Nanocasting of fibrous morphology on a substrate for long-term propagation of human induced pluripotent stem cells.

Human-induced pluripotent stem cells (hiPSCs) can be self-renewed for many generations on nanofibrous substrates. Herein, a casting method is developed to replicate the nanofibrous morphology into a thin layer of polymethylsiloxane (PDMS). The template is obtained by electrospinning and chemical crosslinking of gelatin nanofibers on a glass slide. The replicas of the template are surface-functionalized by gelatin and used for propagation of hiPSCs over tenth generations. The performance of the propagated hiPSCs is checked by immunofluorescence imaging, flowcytometry, and RT-PCR, confirming the practicability of this method. The results are also compared to those obtained using electrospun nanofiber substrates. Inherently, the PDMS replica is of low stiffness and can be reproduced easily. Compared to other patterning techniques, casting is more flexible and cost-effective, suggesting that this method might find applications in cell-based assays that rely on stringent consideration of both substrate stiffness and surface morphology.

The role of chaperone complex HSP90-SGT1-RAR1 as the associated machinery for hybrid inviability between Nicotiana gossei Domin and N. tabacum L.

Two cultured cell lines (GTH4 and GTH4S) of a Nicotiana interspecific F1 hybrid (N. gossei × N. tabacum) were comparatively analyzed to find genetic factors related to hybrid inviability. Both cell lines proliferated at 37 °C, but after shifting to 26 °C, GTH4 started to die similar to the F1 hybrid seedlings, whereas GTH4S survived. As cell death requires de novo expression of genes and proteins, we compared expressed protein profiles between the two cell lines, and found that NgSGT1, a cochaperone of the chaperone complex (HSP90-SGT1-RAR1), was expressed in GTH4 but not in GTH4S. Agrobacterium-mediated transient expression of NgSGT1, but not NtSGT1, induced cell death in leaves of N. tabacum, suggesting its possible role in hybrid inviability. Cell death in N. tabacum was also induced by transient expression of NgRAR1, but not NtRAR1. In contrast, transient expression of any parental combinations of three components revealed that NgRAR1 promoted cell death, whereas NtRAR1 suppressed it in N. tabacum. A specific inhibitor of HSP90, geldanamycin, inhibited the progression of hypersensitive response-like cell death in GTH4 and leaf tissue after agroinfiltration. The present study suggested that components of the chaperone complex are involved in the inviability of Nicotiana interspecific hybrid.

Three-dimensional cultured liver-on-a-Chip with mature hepatocyte-like cells derived from human pluripotent stem cells.

Liver-on-a-Chip technology holds considerable potential for applications in drug screening and chemical-safety testing. To establish such platforms, functional hepatocytes are required; however, primary hepatocytes are commonly used, despite problems involving donor limitations, lot-to-lot variation, and unsatisfactory two-dimensional culture methods. Although human pluripotent stem cells (hPSCs) may represent a strong alternative contender to address the aforementioned issues, remaining technological challenges include the robust, highly efficient production of high-purity hepatic clusters. In addition, current Liver-on-a-Chip platforms are relatively complicated and not applicable for high-throughput experiments. Here, we develop a very simple Liver-on-a-Chip platform with mature and functional hepatocyte-like cells derived from hPSCs. To establish a method for hepatic differentiation of hPSCs, cells were first treated by inhibiting the phosphoinositide 3-kinase- and Rho-associated protein kinase-signaling pathways to stop self-renewal and improve survival, respectively, which enabled the formation of a well-defined endoderm and facilitated hepatocyte commitment. Next, a simple microfluidic device was used to create a three-dimensional (3D) culture environment that enhanced the maturation and function of hepatocyte-like cells by increasing the expression of both hepatic maturation markers and cytochrome P450. Finally, we confirmed improvements in hepatic functions, such as drug uptake/excretion capabilities, in >90% of 3D-matured hepatocyte-like cells by indocyanin green assay. These results indicated that the incorporation of hPSC-derived hepatocytes on our Liver-on-a-Chip platform may serve to enhance the processes involved in drug screening and chemical-safety testing.

