Workshop 2: Cell & Tissue Engineering

(October 22,2007 - October 24,2007 )

Organizers


Melissa Knothe Tate
Biomedical Engineering, Case Western Reserve University
Stanislav Shvartsman
Chemical Engineering, Princeton University

The challenges of tissue engineering include the need to overcome mass transport limitations, to exploit biophysical stimuli in enhancing engineered tissue function, to address anisotropic structural requirements in "manufacturing" of the tissue, immunological considerations, as well as the challenge of designing "off the shelf technology" that will be applicable for the needs of specific patients. Engineered tissues of the future will serve as multifunctional molecular delivery devices that provide a microenvironment conducive to cell infiltration and cultivation. To address and optimize these multiple functions, tissue engineers have begun to exploit principles of transport and biophysical stimuli as well as to hone knowledge of cell recruitment, adhesion, migration, proliferation, and differentiation to improve success of cell cultivation in tissue scaffolds. Interestingly, the focus of many tissue engineering symposia appears to be specific to tissue type or a clinical problem; yet, by organizing symposia that bridge across length and time scales, diverse organisms and tissue types, experimental models (in vivo, in vitro, in silico), as well as bioscience and engineering disciplines, it may be possible for the sub-specialists to recognize common themes and solutions that have wide applicability across a variety of tissues. Hence, the goal of this workshop is to bring developmental biologists, cell and tissue engineers, as well as computational modelers together at a joint forum, bridging across specific cell and tissue types as well as model platforms, to recognize common challenges and relevant strategies for addressing these challenges in tissues from diverse organisms, including plants, drosophila, zebrafish and humans. The promise of predictive modeling in accelerating advances in the field of tissue engineering will be highlighted.

Accepted Speakers

Dany Adams
Forsyth Center for Regenerative and Developmental Biology, Harvard University
David Arnosti
Dept. of Biochemistry and Molecular Biology, Michigan State University
Scott Barolo
Dept. of Cell & Developmental Biology, University of Michigan
Celeste Berg
Department of Genome Sciences, University of Washington
Siobhan Brady
Biology Department, Duke University
Farshid Guilak
Department of Surgery, Duke University
Nathalia Holtzman
Department of Biology, Queens College, CUNY
Lars Hufnagel
Kavli Institute for Theoretical Physics, University of California, Santa Barbara
Melissa Knothe Tate
Biomedical Engineering, Case Western Reserve University
Young Kwon
Department of Biomedical Engineerin, Case Western Reserve University
Ed Munro
Center for Cell Dynamics, University of Washington
Stanislav Shvartsman
Chemical Engineering, Princeton University
Yvonne Stokes
Applied Math, University of Adelaide
Rocky Tuan
National Institute of Arthritis, and Musculoskeletal & Skin Diseases, National Institutes of Health
Monday, October 22, 2007
Time Session
09:00 AM
09:45 AM
Stanislav Shvartsman - Engineering models of epithelial patterning in Drosophila oogenesis

N/A

10:00 AM
10:45 AM
David Arnosti - Identification of a transcriptional cis-regulatory grammar in the Drosophila embryo by quantitative modeling

Control of gene expression in eukaryotic organisms is directed by regulatory sequences in the DNA that serve as molecular switches to activate and repress transcription. Unlike the canonical genetic code that specifies protein structure, the regulatory code that dictates the design of transcription control sequences is loosely constrained and difficult to identify using simple bioinformatic methods. We have identified specific aspects of a transcriptional regulatory grammar that dictates how patterning genes are controlled by short-range repressors such as Giant and Knirps in the developing Drosophila embryo. Confocal laser imaging allows us to quantitatively analyze gene expression, leading to "maps" of gene regulatory surfaces associated with particular arrays of regulatory sequences carried by transgenic lines. We use this information in the development of a three-tier quantitative mathematical model that associates potential functions with subelements of the enhancer, and combines them to provide predictions of the functional output of novel transcriptional elements. With this approach, we have accurately predicted the effects of spacing between activators and repressors, a key feature of short-range repression. This modeling is now being extended to analyze modifications in factor stoichiometry, binding affinity, and arrangement, providing detailed insight into cis regulatory element architecture. We are also adapting a fractional occupancy model that considers specific features of spacing and cooperativity in predicting gene activity. Our work complements "top down" approaches, and is aimed at informing and extending the power of current models of endogenous cis-regulatory elements to develop powerful bioinformatic tools applicable to population and evolutionary studies.

11:00 AM
11:45 AM
Jeremy Zartman - The Expression and Regulation of Cad74A in Drosophila Oogenesis

During Drosophila oogenesis, a two-dimensional follicular epithelium gives rise to an elaborate three-dimensional eggshell. Eggshell morphogenesis critically depends on the patterning of the follicle cells, but the connection between signaling pathways, pattern formation, and eggshell morphogenesis is not well understood. Recently we have identified the non-classical cadherin, Cad74A, as a key molecule that bridges epithelial patterning and the morphogenetic dynamics in this system. Starting in mid-oogenesis, Cad74A is expressed in all the columnar cells except for two dorsolateral patches, in the border cells, and later in the future operculum domain. Using a number of mutant backgrounds and related fly species, we show that this pattern is correlated with the formation of multiple structural features of the eggshell, suggesting that Cad74A plays an important functional role on oogenesis. We provide data that strongly suggest that the dynamic pattern of Cad74A expression is partially controlled by the EGFR and Dpp signaling pathways, two of the key regulators of follicle cell patterning. On the basis of these results, we propose that Cad74A provides an important link between signaling, pattern formation and morphogenesis during egg development.


Work done in collaboration with N. Yakoby, C.A. Bristow, and S.Y. Shvartsman.


 

11:00 AM
12:00 AM
Ahmet Ay - Computational cis-regulatory Transcriptional Grammar and Developing Predictive Mathematical Models

Cis-regulatory information comprises a key portion of genetic coding, yet despite the abundance of genomic sequences now available, identifying and characterizing this information remains a major challenge. We are pursuing a unique "bottom up" approach to understand the mechanistic processing of regulatory elements (input codes) by the transcriptional machinery, using a well defined and characterized set of repressors and activators in Drosophila blastoderm embryos. We are identifying quantitative values for parameters affecting transcriptional regulation in vivo, and these parameters are used to build and test mathematical models that predict the outputs of novel cis-regulatory elements.


Giant, Kruppel, Knirps are short-range transcriptional repressors involved in the developmental patterning of Drosophila blastoderm embryo. Using defined regulatory modules tested in germ line transformed embryos, we are measuring quantitative parameters describing the effects of spacing, stoichiometry, arrangement and binding site affinities of these repressors on cassettes driven by endogenous activators. To develop predictive models we employ a hybrid thermodynamic gene regulation model in which we included variables for the spacing effect between activators and repressors. This model is being used to predict the output of novel permutations of binding sites, which will allow us test and refine parameters used. In one line of investigation, fluorescence quantization of lacZ reporter gene expression was used to measure the effect of moving Giant repressor binding sites from a position adjacent to Twist/Dorsal activator sites to a distal site 125bp away. Our mathematical model successfully predicted the distance effect of intermediate positions, such as 25, 50, 75 and 100bp compared to experimental results. Further tests will illustrate the effects of site permutation, transcription factor concentration and activator/repressor stoichiometry and arrangement. Extension of these predictive models to endogenous cis elements will provide novel insights on regulatory element design and evolution, and should provide a bioinformatics method for predicting quantitative output of novel regulatory elements.


Work done in collaboration with Walid D. Fakhouri, Chichia Chiu, and David N. Arnosti.

