Mechanobiological stimulation in organ-on-a-chip systems reduces hepatic drug metabolic capacity in favor of regenerative specialization.

Hepatic physiology depends on the liver's complex structural composition which among others, provides high oxygen supply rates, locally differential oxygen tension, endothelial paracrine signaling, as well as residual hemodynamic shear stress to resident hepatocytes. While functional improvements were shown by implementing these factors into hepatic culture systems, direct cause-effect relationships are often not well characterized-obfuscating their individual contribution in more complex microphysiological systems. By comparing increasingly complex hepatic in vitro culture systems that gradually implement these parameters, we investigate the influence of the cellular microenvironment to overall hepatic functionality in pharmacological applications. Here, hepatocytes were modulated in terms of oxygen tension and supplementation, endothelial coculture, and exposure to fluid shear stress delineated from oxygen influx. Results from transcriptomic and metabolomic evaluation indicate that particularly oxygen supply rates are critical to enhance cellular functionality-with cellular drug metabolism remaining comparable to physiological conditions after prolonged static culture. Endothelial signaling was found to be a major contributor to differential phenotype formation known as metabolic zonation, indicated by WNT pathway activity. Lastly, oxygen-delineated shear stress was identified to direct cellular fate towards increased hepatic plasticity and regenerative phenotypes at the cost of drug metabolic functionality - in line with regenerative effects observed in vivo. With these results, we provide a systematic evaluation of critical parameters and their impact in hepatic systems. Given their adherence to physiological effects in vivo, this highlights the importance of their implementation in biomimetic devices, such as organ-on-a-chip systems. Considering recent advances in basic liver biology, direct translation of physiological structures into in vitro models is a promising strategy to expand the capabilities of pharmacological models.

A highly efficient cell culture method using oxygen-permeable PDMS-based honeycomb microwells produces functional liver organoids from human induced pluripotent stem cell-derived carboxypeptidase M liver progenitor cells.

Recent advancements in bioengineering have introduced potential alternatives to liver transplantation via the development of self-assembled liver organoids, derived from human-induced pluripotent stem cells (hiPSCs). However, the limited maturity of the tissue makes it challenging to implement this technology on a large scale in clinical settings. In this study, we developed a highly efficient method for generating functional liver organoids from hiPSC-derived carboxypeptidase M liver progenitor cells (CPM+ LPCs), using a microwell structure, and enhanced maturation through direct oxygenation in oxygen-permeable culture plates. We compared the morphology, gene expression profile, and function of the liver organoid with those of cells cultured under conventional conditions using either monolayer or spheroid culture systems. Our results revealed that liver organoids generated using polydimethylsiloxane-based honeycomb microwells significantly exhibited enhanced albumin secretion, hepatic marker expression, and cytochrome P450-mediated metabolism. Additionally, the oxygenated organoids consisted of both hepatocytes and cholangiocytes, which showed increased expression of bile transporter-related genes as well as enhanced bile transport function. Oxygen-permeable polydimethylsiloxane membranes may offer an efficient approach to generating highly mature liver organoids consisting of diverse cell populations.

Optimization of physical microenvironment to maintain the quiescence of human induced pluripotent stem cell-derived hepatic stellate cells.

Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis by producing excessive extracellular matrix (ECM) following chronic inflammation. However, studying HSC function has been challenging due to the limited availability of primary human quiescent HSCs (qHSCs) in vitro, and the fact that primary qHSCs quickly activate when cultured on plastic plates. Advances in stem cell technology have allowed for the generation of qHSCs from human induced pluripotent stem cells (hiPSCs) with the potential to provide an unlimited source of cells. However, differentiated quiescent-like HSCs (iqHSCs) also activate spontaneously on conventional plastic plates. In this study, we generated iqHSCs from hiPSCs and developed a culture method to maintain such iqHSCs in a lowly activated state for up to 5 days by optimizing their physical culture microenvironment. We observed that three-dimensional (3D) culture of iqHSCs in soft type 1 collagen hydrogels significantly inhibited their spontaneous activation in vitro while maintaining their ability to convert to activated state. Activation of iqHSC was successfully modeled by stimulating them with the fibrotic cytokine TGFß1. Hence, our culture method can be used to generate HSCs with functions comparable to those in a healthy liver, facilitating the development of accurate in vitro liver models for identifying novel therapeutic agents.

