Abstract
With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.
Highlights
Stem cells have been studied for nearly 50 years and while our understanding of them has increased immensely, major questions such as their molecular identity, level of plasticity, and role in pathological conditions and aging, still remain to be answered
Culture conditions vary and embryonic stem (ES) cells are often cultured adherently on gelatin coatings or fibroblast feeder cells, whereas adult neural stem cells are maintained as free-floating sphere-like clusters, called neurosphere (NS) cultures
Maintenance of pluri- or multipotency in different stem cell populations depends on various factors such as leukemia inhibitory factor (LIF) [3,4], epidermal growth factor (EGF), and basic fibroblast growth factor [2,5,6]
Summary
Stem cells have been studied for nearly 50 years and while our understanding of them has increased immensely, major questions such as their molecular identity, level of plasticity, and role in pathological conditions and aging, still remain to be answered. Stem cells are identified by their functional characteristics; multipotency and self-renewing capability. Adult neural stem cells can be maintained in culture for several passages and display multipotency by generating neurons, astrocytes, and oligodendrocytes upon differentiation. Mammalian embryonic and adult neural stem cells have been successfully isolated and maintained in vitro [1,2]. Maintenance of pluri- or multipotency in different stem cell populations depends on various factors such as leukemia inhibitory factor (LIF) [3,4], epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) [2,5,6]. New factors are constantly connected to stem cells regulating their maintenance or differentiation, e.g. growth factors, epigenetic modifiers, neurotransmitters, and extracellular matrix proteins
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