11:00 AM
11:45 AM
- Integration of intrinsic and extrinsic cues to achieve appropriate scale in developing organisms

Organ size depends on two developmental forces: an intrinsic system comprised of intercellular patterning molecules, and an extrinsic system that regulates body size in response to nutrition. The two systems must be integrated, so that the sizes and patterns of individual organs scale appropriately with body size. I am studying the nature of this integration, using the Drosophila wing. My approach addresses how systemic Insulin signalling - the level of which determines adult body size - affects the Decapentaplegic and Wingless morphogen gradients that pattern the wing. I have evidence suggesting that insulin signalling affects all 3 processes involved in gradient formation: the level of morphogen, the rate of morphogen movement across the developing wing, and the degradation of the morphogen in recipient cells. The hope is to obtain a picture of how the wing's patterning system can systematically incorporate nutrient variability, enabling a perfect wing pattern to arise across a range of wing sizes. One objective of this work is to delineate the system with sufficient clarity for the purposes of mathematical modelling. It is hoped that such an in silico study would permit us to predict the theoretical size range permitted by the wing plasticity mechanism, help us understand the basis of wing allometry (size-dependent shape variation), and discover potential developmental constraints that may limit wing size evolution.

01:00 PM
01:45 PM
Siobhan Brady - Transcriptionally rich and complex spatiotemporal programs in Arabidopsis root development

Transcriptional programs that regulate development are exquisitely controlled in space and time. Elucidating these programs that underlie development is essential to understanding the acquisition of cell and tissue identity. We present microarray expression profiles of a high resolution set of developmental time points within a single Arabidopsis root, and a comprehensive map of nearly all root cell-types. These cell-type specific transcriptional signatures often predict novel cellular functions. A computational pipeline identified dominant expression patterns that demonstrate transcriptional similarity between disparate cell-types. Dominant expression patterns along the root's longitudinal axis do not strictly correlate with previously defined developmental zones, and in many cases, expression fluctuation along this axis was observed. Both robust co-regulation of gene expression and potential phasing of gene expression were identified between individual roots. Methods that combine these profiles demonstrate transcriptionally rich and complex programs that define Arabidopsis root development in both space and time.

02:00 PM
02:45 PM
Scott Barolo - How to Build an Enhancer: Principles of Cell- and Tissue-Specific Gene Expression

Enhancers, or cis-regulatory elements, control gene expression both qualitatively and quantitatively (i.e., patterns and levels). Enhancers contain clusters of binding sites for transcription factors (TFs), which act as activators or repressors of transcription. Despite 25 years of work on enhancers, we are still not able to build "synthetic" elements (combinations of TF binding sites) that function as normal enhancers in vivo, and must rely on "found" enhancers to drive targeted gene expression, both in model organism research and in potential disease therapy/tissue engineering strategies. The fact that we can't build an enhancer from scratch suggests that we don't understand the basic functional principles of the enhancer, its basic components, or its structure.


We have undertaken a fine-scale transgenic structure-function analysis of a signal-regulated, cell-type-specific enhancer of a gene essential for normal eye development. The sparkling enhancer of the dPax2 gene is a direct target of Notch and EGFR signaling in cone cells of the developing Drosophila eye (Flores et al., Cell 103:75). We find that a synthetic sparkling element, made by simply combining the twelve known transcription factor binding sites within sparkling, is unable to activate gene expression in vivo, even when the sites are placed in their native arrangement and spacing. Our in vivo analysis of the sparkling enhancer has revealed several interesting features of its transcriptional activity in vivo:



  • Sequence: A surprisingly large number and high density of novel regulatory sites; rapid evolutionary turnover of critical regulatory sequences.

  • Structure: Extreme structural flexibility; a role for binding site arrangement in patterning of gene expression.

  • Mechanism: Evidence for a novel "promoter communication element" within the enhancer, which is required only for gene activation at a distance.


 


If time permits, I will briefly describe another project, in which we are finding a link between low-affinity transcription factor binding sites and tissue-specificity of the transcriptional response to the Hedgehog pathway.


The work was done primarily by Christina I. Rogers, with assistance from David Schwimmer, Nicole C. Evans, and me.

03:15 PM
04:15 PM
Alejandro Almarza - Changes in Gene Expression of Passaged Bone Marrow Derived Cells in Culture

Introduction: Functional tissue engineering (FTE) efforts to regenerate musculoskeletal tissues require large numbers of cells, often obtained through cell passage. One study involving MCL fibroblasts showed an increase of collagen type I gene expression and relative consistence of collagen type III gene expression throughout passage, indicating that MCL fibroblasts may be a good cell source for FTE approaches involving ligaments and tendons whose main constituent is collagen type I (1). Bone marrow derived cells (BMDCs) may be a more attractive cell source because unlike MCL fibroblasts, BMDCs can be obtained from the bone marrow by needles without any noticeable harm to the body, are auto-transplantable, easily expandible and have the potential to differentiate into various lineages (2). The objective of this study was to quantify gene expression of Collagen I, Collagen III, and GAPDH through passages in rat BMDCs.


Methods: Isolated BMDCs of three rats and cells were centrifuged, counted, and plated on 2 cm^2 wells until 95% confluency (P1). Ascorbic acid (50 mg/ml) added to medium and cells plated at 300,000/well for one more passage. Cells counted with hemacytometer and RNA isolated with a kit after each passage (n=3). Real time RT-PCR was performed to determine gene expression of Collagen I, Collagen III, and GAPDH. An ANOVA was performed to determine differences between passages (P < 0.05) with Fisher's post hoc test.


Results: Collagen type I gene expression significantly increased at P2 compared to native tissue (P0) or P1 cells (p < 0.05). Collagen type III gene expression significantly increased at P2 when compared to native tissue (P0) or P1 cells and magnitude of collagen type III gene expression is much greater than that of collagen type I gene expression in later passages (p < 0.05).


Discussion: Despite the increase in collagen type I gene expression, the drastic increase of collagen type III gene expression indicates that BMDCs may not be an ideal cell source for FTE approaches to regenerate tendons and ligaments. Future studies may include quantifying collagen type I and III gene expressions from P2 to P5 and measuring the gene expression of other important molecules such as growth factors and metalloproteases.



  1. Almarza, AA., Augustine, S., Woo, S L-Y. (2007) Effect of Passage on Ligament Fibroblasts: Implications for Functional Tissue Engineering. 7th International Symposium on Ligaments and Tendons. San Diego, CA. Febraury 2007.

  2. Pittenger, MF et al. (1999). Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science 284, 143.


 


Work done in collaboration with Augustine S., and Woo S.L-Y.

03:15 PM
04:15 PM
Sara McBride - Cell seeding conditions exert a profound influence on the developmental context of embryonic mesenchymal stem cells for subsequent tissue generation

Cell density, cell shape, and substrate elasticity are powerful modulators of stem cell fate [1-3].Our working hypothesis is that the manner by which cell densities are achieved, i.e. cell seeding conditions, is also a powerful modulator of cell differentiation. Specifically, we hypothesize that, for a target density to be achieved (i) cells seeded at a relatively low density (5,000 cells/cm^2) and allowed to mature to a high target density through population doublings will preferentially form sheet-like epithelial structures and (ii) cells seeded immediately at the target high density (86,500 cells/cm^2) will preferentially form 3D mesenchymal structures. (iii) Furthermore, while the target high densities can be achieved with both seeding methods, initial cell seeding conditions influence the commitment of cells to specific lineages by defining the developmental context of embryonic mesenchymal stem cells for subsequent tissue generation.C3H10T1/2 multipotent progenitor cells were seeded on glass coverslips at 5,000 cells/cm^2 and given 48, 72, or 96 hours to proliferate to low (16,500 cells/cm^2), high (35,000 cells/cm^2), and very high (86,500 cells/cm^2) density cultures. An additional set of coverslips was seeded immediately at these three densities and given 24 hours to attach. In this way, we created density-matched groups that differ in maturity or developmental context. For each independent group, three sets of data were analyzed with real time RT-PCR (n=3 per group) and analyzed for genes, including Collagen type IaI, Osterix, Runx2, Msx2, Collagen type II, Sox9, Aggrecan, and Ppar?. Additional samples were fixed and stained to assess the structure of the seeded cell constructs with microscopy. All density groups, from LD to VHD, show similar patterns of gene up- and down-regulation for all genes studied. However, the magnitude of change in activity from baseline is highest for groups where density is achieved by population doublings. Nonetheless, LD groups show little modulation of gene activity attributable to seeding conditions. Cell seeding conditions exert a profound influence on the developmental context of embryonic mesenchymal stem cells for subsequent differentiation and tissue generation. This study shows, for the first time to our knowledge, that not only the density at which cells are seeded is important in determining subsequent gene activity related to lineage commitment, but also the manner by which cell densities are achieved. Histological studies of cell constructs provide further support regarding the nature of the cell constructs formed as a consequence of cell seeding conditions.