Transcriptomic and proteomic studies suggest the establishment of advanced zonation-like profiles in human-induced pluripotent stem cell-derived liver sinusoidal endothelial cells and carboxypeptidase M-positive liver progenitor cells cocultured in a fluidic microenvironment.

AIM: Hepatic zonation is a physiological feature of the liver, known to be key in the regulation of the metabolism of nutrients and xenobiotics and the biotransformation of numerous substances. However, the reproduction of this phenomenon remains challenging in vitro as only part of the processes involved in the orchestration and maintenance of zonation are fully understood. The recent advances in organ-on-chip technologies, which allow for the integration of multicellular 3D tissues in a dynamic microenvironment, could offer solutions for the reproduction of zonation within a single culture vessel. METHODS: An in-depth analysis of zonation-related mechanisms observed during the coculture of human-induced pluripotent stem cell (hiPSC)-derived carboxypeptidase M-positive liver progenitor cells and hiPSC-derived liver sinusoidal endothelial cells within a microfluidic biochip was carried out. RESULTS: Hepatic phenotypes were confirmed in terms of albumin secretion, glycogen storage, CYP450 activity, and expression of specific endothelial markers such as PECAM1, RAB5A, and CD109. Further characterization of the patterns observed in the comparison of the transcription factor motif activities, the transcriptomic signature, and the proteomic profile expressed at the inlet and the outlet of the microfluidic biochip confirmed the presence of zonation-like phenomena within the biochips. In particular, differences related to Wnt/ß-catenin, transforming growth factor-ß, mammalian target of rapamycin, hypoxia-inducible factor-1, and AMP-activated protein kinase signaling, to the metabolism of lipids, and cellular remolding were observed. CONCLUSIONS: The present study shows the interest in combining cocultures of hiPSC-derived cellular models and microfluidic technologies for reproducing in vitro complex mechanisms such as liver zonation and further incites the use of those solutions for accurate reproduction of in vivo situations.

A novel agonist for the HGF receptor MET promotes differentiation of human pluripotent stem cells into hepatocyte-like cells.

Hepatocyte growth factor (HGF) is the natural ligand of the MET receptor tyrosine kinase. This ligand-receptor couple is essential for the maturation process of hepatocytes. Previously, the rational design of a synthetic protein based on the assembly of two K1 domains from HGF led to the production of a potent and stable MET receptor agonist. In this study, we compared the effects of K1K1 with HGF during the differentiation of hepatocyte progenitors derived from human induced pluripotent stem cells (hiPSCs). In vitro, K1K1, in the range of 20 to 200 nM, successfully substituted for HGF and efficiently activated ERK downstream signaling. Analysis of the levels of hepatocyte markers showed typical liver mRNA and protein expression (HNF4α, albumin, alpha-fetoprotein, CYP3A4) and phenotypes. Although full maturation was not achieved, the results suggest that K1K1 is an attractive candidate MET agonist suitable for replacing complex and expensive HGF treatments to induce hepatic differentiation of hiPSCs.

Characterization of the proteome and metabolome of human liver sinusoidal endothelial-like cells derived from induced pluripotent stem cells.

The liver is a complex organ composed of several cell types organized hierarchically. Among these, liver sinusoidal endothelial cells (LSECs) are specialized vascular cells known to interact with hepatocytes and hepatic stellate cells (HSCs), and to be involved in the regulation of important hepatic processes in healthy and pathological situations. Protocols for the differentiation of LSECs from human induced pluripotent stem cells, hiPSCs, have been proposed and in-depth analysis by transcriptomic profiling of those cells has been performed. In the present work, an extended analysis of those cells in terms of proteome and metabolome has been implemented. The proteomic analysis confirmed the expression of important endothelial markers and pathways. Among them, the expression of patterns typical of LSECs such as PECAM1, VWF, LYVE1, STAB1 (endothelial markers), CDH13, CDH5, CLDN5, ICAM1, MCAM-CD146, ICAM2, ESAM (endothelial cytoskeleton), NOSTRIN, NOS3 (Nitric Oxide endothelial ROS), ESM1, ENG, MMRN2, THBS1, ANGPT2 (angiogenesis), CD93, MRC1 (mannose receptor), CLEC14A (C-type lectin), CD40 (antigen), and ERG (transcription factor) was highlighted. Besides, the pathway analysis revealed the enrichment of the endocytosis, Toll-like receptor, Nod-like receptor, Wnt, Apelin, VEGF, cGMP-PCK, and PPAR related signaling pathways. Other important pathways such as vasopressin regulated water reabsorption, fluid shear stress, relaxin signaling, and renin secretion were also highlighted. At confluence, the metabolome profile appeared consistent with quiescent endothelial cell patterns. The integration of both proteome and metabolome datasets revealed a switch from fatty acid synthesis in undifferentiated hiPSCs to a fatty oxidation in LSECs and activation of the pentose phosphate pathway and polyamine metabolism in hiPSCs-derived LSECs. In conclusion, the comparison between the signature of LSECs differentiated following the protocol described in this work, and data found in the literature confirmed the particular relevance of these cells for future in vitro applications.

