Lecture 10 - BioE10: Lecture 10 Professor Irina Conboy...

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Unformatted text preview: BioE10: Lecture 10 Professor Irina Conboy Tissue engineering and Regenerative Medicine Key concepts of physiological and artificial organogenesis Objectives: Tissue engineering and Regenerative Medicine are focused on deliberate control of adult tissue repair combating injuries and degenerative disorders Biological fundamentals: Zygote; embryo, gametes, blastocyst Ectoderd, mesoderm, endoderm Maternal determinants, morphogens Scheme of embryonic organogenesis Infinite I fi it capacity for embryonic organogenesis it f b i i gametes adult + adult Current Opinion in Genetics & Development 2006, 16:455–462 Finite capacity for adult tissue regeneration More on this topic in Stem cell module p Regenerative medicine Tissue engineering 3rd degree burn Stem cell technologies Spinal cord injury Chemical burn The goal of tissue engineering is to g g g g generate synthetic tissue-like materials able y to replace dysfunctional damaged physiological tissues; e.g. skin and cornea of burn victims, neurons lost due to spinal cord injury, clogged blood vessels, etc. Control of cell fate determination = recapitulate specific molecular signals in space and time Generally speaking, what parameters should we id tif and f G ll ki h t t h ld identify d focus on? ? Mechanical, physical, chemical, biochemical? What are these signals? Proteins interacting signaling networks. How many are the signals that control cell fate? Is it one pathway or condition? Epigenetic modifications (methylation, acetylation, deacytilation)=chromatin silencing and activation ND at the level of whole genome. Manufacturing tissues and organs Embryonic development Establishing divergence from initially equivalent cells=embryonic development, or organogenesis d l i First asymmetry: Ca 2+ wave at the site of sperm entry. Are these cells dividing symmetrically or asymmetrically? First division=2 cells Ectoderm Mesoderm Endoderm Morula 16 cells Maternal determinants are asymmetrically localized Initial small difference in gene expression and cell properties will be later amplified TF or DNA binding proteins Networks of regulatory genes can be orchestrated by initial maternal determinant, such as mRNA Generally speaking, morphogen gradients establish gene expression and thus, cell fate during embryonic development. Nuclear gradient of Dorsal in Drosophila Regulation of cell fate by timing and dosage of gene expression Initially equivalent cells C. elegans vulval development. (A) The six vulval precursor cells (VPCs) specifically named P3 p P8 p (VPCs), P3.p–P8.p, are ventral epidermal cells. The gonadal anchor cell (AC) is positioned dorsally above P6.p. The AC expresses LIN-3/EGF to activate the EGFR/ Ras/ERK pathway strongly in P6.p and induce the 1° vulval fate (yellow). P6.p expresses a combination of DSL ligands to Different cells in an organ activate LIN-12/Notch in P5.p and P7.p and induce the 2° vulval fate (blue). Both Ras and Notch activities are low in P3.p, P4.p, and P8.p, which adopt the 3° fate (black). P(5–7).p, (black) In P(5–7) p the Ras and Notch pathways antagonize each other so that only one of the two pathways is highly active. Subsequent to Ras and Notch signaling, the VPCs go through one or three rounds of cell division, depending on which fate they’ve adopted. Descendants of the 1° and 2° VPCs form the vulva. (B) Ras–Notch interactions in P(5–7).p in more detail. DSL=Delta, Serrate..=Notch ligands Scheme of gene regulatory networks in embryonic development; Interactions between Notch and Ras, or how cells instruct each other. The Notch pathway. Binding of a DSL ligand to Notch triggers its proteolysis and releases the NICD fragment. NICD translocates to the nucleus and interacts with a CSL transcription factor to promote target gene transcription. Contextual factors (purple) determine the specific targets used. The RTK–Ras–ERK pathway. Binding of an EGF ligand to EGFR causes the receptor to dimerize and autophosphorylate tyrosine residues in its cytoplasmic domain. These phospho-tyrosine residues then serve as docking sites for an adaptor protein that recruits the guanine nucleotide exchange factor Sos to activate Ras. Ras-GTP binds to the serine/threonine kinase Raf and facilitates Raf kinase activation. activation Raf phosphorylates and activates the dual specificity kinase MEK, and MEK phosphorylates and activates the serine/threonine kinase ERK. The scaffold protein KSR is essential