  1. McBeath et al. Dev Cell 2004

  2. Chen et al. Science 1997

  3. Engler et al. Cell 2006

  4. Johnstone et al. Exp Cell Res 1998


 


Work done in collaboration with Melissa Knothe Tate.

03:15 PM
04:15 PM
Rui Liang - Effects of a bioscaffold on the fibrillogenesis in the healing medial collateral ligament

Porcine small intestine submucosa (SIS) has been successfully used to improve the healing of ligaments and tendons [1]. In a rabbit model, the healing medial collateral ligament (MCL) with SIS treatment was found to have larger collagen fibril formation with concomitant reduction in collagen type V/I ratio [2]. Thus, we hypothesized that the SIS bioscaffold could affect the regulation of the gene expressions of collagen fibrillogenesis-related molecules at the early stage and result in improved fibril morphology and organization. In the current study, our objective was to examine the organization of the extracellular matrix, measure the collagen fibril diameter, and analyze the gene expressions of the fibrillogenesis-related molecules, specifically, collagen types I, III, V and small leucine-rich proteoglycans including decorin, biglycan, lumican and fibromodulin, in the healing rabbit MCL treated with SIS at 6 weeks post-injury. Twenty rabbits were equally divided into two groups. In the SIS-treated group, a 6 mm gap was surgically created in the right MCL and a layer of SIS was sutured covering the gap. For the non-treated group, the gap-injured MCLs remained untreated. All the left MCLs were sham operated and used as controls. At 6 weeks, Masson trichrome's staining was used to examine the organization of collagen-based extracellular matrix. Collagen fibril diameters and distribution were calculated and analyzed from the transmission electron micrographs. Gene expressions of the fibrillogenesis-related molecules were detected with quantitative real time PCR. The results revealed that in the SIS-treated group the collagen fibers were more regularly aligned as well as the cell nuclei than that for the non-treated group. Additionally, in the SIS-treated group larger collagen fibrils (90 - 120nm) appeared around the fibroblasts and the range of the fibril diameter distribution was from 24 to 120nm while the collagen fibrils in the non-treated group were uniformly small and the distribution of fibril diameter was limited from 26 to 87 nm. Simultaneously, the gene expressions of collagen type V, decorin, biglycan and lumican were downregulated by 41%, 58%, 43% and 51%, respectively (p < 0.05). The present results suggested that the significant reduction in the gene expressions of fibrillogenesis-related molecules in the healing MCLs following SIS treatment are closely related to the improved morphological characteristics, which are known to be coupled to the enhanced mechanical properties observed in the long term as reported in published studies.


References: 1) Woo et al. AJSM, 15: 22-29, 1987; 2) Liang et al, JOR, 24: 811-9, 2006.


Acknowledgments: Support form NIH Grant #AR41820 is gratefully acknowledged. Also, Cook Biotech, Inc. for supplying the SIS.


Work done in collaboration with T. Nguyen, R. Nola, A. Almarza, and S. L-Y Woo.

Tuesday, October 23, 2007
Time Session
09:00 AM
09:45 AM
Ed Munro - Cytomechanics of polarization and morphogenesis in early embryos

The cellular rearrangements and tissue deformations that shape embryos during animal development emerge from the local force generating behaviors of many individual cells. Molecular genetics and biochemistry has identified much of the basic molecular machinery responsible for the production, regulation and transmission of cellular forces. But to understand tissue-level morphogenesis we must comprehend: (a) how this machinery is organized dynamically within individual cells through an interplay between biochemistry and mechanics, and (b) how local forces generated within each of many individual cells are integrated across whole tissues to produce stereotyped patterns of cell shape change and rearrangement. Our approach to addressing these problems combines experimental analyses of cellular and subcellular dynamics with detailed computer simulations. I will illustrate this approach using several examples drawn from our recent work: The first concerns how a robust mechanism for convergent extension of embryonic tissues emerges from the conserved machinery underlying protrusive, contractile and adhesive force generation in single motile cells. The second involves new insights into the cellular mechanisms of invagination gained from the study of a stripped-down version of invagination in ascidian embryos. The third concerns how a mechanism for polarization of single cells emerges from interactions among the PAR proteins - conserved regulators of cell polarity - and the actomyosin cytoskeleton.

10:00 AM
10:45 AM
Celeste Berg - Setting up and interacting across boundaries: Tube formation in Drosophila egg chambers

The reorganization of flat epithelial sheets into tubes is a fundamental process in the formation of many organs, such as the lungs, kidneys, gut, and neural tube. This process involves the patterning of distinct cell types and the coordination of those cells during shape changes, rearrangements, and other behaviors essential to the function of the tube. To study tube formation, we are analyzing the signaling pathways that define and control sub populations within the dorsal-anterior region of the Drosophila follicular epithelium. These cells reorganize to form two closed tubes; secretion of chorion proteins into the tube lumens creates two dorso-lateral eggshell appendages, which facilitate respiration in the developing embryo. EGF and BMP signals define the two primordia and then Notch signaling subdivides the region further and establishes a boundary between "roof" and "floor" cells. Roof cells constrict their apices and converge toward the dorsal midline, lengthening the tube, while floor cells elongate underneath the roof cells and fuse apically to seal the tube. Our goal is to understand how these cells coordinate their behaviors to generate tubes of specific size and shape. Work done in collaboration with Jennie B. Dorman and Ellen J. Ward.

11:15 AM
12:00 PM
Nathalia Holtzman - Endocardial-myocardial interactions direct cell migration during heart tube formation

The embryonic heart tube develops from a simple cylinder, who's foundation is built through the union of bilateral populations of cardiomyocytes. Cardiomyocyte interactions with the neighboring gut endoderm are crucial for recruitment of cardiomyocytes toward the embryonic midline. However, it is not clear if the endoderm is sufficient to direct all the cell movements required for heart formation. In addition, little is known about the morphogenetic and cellular mechanisms that direct heart tube assembly once cardiomyocytes reach the midline. Using time-lapse confocal microscopy to track individual cardiomyocyte movements in the zebrafish embryo, we identify two morphologically and genetically separable phases of cell movement that underlie tube assembly. Specifically, we find an initial phase of endoderm dependent movement during which cardiomyocytes undergo coherent medial movement bringing cells to the midline. This is followed by an endoderm independent phase of movement, whereby groups of peripherally located cardiomyocytes change their direction of movement and angle toward the centrally located vascular endocardium. This pattern of cell behavior suggests that the endocardium influences cardiomyocyte behavior. Indeed, through examination of zebrafish mutants, we find that the induction, direction, and duration of cardiomyocyte movement depend upon myocardial-endocardial interactions. Thus, our studies indicate that the endoderm is required primarily to bring the cardiomyocytes to the midline at which point interactions with the endocardium plays a critical early role in further myocardial morphogenesis, positioning cardiomyocytes into a configuration appropriate for heart tube assembly. To begin to address the role of the endocardium directly, we have begun a molecular and cellular analysis of this poorly understood tissue. Our current findings will be presented.


Work done in collaboration with Huai-Jen Tsai and Deborah Yelon.

02:30 PM
03:15 PM
Dany Adams - Biophysical Regulation of Morphogenesis in Development and Regeneration: A New Target for Clinical Intervention and the Need for Modeling

Data from the Forsyth Center for Regenerative and Developmental Biology is bringing to light the fundamental importance of ion flux during early stages of Development and Regeneration. Currently, there is exploding interest in using research from these fields to drive discovery of drugs and other medical interventions. Thus, now is a particularly exciting time to be studying biophysical signaling, in part because the vast majority of research focuses on genetic regulation of biochemical pathways. Our approach has been to apply the tools of molecular biology and mathematical modeling to long recognized, but understudied bioelectric phenomena, and that approach has proved very rewarding. Our data on early patterning of vertebrate embryos, regulation of planarian stem cells, and induction of regeneration in tadpole tails, indicate that understanding of ion-flux mediated signaling pathways could result in brand new categories of biomedical approaches as well as new paradigms for understanding the interplay of genetic and epigenetic phenomena. We believe that thinking in terms of functional relatedness (physiological parameter space) rather than genetic relatedness, will bring important, complimentary insight to our understanding of biological phenomena.

