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FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA factors dictate human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in controlling hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA mediated knockdown of FOXA1/2 in HepG2 cells significantly downregulated albumin (p < .05) and GRN TF gene expression (HNF4α, HEX, HNF1ß, TBX3) (p < .05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid [GSC], FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < .05), all consistent with partial/transient reprograming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility of phenotype. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, with concomitant accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During human pluripotent stem cells (hPSC)-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate that FOXA1/2 controls hepatic and developmental GRN, and their knockdown leads to reprogramming of both differentiation and metabolism, with applications in studies of cancer, differentiation, and organogenesis., (© 2022 Federation of American Societies for Experimental Biology.)

Background: The process of liver organogenesis has served as a paradigm for organ formation. However, there remains a lack of understanding regarding early mouse and human liver bud morphogenesis and early liver volumetric growth. Elucidating dynamic changes in liver volumes is critical for understanding organ development, implementing toxicological studies, and for modeling hPSC-derived liver organoid growth. New visualization, analysis, and experimental techniques are desperately needed., Results: Here, we combine observational data with digital resources, new 3D imaging approaches, retrospective analysis of liver volume data, mathematical modeling, and experiments with hPSC-derived liver organoids. Mouse and human liver organogenesis, characterized by exponential growth, demonstrate distinct spatial features and growth curves over time, which we mathematically modeled using Gompertz models. Visualization of liver-epithelial and septum transversum mesenchyme (STM) interactions suggests extended interactions, which together with new spatial features may be responsible for extensive exponential growth. These STM interactions are modeled with a novel in vitro human pluripotent stem cell (hPSC)-derived hepatic organoid system that exhibits cell migration., Conclusions: Our methods enhance our understanding of liver organogenesis, with new 3D visualization, analysis, mathematical modeling, and in vitro models with hPSCs. Our approach highlights mouse and human differences and provides potential hypothesis for further investigation in vitro and in vivo., (© 2021 American Association for Anatomy.)

Three-dimensional (3D) collective cell migration (CCM) is critical for improving liver cell therapies, eliciting mechanisms of liver disease, and modeling human liver development and organogenesis. Mechanisms of CCM differ in 2D vs. 3D systems, and existing models are limited to 2D or transwell-based systems, suggesting there is a need for improved 3D models of CCM. To recreate liver 3D CCM, we engineered in vitro 3D models based upon a morphogenetic transition that occurs during liver organogenesis, which occurs rapidly between E8.5 and E9.5 (mouse). During this morphogenetic transition, 3D CCM exhibits co-migration (multiple cell types), thick-strand interactions with surrounding septum transversum mesenchyme (STM), branching morphogenesis, and 3D interstitial migration. Here, we engineer several 3D in vitro culture systems, each of which mimics one of these processes in vitro . In mixed spheroids bearing both liver cells and uniquely MRC-5 (fetal lung) fibroblasts, we observed evidence of co-migration, and a significant increase in length and number of liver spheroid protrusions, which was highly sensitive to transforming growth factor beta 1 (TGFβ1) stimulation. In MRC-5-conditioned medium (M-CM) experiments, we observed dose-dependent branching morphogenesis associated with an upregulation of Twist1, which was inhibited by a broad TGFβ inhibitor. In models in which liver spheroids and MRC-5 spheroids were co-cultured, we observed complex strand morphogenesis, whereby thin, linear, 3D liver cell strands attach to the MRC-5 spheroid, anchor and thicken to form permanent and thick anchoring contacts between the two spheroids. In these spheroid co-culture models, we also observed spheroid fusion and strong evidence for interstitial migration. In conclusion, we present several novel cultivation systems that recreate distinct features of liver 3D CCM. These methodologies will greatly improve our molecular, cellular, and tissue-scale understanding of liver organogenesis, liver diseases like cancer, and liver cell therapy, and will also serve as a tool to bridge conventional 2D studies and preclinical in vivo studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ogoke, Yousef, Ott, Kalinousky, Lin, Shamul, Ross and Parashurama.)

