Natesh Parashurama

Homogeneous differentiation of hepatocyte-like cells from embryonic stem cells: applications for the treatment of liver failure

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 hepa‐tocytes 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.—Cho, C. H., Parashurama, N., Park, E. Y. H., Suganuma, K., Nahmias, Y., Park, J., Tilles, A. W., Berthiaume, F., Yarmush, M. L. Homogeneous differentiation of hep‐atocyte‐like cells from embryonic stem cells: applications for the treatment of liver failure. FASEB J. 22, 898–909 (2008)

Implantable semiconductor biosensor for continuous in vivo sensing of far-red fluorescent molecules

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.

Remodeling of Endogenous Mammary Epithelium by Breast Cancer Stem Cells

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. STEM Cells2012;30:2114–2127

Unexpected dissemination patterns in lymphoma progression revealed by serial imaging within a murine lymph node.

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.

Real-time, continuous, fluorescence sensing in a freely-moving subject with an implanted hybrid VCSEL/CMOS biosensor

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.

A low noise current readout architecture for fluorescence detection in living subjects

Optical molecular imaging is emerging as a powerful preclinical research tool for investigating and quantifying molecular events in living subjects, with applications including earlier detection of disease, therapeutic monitoring and understanding fundamental biology [1]. For example, imaging the fluorescent molecular probe RGD-Cy5.5, which specifically binds to molecules (αvβ3 integrin receptors) that regulate new blood vessel growth in tumors, can be used to quantify this growth [2]. Capturing the fluorescent signal in living subjects with an implanted biosensor would enable continuous monitoring of tumors in freely moving subjects. Continuous monitoring in the setting of cancer would give valuable information on tumor progression, both in assessing drug efficacy and detecting recurrent tumor growth after treatment. Presently, fluorescence imaging in living subjects is performed with bulky instrumentation that does not permit continuous monitoring of freely moving subjects over long time periods. In order to make a fluorescence-detection system implantable, and portable, a laser excitation source, a photodetector and a readout circuit for measuring and digitizing photocurrents are integrated in a single package, and continuous fluorescence detection is demonstrated in live animals.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction.

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.

An integer programming formulation to identify the sparse network architecture governing differentiation of embryonic stem cells

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.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part II. In Vivo Imaging of Bone Marrow Stromal Cells in Swine with PET/CT and MR Imaging

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.

Implantable optical biosensor for in vivo molecular imaging

We present the design and fabrication of an implantable fluorescence biosensor suitable for continuously monitored, freely-moving in vivo rodent studies. The GaAs-based semiconductor sensor incorporates an un-cooled photodetector with a 670nm vertical-cavity surface-emitting laser (VCSEL) optimized for sensing fluorescent Cy5.5 dye. For filtering unwanted spectra, a combination of physical and spectral blocking layers yields OD5 excitation rejection at the detector. The sensor detects near-IR fluorescent Cy5.5 molecules in vitro at 100nM concentration (in a 100μL volume) with linear response for concentrations up to 25μM. In a preliminary study in a living mouse, subcutaneously injected dye (1μM Cy5.5 in 50μL) was detected. This technology has the potential to enable new studies of living systems in applications that require long-term, continuous fluorescence sensing.