Assoc.Prof. Parinya Noisa

Year	Title	Journal
2011	A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli Imsoonthornruksa S., Noisa P., Parnpai R., Ketudat-Cairns M.	Journal of Biotechnology
Abstract: Mouse embryonic stem cells (mESCs) rely on a cytokine named leukemia inhibitory factor (LIF) to maintain their undifferentiated state and pluripotency. However, the progress of mESC research is restricted and limited to highly funded laboratories due to the cost of commercial LIF. Here we presented the homemade hLIF which is biologically active. The hLIF cDNA was cloned into two different vectors in order to produce N-terminal His 6-tag and Trx-His 6-tag hLIF fusion proteins in Origami(DE3) Escherichia coli. The His 6-hLIF fusion protein was not as soluble as the Trx-His 6-hLIF fusion protein. One-step immobilized metal affinity chromatography (IMAC) was done to recover high purity (>95% pure) His 6-hLIF and Trx-His 6-hLIF fusion proteins with the yields of 100 and 200mg/l of cell culture, respectively. The hLIF fusion proteins were identified by Western blot and verified by mass spectrometry (LC/MS/MS). The hLIF fusion proteins specifically promote the proliferation of TF-1 cells in a dose-dependent manner. They also demonstrate the potency to retain the morphology of undifferentiated mESCs, in that they were positive for mESC markers (Oct-4, Sox-2, Nanog, SSEA-1 and alkaline phosphatase activity). These results demonstrated that the N-terminal fusion tags of the His 6-hLIF and Trx-His 6-hLIF fusion proteins do not interfere with their biological activity. This expression and purification approach to produce recombinant hLIF is a simple, reliable, cost effective and user-friendly method. © 2011 Elsevier B.V. Keyword: Fusion protein; Histidine tag; HLIF; Recombinant protein; Solubility; Stem cells Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-79951551289&doi=10.1016%2fj.jbiotec.2010.12.020&partnerID=40&md5=09c2a8b90a185d48bbf040897dd1111a DOI: 10.1016/j.jbiotec.2010.12.020
2011	Generation of Human Embryonic Stem Cells Carrying Lineage Specific Reporters Noisa P., Urrutikoetxea-Uriguen A., Cui W.	Methods in Molecular Biology
Abstract: The distinctive properties of human embryonic stem cells (hESCs) enable them to provide unique models to study the network of signaling pathways that regulate organogenesis, generate disease models, produce cells and tissues for therapies, and identify new drugs for treatment. Genetic modification of hESCs is a powerful tool to assist the above studies. Generation of lineage-specific fluorescent protein reporter hESC lines will greatly benefit investigators to monitor specific cell lineages in a live, easy, and timely manner. This technique will facilitate high throughput screening to identify molecules important in regulating specific cell fate commitment. In addition, such reporter cell lines enable researchers to enrich certain cell populations by fluorescent activated cell sorting (FACS) for either downstream biological analysis or in vivo applications. We have shown that hESCs can be stably transfected with a plasmid in which expression of the green fluorescent protein (GFP) is under the control of the Oct-4 promoter using chemical transfection. The expression pattern of transgenic Oct-4-GFP reflects that of endogenous Oct-4. © 2011, Springer Science+Business Media, LLC. Keyword: Cell culture; Differentiation; Human embryonic stem cell; Reporter cell line; Stable transfection Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-79952199092&doi=10.1007%2f978-1-60761-962-8_6&partnerID=40&md5=e99a5ec5409bfda2c138763e3c9c1e62 DOI: 10.1007/978-1-60761-962-8_6
2010	Generation of Human Embryonic Stem Cell Reporter Lines Expressing GFP Specifically in Neural Progenitors Noisa P., Urrutikoetxea-Uriguen A., Li M., Cui W.	Stem Cell Reviews and Reports
Abstract: Generation of lineage-specific human embryonic stem cell (hESC) reporter lines will facilitate the real time monitoring of differentiation in live cells and the identification of factors governing these processes. It will also enable researchers to purify specific cell populations from heterogeneous differentiated hESC progeny. Here we report the generation of clonally derived nestin-EGFP reporter hESC lines that express GFP under the control of the neuroepithelial specific nestin 2nd intron enhancer. We show that the nestin-EGFP hESC reporter lines retain the features of undifferentiated hESCs, are able to self-renew in hESC culture conditions and to differentiate into cells of all three germ layers. The nestin-EGFP reporter exhibited high expression in neural progenitor cells upon differentiation, although it is detectable at a low level in the undifferentiated state. Furthermore, the expression of the transgene is exclusively confined to the neural progenitors after differentiation. The specific expression of the transgene is determined by collaborative binding motifs of POU and SOX transcription factors in the nestin enhancer. Deletion of either of the binding elements resulted in a significant reduction of enhancer/promoter activity. Taken together, the nestin-EGFP reporter hESC lines are invaluable not only for the study of the neural differentiation process from hESCs but also for the enrichment of neural progenitor cells from other cell lineages. © 2010 Springer Science+Business Media, LLC. Keyword: GFP reporter; Human embryonic stem cells; Nestin; Neural differentiation; Neural progenitor Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-77955853858&doi=10.1007%2fs12015-010-9159-9&partnerID=40&md5=80ca1fb62be87c3d44d8b891611132b0 DOI: 10.1007/s12015-010-9159-9
2010	Dynamic transcriptomes during neural differentiation of human embryonic stem cells revealed by short, long, and paired-end sequencing Wu J.Q., Habegger L., Noisa P., Szekely A., Qiu C., Hutchison S., Raha D., Egholm M., Lin H., Weissman S., Cui W., Gerstein M., Snyder M.	Proceedings of the National Academy of Sciences of the United States of America
Abstract: To examine the fundamental mechanisms governing neural differentiation, we analyzed the transcriptome changes that occur during the differentiation of hESCs into the neural lineage. Undifferentiated hESCs as well as cells at three stages of early neural differentiation - N1 (early initiation), N2 (neural progenitor), and N3 (early glial-like) - were analyzed using a combination of single read, paired-end read, and long read RNA sequencing. The results revealed enormous complexity in gene transcription and splicing dynamics during neural cell differentiation. We found previously unannotated transcripts and spliced isoforms specific for each stage of differentiation. Interestingly, splicing isoform diversity is highest in undifferentiated hESCs and decreases upon differentiation, a phenomenon we call isoform specialization. During neural differentiation, we observed differential expression of many types of genes, including those involved in key signaling pathways, and a large number of extracellular receptors exhibit stage-specific regulation. These results provide a valuable resource for studying neural differentiation and reveal insights into the mechanisms underlying in vitro neural differentiation of hESCs, such as neural fate specification, neural progenitor cell identity maintenance, and the transition from a predominantly neuronal state into one with increased gliogenic potential. Keyword: Glial; Neuron; RNA-Seq; Splicing isoforms; Unannotated transcripts Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-77950396799&doi=10.1073%2fpnas.0914114107&partnerID=40&md5=af0248a9700a20ffc4010ad533b7e6a1 DOI: 10.1073/pnas.0914114107