The field of organoid engineering promises to revolutionize medicine with wide-ranging applications of scientific, engineering, and clinical interest, including precision and personalized medicine, gene editing, drug development, disease modelling, cellular therapy, and human development. Organoids are a three-dimensional (3D) miniature representation of a target organ, are initiated with stem/progenitor cells, and are extremely promising tools with which to model organ function. The biological basis for organoids is that they foster stem cell self-renewal, differentiation, and self-organization, recapitulating 3D tissue structure or function better than two-dimensional (2D) systems. In this review, we first discuss the importance of epithelial organs and the general properties of epithelial cells to provide a context and rationale for organoids of the liver, pancreas, and gall bladder. Next, we develop a general framework to understand self-organization, tissue hierarchy, and organoid cultivation. For each of these areas, we provide a historical context, and review a wide range of both biological and mathematical perspectives that enhance understanding of organoids. Next, we review existing techniques and progress in hepatobiliary and pancreatic organoid engineering. To do this, we review organoids from primary tissues, cell lines, and stem cells, and introduce engineering studies when applicable. We discuss non-invasive assessment of organoids, which can reveal the underlying biological mechanisms and enable improved assays for growth, metabolism, and function. Applications of organoids in cell therapy are also discussed. Taken together, we establish a broad scientific foundation for organoids and provide an in-depth review of hepatic, biliary and pancreatic organoids., (© 2020 Cambridge Philosophical Society.)

Type 1 diabetes mellitus (T1DM) is an autoimmune disease that results from the loss of the pancreatic β-cells. The autoimmune destruction of the β-cells causes the loss of insulin production from the islets of the pancreas, resulting in the loss of blood glucose regulation. This loss of regulation, if not treated, can lead to a plethora of long-term complications in patients. Subsequently, T1DM patients rely on the administration of exogenous insulin sources to maintain their blood glucose levels. In this review, we summarize the history of T1DM therapy and current treatment options. Although treatments for T1DM have progressed substantially, none of the available treatment options allow the patient to live autonomously. Therefore, the challenge to develop a therapy that will fully reverse the disease still remains. A promising field of T1DM therapies is cell replacement therapies derived from human pluripotent stem cells. Here, we specifically review studies that employ stem-cell derived pancreatic progenitors transplanted for in vivo differentiation/maturation and discuss, in detail, the complications that arise post transplantation, including heterogeneity, graft immaturity, and host foreign bodyresponse. We also discuss efforts to induce human stem cell-derived mature β-cells in vitro and compare strategies regarding transplantation of pancreatic progenitors versus mature β-cells cells. Finally, we review key approaches that address critical limitations of in vivo progenitor differentiation including vascularization, oxygenation, and transplant location. The field of islet replacement therapy has made tremendous progress in the last two decades. If the strengths and limitations of the field continue to be identified and addressed, future studies will lead to an ideal treatment for T1DM. Graphical abstract.

SMAD pathways govern epithelial proliferation, and transforming growth factor β (TGF-β and BMP signaling through SMAD members has distinct effects on mammary development and homeostasis. Here, we show that LEFTY1, a secreted inhibitor of NODAL/SMAD2 signaling, is produced by mammary progenitor cells and, concomitantly, suppresses SMAD2 and SMAD5 signaling to promote long-term proliferation of normal and malignant mammary epithelial cells. In contrast, BMP7, a NODAL antagonist with context-dependent functions, is produced by basal cells and restrains progenitor cell proliferation. In normal mouse epithelium, LEFTY1 expression in a subset of luminal cells and rare basal cells opposes BMP7 to promote ductal branching. LEFTY1 binds BMPR2 to suppress BMP7-induced activation of SMAD5, and this LEFTY1-BMPR2 interaction is specific to tumor-initiating cells in triple-negative breast cancer xenografts that rely on LEFTY1 for growth. These results suggest that LEFTY1 is an endogenous dual-SMAD inhibitor and that suppressing its function may represent a therapeutic vulnerability in breast cancer., Competing Interests: Declaration of Interests M.Z. is a co-founder and shareholder of Lefty Labs and Onena Medicines. N.A.L. is a co-founder and shareholder of Lefty Labs and Onena Medicines., (Copyright © 2020. Published by Elsevier Inc.)

Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody-binding protein as an adapter can simplify antibody functionalization by forming a specific antibody-bound complex and introducing site-specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody-binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker. The linker is essential for the increase in affinity because it allows simultaneous binding of both domains. The molecule is further circularized using a split intein, creating a novel adapter protein ("lasso"), which binds human immunoglobulin G1 (IgG1) with K D  = 0.53 n m and a dissociation rate that is 55- to 84-fold slower than Z. The lasso contains a unique cysteine for conjugation with a reporter and may be engineered to introduce other functional groups, including a biotin tag and protease recognition sequences. When used in enzyme-linked immunosorbent assay (ELISA), the lasso generates a stronger reporter signal compared to a secondary antibody and lowers the limit of detection by 12-fold. The small size of the lasso and a long half-life of dissociation make the peptide a useful tool in antibody detection and immobilization., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The reshaping of the world's aging population has created an urgent need for therapies for chronic diseases. Regenerative medicine offers a ray of hope, and its complex solutions include material, cellular, or tissue systems. We review basics of regenerative medicine/stem cells and describe how the field of molecular imaging, which is based on quantitative, noninvasive, imaging of biological events in living subjects, can be applied to regenerative medicine in order to interrogate tissues in innovative, informative, and personalized ways. We consider aspects of regenerative medicine for which molecular imaging will benefit. Next, genetic and nanoparticle-based cell imaging strategies are discussed in detail, with modalities like magnetic resonance imaging, optical imaging (near infra-red, bioluminescence), raman microscopy, and photoacoustic microscopy), ultrasound, computed tomography, single-photon computed tomography, and positron emission tomography. We conclude with a discussion of "next generation" molecular imaging strategies, including imaging host tissues prior to cell/tissue transplantation.

Hippo signaling pathway is an evolutionarily conserved pathway that controls organ size by regulating cell proliferation, apoptosis and stem cell self-renewal. TAZ (transcriptional coactivator with the PDZ-binding motif) is a key downstream effector of the mammalian Hippo pathway. Here, using a transgenic mouse model with mammary-gland-specific expression of constitutively active TAZ, we found that TAZ induction in mammary epithelial cells was associated with an increase in mammary glandular size, which probably resulted from adipocyte hypertrophy. Consistent with its known oncogenic potential, we observed tumor formation in TAZ transgenic mice after administration of the carcinogen 7,12-dimethylbenzanthracene (DMBA) and demonstrated that tumorigenesis was reliant on the presence of TAZ. Our findings establish a previously unknown roles of TAZ in regulating both mammary gland morphogenesis as well as carcinogen-induced mammary tumor formation.

Background: Liver disease contributes significantly to global disease burden and is associated with rising incidence and escalating costs. It is likely that innovative approaches, arising from the emerging field of liver regenerative medicine, will counter these trends., Main Body: Liver regenerative medicine is a rapidly expanding field based on a rich history of basic investigations into the nature of liver structure, physiology, development, regeneration, and function. With a bioengineering perspective, we discuss all major subfields within liver regenerative medicine, focusing on the history, seminal publications, recent progress within these fields, and commercialization efforts. The areas reviewed include fundamental aspects of liver transplantation, liver regeneration, primary hepatocyte cell culture, bioartificial liver, hepatocyte transplantation and liver cell therapies, mouse liver repopulation, adult liver stem cell/progenitor cells, pluripotent stem cells, hepatic microdevices, and decellularized liver grafts., Conclusion: These studies highlight the creative directions of liver regenerative medicine, the collective efforts of scientists, engineers, and doctors, and the bright outlook for a wide range of approaches and applications which will impact patients with liver disease.

In contrast to conventional, molecular medicine that focuses on targeting specific pathways, stem cell therapy aims to perturb many related mechanisms in order to derive therapeutic benefit. This emerging modality is inherently complex due to the variety of cell types that can be used, delivery approaches that need to be optimized in order to target the cellular therapeutic to specific sites in vivo, and non-invasive imaging methods that are needed to monitor cell fate. This review highlights advancements in the field, with focus on recent publications that use preclinical animal models for cardiovascular stem cell therapy. It highlights studies where cell adhesion engineering (CAE) has been used to functionalize stem cells to home them to sites of therapy, much like peripheral blood neutrophils. It also describes the current state of molecular imaging approaches that aim to non-invasively track the spatio-temporal pattern of stem cell delivery in living subjects.

Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow-derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. (©) RSNA, 2016 Online supplemental material is available for this article.

Purpose To quantitatively determine the limit of detection of marrow stromal cells (MSC) after cardiac cell therapy (CCT) in swine by using clinical positron emission tomography (PET) reporter gene imaging and magnetic resonance (MR) imaging with cell prelabeling. Materials and Methods Animal studies were approved by the institutional administrative panel on laboratory animal care. Seven swine received 23 intracardiac cell injections that contained control MSC and cell mixtures of MSC expressing a multimodality triple fusion (TF) reporter gene (MSC-TF) and bearing superparamagnetic iron oxide nanoparticles (NP) (MSC-TF-NP) or NP alone. Clinical MR imaging and PET reporter gene molecular imaging were performed after intravenous injection of the radiotracer fluorine 18-radiolabeled 9-[4-fluoro-3-(hydroxyl methyl) butyl] guanine ((18)F-FHBG). Linear regression analysis of both MR imaging and PET data and nonlinear regression analysis of PET data were performed, accounting for multiple injections per animal. Results MR imaging showed a positive correlation between MSC-TF-NP cell number and dephasing (dark) signal (R(2) = 0.72, P = .0001) and a lower detection limit of at least approximately 1.5 × 10(7) cells. PET reporter gene imaging demonstrated a significant positive correlation between MSC-TF and target-to-background ratio with the linear model (R(2) = 0.88, P = .0001, root mean square error = 0.523) and the nonlinear model (R(2) = 0.99, P = .0001, root mean square error = 0.273) and a lower detection limit of 2.5 × 10(8) cells. Conclusion The authors quantitatively determined the limit of detection of MSC after CCT in swine by using clinical PET reporter gene imaging and clinical MR imaging with cell prelabeling. (©) RSNA, 2016 Online supplemental material is available for this article.

Purpose: Human pluripotency gene networks (PGNs), controlled in part by Oct4, are central to understanding pluripotent stem cells, but current fluorescent reporter genes (RGs) preclude noninvasive assessment of Oct4 dynamics in living subjects., Procedures: To assess Oc4 activity noninvasively, we engineered a mouse embryonic stem cell line which encoded both a pOct4-hrluc (humanized renilla luciferase) reporter and a pUbi-hfluc2-gfp (humanized firefly luciferase 2 fused to green fluorescent protein) reporter., Results: In cell culture, pOct4-hRLUC activity demonstrated a peak at 48 h (day 2) and significant downregulation by 72 h (day 3) (p=0.0001). Studies in living subjects demonstrated significant downregulation in pOct4-hRLUC activity between 12 and 144 h (p = 0.001) and between 12 and 168 h (p = 0.0003). pOct4-hRLUC signal dynamics after implantation was complex, characterized by transient upregulation after initial downregulation in all experiments (n = 10, p = 0.01). As expected, cell culture differentiation of the engineered mouse embryonic stem cell line demonstrated activation of mesendodermal, mesodermal, endodermal, and ectodermal master regulators of differentiation, indicating potency to form all three germ layers., Conclusions: We conclude that the Oct4-hrluc RG system enables noninvasive Oct4 imaging in cell culture and in living subjects.

Performance improvements in instrumentation for optical imaging have contributed greatly to molecular imaging in living subjects. In order to advance molecular imaging in freely moving, untethered subjects, we designed a miniature vertical-cavity surface-emitting laser (VCSEL)-based biosensor measuring 1cm(3) and weighing 0.7g that accurately detects both fluorophore and tumor-targeted molecular probes in small animals. We integrated a critical enabling component, a complementary metal-oxide semiconductor (CMOS) read-out integrated circuit, which digitized the fluorescence signal to achieve autofluorescence-limited sensitivity. After surgical implantation of the lightweight sensor for two weeks, we obtained continuous and dynamic fluorophore measurements while the subject was un-anesthetized and mobile. The technology demonstrated here represents a critical step in the path toward untethered optical sensing using an integrated optoelectronic implant.

