September 23, 2014

Frontiers in Bioengineering Conference: The Farthest Out in Front

Earlier this month, I attended the Frontiers in Bioengineering conference at the Beckman Institute (UIUC). It was an interesting event with diverse perspectives on the field of Bioengineering. The two-day event featured researchers from all over the world, but focused on the synergies between Engineering and Cell/Molecular Biology.

One way to summarize the proceedings is to discuss the three most interesting (at least to me) technological advances. So here they are, in no particular order:

1) tissue co-cultures: Roger Kamm and Kevin Healey discussed the use of co-cultures to synthetically grow new organs and/or repair scaffolds. Co-cultures [1] are ex vivo systems within which multiple cell types are established and grown in media. The benefit of this artificial system involves the endogenous production of growth factors and a microenvironment. Exogenously-delivered factors apparently do not have the same efficacy for applications such as nerve grafts and cardiac repair. In the case of nerve grafts, supplying a bona-fide microenvironment can increase the distance of nerve innervation across a denervated gap. Co-cultures can also provide target tissues and anterograde cells to better approximate neuronal communication.

2) SHAPEseq: this an up-and-coming technique that has been used by a number of research groups to sequence the secondary structure of RNA [2]. SHAPEseq, or Selective 2'-hydroxyl acylation analyzed by primer extension, involves several steps that are similar to or go beyond the basic RNAseq technology. These include: preparing a barcoded RNA library, preparing a structure-specific cDNA library, aligning the corresponding reads, and calculating shape reactivities [3]. As with RNAseq, the objective is to build sequence libraries. Unlike with RNAseq, these libraries are structure-dependent. This allows for important structural information (e.g. hairpins, loops) to be estimated from a sample with single-nucleotide resolution.

A graphical summary of the SHAPEseq protocol. COURTESY: protocol description in [3].

3) NiN (nonviral induced neuronal) cells: this is a technique that was presented by Kam Leong at Duke. The idea is to use a non-viral genetic engineering approach (such as CRISPR) to introduce reprogramming factors into a cell. Non-viral factor delivery, as opposed to viral-mediated delivery using polycistronic vectors (genetic elements), is supposedly safer for transplantation and other therapeutic uses [4]. Other non-viral techniques (such as RNA-mediated reprogramming) have been tried with a mixed record of success. But by using the gene editing method [5], a cell population can be reprogrammed to a level of efficiency approximating viral-mediated reprogramming techniques. Despite various issues with estimating reprogramming efficiency and diversity across source cells [6], NiN techniques might be a easy and relatively controllable way to produce highly-specialized types of induced Neurons.

Honorable mention by association: The technology enabling the NiN advance is called CRISPR, or clustered, regularly interspaced, short palindromic repeat technology [7]. By using RNA-guided nucleases such as members of the Cas protein family (Cas9 in particular) [8], CRISPR technology can enable precise targeting of gene regulation. This includes the introduction and control of transgenes, something for which CRISPR has a lot of potential. To be fair, there are other, similar methods such as Transcription Activator-Like Effector Nucleases (TALENs) and Zinc Finger Nucleases (ZFNs) [9]. So congratulations to all of our gene editing technologies as we look to the future.

A diagram of the Cas-mediated CRISPR protocol. COURTESY: James Atmos, Wikipedia.

[1] For examples, please see the following articles:
a) Paschos, N.K., Brown, W.E., Eswaramoorthy, R., Hu, J.C., and Athanasiou, K.A.   Advances in tissue engineering through stem cell-based co-culture. Journal of Tissue Engineering and Regenerative Medicine, doi:10.1002/term.1870 (2014) AND

b) Ma, J., Both, S.K., Yang, F., Cui, F-Z., Pan, J., Meijer, G.J., Jansen, J.A., and van den Beucken, J.J.J.P.   Cell-Based Strategies in Bone Tissue Engineering and Regenerative Medicine. Stem Cells and Translational Medicine, sctm.2013-0126 (2013).

c) Meijer, G.J., de Bruijn, J.D., Koole, R., van Blitterswijk, C.A.   Cell-Based Bone Tissue Engineering. PLoS Medicine, 4(2), e9. doi:10.1371/journal.pmed.0040009 (2007).

[2] For examples, please see the following articles:

a) Lucks, J.B., Mortimer, S.A., Trapnell, C., Luof, S., Aviran, S., Schroth, G.P., Pachter, L., Doudna, J.A., and Arkin, A.P.   Multiplexed RNA structure characterization with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). PNAS, 108(27), 11063-11068 (2011)

b) Steen, K.A., Malhotra, A., Weeks, K.M.   Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease. Journal of the American Chemical Society, 132(29), 9940-9943 (2010).

[3] Mortimer, S.A., Trapnell, C., Aviran, C., Pachter, L., and Lucks, J.B.   SHAPE–Seq: High‐Throughput RNA Structure Analysis. Current Protocols in Chemical Biology, 10.1002/
9780470559277.ch120019 (2012).

[4] Park, H.J., Shin, J., Kim, J., and Cho, S.W.   Nonviral delivery for reprogramming to pluripotency and differentiation. Archives of Pharmacology Research, 37(1), 107-119 (2014).

[5] Perez-Pinera, P., Kocak, D.D., Vockley, C.M., Adler, A.F., Kabadi, A.M., Polstein, L.R., Thakore, P.I., Glass, K.A., Ousterout, D.G., Leong, K.W., Guilak, F., Crawford, G.E., Reddy, T.E., and Gersbach, C.A.   RNA-guided gene activation by CRISPR-Cas9–based transcription factors. Nature Methods, 10, 973-976 (2013).

[6] Alicea, B., Murthy, S., Keaton, S.A., Cobbett, P., Cibelli, J.B., and Suhr, S.T.   Defining phenotypic respecification diversity using multiple cell lines and reprogramming regimens. Stem Cells and Development, 22(19), 2641-2654.

[7] Hsu, P.D., Lander, E.S., and Zhang, F.   Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell, 157(6), 1262-1278 (2014).

[8] Sander, J.D. and Joung, J.K.   CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology, 32, 347-355 (2014).

[9] Gaj, T., Gersbach, C.A., and Barbas, C.F.   ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Bioengineering, 31(7), 397-405 (2013).

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