Year 2

Proposed Plan: We have assembled a team of investigators with complementary expertise in applying the state-of-the-art “one-bead-one-compound” (OBOC) combinatorial library methods to identify synthetic chemical molecules that bind to unique receptors (protein molecules) on the surface of human embryonic stem cells and induced pluripotent stem cells. In this technology, stem cells will be mixed with huge number of chemical-beads (1,000,000 or more), and those 100 micron beads coated by the stem cells will be isolated for chemical analysis. We believe some of the chemical molecules identified by this method will support the growth and proliferation of stem cells while maintaining their “stemness” nature (pluripotency, self-renewal). Other molecules may induce directed-differentiation into specific desirable cell types such as heart cells for damaged heart and brain cells for patients who suffer stroke. Once these molecules are identified, we shall incorporate them into an artificial gel that can support large scale stem cell growth and directed-differentiation. Such artificial gels are free of animal products and viruses, making them safe for therapeutic use in human. We shall take advantage of these novel molecules and gels to study and therefore understand how stem cells work. These molecules may also be used as imaging probes to track or localized stem cells inside patients. For tissue regeneration, we may incorporate one or more of these molecules onto surfaces of biodegradable scaffolding with predetermined shape, so that simple artificial organs with various cell types can be developed. These molecules may also serve as very important tools and reagents for basic stem cell research.

In year 2, We have further optimized methods for using OB2C combinatorial methods and applied it successfully to iPSC and discovered protein mimic ligand that promote directed-differentiation of murine ESCs (mESC) towards oligodendrocyte precursor cells (OPCs). In addition, we have further optimize methods for growing iPSCs and ESCs in our novel 3-D gel matrix. We have also developed a releasable solution phase assays for OBOC combinatorial libraries such that directed-differentiation of stem cells can be screened rapidly. Such a library was designed, synthesized, and screened small molecules for directed-differentiation of stem cells into neuronal precursor cells. These compounds identified in year 2 have great potential to be further developed into molecules that can be used in regeneration of brain cells.

Based on the results obtained form the CIRM grant, we have submitted a R21 grant proposal to NIH entitled: “Discovery of ligands for directed-differentiation of stem cells”. I am pleased that the grant proposal has just received an impact score of 14 at four percentile. It is likely to be funded starting this fall.

Progress in the last two year: In the past two year, we have prepared several random OBOC linear and cyclic peptide libraries and one-bead-two-compound (OB2C) combinatorial libraries. We use embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) to screen these libraries for cell binding and also for activation or inactivation of specific intracellular pathways pertinent to stem cell maintenance and differentiation. We have also screen the OB2C libraries for molecules that can maintain the growth and pluripotency (“stemness nature”) of the stem cells. Through these studies, we have identified several biologically active peptides, some of which have been confirmed to bind to human and mouse ESC. Some of these molecules appeared to be able to maintain the stem cell growth on the bead for over two weeks.

Discovery of cell surface acting ligands that induce the differentiation of iPSCs to oligodendrocyte precursor cells. Genetically-labeled stem cell lines were created such that when they differentiate to brain precursor cells, they will turn green. Such cell line was obtained from Dr. Deng (Colleague at Shriner Hospital, Sacramento, faculty of Cell biology, UC Davis) and used to scsreen our combinatorial libraries. Using two distinct assay systems were were able to discover several chemical molecules that induce the differentiation of stem cells into neuronal precursor cells. These chemical agents will undoubtedly be useful for brain cell regeneration.

3-D gel matrix for stem cell growth. We have also succeeded in the development and testing of a novel, biocompatible and highly versatile synthetic self-assemble 3-D hydrogel matrix for supporting stem cell growth, maintenance or directed-differentiation. Our novel polymeric hydrogels is comprised of polyvinyl alcohol (PVA), crosslinked by linkers with boronic acids at both ends. The cell adhesion ligands LLP2A, LXY3, HYD-1, and LXW7 previously identified using the OBOC method, and shown to bind alpha4beta1, alpha3beta1, alpha6beta1, alphavbeta3 integrin, respectively were prepared with two boronic acids at one end. These functionalized ligands can be incorporated into a preformed 3-D gel matrix or prepared fresh by mixing PVA with the functionalized ligands followed by the crossinker. Stem cells can be seeded either on top of these gels or added to the chemical solution during the geling process so that the stem cells are immobilized within the 3-D gel matrix. The resulting gel matrix is non-toxic and totally compatible with the culture of stem cells.

We have tested the growth of iPSC and hESC in these 3-D gel matrix. Cells were maintained in gels for 3 to 13 days, and fed by removing and replacing overlaid spent media. At the end of culture period, cells were fixed in situ using formalin, extracted from the gels after dissolution of gels with dopamine or fructose, cytospun to microscope slides, stained with indirect immunofluorescence method, and analyzed via a confocal microscope.

Induced pluripotent stem cells cultured on these 3-D gel matrix formed clusters of cells reminiscent of embryoid bodies (EBs) after 24h in culture, both in the presence and absence of growth factor-supplemented medium, and with or without polymer-tethered integrin ligands present. Embryoid bodies varied in size and shape, with the spheroid being the most predominant structure. Formed embryoid bodies grew larger in size with additional days in culture, reaching an average diameter of 85 microm by day 7. Cells remained viable for the entire duration of the culture period (up to 13 days).

To investigate the effect of culture medium components on iPSCs in 3-D culture, cells were seeded atop integrin ligand-free hydrogels prepared in either basal stem cell medium, or complete serum-free, growth factor-supplemented stem cell culture medium, and maintained in 3-D culture for seven days. iPSCs grown in basal medium hydrogels formed hollow spheroids with a marked central cavity similar to the appearance of a gland. In smaller spheroids, the central cavity was surrounded by a single layer of cells, while larger spheroids tended towards incomplete cavitation, with a number of cells still present within the lumen. This finding was in stark contrast to the observed non-hollow structures formed by these cells in the gel matrix with growth factor-supplemented culture medium. Here, cells were contained within the full thickness of both small and larger spheroids. Thus the influence of the culture medium on iPSC fate in 3-D culture is most pronounced in cells within the center of the embryoid bodies, where they are either rescued or delayed from undergoing cell death.

The influence of cell-matrix (biomaterial) interactions on iPSC fate was probed in 3-D culture of iPSCs seeded atop the gel matrix comprised of polymer-tethered integrin-binding ligands in the absence of growth factors or other culture supplements. Here, the sole biochemical cell signaling components are the integrin-binding ligands. Cells formed spheroidal structures with central cavity. Using immunofluorrescent technique, we were able to detect markers of all three germ layers, including the ectodermal nestin, beta1 integrin found in both mesoderm and endoderm, and c-Kit (mesoderm), but weak expression of pluripotency markers Sox2 and TRA-1-60.

Conclusion: We were able to use OBOC and OB2C combinatorial technology to identify biologically active ligands that can attach to or interact with stem cell surface. We have developed and used two novel screening methods to screen chemical libraries for chemical molecules that induce directed differentiation of stem cells to neuronal precursor cells. These chemical molecules undoubtedly will be useful in regeneration of neuronal cells. In addition, we have demonstrated the use of a purely synthetic, well-defined 3D culture platform as suitable for maintenance of pluripotent stem cell viability, embryoid body formation, and multilineage differentiation.