03:30 PM
04:15 PM
Lars Hufnagel - Regulation of Growth during Development: Role of Mechanics

A fundamental and unresolved problem in animal development is the question of how a growing tissue knows when it has achieved its correct final size. A widely held view suggests that this process is controlled by morphogen gradients, which adapt to tissue size and become flatter as tissue grows, leading eventually to growth arrest. I will discuss the spatio-temporal dynamics of that the decapentaplegic (Dpp) morphogen distribution in the developing Drosophila wing imaginal disk and present an alternative model for wing size determination and proliferation control in tissues. I will also present results on the transition of an irregularly packed epithelium to a hexagonally ordered geometry.



 
04:30 PM
05:00 PM
Yvonne Stokes - Mathematical modeling towards successful in vitro maturation of mammalian oocytes

Better understanding of the nutritional environments seen by eggs and embryos in the female reproductive system, and better replication of these in the laboratory, would increase the success of IVF technologies. Here we focus on the in-vitro maturation (IVM) of initially immature oocytes (eggs) for subsequent use in standard IVF procedures. IVM allows a reduction in the drugs administered to stimulate oocyte maturation prior to harvesting from the ovary, with health and cost benefits. However, it also requires keeping the developing oocyte in vitro for a longer period of time than seen with standard IVF, because of which there is greater negative impact from non-ideal nutrient environments and a much lower chance of success.


In vivo, prior to ovulation, an oocyte is surrounded by cells and fluid within a follicle in the ovary, and the nutritional environment seen by the oocyte is difficult to determine experimentally. We describe mathematical modelling, using experimental micronutrient data, which is aimed at increasing our understanding of the in-vivo oocyte environment. We will specifically look at glucose concentration but the basic model should have application to other nutrients also.

05:15 PM
05:45 PM
Young Kwon - Quantitative Analysis of Retroviral Gene Delivery

Gene therapy including cell-based therapy using genetically manipulated stem cells has been studied as one of the most feasible strategies in curing fatal diseases and transforming malfunctioning tissues to healthy ones by correcting existing disordered genes or introducing new therapeutic genes. Among many different ways of gene delivery, retroviral vector has been widely used due to its stable long-term effects. There are two major questions in retroviral transduction systems: Concentration of infectious virus and quantification of transduction conditions. Retroviral transduction systems were analyzed and infectious retroviral concentration and transduction rate constant were determined by curve-fitting experimental data to a mathematical model. The result showed two-order of magnitude difference of infectious retroviral concentration from the titer, a commonly used quantity of infectious retrovirus. From the analysis developed in this study, an efficiency parameter representing tranduction rate constant was extracted and showed its usefulness in quantifying various retroviral transduction systems under various conditions. For example, VSV-G pseudotyped retrovirus has shown to be more efficient vector than ecotropic and amphotropic retroviruses by comparing transductin rate constants. By analyzing the effect of factors involved in retroviral transduction, it was shown that cationic polymers (e.g., Polybrene) increased the transduction efficiency with higher concentration, while glycosaminoglycans (e.g., chondroitin sulfate C) increases nonspecific binding efficiency at low concentrations but decreases the efficiency at high concentrations. Furthermore, the correlation of amphotropic retroviral transduction efficiency with receptor expression was verified. This study clearly showed the usefulness of mathematical approach to understanding and maximizing retroviral gene delivery for therapeutic cell and tissue engineering.



 
Wednesday, October 24, 2007
Time Session
09:00 AM
09:45 AM
Farshid Guilak - Functional Tissue Engineering: The Role of Biomechanics in Cartilage Repair

Tissue engineering is a rapidly growing field that seeks to repair or replace tissues and organs using combinations of cells, biomaterials, and/or biologically active molecules. Despite many advances, significant challenges still remain in the replacement or repair of tissues that primarily serve a biomechanical function, such as articular cartilage. An important consideration in the long-term success of such biomechanically functional tissue replacements is a more thorough investigation of the influence of biomechanical factors, such as the design and characterization of the mechanical properties of biomaterial scaffolds and the use of biophysical stimuli to control cell differentiation and metabolism [1]. Using principles of "functional tissue engineering" [2], we have developed novel biomimetic scaffold designs based on techniques for three-dimensional weaving of biocompatible fibers. A microscale weaving technique was utilized to generate anisotropic 3-D woven structures as the basis for composite scaffolds [3]. These scaffolds exhibited mechanical properties that were similar to those of native articular cartilage, as measured by compressive, tensile, and shear testing. Such porous composite scaffolds can be engineered with initial properties that reproduce the anisotropy, viscoelasticity, and tension-compression nonlinearity of native articular cartilage. In combination with human adipose-derived adult stem (hADAS) cells [4], we have developed cartilage constructs with the potential for load-bearing immediately after implantation in vivo, with biological support for cell-based tissue regeneration without requiring cultivation in vitro. Recapitulating the biomechanical properties of the tissue, in addition to providing developmental biological cues to the stem cells, may provide new advances in the engineering of functional tissue replacements.


Acknowledgments: NIH AR49294, AR50245; NASA NNJ04HC72G; and the Coulter Foundation.


References:



  1. Butler DL, Goldstein SA, Guilak F. J Biomech Eng. 122:570, 2000.

  2. Guilak F, Butler DL, Goldstein SA. Clin Orthop Rel Res. 391S:295, 2001.

  3. Moutos FT, Freed LE, Guilak F. Nat Mat, in press, 2007.

  4. Estes BT, Wu AW, Guilak F. Arthritis Rheum. 54:1222, 2006.


 


Work done in collaboration with F.T. Moutos, B.T. Estes, and L.E. Freed.

10:00 AM
10:45 AM
Rocky Tuan - Application of Adult Stem Cells and Nanomaterials for Skeletal Tissue Engineering

Nanoscale materials are the fundamental building blocks and functional subunits of cells, including subcellular organelles and extracellular matrix components. Currently, there is growing recognition of the importance of understanding and incorporating nanobiology into biomedical applications. This issue is of particular importance in the emerging field of regenerative medicine, the goal of which is to develop methods to repair, replace, and regenerate diseased, injured, or non-functional tissues. Towards this goal, stem or progenitor cells have been considered a highly desirable candidate cell type, because of their expandability and potential to be induced toward specific cell differentiation lineages. A key requirement in tissue engineering and regenerative medicine is that ultimately the "regenerate tissue" needs to be a three-dimensional structure. In weight-bearing musculoskeletal tissues, this requirement is particularly critical. Musculoskeletal disorders affect one out of seven Americans. This severe disease burden underscores the need to develop novel and effective treatment protocols. This talk will present the excitement as well as the challenges in the field of skeletal tissue engineering and regeneration, specifically the application of adult stem cells and nanomateiral scaffolds. The biology of adult stem cells, particularly the mechanisms regulating their proliferation versus differentiation into specific lineages, is intricately regulated by cell-cell interactions, signaling by extracellular bioactive factors, and transcriptional and epigenetic activities. More importantly, the extracellular matrix milieu provides critical cues, both architectural and structure-dependent, to guide cell-based tissue morphogenesis. We have developed biomimetic and biodegradable nanofibrous biomaterials to serve as scaffolds for cell-based tissue engineering. Information on the fabrication and biological basis of the scale-dependent bioactivities of the nanofibrous scaffold will be presented. Cell-nanofibrous constructs are currently being tested in animal models for their cartilage reparative potential in vivo. In conclusion, tissue engineering represents a unique, emerging inter-disciplinary research field that is a natural platform for life scientists, engineers, and clinicians working together to advance regenerative medicine.

