A simple tool for the synchronous differentiation of human induced pluripotent stem cells into pancreatic progenitors.

Efficient differentiation of human induced pluripotent stem cells (hiPSCs) into functional pancreatic cells holds great promise for diabetes research and treatment. However, a robust culture strategy for producing pancreatic progenitors with high homogeneity is lacking. Here, we established a simple differentiation strategy for generating synchronous iPSC-derived pancreatic progenitors via a two-step method of sequential cell synchronization using botulinum hemagglutinin (HA), an E-cadherin function-blocking agent. Of the various methods tested, the first-step synchronization method with HA exposure induces a synchronous switch from E- to N-cadherin and N- to E-cadherin expression by spatially controlling heterogeneous cell distribution, subsequently improving their competency for directed differentiation into definitive endodermal cells from iPSCs. The iPSC-derived definitive endodermal cells can efficiently generate PDX1+ and NKX6.1+ pancreatic progenitor cells in high yields. The PDX1+ and PDX1+ /NKX6.1+ cell densities showed 1.6- and 2.2-fold increases, respectively, compared with those from unsynchronized cultures. The intra-run and inter-run coefficient of variation were below 10%, indicating stable and robust differentiation across different cultures and runs. Our approach is a simple and efficient strategy to produce large quantities of differentiated cells with the highest homogeneity during multistage pancreatic progenitor differentiation, providing a potential tool for guided differentiation of iPSCs to functional insulin-producing cells.

Detachable-dissolvable-microneedle as a potent subunit vaccine delivery device that requires no cold-chain.

Although vaccine administration by microneedles has been demonstrated, delivery reliability issues have prevented their implementation. Through an ex vivo porcine skin experiment, we show visual evidence indicating that detachable dissolvable microneedles (DDMN) can deposit cargo into the dermis with insignificant loss of cargo to the stratum corneum. Using ovalbumin (OVA), a model antigen vaccine, as a cargo, the ex vivo experiments yielded a delivery efficiency of 86.08 ± 4.16 %. At room temperature, OVA could be stabilized for up to 35 days in DDMN made from hyaluronic acid and trehalose. The DDMN matrix could improve the denaturation temperature of the OVA from around 70-120 °C to over 150 °C, as demonstrated by differential scanning calorimetric analysis. In vivo delivery of OVA antigen into the mice's skin via DDMN elicited 10 times higher specific antibody responses compared to conventional intramuscular injection. We envision DDMN as an effective, precise dosing, intradermal vaccine delivery system that may require no cold-chain, offers a dose-sparing effect, and can be administered easily.

An in vitro culture platform for studying the effect of collective cell migration on spatial self-organization within induced pluripotent stem cell colonies.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) provide an in vitro system to identify the impact of cell behavior on the earliest stages of cell fate specification during human development. Here, we developed an hiPSC-based model to study the effect of collective cell migration in meso-endodermal lineage segregation and cell fate decisions through the control of space confinement using a detachable ring culture system. RESULTS: The actomyosin organization of cells at the edge of undifferentiated colonies formed in a ring barrier differed from that of the cells in the center of the colony. In addition, even in the absence of exogenous supplements, ectoderm, mesoderm, endoderm, and extraembryonic cells differentiated following the induction of collective cell migration at the colony edge by removing the ring-barrier. However, when collective cell migration was inhibited by blocking E-cadherin function, this fate decision within an hiPSC colony was altered to an ectodermal fate. Furthermore, the induction of collective cell migration at the colony edge using an endodermal induction media enhanced endodermal differentiation efficiency in association with cadherin switching, which is involved in the epithelial-mesenchymal transition. CONCLUSIONS: Our findings suggest that collective cell migration can be an effective way to drive the segregation of mesoderm and endoderm lineages, and cell fate decisions of hiPSCs.

Novel strategy to improve hepatocyte differentiation stability through synchronized behavior-driven mechanical memory of iPSCs.

Cellular homeostasis is assumed to be regulated by the coordination of dynamic behaviors. Lack of efficient methods for synchronizing large quantities of cells makes studying cell culture strategies for bioprocess development challenging. Here, we demonstrate a novel application of botulinum hemagglutinin (HA), an E-cadherin function-blocking agent, to synchronize behavior-driven mechanical memory in human induced pluripotent stem cell (hiPSC) cultures. Application of HA to hiPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration-and time-dependent manner. Interestingly, cytoskeleton rearrangement in cells with prolonged exposure to HA resulted in mechanical memory synchronization with Yes-associated protein, which increased pluripotent cell homogeneity. Synchronized hiPSCs have higher capability to differentiate into functional hepatocytes than unsynchronized hiPSCs, resulting in improved efficiency and robustness of hepatocyte differentiation. Thus, our strategy for cell behavior synchronization before differentiation induction provides an approach against the instability of differentiation of pluripotent cells.

Stable and efficient generation of functional iPSC-derived neural progenitor cell rosettes through regulation of collective cell-cell behavior.

