Parsing Biomaterials and Small Molecules for Stem Cell Activity
The goal of this area is to identify synthetic combinatorially varied substrates and small molecules that control the differentiation of human embryonic stem cells (hESCs) in defined environments. The following established methods (Fig. 7) will be applied by IGERT Trainees in the laboratories of K. Lee (CCB), G. Brewer (Pharmacology), and S. Gunderson (BMB) (i) high throughput screening of diverse molecular libraries, (ii) affinity/biochemical approaches to target identification and (iii) characterization of the target protein(s) and the signaling pathways via gene chip profiling and genetic complementation experiments. A high-throughput screening strategy using synthetic small molecule has been demonstrated to be a valuable tool in recent stem cell research where signaling pathways and protein targets involved in stem cell regeneration, differentiation, and dedifferentiation are not clearly defined.
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Fig. 7. Chemical and functional genomic approaches to regulate stem cell fate |
A specific project goal is to identify small molecules that differentiate hESCs into subtype specific neurons, such as neural epithelial precursor (NEP) using NEP specific markers (Fig. 8). Once NEP cells are selectively induced and sorted, they will be sequentially used for a second screen to identify small molecules that can further commit NEP cells into different subtype-specific neuronal cells.
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Fig 8. An example of the control of stem cell fate by a synthetic small molecule. Gametogenol (hit compound identified from high throughput chemical screens) induced primordial germ cell (PGC) differentiation of murine ESCs. A. RT-PCR analysis confirmed PGC formation, A1. Gametogenol treatment (7D) ; A2. No treatment (negative control, 7D); A3. ESC at day 0 B. Western blot for vasa, gcOct4-GFP, and dapi. gc-oct4 and vasa are germ cell specific markers.
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A parallel set of projects will focus on elucidating the role of defined, combinatorially varied synthetic, biocompatible polymers as substrates for controllably accelerating the induction of differentiation of human mesenchymal stem cells (hMSCs) toward bone vs. fat vs neuronal lineages. A large number of polymeric substrates can be combinatorially designed in the J. Kohn laboratory (CCB), including libraries of tyrosine-derived polycarbonates and polymethacrylates. IGERT Trainees in the Moghe lab (BME) will probe hMSC interactions with these polymers. They will use high resolution multiphoton microscopy and high content imaging to discern minute changes in the cytoskeletal organization of hMSCs via genetically transfected fluororeporters, which are quantified in terms of descriptors (see color coded "heat map" in Fig. 9). Through decision tree modeling in the laboratories of D. Knight (MAE) and I. Androulakis (BME), such descriptors can be correlated to cell functions. Ultimately, short term descriptors will be used to seek predictive roles of biomaterials on longer term stem cell fates.
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Fig. 9. Screening combinatorially designed polymeric substrates via high content imaging of cytoskeletal organization and by modeling correlations between intracellular descriptors and cell adhesion and differentiation. A goal is to predict long-term stem cell differentiation based upon early intracellular descriptors as "sensors" of the synthetic substrates. Two "decision trees" illustrate how two distinct "actin" descriptors correlate with various components of polymer composition |
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