Automated generation of embryoid bodies of specific sizes powered by LEAP

IntroductionDownload as PDF

Embryoid body (EB) formation is a common intermediate during the in vitro differentiation of human embryonic and induced pluripotent stem cells (ESCs and iPSCs) into specialized cell types. EBs are typically generated by removing stem cell colonies from feeder/matrix with collagenase treatment after 5-6 days of culture. Removed colonies are then grown in suspension culture on low attachment plates in differentiation medium. EBs are formed within a few hours to days of suspension culture after which various differentiation strategies are employed. Cultures maintained by enzymatic passage contain variable sized colonies and result in formation of a heterogeneous EB population, varying in size and morphology. Heterogeneous EBs exhibit limited differentiation potential and low production yields (Dang et al. 2004; Ng et al. 2005; Falconnet et al. 2006).

Recent efforts to enrich EBs of preferred size have used biocompatible coatings (Valamehr et al. 2008), microcontact printing (Bauwens et al, 2008), and forced aggregation systems (Burridge et al. 2007). Forced aggregation and microcontact printing are subject to significant variability associated with disaggregating hESCs/hiPSCs into single cell suspensions. In addition, all these techniques are not automated and are limited to enriching for one particular EB size group.

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  Fig. 1. EB cultures produced from confluent human iPSCl cultures using the Automated EB Generator Application. (A, B) iPSC cultures sectioned into 125 µm and 500 µm clumps, respectively. (C, D, E) Resulting EB populations generated from 125 µm, 300 µm, and 500 µm clumps, respectively. EBs produced using this application were more homogeneous than typical EB cultures. (F) Resulting EB population generated by standard methodology using day 5 collagenase passaged iPSC cultures.

The Automated EB Generation Application automates the generation of EBs of specific sizes. This application is powered by the Laser-Enabled Analysis and Processing (LEAP™) platform using laser manipulation of stem cell cultures to produce EB cultures. LEAP operates through laser-mediated in situ elimination of undesired cells without physically manipulating the cells that are preserved.

Approach & Results

Human iPSCs (Kan and Mercola 2009) were cultured in normal growth medium on murine embryonic feeders (MEFs) in 96-well plates to confluence. EBs were generated using the Automated EB Generation Application and EB Generation Kit on LEAP. This application systematically sections confluent iPSC cultures into defined sizes using the laser. Section sizes range from 125 µm (Fig. 1A) to 1000 µm (500 µm shown in Fig. 1B). After sectioning, the cultures were treated with collagenase for 0.5-1.0 hr to dislodge the clumps which were then transferred to low attachment cell culture plates in differentiation medium. Well-defined EBs were formed within a few days of suspension culture.

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  Fig. 2. EB generation using the Automated EB Generator Application results in more uniform EB populations of specific sizes as compared with EBs derived from enzymatically passaged iPSC cultures.

The Automated EB Generation Application produced more consistently sized and morphologically similar EBs (Figs. 1C, D, E) as compared with typical EBs generated from enzymatically passaged iPSCs (i.e., collagenase, Fig. 1F). Resulting EB populations were imaged in brightfield on LEAP after 4 days of suspension culture and the diameter of each EB was determined. Increasing the size of iPSC clumps (section size) resulted in progressively larger EBs 4 days after EB generation. In addition, EBs prepared using the Automated EB Generation Application were significantly more consistent in size as compared with typical EBs prepared using enzymatically passaged cultures. Collagenase-passaged iPSC cultures formed variable sized EBs ranging from 112-736 µm in diameter (335 µm mean, 43% CV). In contrast, EBs generated using the Automated EB Generation Application were significantly more uniform in size; 125 µm iPSC clumps formed 154 µm mean sized EBs (18% CV), 300 µm iPSC clumps formed 291 µm mean sized EBs (14% CV), and 500 µm iPSC clumps formed 392 µm mean sized EBs (14% CV) (Fig. 2).

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  Fig. 3. The Automated EB Generator Application was used to section iPSC cultures into 500 µm clumps, resulting in EB populations with >90% of EBs containing neural rosettes. Rosettes were composed of neural progenitor cells that expressed Pax6.

EBs produced from 125, 200, 300, 400, and 500 µm iPSC clumps were induced to differentiate into neural progenitor cells using standard differentiation protocols. Induction of neural differentiation resulted in EBs containing neural rosettes that expressed Nestin, Sox2, and Pax 6 on days 9-12 in culture (Fig. 3). The number of EBs containing neural rosettes was visually quantified. Greater than 90% of EBs produced from 500 µm iPSC sections contained neural rosettes as compared with <20% of EBs produced from either 125-400 µm iPSC sections or EBs derived from enzymatically passaged iPSC cultures.

Similarly, EBs of varying sizes were induced to differentiate into cardiomyocytes using standard differentiation protocols. Induction of cardiac differentiation resulted in contracting EBs starting on day 8 in culture. The number of contracting EBs was counted visually. Greater than 35% of EBs produced from 400 µm iPSC sections contained beating cardiomyocytes as compared with <2% of EBs produced from 125 and 200 µm iPSC sections, 12% of EBs produced from 300 µm iPSC sections, and 7% of EBs derived from enzymatically passaged iPSC cultures. Mature cardiomyocytes derived from the Automted EB Generator Application expressed cardiac troponin, -actinin, NPPA, and MHC (Fig. 4).

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  Fig. 4. The Automated EB Generator Application was used to section iPSC cultures into 400 µm clumps, resulting in EB populations with >35% of EBs contracting. Beating EBs expressed MHC.

All EB cultures produced by the Automated EB Generator Application maintained a normal growth rate and were capable of forming well-defined EBs that could be differentiated into a variety of cell types derived from all three primary germ layers.

Conclusion

The Automated EB Generation Application on LEAP provides an efficient, automated method of generating uniform EB populations of desired size. EBs produced using this application from human stem cells were successfully differentiated into mature differentiated cell types with higher efficiency than typical heterogeneous EB cultures. The ability to produce homogeneous populations of EBs of particular sizes should dramatically increase the differentiation potential of ESCs and iPSCs into many specialized cell types. The Automated EB Generation Application provides a reproducible method for production of high-quality, large-scale homogeneous EB cultures of any specific size.

References

1. Dang et al, Stem Cells 2004; 22:275.
2. Ng et al, Blood 2005; 106:1601.
3. Falconnet et al, Biomaterials 2006, 27:3044.
4. Valamehr et al, PNAS 2008, 105:14459.
5. Bauwens et al, Stem Cells 2008, 26:2300.
6. Burridge et al, Stem Cells 2007, 25:929.
7. Kan and Mercola. Human iPS cell lines. In prep. 2009.