Integrating Oxygen and 3D Cell Culture System: A Simple Tool to Elucidate the Cell Fate Decision of hiPSCs.

Oxygen, as an external environmental factor, plays a role in the early differentiation of human stem cells, such as induced pluripotent stem cells (hiPSCs). However, the effect of oxygen concentration on the early-stage differentiation of hiPSC is not fully understood, especially in 3D aggregate cultures. In this study, we cultivated the 3D aggregation of hiPSCs on oxygen-permeable microwells under different oxygen concentrations ranging from 2.5 to 20% and found that the aggregates became larger, corresponding to the increase in oxygen level. In a low oxygen environment, the glycolytic pathway was more profound, and the differentiation markers of the three germ layers were upregulated, suggesting that the oxygen concentration can function as a regulator of differentiation during the early stage of development. In conclusion, culturing stem cells on oxygen-permeable microwells may serve as a platform to investigate the effect of oxygen concentration on diverse cell fate decisions during development.

Early C-reactive protein kinetics predict survival of patients with advanced urothelial cancer treated with pembrolizumab.

OBJECTIVE: To assess the prognostic and predictive ability of early C-reactive protein (CRP) kinetics, dynamic changes in CRP levels, in patients with advanced urothelial cancer treated with pembrolizumab. PATIENTS AND METHODS: We retrospectively evaluated 97 patients with advanced urothelial cancer treated with pembrolizumab in second-line or later settings. Patients were divided into three early CRP kinetics groups: non-elevated (baseline CRP < 5 mg/L), responder (baseline CRP ≥ 5 mg/L and CRP decreased below baseline at least once within 30 days), and non-responder (baseline CRP ≥ 5 mg/L and CRP never decreased to baseline within 30 days). Association between early CRP kinetics and pembrolizumab efficacy including objective response rate (ORR), disease control rate (DCR), and overall survival (OS) were evaluated. RESULTS: Based on early CRP kinetics, 40, 27, and 30 patients were classified as non-elevated, responder, and non-responder, respectively. ORR and DCR were 33% and 60% in non-elevated, 30% and 48% in responder, and 17% and 40% in non-responder; without a statistically significant difference. OS was significantly different among the non-elevated, responder, and non-responder groups (p < 0.01), with 1-year survival rates of 69%, 61%, and 31%, respectively. Early CRP kinetics could discriminate the OS of patients without objective response. Non-responder was an independent predictor for OS (HR 3.65, p < 0.01), as well as liver metastasis and ECOG PS ≥ 2. CONCLUSION: Early CRP kinetics is associated with survival of advanced urothelial cancer patients treated with pembrolizumab and could be a potential biomarker for clinical benefit from immune checkpoint inhibitors.

Multi-omics analysis of hiPSCs-derived HLCs matured on-chip revealed patterns typical of liver regeneration.

Maturation of human-induced pluripotent stem cells (hiPSCs)-derived hepatocytes-like cells (HLCs) toward a complete hepatocyte phenotype remains a challenge as primitiveness patterns are still commonly observed. In this study, we propose a modified differentiation protocol for those cells which includes a prematuration in Petri dishes and a maturation in microfluidic biochip. For the first time, a large range of biomolecular families has been extracted from the same sample to combine transcriptomic, proteomic, and metabolomic analysis. After integration, these datasets revealed specific molecular patterns and highlighted the hepatic regeneration profile in biochips. Overall, biochips exhibited processes of cell proliferation and inflammation (via TGFB1) coupled with anti-fibrotic signaling (via angiotensin 1-7, ATR-2, and MASR). Moreover, cultures in this condition displayed physiological lipid-carbohydrate homeostasis (notably via PPAR, cholesterol metabolism, and bile synthesis) coupled with cell respiration through advanced oxidative phosphorylation (through the overexpression of proteins from the third and fourth complex). The results presented provide an original overview of the complex mechanisms involved in liver regeneration using an advanced in vitro organ-on-chip technology.