11:00 AM
11:45 AM
Melissa Knothe Tate - Taking Cues from Nature's Paradigm to Build Tissue in the Lab and the O.R.

N/A

Name Email Affiliation
Adams, Dany dadams@drmichaellevin.org Forsyth Center for Regenerative and Developmental Biology, Harvard University
Agarwal, Sudha agarwal.61@osu.edu Oral Biology, The Ohio State University
Aguda, Baltazar bdaguda@gmail.com Mathematical Biosciences Institute, The Ohio State University
Almarza, Alejandro ajal9@pitt.edu Musculoskeletal Research Center, University of Pittsburgh
Arnosti, David arnosti@msu.edu Dept. of Biochemistry and Molecular Biology, Michigan State University
Ay, Ahmet ayahmet@msu.edu Department of Mathematics, Michigan State University
Barolo, Scott sbarolo@umich.edu Dept. of Cell & Developmental Biology, University of Michigan
Bell, Jonathan jbell@umbc.edu MBI - Long term visitor, The Ohio State University
Berg, Celeste caberg@u.washington.edu Department of Genome Sciences, University of Washington
Best, Janet jbest@mbi.osu.edu
Brady, Siobhan philip.benfey@duke.edu Biology Department, Duke University
Chiu, Chichia chiu@math.msu.edu Dept. of Biochemistry and Molecular Biology, Michigan State University
Coskun, Huseyin hcusckun@mbi.osu.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Day, Judy jday@mbi.osu.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Dayringer, Evan evan.dayringer@gmail.com Dept. of Biochemistry and Molecular Biology, Michigan State University
Djordjevic, Marko mdjordjevic@mbi.osu.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Enciso, German German_Enciso@hms.harvard.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Grajdeanu, Paula pgrajdeanu@mbi.osu.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Green, Edward egreen@mbi.osu.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Grotewold, Erich grotewold.1@osu.edu MBI-Long Term Visitor, The Ohio State University
Guilak, Farshid guilak@duke.edu Department of Surgery, Duke University
Hansford, Derek hansford.4@osu.edu Biomedical Engineering, The Ohio State University
Holtzman, Nathalia nathalia.holtzman@qc.cuny.edu Department of Biology, Queens College, CUNY
Hufnagel, Lars hufnagel@kitp.ucsb.edu Kavli Institute for Theoretical Physics, University of California, Santa Barbara
Kao, Chiu-Yen kao.71@osu.edu MBI - Long Term Visitor, The Ohio State University
Kim, Yangjin ykim@mbi.osu.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Kniss, Douglas kniss.1@osu.edu OB&GYN, The Ohio State University
Knothe Tate, Melissa knothetate@gmail.com Biomedical Engineering, Case Western Reserve University
Kwon, Young young.kwon@case.edu Department of Biomedical Engineerin, Case Western Reserve University
Li, Ruth li.538@osu.edu IBGP, The Ohio State University
Liang, Rui rul3@pitt.edu Dept. of Bioengineering, University of Pittsburgh
Lou, Yuan lou@math.ohio-state.edu MBI - Long Term Visitor, The Ohio State University
Machiraju, Raghu machiraju@math.ohio-state.edu Computer Science and Engineering, The Ohio State University
Matzavinos, Tasos tasos@math.ohio-state.edu MBI - Long term visitor, The Ohio State University
McBride, Sara sara.mcbride@alumni.clemson.edu Department of Biomedical Engineering, Case Western Reserve University
Munro, Ed munroem@u.washington.edu Center for Cell Dynamics, University of Washington
Nam, Jin nam.27@osu.edu Oral Biology, The Ohio State University
Nevai, Andrew anevai@mbi.osu.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Oster, Andrew aoester@mbi.osu.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Rath, Bjoern rath.27@osu.edu Oral Biology, The Ohio State University
Rempe, Michael mrempe@mbi.osu.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Sample, Christine csample@princeton.edu Engineering Sciences and Applied Mathematics, J. L. Kellogg Graduate School of Management
Schugart, Richard richard.schugart@wku.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Shih, Chih-Wen shih@math.ohio-state.edu MBI - Long term visitor, The Ohio State University
Shvartsman, Stanislav stas@princeton.edu Chemical Engineering, Princeton University
Smith, Greg greg@as.wm.edu MBI - Long term visitor, The Ohio State University
Srinivasan, Partha p.srinivasan35@csuohio.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Stigler, Brandy bstigler@mbi.osu.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Stokes, Yvonne Yvonne.Stokes@adelaide.edu.au Applied Math, University of Adelaide
Sun, Jianxin sun.187@osu.edu Department of Chemical Engineering, The Ohio State University
Sun, Shuying ssun@mbi.osu.edu Mathematical Biosciences Institute (MBI), The Ohio State University
Szomolay, Barbara b.szomolay@imperial.ac.uk Mathematical Biosciences Institute (MBI), The Ohio State University
Tuan , Rocky Tuanr@mail.nih.gov National Institute of Arthritis, and Musculoskeletal & Skin Diseases, National Institutes of Health
Zartman, Jeremy jzartman@princeton.edu Lewis-Sigler Institute for Integrative Genomics, Princeton University
Biophysical Regulation of Morphogenesis in Development and Regeneration: A New Target for Clinical Intervention and the Need for Modeling

Data from the Forsyth Center for Regenerative and Developmental Biology is bringing to light the fundamental importance of ion flux during early stages of Development and Regeneration. Currently, there is exploding interest in using research from these fields to drive discovery of drugs and other medical interventions. Thus, now is a particularly exciting time to be studying biophysical signaling, in part because the vast majority of research focuses on genetic regulation of biochemical pathways. Our approach has been to apply the tools of molecular biology and mathematical modeling to long recognized, but understudied bioelectric phenomena, and that approach has proved very rewarding. Our data on early patterning of vertebrate embryos, regulation of planarian stem cells, and induction of regeneration in tadpole tails, indicate that understanding of ion-flux mediated signaling pathways could result in brand new categories of biomedical approaches as well as new paradigms for understanding the interplay of genetic and epigenetic phenomena. We believe that thinking in terms of functional relatedness (physiological parameter space) rather than genetic relatedness, will bring important, complimentary insight to our understanding of biological phenomena.

Changes in Gene Expression of Passaged Bone Marrow Derived Cells in Culture

Introduction: Functional tissue engineering (FTE) efforts to regenerate musculoskeletal tissues require large numbers of cells, often obtained through cell passage. One study involving MCL fibroblasts showed an increase of collagen type I gene expression and relative consistence of collagen type III gene expression throughout passage, indicating that MCL fibroblasts may be a good cell source for FTE approaches involving ligaments and tendons whose main constituent is collagen type I (1). Bone marrow derived cells (BMDCs) may be a more attractive cell source because unlike MCL fibroblasts, BMDCs can be obtained from the bone marrow by needles without any noticeable harm to the body, are auto-transplantable, easily expandible and have the potential to differentiate into various lineages (2). The objective of this study was to quantify gene expression of Collagen I, Collagen III, and GAPDH through passages in rat BMDCs.


Methods: Isolated BMDCs of three rats and cells were centrifuged, counted, and plated on 2 cm^2 wells until 95% confluency (P1). Ascorbic acid (50 mg/ml) added to medium and cells plated at 300,000/well for one more passage. Cells counted with hemacytometer and RNA isolated with a kit after each passage (n=3). Real time RT-PCR was performed to determine gene expression of Collagen I, Collagen III, and GAPDH. An ANOVA was performed to determine differences between passages (P < 0.05) with Fisher's post hoc test.


Results: Collagen type I gene expression significantly increased at P2 compared to native tissue (P0) or P1 cells (p < 0.05). Collagen type III gene expression significantly increased at P2 when compared to native tissue (P0) or P1 cells and magnitude of collagen type III gene expression is much greater than that of collagen type I gene expression in later passages (p < 0.05).


Discussion: Despite the increase in collagen type I gene expression, the drastic increase of collagen type III gene expression indicates that BMDCs may not be an ideal cell source for FTE approaches to regenerate tendons and ligaments. Future studies may include quantifying collagen type I and III gene expressions from P2 to P5 and measuring the gene expression of other important molecules such as growth factors and metalloproteases.