Non-Hodgkin lymphoma (NHL) is a heterogeneous and highly disseminated disease, but the mechanisms of its growth and dissemination are not well understood. Using a mouse model of this disease, we used multimodal imaging, including intravital microscopy (IVM) combined with bioluminescence, as a powerful tool to better elucidate NHL progression. We injected enhanced green fluorescent protein and luciferase-expressing Eμ-Myc/Arf(-/-) (Cdkn2a(-/-)) mouse lymphoma cells (EL-Arf(-/-)) into C57BL/6NCrl mice intravenously. Long-term observation inside a peripheral lymph node was enabled by a novel lymph node internal window chamber technique that allows chronic, sequential lymph node imaging under in vivo physiologic conditions. Interestingly, during early stages of tumor progression we found that few if any lymphoma cells homed initially to the inguinal lymph node (ILN), despite clear evidence of lymphoma cells in the bone marrow and spleen. Unexpectedly, we detected a reproducible efflux of lymphoma cells from spleen and bone marrow, concomitant with a massive and synchronous influx of lymphoma cells into the ILN, several days after injection. We confirmed a coordinated efflux/influx of tumor cells by injecting EL-Arf(-/-) lymphoma cells directly into the spleen and observing a burst of lymphoma cells, validating that the burst originated in organs remote from the lymph nodes. Our findings argue that in NHL an efflux of tumor cells from one disease site to another, distant site in which they become established occurs in discrete bursts.

Molecular optical imaging is a widespread technique for interrogating molecular events in living subjects. However, current approaches preclude long-term, continuous measurements in awake, mobile subjects, a strategy crucial in several medical conditions. Consequently, we designed a novel, lightweight miniature biosensor for in vivo continuous optical sensing. The biosensor contains an enclosed vertical-cavity surface-emitting semiconductor laser and an adjacent pair of near-infrared optically filtered detectors. We employed two sensors (dual sensing) to simultaneously interrogate normal and diseased tumor sites. Having established the sensors are precise with phantom and in vivo studies, we performed dual, continuous sensing in tumor (human glioblastoma cells) bearing mice using the targeted molecular probe cRGD-Cy5.5, which targets αVβ3 cell surface integrins in both tumor neovasculature and tumor. The sensors capture the dynamic time-activity curve of the targeted molecular probe. The average tumor to background ratio after signal calibration for cRGD-Cy5.5 injection is approximately 2.43±0.95 at 1 h and 3.64±1.38 at 2 h (N=5 mice), consistent with data obtained with a cooled charge coupled device camera. We conclude that our novel, portable, precise biosensor can be used to evaluate both kinetics and steady state levels of molecular probes in various disease applications.

Poorly regulated tissue remodeling results in increased breast cancer risk, yet how breast cancer stem cells (CSC) participate in remodeling is unknown. We performed in vivo imaging of changes in fluorescent, endogenous duct architecture as a metric for remodeling. First, we quantitatively imaged physiologic remodeling of primary branches of the developing and regenerating mammary tree. To assess CSC-specific remodeling events, we isolated CSC from MMTV-Wnt1 (mouse mammary tumor virus long-term repeat enhancer driving Wnt1 oncogene) breast tumors, a well studied model in which tissue remodeling affects tumorigenesis. We confirm that CSC drive tumorigenesis, suggesting a link between CSC and remodeling. We find that normal, regenerating, and developing gland maintain a specific branching pattern. In contrast, transplantation of CSC results in changes in the branching patterns of endogenous ducts while non-CSC do not. Specifically, in the presence of CSC, we identified an increased number of branches, branch points, ducts which have greater than 40 branches (5/33 for CSC and 0/39 for non-CSC), and histological evidence of increased branching. Moreover, we demonstrate that only CSC implants invade into surrounding stroma with structures similar to developing mammary ducts (nine for CSC and one for non-CSC). Overall, we demonstrate a novel approach for imaging physiologic and pathological remodeling. Furthermore, we identify unique, CSC-specific, remodeling events. Our data suggest that CSC interact with the microenvironment differently than non-CSC, and that this could eventually be a therapeutic approach for targeting CSC., (Copyright © 2012 AlphaMed Press.)