Antiresorptive agent-related osteonecrosis of the jaw (ARONJ) in urological malignancies: a multi-center retrospective study.

INTRODUCTION: We evaluated the incidence and risk factors for antiresorptive agent-related osteonecrosis of the jaw (ARONJ) in prostate and kidney cancer patients. MATERIALS AND METHODS: We retrospectively reviewed the clinical data of 547 patients from 13 hospitals. Prostate and kidney cancer patients with bone metastases who were treated with a bone-modifying agent (BMA) between January 2012 and February 2019 were enrolled. Exclusion criteria were BMA use for hypercalcemia, a lack of clinical data, a follow-up period of less than 28 days and a lack of evaluation by dentists before BMA administration. The diagnosis and staging of ARONJ were done by dentists. RESULTS: Two-hundred eighteen patients were finally enrolled in the study, including 168 prostate cancer patients and 50 kidney cancer patients. Of them, 49 (29%) prostate cancer patients and 18 (36%) kidney cancer patients needed tooth extraction prior to BMA initiation. The mean follow-up period after BMA initiation was 552.9 ± 424.7 days (mean ± SD). In the cohort, 23% of the patients were diagnosed with ARONJ in the follow-up period. The 1-year cumulative incidences of ARONJ were 9.4% and 15.4% in prostate and kidney cancer patients, respectively. Multivariate analysis indicated that kidney cancer, tooth extraction before BMA and a body mass index (BMI) ≥ 25 kg/m2 were significant predictors for ARONJ. CONCLUSION: ARONJ is not a rare adverse event in urological malignancies. Especially, kidney cancer, high BMI patients and who needed tooth extraction before BMA were high risk for developing ARONJ.

Ab Initio and RRKM/Master Equation Analysis of the Photolysis and Thermal Unimolecular Decomposition of Bromoacetaldehyde.

Bromoacetaldehyde (BrCH2CHO) is a major stable brominated organic intermediate of the bromine-ethylene addition reaction during the arctic bromine explosion events. Similar to acetaldehyde, which has been recently identified as a source of organic acids in the troposphere, it may be subjected to photo-tautomerization initially forming brominated vinyl compounds. In this study, we investigate the unimolecular reactions of BrCH2CHO under both photolytic and thermal conditions using high-level quantum chemical calculations and Rice-Ramsperger-Kassel-Marcus (RRKM)/master equation analysis. The unimolecular decomposition of BrCH2CHO takes place through 14 dissociation and isomerization channels along a potential energy surface involving eight wells. Under the assumption of singlet ground-state potential energy surface-dominated photodynamics, the primary photodissociation yields of BrCH2CHO are investigated under both collision-free and collision energy transfer conditions. At atmospheric pressure and under tropospheric actinic flux conditions at ground level, depending on the assumed collisional energy transfer parameter, 150 cm-1 < ⟨ΔEdown⟩ < 450 cm-1, 78-33% of BrCH2CHO undergoes direct photodissociation instead of collisional deactivation at an excitation wavelength of 320 nm. This is significantly higher than the 14% reported for acetaldehyde, hence indicating a strong effect of bromine substitution on the product photolysis yield that is related to additional favorable Br and HBr forming dissociation channels. In contrast to the overall photodissociation quantum yield, the relative branching fractions of the photodissociation products are less dependent on the collisional energy transfer parameter. For a representative value of ⟨ΔEdown⟩ = 300 cm-1 and an excitation wavelength of 320 nm, with 27% for C-C bond fission, 11% for C-Br bond fission, 7% for HBr elimination, and only below 2% each for a consecutive O-Br fission reaction and the photo-tautomerization channel yielding brominated vinyl alcohol, the photodissociation is markedly different from the acetaldehyde case. Finally, as brominated halogenated compounds are of interest for flame inhibition purposes, thermal multichannel unimolecular rate constants were calculated for temperatures in the range from 500 to 2000 K. At a temperature of 2000 K and ambient pressure, the two main reaction channels are the C-Br and C-C bond fissions, contributing 35 and 43% to the total reaction flux, respectively.

Screening of chronic radiation proctitis and colorectal cancer using periodic total colonoscopy after external beam radiation therapy for prostate cancer.

