Toward Defined Culture Conditions for Pluripotent Stem Cells, Part 2

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At the UK National Stem Cell Network ‘s annual meeting in York, UK on 31 March 2011, a workshop organized by STEMCELL Technologies workshop addressed defined media for human stem cell culture. As illustrated in Part 1 (October 2011), it is critical to understand the pathways that maintain genetic stability during hES self-renewal, which is a prerequisite for all clinical applications. Because physiological DNA damage can take place during normal cellular proliferation, and accumulation of unrepaired DNA could encourage hESC transformation, it is imperative to undertake high-resolution SNP analysis when studying genetic stability. Furthermore, most hESCs exhibit normal karyotypes after long-term culture, so high-resolution DNA analysis can identify copy-number variations 5,. And culture-associated genetic aberrations are more likely due to suboptimal culture conditions that select and amplify mutated cells with growth and survival advantages than to an intrinsic property of hESCs.

 

On Differentiation

 

Sue Kimber (University of Manchester) discussed key issues surrounding hESC/hiPSC differentiation in defined conditions by citing an example: generating cells of musculoskeletal lineage 6,. She described an in-house, chemically def ined formulation for hESC maintenance, expansion, and differentiation toward chondrocytes. Stem cells are directed through intermediate developmental stages (primitive streak to mesoderm) before differentiating into chondrocytic cell aggregates in which >95% of cells express the SOX-9 chondrogenic transcription factor with positive CD-44 expression. Importantly, cell aggregates demonstrated negligible expression of genes/proteins linked with nontarget cell types. Kimber reported a simple and eff icient protocol for controlled differentiation that can serve as an experimental tool to study specif ic factors and processes in human cartilage development under def ined in vitro conditions. Such reproducible and scalable protocols are key to studies in developmental biology, disease modeling, drug development, and cell transplantation.

Alexandra Blak highlighted the eff iciency, reproducibility, and speed of a latest development from STEMCELL Technologies: a novel, def ined, and serum-free neural-induction system for hESCs and hiPSCs. This complete system encompasses STEMdiff neural induction medium, AggreWell plates, and STEMdiff neural rosette selection reagent In conjunction with AggreWell plates, the medium is used for neural induction of hPSCs, producing neural rosettes.

Readily recognizable neural rosette structures are a commonly accepted morphological indicator of early neural induction. Expression of early markers such as Pax6, SOX1, and Nestin reveals that rosettes are aggregations of early neural progenitor cells (NPCs). With the system Blak described, neural aggregates are formed and cultured in plates for f ive days, then harvested and plated onto precoated poly-l-ornithine/laminin (PLO/L) tissue culture plates. Rosettes prominently appear, and after a short but critical culture period, the neural rosette selection reagent is used to selectively detach clusters of them.The reagent eliminates laborious manual rosette harvesting techniques, so NPCs can be obtained in ≥12 days for further subculture or differentiation.

So hPSCs adapted to a defined-media system for an extended period can differentiate into highly enriched, functional, neural progenitors under serum-free defined conditions through a straightforward and efficient in vitro protocol. That approach should facilitate development of protocols for controlled differentiation to generate functional neurons, astrocytes, and oligodendrocytes by exposing NPCs to different signaling factors. Both NPCs and end-stage, terminally differentiated neural cells can serve as experimental tools for studying specific factors and processes involved in human neural development under defined in vitro conditions. This potentially scalable system can provide useful insights in studies of early developmental biology, terminal differentiation, and disease modeling.

 

Take-Home message

 

A lively debate followed the scientific presentations. Speakers and attendees discussed their own perspectives, citing personal experiences and challenges encountered during laboratory experiments. Thoughts were again brought up about a universal medium for maintenance and expansion of different hESC lines. Healy said, “We need different formulations for different applications. However, just to grow and expand the cells as pluripotent and self-renewing, we don’t find great batch-to-batch variation with commercially available media.” On in-house media formulations, Andrews commented that because of the batch variation in medium components, researchers in different labs making the same medium often report considerably different results. For instance, albumin variation is challenging because of albumin’s high concentration in culture media and inherent capacity to bind lipids and other impurities. Healy added, “Experience of personnel handling cells also matters: We have to be patient while working with these cells. The overall idea is to transfer media formulations and protocols across different laboratories, so reproducibility of results is of prime importance.”

All participants agreed that this workshop was highly informative and insightful. It provided an excellent platform for academic and industrial scientists to interact and network while delivering on its principal goal of increasing understanding among all in attendance.

About the Author

Author Details
Pawanbir Singh, MBBS, MS, PhD is a product manager for STEMCELL Technologies Inc., Rutherford House, Manchester Science Park, Pencroft Way, Manchester, M15 6SZ, United Kingdom; 44-1616-600325, fax 44-1616-6003-81; pawanbir.singh@stemcell.com; www.stemcell.com.

REFERENCES

5.) Narva, E. 2011. High-Resolution DNA Analysis of Human Embryonic Stem Cell Lines Reveals Culture-Induced Copy Number Changes and Loss of Heterozygosity. Nat. Biotechnol. 28:371-377.

 

6.) Oldershaw, R A Directed Differentiation of Human Embryonic Stem Cells Toward Chondrocytes. Nature Biotech.

 

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