Large T antigen. VIT1 cells exhibit several properties of mesenchymal stem cells including self-renewal, expression of key genes of the hedgehog and Wnt pathways, genes that are characteristic of hematopoietic stem cells together with numerous homeobox genes that are known to control cell lineage and several other markers characteristic of mesenchymal cells. Furthermore, epithelial markers could be induced within VIT1 cells by exposure to the incretin hormone, glucagon-like peptide 1, and subsequent isolation of Ep-CAM positive cells led to establishment of several stem cell lines with apparent capacity to differentiate into pancreatic beta islet cells. Immortality of LT2 cells may involve other mechanisms rather than expression of Large T antigen. (See US Patent Application, S/N 20060093586 for additional details, above link).

Potential applications of these cell lines include: studies of differentiation capacity of adult stem cells, mesenchymal stem cell research, stem cell self-renewal mechanisms, mesenchymal to epithelium transition (MET), toxicology studies, drug discovery studies involving the hedgehog and Wnt pathways, G-coupled membrane receptors, etc.

Optimized Growth Medium

We also manufacture a medium optimized to support growth and maintenance of VIT1 and LT2 cells in in-vitro cell culture. Complete medium includes a basal medium and a proprietary low serum-containing supplement provided as a concentrate. This medium was used to expand cell cultures shown above, including the VIT4-G9, VIT1 and LT2 cell lines and may have application to growth optimization of other mesenchymal stem cell lines.

These products are manufactured to GMP standards and QC release requires specific performance standards and negative tests for common contaminants including bacteria, fungi, viruses and mycoplasma. The cell lines VIT1 and LT2 are provided for research purposes only through a material transfer agreement. Please contact the Company regarding availability of these products and licensing opportunities.