  1. Almarza, AA., Augustine, S., Woo, S L-Y. (2007) Effect of Passage on Ligament Fibroblasts: Implications for Functional Tissue Engineering. 7th International Symposium on Ligaments and Tendons. San Diego, CA. Febraury 2007.

  2. Pittenger, MF et al. (1999). Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science 284, 143.


 


Work done in collaboration with Augustine S., and Woo S.L-Y.

Identification of a transcriptional cis-regulatory grammar in the Drosophila embryo by quantitative modeling

Control of gene expression in eukaryotic organisms is directed by regulatory sequences in the DNA that serve as molecular switches to activate and repress transcription. Unlike the canonical genetic code that specifies protein structure, the regulatory code that dictates the design of transcription control sequences is loosely constrained and difficult to identify using simple bioinformatic methods. We have identified specific aspects of a transcriptional regulatory grammar that dictates how patterning genes are controlled by short-range repressors such as Giant and Knirps in the developing Drosophila embryo. Confocal laser imaging allows us to quantitatively analyze gene expression, leading to "maps" of gene regulatory surfaces associated with particular arrays of regulatory sequences carried by transgenic lines. We use this information in the development of a three-tier quantitative mathematical model that associates potential functions with subelements of the enhancer, and combines them to provide predictions of the functional output of novel transcriptional elements. With this approach, we have accurately predicted the effects of spacing between activators and repressors, a key feature of short-range repression. This modeling is now being extended to analyze modifications in factor stoichiometry, binding affinity, and arrangement, providing detailed insight into cis regulatory element architecture. We are also adapting a fractional occupancy model that considers specific features of spacing and cooperativity in predicting gene activity. Our work complements "top down" approaches, and is aimed at informing and extending the power of current models of endogenous cis-regulatory elements to develop powerful bioinformatic tools applicable to population and evolutionary studies.

Computational cis-regulatory Transcriptional Grammar and Developing Predictive Mathematical Models

Cis-regulatory information comprises a key portion of genetic coding, yet despite the abundance of genomic sequences now available, identifying and characterizing this information remains a major challenge. We are pursuing a unique "bottom up" approach to understand the mechanistic processing of regulatory elements (input codes) by the transcriptional machinery, using a well defined and characterized set of repressors and activators in Drosophila blastoderm embryos. We are identifying quantitative values for parameters affecting transcriptional regulation in vivo, and these parameters are used to build and test mathematical models that predict the outputs of novel cis-regulatory elements.


Giant, Kruppel, Knirps are short-range transcriptional repressors involved in the developmental patterning of Drosophila blastoderm embryo. Using defined regulatory modules tested in germ line transformed embryos, we are measuring quantitative parameters describing the effects of spacing, stoichiometry, arrangement and binding site affinities of these repressors on cassettes driven by endogenous activators. To develop predictive models we employ a hybrid thermodynamic gene regulation model in which we included variables for the spacing effect between activators and repressors. This model is being used to predict the output of novel permutations of binding sites, which will allow us test and refine parameters used. In one line of investigation, fluorescence quantization of lacZ reporter gene expression was used to measure the effect of moving Giant repressor binding sites from a position adjacent to Twist/Dorsal activator sites to a distal site 125bp away. Our mathematical model successfully predicted the distance effect of intermediate positions, such as 25, 50, 75 and 100bp compared to experimental results. Further tests will illustrate the effects of site permutation, transcription factor concentration and activator/repressor stoichiometry and arrangement. Extension of these predictive models to endogenous cis elements will provide novel insights on regulatory element design and evolution, and should provide a bioinformatics method for predicting quantitative output of novel regulatory elements.


Work done in collaboration with Walid D. Fakhouri, Chichia Chiu, and David N. Arnosti.

How to Build an Enhancer: Principles of Cell- and Tissue-Specific Gene Expression

Enhancers, or cis-regulatory elements, control gene expression both qualitatively and quantitatively (i.e., patterns and levels). Enhancers contain clusters of binding sites for transcription factors (TFs), which act as activators or repressors of transcription. Despite 25 years of work on enhancers, we are still not able to build "synthetic" elements (combinations of TF binding sites) that function as normal enhancers in vivo, and must rely on "found" enhancers to drive targeted gene expression, both in model organism research and in potential disease therapy/tissue engineering strategies. The fact that we can't build an enhancer from scratch suggests that we don't understand the basic functional principles of the enhancer, its basic components, or its structure.


We have undertaken a fine-scale transgenic structure-function analysis of a signal-regulated, cell-type-specific enhancer of a gene essential for normal eye development. The sparkling enhancer of the dPax2 gene is a direct target of Notch and EGFR signaling in cone cells of the developing Drosophila eye (Flores et al., Cell 103:75). We find that a synthetic sparkling element, made by simply combining the twelve known transcription factor binding sites within sparkling, is unable to activate gene expression in vivo, even when the sites are placed in their native arrangement and spacing. Our in vivo analysis of the sparkling enhancer has revealed several interesting features of its transcriptional activity in vivo:



  • Sequence: A surprisingly large number and high density of novel regulatory sites; rapid evolutionary turnover of critical regulatory sequences.

  • Structure: Extreme structural flexibility; a role for binding site arrangement in patterning of gene expression.

  • Mechanism: Evidence for a novel "promoter communication element" within the enhancer, which is required only for gene activation at a distance.


 


If time permits, I will briefly describe another project, in which we are finding a link between low-affinity transcription factor binding sites and tissue-specificity of the transcriptional response to the Hedgehog pathway.


The work was done primarily by Christina I. Rogers, with assistance from David Schwimmer, Nicole C. Evans, and me.

Setting up and interacting across boundaries: Tube formation in Drosophila egg chambers

The reorganization of flat epithelial sheets into tubes is a fundamental process in the formation of many organs, such as the lungs, kidneys, gut, and neural tube. This process involves the patterning of distinct cell types and the coordination of those cells during shape changes, rearrangements, and other behaviors essential to the function of the tube. To study tube formation, we are analyzing the signaling pathways that define and control sub populations within the dorsal-anterior region of the Drosophila follicular epithelium. These cells reorganize to form two closed tubes; secretion of chorion proteins into the tube lumens creates two dorso-lateral eggshell appendages, which facilitate respiration in the developing embryo. EGF and BMP signals define the two primordia and then Notch signaling subdivides the region further and establishes a boundary between "roof" and "floor" cells. Roof cells constrict their apices and converge toward the dorsal midline, lengthening the tube, while floor cells elongate underneath the roof cells and fuse apically to seal the tube. Our goal is to understand how these cells coordinate their behaviors to generate tubes of specific size and shape. Work done in collaboration with Jennie B. Dorman and Ellen J. Ward.

Transcriptionally rich and complex spatiotemporal programs in Arabidopsis root development

Transcriptional programs that regulate development are exquisitely controlled in space and time. Elucidating these programs that underlie development is essential to understanding the acquisition of cell and tissue identity. We present microarray expression profiles of a high resolution set of developmental time points within a single Arabidopsis root, and a comprehensive map of nearly all root cell-types. These cell-type specific transcriptional signatures often predict novel cellular functions. A computational pipeline identified dominant expression patterns that demonstrate transcriptional similarity between disparate cell-types. Dominant expression patterns along the root's longitudinal axis do not strictly correlate with previously defined developmental zones, and in many cases, expression fluctuation along this axis was observed. Both robust co-regulation of gene expression and potential phasing of gene expression were identified between individual roots. Methods that combine these profiles demonstrate transcriptionally rich and complex programs that define Arabidopsis root development in both space and time.