We have fabricated miniature implantable fluorescence sensors for continuous fluorescence sensing applications in living subjects. These monolithically integrated GaAs-based sensors incorporate a 675 nm vertical-cavity surface-emitting laser (VCSEL), a GaAs PIN photodiode, and a fluorescence emission filter. We demonstrate high detection sensitivity for Cy5.5 far-red dye (50 nanoMolar) in living tissue, limited by the intrinsic background autofluorescence. These low cost, sensitive and scalable sensors are promising for long-term continuous monitoring of molecular dynamics for biomedical studies in freely moving animals.

Motivation: Primary purpose of modeling gene regulatory networks for developmental process is to reveal pathways governing the cellular differentiation to specific phenotypes. Knowledge of differentiation network will enable generation of desired cell fates by careful alteration of the governing network by adequate manipulation of cellular environment., Results: We have developed a novel integer programming-based approach to reconstruct the underlying regulatory architecture of differentiating embryonic stem cells from discrete temporal gene expression data. The network reconstruction problem is formulated using inherent features of biological networks: (i) that of cascade architecture which enables treatment of the entire complex network as a set of interconnected modules and (ii) that of sparsity of interconnection between the transcription factors. The developed framework is applied to the system of embryonic stem cells differentiating towards pancreatic lineage. Experimentally determined expression profile dynamics of relevant transcription factors serve as the input to the network identification algorithm. The developed formulation accurately captures many of the known regulatory modes involved in pancreatic differentiation. The predictive capacity of the model is tested by simulating an in silico potential pathway of subsequent differentiation. The predicted pathway is experimentally verified by concurrent differentiation experiments. Experimental results agree well with model predictions, thereby illustrating the predictive accuracy of the proposed algorithm., Contact: ipb1@pitt.edu, Supplementary Information: Supplementary data are available at Bioinformatics online.

One of the major hurdles of cellular therapies for the treatment of liver failure is the low availability of functional human hepatocytes. While embryonic stem (ES) cells represent a potential cell source for therapy, current methods for differentiation result in mixed cell populations or low yields of the cells of interest. Here we describe a rapid, direct differentiation method that yields a homogeneous population of endoderm-like cells with 95% purity. Mouse ES cells cultured on top of collagen-sandwiched hepatocytes differentiated and proliferated into a uniform and homogeneous cell population of endoderm-like cells. The endoderm-like cell population was positive for Foxa2, Sox17, and AFP and could be further differentiated into hepatocyte-like cells, demonstrating hepatic morphology, functionality, and gene and protein expression. Incorporating the hepatocyte-like cells into a bioartificial liver device to treat fulminant hepatic failure improved animal survival, thereby underscoring the therapeutic potential of these cells.

Embryonic stem cell-derived endoderm is critical for the development of cellular therapies for the treatment of disease such as diabetes, liver cirrhosis, or pulmonary emphysema. Here, we describe a novel approach to induce endoderm from mouse embryonic stem (mES) cells using fibronectin-coated collagen gels. This technique results in a homogeneous endoderm-like cell population, demonstrating endoderm-specific gene and protein expression, which remains committed following in vivo transplantation. In this system, activin, normally an endoderm inducer, caused an 80% decrease in the Foxa2-positive endoderm fraction, whereas follistatin increased the Foxa2-positive endoderm fraction to 78%. Our work suggests that activin delays the induction of endoderm through its transient precursors, the epiblast and mesendoderm. Long-term differentiation displays a twofold reduction in hepatic gene expression and threefold reduction in hepatic protein expression of activin-treated cells compared with follistatin-treated cells. Moreover, subcutaneous transplantation of activin-treated cells in a syngeneic mouse generated a heterogeneous teratoma-like mass, suggesting that these were a more primitive population. In contrast, follistatin-treated cells resulted in an encapsulated epithelial-like mass, suggesting that these cells remained committed to the endoderm lineage. In conclusion, we demonstrate a novel technique to induce the direct differentiation of endoderm from mES cells without cell sorting. In addition, our work suggests a new role for activin in induction of the precursors to endoderm and a new endoderm-enrichment technique using follistatin.