OBJECTIVE: To investigate the incidence of colorectal cancer and chronic radiation proctitis after prostate radiotherapy using periodic total colonoscopy screening. METHODS: From February 2013 to January 2018, 270 patients who underwent external beam radiation therapy for prostate cancer were advised to receive periodic total colonoscopy screening annually. We evaluated the incidence and characteristics of colorectal cancer and chronic radiation proctitis. RESULTS: First, second, third, fourth and fifth total colonoscopy were performed in 256 (95%), 151 (56%), 60 (22%), 23 (8.5%) and 7 (2.6%) patients at a median of 14, 31, 42, 54 and 72 months after radiotherapy, respectively. The prevalence proportion of colorectal cancer in the first colonoscopy since radiotherapy was 3.9%. Twelve (4.4%) patients were diagnosed with colorectal cancer, including four invasive cancers, during a follow-up period. Eight of these 12 patients had not experienced rectal bleeding. The median time to diagnosis of colorectal cancer was 21 months. Chronic radiation proctitis was observed in 136 (50%) patients, including 67 (25%) patients with symptomatic bleeding. CONCLUSIONS: The high detection rate of asymptomatic radiation proctitis suggests the utility of total colonoscopy to screen for early-stage colorectal cancer prior to or following radiotherapy for prostate cancer. Considering the longevity after localized prostate cancer treatment, the awareness of chronic radiation-induced proctitis and the risk of colorectal cancer masked by bleeding is needed in treatment decision -making.

Metabolomic profiling during the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

Human induced pluripotent stem cells (hiPSCs) are potentially an invaluable source of cells for regenerative medicine, disease modeling and drug discovery. However, the differentiation of hiPSCs into fully functional hepatocytes remains a major challenge. Despite the importance of the information carried by metabolomes, the exploitation of metabolomics for characterizing and understanding hiPSC differentiation remains largely unexplored. Here, to increase knowledge of hiPSC maturation into mature hepatocytes, we investigated their metabolomics profiles during sequential step-by-step differentiation: definitive endoderm (DE), specification into hepatocytes (HB-pro (hepatoblast progenitors)), progenitor hepatocytes (Pro-HEP) and mature hepatocyte-like cells (HLCs). Metabolomics analysis illustrated a switch from glycolysis-based respiration in DE step to oxidative phosphorylation in HLCs step. DE was characterized by fatty acid beta oxidation, sorbitol metabolism and pentose phosphate pathway, and glutamine and glucose metabolisms as various potential energy sources. The complex lipid metabolism switch was monitored via the reduction of lipid production from DE to HLCs step, whereas high glycerol production occurred mainly in HLCs. The nitrogen cycle, via urea production, was also a typical mechanism revealed in HLCs step. Our analysis may contribute to better understanding of differentiation and suggest new targets for improving iPSC maturation into functional hepatocytes.

Analysis of hiPSCs differentiation toward hepatocyte-like cells upon extended exposition to oncostatin.

The capability to produce and maintain functional human adult hepatocytes remains one of the major challenges for the use of in-vitro models toward liver cell therapy and industrial drug-screening applications. Among the suggested strategies to solve this issue, the use of human-induced pluripotent stem cells (hiPSCs), differentiated toward hepatocyte-like cells (HLCs) is promising. In this work, we propose a 31-day long protocol, that includes a final 14-day long phase of oncostatin treatment, as opposed to a 7-day treatment which led to the formation of a hepatic tissue functional for CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP3A4. The production of albumin, as well as bile acid metabolism and transport, were also detected. Transcriptome profile comparisons and liver transcription factors (TFs) motif dynamics revealed increased expression of typical hepatic markers such as HNF1A and of important metabolic markers like PPARA. The performed analysis has allowed for the extraction of potential targets and pathways which would allow enhanced hepatic maturation in-vitro. From this investigation, NRF1 and SP3 appeared as transcription factors of importance. Complex epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) patterns were also observed during the differentiation process. Moreover, whole transcriptome analysis highlighted a response typical of the one observed in liver regeneration and hepatocyte proliferation. While a complete maturation of hepatocytes was yet to be obtained, the results presented in this work provide new insights into the process of liver development and highlight potential targets aimed to improve in-vitro liver regeneration.

The Reaction NCN + H2: Quantum Chemical Calculations, Role of 1NCN Chemistry, and 3NCN Absorption Cross Section.