Functional Tissue Engineering: The Role of Biomechanics in Cartilage Repair

Tissue engineering is a rapidly growing field that seeks to repair or replace tissues and organs using combinations of cells, biomaterials, and/or biologically active molecules. Despite many advances, significant challenges still remain in the replacement or repair of tissues that primarily serve a biomechanical function, such as articular cartilage. An important consideration in the long-term success of such biomechanically functional tissue replacements is a more thorough investigation of the influence of biomechanical factors, such as the design and characterization of the mechanical properties of biomaterial scaffolds and the use of biophysical stimuli to control cell differentiation and metabolism [1]. Using principles of "functional tissue engineering" [2], we have developed novel biomimetic scaffold designs based on techniques for three-dimensional weaving of biocompatible fibers. A microscale weaving technique was utilized to generate anisotropic 3-D woven structures as the basis for composite scaffolds [3]. These scaffolds exhibited mechanical properties that were similar to those of native articular cartilage, as measured by compressive, tensile, and shear testing. Such porous composite scaffolds can be engineered with initial properties that reproduce the anisotropy, viscoelasticity, and tension-compression nonlinearity of native articular cartilage. In combination with human adipose-derived adult stem (hADAS) cells [4], we have developed cartilage constructs with the potential for load-bearing immediately after implantation in vivo, with biological support for cell-based tissue regeneration without requiring cultivation in vitro. Recapitulating the biomechanical properties of the tissue, in addition to providing developmental biological cues to the stem cells, may provide new advances in the engineering of functional tissue replacements.


Acknowledgments: NIH AR49294, AR50245; NASA NNJ04HC72G; and the Coulter Foundation.


References:



  1. Butler DL, Goldstein SA, Guilak F. J Biomech Eng. 122:570, 2000.

  2. Guilak F, Butler DL, Goldstein SA. Clin Orthop Rel Res. 391S:295, 2001.

  3. Moutos FT, Freed LE, Guilak F. Nat Mat, in press, 2007.

  4. Estes BT, Wu AW, Guilak F. Arthritis Rheum. 54:1222, 2006.


 


Work done in collaboration with F.T. Moutos, B.T. Estes, and L.E. Freed.

Endocardial-myocardial interactions direct cell migration during heart tube formation

The embryonic heart tube develops from a simple cylinder, who's foundation is built through the union of bilateral populations of cardiomyocytes. Cardiomyocyte interactions with the neighboring gut endoderm are crucial for recruitment of cardiomyocytes toward the embryonic midline. However, it is not clear if the endoderm is sufficient to direct all the cell movements required for heart formation. In addition, little is known about the morphogenetic and cellular mechanisms that direct heart tube assembly once cardiomyocytes reach the midline. Using time-lapse confocal microscopy to track individual cardiomyocyte movements in the zebrafish embryo, we identify two morphologically and genetically separable phases of cell movement that underlie tube assembly. Specifically, we find an initial phase of endoderm dependent movement during which cardiomyocytes undergo coherent medial movement bringing cells to the midline. This is followed by an endoderm independent phase of movement, whereby groups of peripherally located cardiomyocytes change their direction of movement and angle toward the centrally located vascular endocardium. This pattern of cell behavior suggests that the endocardium influences cardiomyocyte behavior. Indeed, through examination of zebrafish mutants, we find that the induction, direction, and duration of cardiomyocyte movement depend upon myocardial-endocardial interactions. Thus, our studies indicate that the endoderm is required primarily to bring the cardiomyocytes to the midline at which point interactions with the endocardium plays a critical early role in further myocardial morphogenesis, positioning cardiomyocytes into a configuration appropriate for heart tube assembly. To begin to address the role of the endocardium directly, we have begun a molecular and cellular analysis of this poorly understood tissue. Our current findings will be presented.


Work done in collaboration with Huai-Jen Tsai and Deborah Yelon.

Regulation of Growth during Development: Role of Mechanics

A fundamental and unresolved problem in animal development is the question of how a growing tissue knows when it has achieved its correct final size. A widely held view suggests that this process is controlled by morphogen gradients, which adapt to tissue size and become flatter as tissue grows, leading eventually to growth arrest. I will discuss the spatio-temporal dynamics of that the decapentaplegic (Dpp) morphogen distribution in the developing Drosophila wing imaginal disk and present an alternative model for wing size determination and proliferation control in tissues. I will also present results on the transition of an irregularly packed epithelium to a hexagonally ordered geometry.



 
Taking Cues from Nature's Paradigm to Build Tissue in the Lab and the O.R.

N/A

Quantitative Analysis of Retroviral Gene Delivery

Gene therapy including cell-based therapy using genetically manipulated stem cells has been studied as one of the most feasible strategies in curing fatal diseases and transforming malfunctioning tissues to healthy ones by correcting existing disordered genes or introducing new therapeutic genes. Among many different ways of gene delivery, retroviral vector has been widely used due to its stable long-term effects. There are two major questions in retroviral transduction systems: Concentration of infectious virus and quantification of transduction conditions. Retroviral transduction systems were analyzed and infectious retroviral concentration and transduction rate constant were determined by curve-fitting experimental data to a mathematical model. The result showed two-order of magnitude difference of infectious retroviral concentration from the titer, a commonly used quantity of infectious retrovirus. From the analysis developed in this study, an efficiency parameter representing tranduction rate constant was extracted and showed its usefulness in quantifying various retroviral transduction systems under various conditions. For example, VSV-G pseudotyped retrovirus has shown to be more efficient vector than ecotropic and amphotropic retroviruses by comparing transductin rate constants. By analyzing the effect of factors involved in retroviral transduction, it was shown that cationic polymers (e.g., Polybrene) increased the transduction efficiency with higher concentration, while glycosaminoglycans (e.g., chondroitin sulfate C) increases nonspecific binding efficiency at low concentrations but decreases the efficiency at high concentrations. Furthermore, the correlation of amphotropic retroviral transduction efficiency with receptor expression was verified. This study clearly showed the usefulness of mathematical approach to understanding and maximizing retroviral gene delivery for therapeutic cell and tissue engineering.



 
Effects of a bioscaffold on the fibrillogenesis in the healing medial collateral ligament

Porcine small intestine submucosa (SIS) has been successfully used to improve the healing of ligaments and tendons [1]. In a rabbit model, the healing medial collateral ligament (MCL) with SIS treatment was found to have larger collagen fibril formation with concomitant reduction in collagen type V/I ratio [2]. Thus, we hypothesized that the SIS bioscaffold could affect the regulation of the gene expressions of collagen fibrillogenesis-related molecules at the early stage and result in improved fibril morphology and organization. In the current study, our objective was to examine the organization of the extracellular matrix, measure the collagen fibril diameter, and analyze the gene expressions of the fibrillogenesis-related molecules, specifically, collagen types I, III, V and small leucine-rich proteoglycans including decorin, biglycan, lumican and fibromodulin, in the healing rabbit MCL treated with SIS at 6 weeks post-injury. Twenty rabbits were equally divided into two groups. In the SIS-treated group, a 6 mm gap was surgically created in the right MCL and a layer of SIS was sutured covering the gap. For the non-treated group, the gap-injured MCLs remained untreated. All the left MCLs were sham operated and used as controls. At 6 weeks, Masson trichrome's staining was used to examine the organization of collagen-based extracellular matrix. Collagen fibril diameters and distribution were calculated and analyzed from the transmission electron micrographs. Gene expressions of the fibrillogenesis-related molecules were detected with quantitative real time PCR. The results revealed that in the SIS-treated group the collagen fibers were more regularly aligned as well as the cell nuclei than that for the non-treated group. Additionally, in the SIS-treated group larger collagen fibrils (90 - 120nm) appeared around the fibroblasts and the range of the fibril diameter distribution was from 24 to 120nm while the collagen fibrils in the non-treated group were uniformly small and the distribution of fibril diameter was limited from 26 to 87 nm. Simultaneously, the gene expressions of collagen type V, decorin, biglycan and lumican were downregulated by 41%, 58%, 43% and 51%, respectively (p < 0.05). The present results suggested that the significant reduction in the gene expressions of fibrillogenesis-related molecules in the healing MCLs following SIS treatment are closely related to the improved morphological characteristics, which are known to be coupled to the enhanced mechanical properties observed in the long term as reported in published studies.


References: 1) Woo et al. AJSM, 15: 22-29, 1987; 2) Liang et al, JOR, 24: 811-9, 2006.


