Biomaterial transfer is used in all biological fields to manipulate cell function and dissect molecular and cellular mechanisms in physiologic and pathologic settings. New, enabling technologies are required to overcome size limitations inherent in all current delivery methods to allow the transfer of large cargo, such as live pathogens, whole chromosomes, and (eventually) bioengineered replacement components, such as genetically modified mitochondria, for new-age therapies. As Nicole Rusk, senior editor Nature Methods, recently opined "surprisingly, no methods for efficiently bringing impermeant large molecules into living cells exist (Nat Methods 8:44, 2011). This proposal is focused on our recently developed cargo transfer technology, which is called the photothermal nanoblade, to deliver whole human chromosomes into human pluripotent stem cells (hPSCs) in order to examine chromosome reprogramming. These transferred chromosomes will be analyzed for epigenetic reprogramming within the hPSC nucleus using standard and cutting-edge molecular techniques and is supported by a strong institutional bioinformatics infrastructure. This proposal also aims to develop a microfluidics-integrated photothermal nanoblade platform for next-generation high- throughput and near simultaneous delivery of large cargo into more than 10,000 cells in seconds. Achieving these goals will provide custom engineered hPSCs for biological investigations, an assessment of the chromosome reprogramming capability of federally-registered H1 and H9 human embryonic stem cells (hESCs), and an advanced yet simple to use platform to overcome potential biological obstacles that, if they exist, would limit success due to an insufficient throughput, should chromosome integration be a low frequency event, which is currently not known.
Biomaterial transfer is used in all biological fields to manipulate cell function and dissect molecular and cellular mechanisms in physiologic and pathologic settings. New, enabling technologies are required to overcome size limitations inherent in all current delivery methods to allow the transfer of large cargo for new-age therapies. Our R21 proposal is to use a novel photothermal nanoblade system (Wu, et al., in press, Analytical Chemistry, 2011- see paper in appendix) to deliver whole human chromosomes into pluripotent human stem cells, a currently impossible task, in order to examine whole chromosome epigenetic reprogramming.
|Wu, Ting-Hsiang; Sagullo, Enrico; Case, Dana et al. (2016) Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells. Cell Metab 23:921-9|
|Wu, Ting-Hsiang; Wu, Yi-Chien; Sagullo, Enrico et al. (2015) Direct Nuclear Delivery of DNA by Photothermal Nanoblade. J Lab Autom 20:659-62|
|Wu, Yi-Chien; Wu, Ting-Hsiang; Clemens, Daniel L et al. (2015) Massively parallel delivery of large cargo into mammalian cells with light pulses. Nat Methods 12:439-44|
|Yamane, Daisuke; Wu, Yi-Chien; Wu, Ting-Hsiang et al. (2014) Electrical impedance monitoring of photothermal porated mammalian cells. J Lab Autom 19:50-9|
|Chen, Yue; Chung, Aram J; Wu, Ting-Hsiang et al. (2014) Pulsed laser activated cell sorting with three dimensional sheathless inertial focusing. Small 10:1746-51|
|Teh, Boon Eng; French, Christopher Todd; Chen, Yahua et al. (2014) Type three secretion system-mediated escape of Burkholderia pseudomallei into the host cytosol is critical for the activation of NFÎºB. BMC Microbiol 14:115|
|Chen, Yue; Wu, Ting-Hsiang; Kung, Yu-Chun et al. (2013) 3D pulsed laser-triggered high-speed microfluidic fluorescence-activated cell sorter. Analyst 138:7308-15|
|Xu, Jianmin; Teslaa, Tara; Wu, Ting-Hsiang et al. (2012) Nanoblade delivery and incorporation of quantum dot conjugates into tubulin networks in live cells. Nano Lett 12:5669-72|
|Wu, Ting-Hsiang; Chen, Yue; Park, Sung-Yong et al. (2012) Pulsed laser triggered high speed microfluidic fluorescence activated cell sorter. Lab Chip 12:1378-83|