Although the potential of stem cells to differentiate into several cell types has shown promise in regenerative medicine, low differentiation efficiency and poor reproducibility significantly limit their practical application. We developed an effective and robust differentiation strategy for the efficient and robust generation of neural progenitor cell rosettes from induced pluripotent stem cells (iPSCs) incorporating botulinum hemagglutinin (HA). Treatment with HA suppressed the spontaneous differentiation of iPSCs cultured under undirected differentiation conditions, resulting in the preservation of their pluripotency. Moreover, treatment with HA during neural progenitor differentiation combined with dual SMAD inhibition generated a highly homogeneous population of PAX6-and SOX1-expressing neural progenitor cells with 8.4-fold higher yields of neural progenitor cells than untreated control cultures. These neural progenitor cells formed radially organized rosettes surrounding the central lumen. This differentiation method enhanced the generation of functional iPSC-derived neural progenitor cell rosettes throughout the culture vessel, suggesting that the regulation of collective cell-cell behavior using HA plays a morphogenetically important role in rosette formation and maturation. These findings show the significance of HA in the suppression of spontaneous differentiation through spatial homogeneity. The study proposes a novel methodology for the efficient derivation of functional iPSC-derived neural progenitor cell rosettes.

Cell Behavioral Dynamics as a Cue in Optimizing Culture Stabilization in the Bioprocessing of Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) are important for future regenerative medicine therapies. However, in the production of PSCs and derivatives, the control of culture-induced fluctuations in the outcome of cell quality remains challenging. A detailed mechanistic understanding of how PSC behaviors are altered in response to biomechanical microenvironments within a culture is necessary for rational bioprocessing optimization. In this review, we discuss recent insights into the role of cell behavioral and mechanical homeostasis in modulating the states and functions of PSCs during culture processes. We delineate promising ways to manipulate the culture variability through regulating cell behaviors using currently developed tools. Furthermore, we anticipate their potential implementation for designing a culture strategy based on the concept of Waddington's epigenetic landscape that may provide a feasible solution for tuning the culture quality and stability in the bioprocessing space.

Effect of initial seeding density on cell behavior-driven epigenetic memory and preferential lineage differentiation of human iPSCs.

Understanding the cellular behavioral mechanisms underlying memory formation and maintenance in human induced pluripotent stem cell (hiPSC) culture provides key strategies for achieving stability and robustness of cell differentiation. Here, we show that changes in cell behavior-driven epigenetic memory of hiPSC cultures alter their pluripotent state and subsequent differentiation. Interestingly, pluripotency-associated genes were activated during the entire cell growth phases along with increased active modifications and decreased repressive modifications. This memory effect can last several days in the long-term stationary phase and was sustained in the aspect of cell behavioral changes after subculture. Further, changes in growth-related cell behavior were found to induce nucleoskeletal reorganization and active versus repressive modifications, thereby enabling hiPSCs to change their differentiation potential. Overall, we discuss the cell behavior-driven epigenetic memory induced by the culture environment, and the effect of previous memory on cell lineage specification in the process of hiPSC differentiation.

The impact of culture dimensionality on behavioral epigenetic memory contributing to pluripotent state of iPS cells.

Three-dimensional (3D) culture platforms have been explored to establish physiologically relevant cell culture environment and permit expansion scalability; however, little is known about the mechanisms underlying the regulation of pluripotency of human induced pluripotent stem cells (hiPSCs). This study elucidated epigenetic modifications contributing to pluripotency of hiPSCs in response to 3D culture. Unlike two-dimensional (2D) monolayer cultures, 3D cultured cells aggregated with each other to form ball-like aggregates. 2D cultured cells expressed elevated levels of Rac1 and RhoA; however, Rac1 level was significantly lower while RhoA level was persisted in 3D aggregates. Compared with 2D monolayers, the 3D aggregates also exhibited significantly lower myosin phosphorylation. Histone methylation analysis revealed remarkable H3K4me3 upregulation and H3K27me3 maintenance throughout the duration of 3D culture; in addition, we observed the existence of naïve pluripotency signatures in cells grown in 3D culture. These results demonstrated that hiPSCs adapted to 3D culture through alteration of the Rho-Rho kinase-phospho-myosin pathway, influencing the epigenetic modifications and transcriptional expression of pluripotency-associated factors. These results may help design culture environments for stable and high-quality hiPSCs.

Investigation of FoxO3 dynamics during erythroblast development in ß-thalassemia major.

The FoxO3 transcription factor is a key regulator of oxidative stress and erythroid maturation during erythropoiesis. In this study, we explored the involvement of FoxO3 in severe ß-thalassemia. Using primary CD34+ hematopoietic progenitor cells from patients with ß-thalassemia major, we successfully developed an in vitro model of ineffective erythropoiesis. Based on this model, FoxO3 activity was quantified in single cells using high throughput imaging flow cytometry. This study revealed a significant reduction of FoxO3 activity during the late stage of erythroblast differentiation in ß-thalassemia, in contrast to erythropoiesis in normal cells that maintain persistent activation of FoxO3. In agreement with the decreased FoxO3 activity in ß-thalassemia, the expression of FoxO3 target genes was also found to decrease, concurrent with elevated phosphorylation of AKT, most clearly at the late stage of erythroid differentiation. Our findings provide further evidence for the involvement of FoxO3 during terminal erythropoiesis and confirm the modulation of the PI3K/AKT pathway as a potential therapeutic strategy for ß-thalassemia.