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

Human Pluripotent Stem Cell-Derived Cardiac Tissue-like Constructs for Repairing the Infarcted Myocardium.

High-purity cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are promising for drug development and myocardial regeneration. However, most hiPSC-derived CMs morphologically and functionally resemble immature rather than adult CMs, which could hamper their application. Here, we obtained high-quality cardiac tissue-like constructs (CTLCs) by cultivating hiPSC-CMs on low-thickness aligned nanofibers made of biodegradable poly(D,L-lactic-co-glycolic acid) polymer. We show that multilayered and elongated CMs could be organized at high density along aligned nanofibers in a simple one-step seeding process, resulting in upregulated cardiac biomarkers and enhanced cardiac functions. When used for drug assessment, CTLCs were much more robust than the 2D conventional control. We also demonstrated the potential of CTLCs for modeling engraftments in vitro and treating myocardial infarction in vivo. Thus, we established a handy framework for cardiac tissue engineering, which holds high potential for pharmaceutical and clinical applications.

Microfluidic-Nanofiber Hybrid Array for Screening of Cellular Microenvironments.

Cellular microenvironments are generally sophisticated, but crucial for regulating the functions of human pluripotent stem cells (hPSCs). Despite tremendous effort in this field, the correlation between the environmental factors-especially the extracellular matrix and soluble cell factors-and the desired cellular functions remains largely unknown because of the lack of appropriate tools to recapitulate in vivo conditions and/or simultaneously evaluate the interplay of different environment factors. Here, a combinatorial platform is developed with integrated microfluidic channels and nanofibers, associated with a method of high-content single-cell analysis, to study the effects of environmental factors on stem cell phenotype. Particular attention is paid to the dependence of hPSC short-term self-renewal on the density and composition of extracellular matrices and initial cell seeding densities. Thus, this combinatorial approach provides insights into the underlying chemical and physical mechanisms that govern stem cell fate decisions.

Nano-on-micro fibrous extracellular matrices for scalable expansion of human ES/iPS cells.

Human pluripotent stem cells (hPSCs) hold great potential for industrial and clinical applications. Clinical-grade scaffolds and high-quality hPSCs are required for cell expansion as well as easy handling and manipulation of the products. Current hPSC culture methods do not fulfill these requirements because of a lack of proper extracellular matrices (ECMs) and cell culture wares. We developed a layered nano-on-micro fibrous cellular matrix mimicking ECM, named "fiber-on-fiber (FF)" matrix, which enables easy handling and manipulation of cultured cells. While non-woven sheets of cellulose and polyglycolic acid were used as a microfiber layer facilitating mechanical stability, electrospun gelatin nanofibers were crosslinked on the microfiber layer, generating a mesh structure with connected nanofibers facilitating cell adhesion and growth. Our results showed that the FF matrix supports effective hPSC culture with maintenance of their pluripotency and normal chromosomes over two months, as well as effective scaled-up expansion, with fold increases of 54.1 ± 15.6 and 40.4 ± 8.4 in cell number per week for H1 human embryonic stem cells and 253G1 human induced pluripotent stem cells, respectively. This simple approach to mimick the ECM may have important implications after further optimization to generate lineage-specific products.

Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions.

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however, although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes, most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here, the authors report a 3D cellular microenvironment plate (3D-CEP), which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-ß-poly(ethylene glycol) hydrogel (HG), which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally, global gene expression analyses are used to elucidate small variations among different test environments. Interestingly, the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine.

Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells.

Nanofibrous gelatin substrates are suited for long-term expansion of human pluripotent stem cells (hPSCs) under feeder- and serum-free culture conditions. A combinatorial library with different sets of processing parameters was established to assess the culture performance of hPSCs on nanofibrous substrates in terms of cell adhesion and growth rate, using Matrigel as control. Then, the optimal conditions were applied to long-term expansion of hPSCs with several cell lines, showing a maintained pluripotency over more than 20 passages without introducing any abnormal chromosome. In addition, this approach allowed us to avoid enzymatic disassociation and mechanic cutting during passages, thereby promoting a better hPSC culture and long-term expansion.