Acknowledgments: Support form NIH Grant #AR41820 is gratefully acknowledged. Also, Cook Biotech, Inc. for supplying the SIS.


Work done in collaboration with T. Nguyen, R. Nola, A. Almarza, and S. L-Y Woo.

Cell seeding conditions exert a profound influence on the developmental context of embryonic mesenchymal stem cells for subsequent tissue generation

Cell density, cell shape, and substrate elasticity are powerful modulators of stem cell fate [1-3].Our working hypothesis is that the manner by which cell densities are achieved, i.e. cell seeding conditions, is also a powerful modulator of cell differentiation. Specifically, we hypothesize that, for a target density to be achieved (i) cells seeded at a relatively low density (5,000 cells/cm^2) and allowed to mature to a high target density through population doublings will preferentially form sheet-like epithelial structures and (ii) cells seeded immediately at the target high density (86,500 cells/cm^2) will preferentially form 3D mesenchymal structures. (iii) Furthermore, while the target high densities can be achieved with both seeding methods, initial cell seeding conditions influence the commitment of cells to specific lineages by defining the developmental context of embryonic mesenchymal stem cells for subsequent tissue generation.C3H10T1/2 multipotent progenitor cells were seeded on glass coverslips at 5,000 cells/cm^2 and given 48, 72, or 96 hours to proliferate to low (16,500 cells/cm^2), high (35,000 cells/cm^2), and very high (86,500 cells/cm^2) density cultures. An additional set of coverslips was seeded immediately at these three densities and given 24 hours to attach. In this way, we created density-matched groups that differ in maturity or developmental context. For each independent group, three sets of data were analyzed with real time RT-PCR (n=3 per group) and analyzed for genes, including Collagen type IaI, Osterix, Runx2, Msx2, Collagen type II, Sox9, Aggrecan, and Ppar?. Additional samples were fixed and stained to assess the structure of the seeded cell constructs with microscopy. All density groups, from LD to VHD, show similar patterns of gene up- and down-regulation for all genes studied. However, the magnitude of change in activity from baseline is highest for groups where density is achieved by population doublings. Nonetheless, LD groups show little modulation of gene activity attributable to seeding conditions. Cell seeding conditions exert a profound influence on the developmental context of embryonic mesenchymal stem cells for subsequent differentiation and tissue generation. This study shows, for the first time to our knowledge, that not only the density at which cells are seeded is important in determining subsequent gene activity related to lineage commitment, but also the manner by which cell densities are achieved. Histological studies of cell constructs provide further support regarding the nature of the cell constructs formed as a consequence of cell seeding conditions.



  1. McBeath et al. Dev Cell 2004

  2. Chen et al. Science 1997

  3. Engler et al. Cell 2006

  4. Johnstone et al. Exp Cell Res 1998


 


Work done in collaboration with Melissa Knothe Tate.

Cytomechanics of polarization and morphogenesis in early embryos

The cellular rearrangements and tissue deformations that shape embryos during animal development emerge from the local force generating behaviors of many individual cells. Molecular genetics and biochemistry has identified much of the basic molecular machinery responsible for the production, regulation and transmission of cellular forces. But to understand tissue-level morphogenesis we must comprehend: (a) how this machinery is organized dynamically within individual cells through an interplay between biochemistry and mechanics, and (b) how local forces generated within each of many individual cells are integrated across whole tissues to produce stereotyped patterns of cell shape change and rearrangement. Our approach to addressing these problems combines experimental analyses of cellular and subcellular dynamics with detailed computer simulations. I will illustrate this approach using several examples drawn from our recent work: The first concerns how a robust mechanism for convergent extension of embryonic tissues emerges from the conserved machinery underlying protrusive, contractile and adhesive force generation in single motile cells. The second involves new insights into the cellular mechanisms of invagination gained from the study of a stripped-down version of invagination in ascidian embryos. The third concerns how a mechanism for polarization of single cells emerges from interactions among the PAR proteins - conserved regulators of cell polarity - and the actomyosin cytoskeleton.

Engineering models of epithelial patterning in Drosophila oogenesis

N/A

Mathematical modeling towards successful in vitro maturation of mammalian oocytes

Better understanding of the nutritional environments seen by eggs and embryos in the female reproductive system, and better replication of these in the laboratory, would increase the success of IVF technologies. Here we focus on the in-vitro maturation (IVM) of initially immature oocytes (eggs) for subsequent use in standard IVF procedures. IVM allows a reduction in the drugs administered to stimulate oocyte maturation prior to harvesting from the ovary, with health and cost benefits. However, it also requires keeping the developing oocyte in vitro for a longer period of time than seen with standard IVF, because of which there is greater negative impact from non-ideal nutrient environments and a much lower chance of success.


In vivo, prior to ovulation, an oocyte is surrounded by cells and fluid within a follicle in the ovary, and the nutritional environment seen by the oocyte is difficult to determine experimentally. We describe mathematical modelling, using experimental micronutrient data, which is aimed at increasing our understanding of the in-vivo oocyte environment. We will specifically look at glucose concentration but the basic model should have application to other nutrients also.

Application of Adult Stem Cells and Nanomaterials for Skeletal Tissue Engineering

Nanoscale materials are the fundamental building blocks and functional subunits of cells, including subcellular organelles and extracellular matrix components. Currently, there is growing recognition of the importance of understanding and incorporating nanobiology into biomedical applications. This issue is of particular importance in the emerging field of regenerative medicine, the goal of which is to develop methods to repair, replace, and regenerate diseased, injured, or non-functional tissues. Towards this goal, stem or progenitor cells have been considered a highly desirable candidate cell type, because of their expandability and potential to be induced toward specific cell differentiation lineages. A key requirement in tissue engineering and regenerative medicine is that ultimately the "regenerate tissue" needs to be a three-dimensional structure. In weight-bearing musculoskeletal tissues, this requirement is particularly critical. Musculoskeletal disorders affect one out of seven Americans. This severe disease burden underscores the need to develop novel and effective treatment protocols. This talk will present the excitement as well as the challenges in the field of skeletal tissue engineering and regeneration, specifically the application of adult stem cells and nanomateiral scaffolds. The biology of adult stem cells, particularly the mechanisms regulating their proliferation versus differentiation into specific lineages, is intricately regulated by cell-cell interactions, signaling by extracellular bioactive factors, and transcriptional and epigenetic activities. More importantly, the extracellular matrix milieu provides critical cues, both architectural and structure-dependent, to guide cell-based tissue morphogenesis. We have developed biomimetic and biodegradable nanofibrous biomaterials to serve as scaffolds for cell-based tissue engineering. Information on the fabrication and biological basis of the scale-dependent bioactivities of the nanofibrous scaffold will be presented. Cell-nanofibrous constructs are currently being tested in animal models for their cartilage reparative potential in vivo. In conclusion, tissue engineering represents a unique, emerging inter-disciplinary research field that is a natural platform for life scientists, engineers, and clinicians working together to advance regenerative medicine.

The Expression and Regulation of Cad74A in Drosophila Oogenesis

During Drosophila oogenesis, a two-dimensional follicular epithelium gives rise to an elaborate three-dimensional eggshell. Eggshell morphogenesis critically depends on the patterning of the follicle cells, but the connection between signaling pathways, pattern formation, and eggshell morphogenesis is not well understood. Recently we have identified the non-classical cadherin, Cad74A, as a key molecule that bridges epithelial patterning and the morphogenetic dynamics in this system. Starting in mid-oogenesis, Cad74A is expressed in all the columnar cells except for two dorsolateral patches, in the border cells, and later in the future operculum domain. Using a number of mutant backgrounds and related fly species, we show that this pattern is correlated with the formation of multiple structural features of the eggshell, suggesting that Cad74A plays an important functional role on oogenesis. We provide data that strongly suggest that the dynamic pattern of Cad74A expression is partially controlled by the EGFR and Dpp signaling pathways, two of the key regulators of follicle cell patterning. On the basis of these results, we propose that Cad74A provides an important link between signaling, pattern formation and morphogenesis during egg development.


Work done in collaboration with N. Yakoby, C.A. Bristow, and S.Y. Shvartsman.