An interview with Dr. Uma Lakshmipathy, principle scientist at Life Technologies

The end of the year is a great time to reflect on the events of the year and predict what might happen next year. I’ve asked Dr. Uma Lakshmipathy, Principle Scientist with Life Technologies, to tell us about her work with stem cells at Life Technologies, her thoughts on the biggest discoveries in stem cell research in 2011 and prospects for 2012.

What projects are you currently working on?

Our team is currently focused on developing simplified solutions that overcome current bottlenecks and bridge gaps to build comprehensive stem cell characterization tools. For example, alkaline phosphatase is a pluripotent marker which is commonly used to monitor emergence of induced Pluripotent Stem cell (iPSC) colonies during reprogramming. Current available substrates are end-point assays and do not preserve cell integrity. This constraint was overcome with the novel alkaline phosphatase substrate that stains pluripotent stem cells and also preserves cell viability and characteristics, thereby enabling further expansion of stained cells.

The end of the year is always a favorite time to make lists of top events, discoveries, etc. What are your five top stem cell advances or discoveries of 2011?

Cell reprogramming with miRNAs: Reports on the use of miRNA either by expression of the miR 302/367 clusters in Lenti vector and Hdac2 suppression (doi: 10.1016/j.stem.2011.03.001), mature synthetic miRNA molecules (doi:10.1016/j.stem.2011.05.001) or in combination with other existing methods to increase efficiency (doi:10.1261/rna.2664111). It remains to be seen if this method will emerge as a preferred method for cell reprogramming.

iPSC Characterization - Predicting and Priming: A few elegant papers reported methods that may well turn out to be the gold standard for characterizing and differentiating iPSC derived from diverse genetic and disease backgrounds. A “Scorecard” approach based on the use of genome-wide gene expression and DNA methylation to confirm pluripotency and predict differentiation potential of ESC and iPSCs was reported (doi: 10.1016/j.cell.2010.12.032). A similar biomarkers approach was reported with “PluriTest” as a potential alternative to teratoma assay (doi: 10.1038/nmeth.1580). A study reporting differentiation potential of iPSC lines demonstrated that despite developmental differences, cells can be primed into neural differentiation (doi:10.1038/nbt1783)

Gene Targeting: Several studies on exogeneous DNA introduction into ESC/iPSC have progressed the field closer to therapeutic applications. Traditional homologous recombination was used to create Knock-In fluorescent reporters specific for cardiac (doi:10.1038/nmeth.1740) and neuronal lineages (doi: 10.1002/stem.587). ZFN (Zinc finger nucleases) were reported to insert or remove a single base pair mutation in the alpha synucelin gene known to cause early onset Parkinson's disease.(doi: 10.1016/j.cell.2011.06.019). TALENs (Transcription activator like effector nucleases) were also reported to alter genes with an efficiency and precision similar to ZFN (doi: 10.1038/nbt.1927).

Immunogenecity and stability of iPSCs: A report on autologous iPSC cells triggering immune reactions in mice as a result of incomplete epigenetic reprogramming generated quite a stir casting shadows on the use of iPSCs in clinical applications (doi: 10.1038/nature10135). In addition, studies reported increased level of mutations in iPSCs compared to starting somatic cell population (doi: 10.1038/nature09805) and increased copy number variation (doi: 10.1038/nature09871).

The first (real!) cloned embryo: An adult human egg cell was reprogrammed to an embryonic state to further create ESCs from the developing embryo (doi: 10.1038/nature10397). While the ESCs are, (a) not normal, (b) do not contain just the patient DNA material and (c) maybe of limiting value as a physiological relevant screening/therapeutic tool, overcoming the hurdle of creating a cloned ESC in vitro is a technical breakthrough! Understanding the active elements in the egg DNA that are required for embryonic development will be useful not only for cloning but may also be beneficial for understanding and improving reprogramming technologies.

Give us your predictions for 2012. What do you think is going to make a big splash in the field of stem cell research?

A combination of different small molecule methods for iPSC generation and/or direct reprogramming providing better control of cell fate.

Modulating media, matrix, hypoxia and other culture conditions for a less harsh, more efficient reprogramming method.

Massively parallel analysis methods to further dissect genomic stability and establish definitive differences between ESCs and iPSCs.

Devices /automated systems for patient material collection, curation, iPSC generation and expansion.

Convergence of platforms for creation of disease-specific iPSCs and their use in screening.

miRNA based generation of induced pluripotent stem cells

In the last decade, miRNAs have been established as one of the primary modulators of biological pathways owing to their ability to impact possibly >30% of all protein-coding genes. Their role as central regulators of stem cell maintenance and differentiation is becoming more prominent in the last two years.

Specific miRNAs, such as the mir302-367 cluster, are preferentially expressed in pluripotent stem cells, their expression is regulated by key transcription factors required for maintenance of pluripotency and they control the expression of key developmental pathways and cell cycle regulators.

As miRNAs play an important role in pluripotent stem cell maintenance and differentiation, several groups have investigated the use of miRNAs for both reprogramming and transdifferentiation. IPSCs hold great promise in cell therapy and drug discovery applications, but the low efficiency of reprogramming and the use of integrating and/or DNA based constructs present significant.

Anokye-Danso et al achieved a greater than 3000-fold improvement in reprogramming efficiency via lentiviral-based transduction of mir302-367 compared to standard lentiviral-based reprogramming with Oct4, Klf4, Sox2 and cMyc. Despite the high efficiencies, this method still relied on integrating viral vectors to induce pluripotency. Miyoshi et al used Lipofectamine-based direct transfection of mir-200c, mir-302s and mir-369s mimics to generate iPSCs at an efficiency comparable to standard retroviral methods. Yang et al, reported that depletion of MEF-enriched mir-21 and mir- 29a enhances reprogramming.

More recently, two groups have successfully converted functional neurons directly from human fibroblasts using a combination of miRNAs and transcription factors introduced by lentivirus. While both groups demonstrated a role for mir-124 in neuronal differentiation from fibroblasts, Yoo et al relied on a combination of mir-124, mir-99 and the transcription factors NEUROD2, ASCL1 and MYT1L to generate neurones from both neonatal and adult fibroblasts. Ambasudhan et al used a combination of mir-124, BRN2 and MYT1L to produce neurons with mature functional synapses in the absence of co-culture with other cell types. Kim et al have recently shown that levels of miR-371-3 in human pluripotent stem cells is inversely correlated to their ability to differentiate into neurons.

miRNA-based reprogramming and transdifferentiation provides a powerful tool for the efficient and safe creation of patient derived cells for basic research, drug discovery and therapeutic applications. Research into the optimal combination of miRNAs mimics and miRNA inhibitors to drive conversion to the desired lineage will help improve efficiency when using direct transfection.