Embryonic stem (ES) cells form spontaneous aggregates during differentiation, and cell-cell communication in the aggregates plays an important role in differentiation. The development of a controlled differentiation scheme for ES cells has been hindered by the lack of a reliable method to produce uniform aggregate sizes. Conventional techniques, such as hanging drop and suspension cultures, do not allow precise control over size of ES cell aggregates. To surmount this problem, we microfabricated adhesive stencils to make mouse ES (mES) cell aggregates of specific sizes ranging from 100 microm to 500 microm in diameter. With this technique, we studied the effect of the initial aggregate size on ES cell differentiation. After 20 days of induction of differentiation, we analyzed the stem cell populations using gene and protein expression assays as well as biochemical functions. Notably, we found that germ layer differentiation depends on the initial size of the ES cell aggregate. Among the ES cell aggregate sizes tested, the aggregates with 300 microm diameter showed similar differentiation profiles of three germ layers as embryoid bodies made using the "hanging drop" technique. The smaller (100 microm) aggregates showed the increased expression of ectodermal markers compared to the larger (500 microm) aggregates, while the 500 microm aggregates showed the increased expression of mesodermal and endodermal markers compared to the 100 microm aggregates. These results indicate that the initial size of the aggregate is an important factor for ES cell differentiation, and can affect germ layer selection as well as the extent of differentiation.

Background: Delayed or nonreepithelialization of the large conducting airway (ie, trachea and bronchus) is a clinically recognized but poorly understood result of airway trauma. This delay results in granulation tissue formation and scarring, which impairs mucocilliary transport and can critically compromise gas exchange. Keratinocyte growth factor (KGF) is a known epithelial cell mitogen that is derived from mesenchymal cells. We previously observed its expression in injured tracheal explants, and in the present study we investigated its origin., Design: Freshly isolated porcine tracheal epithelial cells were cytospun onto glass slides for immunohistochemical identification and localization of KGF and for in situ hybridization localization of its messenger RNA. Polymerase chain reaction analysis for KGF was also performed., Results: Freshly isolated respiratory epithelial cells were identified as being of epithelial origin and uncontaminated by fibroblasts, as evidenced by stains that were positive for AE3 and negative for vimentin. Immunohistochemical analysis and in situ hybridization revealed a subset of cells that were positive for both the protein and the message for KGF., Conclusion: This subset of KGF-expressing respiratory epithelial cells may participate in a hitherto undescribed autocrine loop for stimulating KGF production in response to injury.

Mouse F9 embryonic carcinoma 5.51 cells that lack the cytoskeletal protein vinculin spread poorly on extracellular matrix compared with wild-type F9 cells or two vinculin-transfected clones (5.51Vin3 and Vin4; Samuels et al., 1993, J. Cell Biol. 121, 909-921). In the present study, we used this model system to determine how the presence of vinculin promotes cytoskeletal alterations and associated changes in cell shape. Microscopic analysis of cell spreading at early times, revealed that 5.51 cells retained the ability to form filopodia; however, they could not form lamellipodia, assemble stress fibers, or efficiently spread over the culture substrate. Detergent (Triton X-100) studies revealed that these major differences in cell morphology and cytoskeletal organization did not result from differences in levels of total polymerized or cross-linked actin. Biochemical studies showed that 5.51 cells, in addition to lacking vinculin, exhibited slightly reduced levels of alpha-actinin and paxillin in their detergent-insoluble cytoskeleton. The absence of vinculin correlated with a decrease in the mechanical stiffness of the integrin-cytoskeleton linkage, as measured using cell magnetometry. Furthermore, when vinculin was replaced by transfection in 5.51Vin3 and 5.51Vin4 cells, the levels of cytoskeletal-associated alpha-actinin and paxillin, the efficiency of transmembrane mechanical coupling, and the formation of actin stress fibers were all restored to near wild-type levels. These findings suggest that vinculin may promote cell spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, rather than by altering the total level of actin polymerization or cross-linking.