The NCN radical plays a key role for modeling prompt-NO formation in hydrocarbon flames. Recently, in a combined shock tube and flame modeling study, the so far neglected reaction NCN + H2 and the related chemistry of the main product HNCN turned out to be significant for NO modeling under fuel-rich conditions. In this study, the reaction has been thoroughly revisited by detailed quantum chemical rate constant calculations both for the singlet 1NCN and triplet 3NCN pathways. Optimized geometries and vibrational frequencies of reactants, products, and transition states were calculated on B3LYP/aug-cc-pVQZ level with single-point energy calculations carried out against the optimized structures using CASPT2/aug-cc-pVQZ. The determined rate constants for the 1NCN + H2 reaction as well as the newly measured high temperature absorption cross section of 3NCN made a reevaluation of the shock tube data of the previous work necessary, finally revealing quantitative agreement between experiment and theory. Moreover, the new directly measured Doppler-limited absorption cross section data, σ(3NCN, λ = 329.1302 nm) = 2.63 × 109 × exp(-1.96 × 10-3 × T/K) cm2/mol (±23%, p = 0 bar, T = 870-1700 K), are in agreement with previously reported values based on detailed spectroscopic simulations. Hence, a long-standing debate about a reliable high temperature 3NCN absorption cross section has been resolved. Whereas 3NCN + H2 resembles a simple abstraction type reaction with the exclusive products HNCN + H, the singlet radical reaction is initiated by the insertion into the H-H bond. Up to pressures of 100 bar, the main products of the subsequent decomposition of the H2NCN intermediate are HNCN + H as well, with minor contributions of CN + NH2 toward higher temperatures. Although much faster than the triplet reaction, the singlet radical insertion is actually rather slow, due to the necessary reorganization of the HOMO electron density in 1NCN that is equally distributed over the two N atom sites. In general, the distinct reactivity differences call for a separate treatment of 1NCN and 3NCN chemistry. However, as the main reaction products in case of the H2 reaction are the same and as the population of the 1NCN in thermal equilibrium remains low, a properly weighted effective rate constant k(NCN + H2 → HNCN + H) = 2.62 × 104 × (T/K)2.78 × exp(-97.6 kJ/mol/RT) cm3 mol-1s-1(±30%, 800 K < T < 3000 K, p < 100 bar) is recommended for inclusion into flame models that, as yet, do not explicitly account for 1NCN chemistry.

Simplest Chemosensor Array for Phosphorylated Saccharides.

We believe that "the simpler we are, the more complete we become" is a key concept of chemical sensing systems. In this work, a "turn-on" fluorescence chemosensor array relying on only two self-assembled molecular chemosensors with ability of both qualitative and quantitative detection of phosphorylated saccharides has been developed. The easy-to-prepare chemosensor array was fabricated by in situ mixing of off-the-shelf reagents (esculetin, 4-methylesculetin, and 3-nitrophenylboronic acid). The fluorescence-based saccharide sensing system was carried out using indicator displacement assay accompanied by photoinduced electron transfer (PeT) under various pH conditions. The simultaneous recognition of 14 types of saccharides including glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) was achieved with a successful classification rate of 100%. We also succeeded in the quantitative analysis of a mixture of glucose (Glc), as an original substrate, G6P and F6P, as enzymatic products in pseudoglycolysis pathway. Finally, levels of Glc and F6P in human induced pluripotent stem (hiPS) cells were indirectly monitored by using our proposed chemosensor array. Glc and F6P in supernatants of hiPS cells were classified by linear discriminant analysis as a pattern recognition model and the observed clusters represent the activity of hiPS cells. The results show the high accuracy of the proposed chemosensor array in detection of phosphorylated and similarly modified saccharides.

Cytoplasmic fusion between an enlarged embryonic stem cell and a somatic cell using a microtunnel device.

Based on a previous finding that fusion of a somatic cell with an embryonic stem (ES) cell reprogrammed the somatic cell, genes for reprogramming transcription factors were selected and induced pluripotent stem (iPS) cell technology was developed. The cell fusion itself produced a tetraploid cell. To avoid nuclear fusion, a method for cytoplasmic fusion using a microtunnel device was developed. However, the ES cell was too small for cell pairing at the device. Therefore, in the present study, ES cell enlargement was carried out with the colchicine derivative demecolcine (DC). DC induced enlargement of ES cells without loss of their stemness. When an enlarged ES cell was paired with a somatic cell in the microtunnel device, cytoplasmic fusion was observed. The present method may be useful for further development of reprogramming techniques for iPS cell preparation without gene transfection.

