Tracking STEMEZ hNP1 Progenitor Cell Fate

We would like to promote an important new discovery using our STEMEZ(TM) hNP1 Human Neural Progenitors Discovery Kit.

Human embryonic stem cell–derived neural progenitors (NP) present an important tool for understanding human development and disease. Optimal utilization of NP cells, however, requires an enhanced ability to monitor these cells in vitro and in vivo. Here we report production of the first genetically modified self-renewing human embryonic stem cell–derived NP cells that express fluorescent proteins under constitutive as well as lineage-specific promoters, enabling tracking and monitoring of cell fate. Nucleofection, transfection, and lentiviral transduction were compared for optimal gene delivery to NP cells. Transduction was most efficient in terms of transgene expression (37%), cell viability (39%), and long-term reporter expression (>3 months). Further, the constitutive gene promoters, cytomegalovirus, elongation factor 1α, and ubiquitin-C, exhibited comparable silencing (20–30%) in NP cells over a 2-month period, suggesting their suitability for long-term reporter expression studies. Transduced NP cells maintained their progenitor state and differentiation potential, as demonstrated by expression of endogenous NP markers and neuronal markers after differentiation. We also detected reporter expression in astrocytes generated from NP cells transduced with an astrocyte-specific gene promoter, glial fibrillary acidic protein, demonstrating the usefulness of this approach. The genetically manipulated NP cells described here offer great potential for live cell–tracking experiments, and a similar approach can as well be used for expression of proteins other than reporters.

Related Publication:
Sujoy K. Dhara, Brian A. Gerwe, Anirban Majumder, Mahesh C. Dodla, Nolan L. Boyd, David W. Machacek, Kowser Hasneen, Steven L. Stice. Genetic Manipulation of Neural Progenitors Derived from Human Embryonic Stem Cells. Tissue Engineering Part A. -Not available-, ahead of print. doi:10.1089/ten.tea.2009.0155

Neuromics' Stem Cell Markers Referenced:
Musashi-1, Rabbit, 1:100 , Catalog#:RA14128
Nestin, Mouse 1:500, Catalog#: MO15012
Tuj 1 (Neuron-specific class III beta-tubulin), Mouse, 1:500, Catalog#: MO15013
TH, Chicken, 1:80, Catalog#: CH23006

survival of efferent synapses on mammalian outer hair cells.

Dr Douglas Vetter (Tufts University School of Medicine) and team recently published a study that amplifies the understanding of the development, function and maintenance of auditory system function.

Their data strongly suggest that hair cell responses induced and/or modulated by Olivocochlear (OC) activation are necessary for the survival of OC innervation and that these responses must involve SK2-mediated hyperpolarization.

It also includes an excellent image of Olivocochlear fibers degeneration in SK2−/− mice. One of the makers used was our Tuj 1 (Neuron-specific class III beta-tubulin).

Vidya Murthy, Stéphane F. Maison, Julián Taranda, Nadeem Haque, Chris T. Bond , A. Belén Elgoyhen, John P. Adelman, M. Charles Liberman, Douglas E. Vetter. SK2 channels are required for function and long-term survival of efferent synapses on mammalian outer hair cells. Molecular and Cellular Neuroscience 40 (2009) 39–49
...TuJ-1 (class III β-tubulin, Neuromics, Northfield, MN; cat. # MO15013) and processedwith Oregon Green labeled secondary antibodies (Molecular Probes/InVitrogen) for confocal microscopy...

Other Pubs Referencing Neuromics' Tuj 1 (Neuron-specific class III beta-tubulin):
Yoshifumi Saisho, Paul E. Harris, Alexandra E. Butler, Ryan Galasso, Tatyana Gurlo,1 Robert A. Rizza, and Peter C. Butler. Relationship between pancreatic vesicular monoamine transporter 2 (VMAT2) and insulin expression in human pancreas. J Mol Histol. 2008 October; 39(5): 543–551. Published online 2008 September 13. doi: 10.1007/s10735-008-9195-9.
Sujoy K. Dhara, Kowser Hasneen, David W. Machacek, Nolan L. Boyd, Raj R. Rao, Steven L. Stice (2008). Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures. Differentiation 76 (5) , 454–464 doi:10.1111/j.1432-0436.2007.00256.x
...NES (1:100, Neuromics, Edina, MN), MSI1 (1:100, Neuromics), Tuj1 (1:500, Neuromics)...
D. Conte, V. Lall, G. Dobson, S. Deuchars, J. Deuchars. Cerebrospinal fluid contacting neurones in the spinal cord of the mouse and rat: small cells with a big purpose? University of Leeds (2008) Proc Physiol Soc 10 PC45
...Preliminary immunohistochemical data supports the concept that the CSFcNs are immature neurones, since they express neuronal markers Tuj1 (Neuromics) ...