Enzymes used for passaging human pluripotent stem cells (hPSCs) digest cell surface proteins, resulting in cell damage. Moreover, cell dissociation using divalent cation-free solutions causes apoptosis. Here we report that Mg2+ and Ca2+ control cell-fibronectin and cell-cell binding of hPSCs, respectively, under feeder- and serum-free culture conditions without enzyme. The hPSCs were detached from fibronectin-, vitronectin- or laminin-coated dishes in low concentrations of Mg2+ and remained as large colonies in high concentrations of Ca2+. Using enzyme-free solutions containing Ca2+ without Mg2+, we successfully passaged hPSCs as large cell clumps that showed less damage than cells passaged using a divalent cation-free solution or dispase. Under the same conditions, the undifferentiated and early-differentiated cells could also be harvested as a cell sheet without being split off. Our enzyme-free passage of hPSCs under a serum- and feeder-free culture condition reduces cell damage and facilitates easier and safer cultures of hPSCs.

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for drug screening. For this application, self-renewal or differentiation of the cells is required, and undefined factors in the culture conditions are not desirable. Microfluidic perfusion culture allows the production of small volume cultures with precisely controlled microenvironments, and is applicable to high-throughput cellular environment screening. Here, we developed a microfluidic perfusion culture system for hiPSCs that uses a microchamber array chip under defined extracellular matrix (ECM) and culture medium conditions. By screening various ECMs we determined that fibronectin and laminin are appropriate for microfluidic devices made out of the most popular material, polydimethylsiloxane (PDMS). We found that the growth rate of hiPSCs under pressure-driven perfusion culture conditions was higher than under static culture conditions in the microchamber array. We applied our new system to self-renewal and differentiation cultures of hiPSCs, and immunocytochemical analysis showed that the state of the hiPSCs was successfully controlled. The effects of three antitumor drugs on hiPSCs were comparable between microchamber array and 96-well plates. We believe that our system will be a platform technology for future large-scale screening of fully defined conditions for differentiation cultures on integrated microfluidic devices.

Human induced pluripotent stem cells (hiPSCs) are a promising source of cells for medical applications. Recently, the development of polydimethylsiloxane (PDMS) microdevices to control the microenvironment of hiPSCs has been extensively studied. PDMS surfaces are often treated with low-pressure air plasma to facilitate protein adsorption and cell adhesion. However, undefined molecules present in the serum and extracellular matrix used to culture cells complicate the study of cell adhesion. Here, we studied the effects of vitronectin and γ-globulin on hiPSC adhesion to plasma-treated and untreated PDMS surfaces under defined culture conditions. We chose these proteins because they have opposite properties: vitronectin mediates hiPSC attachment to hydrophilic siliceous surfaces, whereas γ-globulin is adsorbed by hydrophobic surfaces and does not mediate cell adhesion. Immunostaining showed that, when applied separately, vitronectin and γ-globulin were adsorbed by both plasma-treated and untreated PDMS surfaces. In contrast, when PDMS surfaces were exposed to a mixture of the two proteins, vitronectin was preferentially adsorbed onto plasma-treated surfaces, whereas γ-globulin was adsorbed onto untreated surfaces. Human iPSCs adhered to the vitronectin-rich plasma-treated surfaces but not to the γ-globulin-rich untreated surfaces. On the basis of these results, we used perforated masks to prepare plasma-patterned PDMS substrates, which were then used to pattern hiPSCs. The patterned hiPSCs expressed undifferentiated-cell markers and did not escape from the patterned area for at least 7 days. The patterned PDMS could be stored for up to 6 days before hiPSCs were plated. We believe that our results will be useful for the development of hiPSC microdevices.

Thalidomide, which was formerly available commercially to control the symptoms of morning sickness, is a strong teratogen that causes fetal abnormalities. However, the mechanism of thalidomide teratogenicity is not fully understood; thalidomide toxicity is not apparent in rodents, and the use of human embryos is ethically and technically untenable. In this study, we designed an experimental system featuring human-induced pluripotent stem cells (hiPSCs) to investigate the effects of thalidomide. These cells exhibit the same characteristics as those of epiblasts originating from implanted fertilized ova, which give rise to the fetus. Therefore, theoretically, thalidomide exposure during hiPSC differentiation is equivalent to that in the human fetus. We examined the effects of thalidomide on undifferentiated hiPSCs and early-differentiated hiPSCs cultured in media containing bone morphogenetic protein-4, which correspond, respectively, to epiblast (future fetus) and trophoblast (future extra-embryonic tissue). We found that only the number of undifferentiated cells was reduced. In undifferentiated cells, application of thalidomide increased the number of apoptotic and dead cells at day 2 but not day 4. Application of thalidomide did not affect the cell cycle. Furthermore, immunostaining and flow cytometric analysis revealed that thalidomide exposure had no effect on the expression of specific markers of undifferentiated and early trophectodermal differentiated cells. These results suggest that the effect of thalidomide was successfully detected in our experimental system and that thalidomide eliminated a subpopulation of undifferentiated hiPSCs. This study may help to elucidate the mechanisms underlying thalidomide teratogenicity and reveal potential strategies for safely prescribing this drug to pregnant women.

Thalidomide was once administered to pregnant women as a mild sedative; however, it was subsequently shown to be strongly teratogenic. Recently, there has been renewed interest in thalidomide because of its curative effects against intractable diseases. However, the teratogenicity of thalidomide is manifested in various ways and is still not fully understood. In the present study, we evaluated the effects of thalidomide on early mesodermal differentiation by examining the differentiation of human induced pluripotent stem cells (hiPSCs). The most common symptom of thalidomide teratogenicity is limb abnormality, which led us to hypothesize that thalidomide prevents early mesodermal differentiation. Therefore, mesodermal differentiation of hiPSCs was induced over a 6-d period. To induce early mesoderm differentiation, 1 d after seeding, the cells were incubated with the small molecule compound CHIR99021 for 3 d. Thalidomide exposure was initiated at the same time as CHIR99021 treatment. After 5 d of thalidomide exposure, the hiPSCs began expressing a mesodermal marker; however, the number of viable cells decreased significantly as compared to that of control cells. We observed that the proportion of apoptotic and dead cells increased on day 2; however, the proportion of dead cells on day 5 had decreased, suggesting that the cells were damaged by thalidomide during early mesodermal differentiation (days 0-2). Our findings may help elucidate the mechanism underlying thalidomide teratogenicity and bring us closer to the safe use of this drug.