Optimized protocol for the hepatic differentiation of induced pluripotent stem cells in a fluidic microenvironment.

In the present study, we evaluated the performance of different protocols for the hepatic differentiation of human-induced pluripotent stem cells (hiPSCs) in microfluidic biochips. Strategies for complete and partial on-chip differentiation were tested. Unlike full on-chip differentiation, the transfer of iPSCs from Petri dishes to biochips during the differentiation process produced a heterogeneous tissue with enhanced hepatic features compared with control cultures in Petri dishes. The tissue in biochips was constituted of cells expressing either stabilin-1 or albumin, while no stabilin-1 was detected in controls. Functional analysis also revealed double the production rate for albumin in biochips (about 2,000 ng per day per 106 cells). Besides this, tissues obtained in biochips and controls exhibited the metabolism of a specific bile acid. Whole transcriptome analysis with nanoCAGE exhibited a differential expression of 302 genes between control and biochip cultures and a higher degree of hepatic differentiation in biochips, together with increased promoter motif activity for typical liver transcription factors such as estrogen related receptor alpha ( ESRRA), hepatic nuclear factor 1 ( HNF1A), hepatic nuclear factor 4 ( HNF4A), transcription factor 4 ( TCF4), and CCAAT enhancer binding protein alpha ( CEBPA). Gene set enrichment analysis identified several pathways related to the extracellular matrix, tissue reorganization, hypoxia-inducible transcription factor, and glycolysis that were differentially modulated in biochip cultures. However, the presence of CK19/ALB-positive cells and the ɑ-fetoprotein levels measured in the cultures still reflect primitive differentiation patterns. Overall, we identified key parameters for improved hepatic differentiation on-chip, including the maturation stage of hepatic progenitors, inoculation density, adhesion time, and perfusion flow rate. Optimization of these parameters further led to establish a protocol for reproducible differentiation of hiPSCs into hepatocyte-like cells in microfluidic biochips with significant improvements over Petri dish cultures.

Size-Controlled Preparation of Microsized Perfluorocarbon Emulsions as Oxygen Carriers via the Shirasu Porous Glass Membrane Emulsification Technique.

We have developed microsized perfluorocarbon (PFC) emulsions with different sizes as artificial oxygen carriers (OCs) via Shirasu porous glass membrane emulsification. Monodispersed PFC emulsions with narrow size distribution were obtained. By changing the membrane pore size, we were able to precisely control the size of emulsions and fabricate emulsions similar in size to human red blood cells. Behaviors of Pluronics with different physiochemical properties (F127, F68, P85, and P103) as surfactants were also investigated, which evidenced that the type and concentration of Pluronics have a major impact on the size of emulsions and the response to different thermal conditions. The F127-stabilized microsized PFC emulsions were stable even during autoclave sterilization. The emulsions were loaded with Ru(ddp)-an oxygen-sensitive probe-on their surfaces to indicate oxygen concentration. Finally, incubations with HeLa cells that show fluorescence in response to hypoxia cultured in 2D and 3D suggested promising potential of our emulsions for OCs.

Switching of Cell Proliferation/Differentiation in Thiol-Maleimide Clickable Microcapsules Triggered by in Situ Conjugation of Biomimetic Peptides.

Extracellular environments significantly affect cell proliferation, differentiation, and functions. The extracellular environment changes during many physiological and pathological processes such as embryo development, wound healing, and tumor growth. To mimic these changes, we developed novel thiol-maleimide clickable alginate microcapsules, which can introduce thiol-containing peptides by " in situ conjugation" with maleimide-modified alginate, even in serum-containing cell culture media. Additive peptides were rapidly concentrated into microcapsules by a diffusion-reaction process in the capsule. The proliferation of encapsulated fibroblasts was accelerated by in situ conjugation of CRGDS, while free RGDS showed no effect. Moreover, encapsulated preosteoblastic cells started osteogenic differentiation via in situ conjugation of BMP-2 mimetic peptides such as CDWIVA and CG-BMP-2 knuckle epitope peptide, while BMP-2 did not induce differentiation of the encapsulated cells. Especially in tissue engineering, accurate and inexpensive methods for inducing cell differentiation are required. We believe that this in situ conjugation approach employing various functional peptides will be useful in biomedical, bioindustrial, and biochemical fields in the future.