for signal propagation through this kinase cascade. Once activated, ERK may phosphorylate numerous targets, including Ets domain transcription factors, to affect target gene transcription. Contextualfactors (purple) determine the specific targets used. PGC migration Developmental origin of pluripotent embryonic stem cell lines of the mouse. The scheme demonstrates the derivation of embryonic stem cells (ESC), embryonic carcinoma cells (ECC), and embryonic germ cells (EGC) from different embryonic stages of the mouse. ECC are derived from malignant teratocarcinomas that originate from t t i th t i i t f embryos (blastocysts or egg cylinder stages) transplanted to extrauterine sites. EGC are cultured from primordial germ cells (PGC) isolated from the genital ridges between embryonic day 9 to 12 5 12.5. [From Boheler et al. Pluripotent ESC cell lines can be established from the INNER CELL MASS (ICM) or the PRIMITIVE ECTODERM (also known as the epiblast) of peri-implantation BLASTOCYSTS. After transplantation into unrelated blastocysts, blastocysts these cells can become incorporated into the embryo and participate in development. Key genetic determinants of germ layer formation in mammals During embryogenesis, cells migrate and encounter changing signaling environments. Transcriptional cascades ligand-receptor regulation of signaling networks. Key discoveries in developmental biology •Hans Spemann and Hilde Mangold transplanted embryonic cells that become back tissue from the animal-pole side of a donor newt embryo to the vegetalpole side of a host newt embryo. •The transplanted cells did not just become back tissue. Instead, a second, twinned developed. “twinned” embryo that was joined to the normal host embryo developed 1924 •mRNAs present during early embryogenesis in the organizer region were tested for their ability to restore organizer function to irradiated frog embryos that could not form the organizer. •Injection of an mRNA encoding a protein called noggin into the arrested embryo could completely b ld l t l rescue the embryo and activate normal developmental processes. Mesoderm induction 1973 Animal cap cells of Xenopus bl t l X blastula embryos normally give rise to ectoderm derivatives, while vegetal cells d t l ll develop l into endoderm, but neither tissue on its own is capable of forming f i mesoderm. By combining vegetal and animal cap explants, Nieuwkoop e plants Nie koop was able to elicit formation of mesodermal structures such as notochord and muscle Morphogen; mesoderm-inducing activity 1990 Different concentrations of activin would elicit distinct responses from Xenopus animal caps, ultimately producing a range of mesodermal fates. Moreover, Activin is able to act directly at a distance, moving through tissue unable to respond to activin. Frontiers in Bioscience, 2006 Conboy Hypothetical synthetic scaffold adapting Notch signaling for ex-vivo myogenesis. Adult stem cells such as quiescent satellite cells (muscle stem cells expressing inactive Notch receptor) can be cultured on synthetic biomaterials (either in 2D or in 3D scaffolds) which release growth factors (i.e. EGF) in a gradient fashion and initiate asymmetric stem cell differentiation via variable Notch signaling In this hypothetical scheme muscle stem cells exposed to high levels of EGF signaling. scheme, begin to express Notch ligands (i.e. Delta) which can activate Notch in neighboring, but not distant cells to commit to an intermediate precursor cell fate (pre-myoblasts). The highest concentration of EGF might also inhibit the expression of Notch in the cells that have been orchestrated to up-regulate Delta most efficiently. Cells exposed to moderate or low levels of EGF, especially those adjacent to Delta-expressing neighbors, will have the highest level of Notch activation. Hence, three populations of cells will be created from one y q group: g periphery, p g g initially equivalent cell g p original satellite cells at the p p y, satellite cells expressing high levels of Delta in the center and more differentiated intermediate progenitor cells with high Notch activity in between. Subsequently, mitogenic growth factors, such as basic FGF (bFGF) can be withdrawn and replaced with differentiation-promoting factors, e.g IGF-1, thus forming the second gradient that directs terminal differentiation of muscle progenitor cells into fused myotubes. Importantly, muscle stem and progenitor cells can be orchestrated to co-exist with the differentiated muscle cells, thus recreating dynamically remodeling living tissue by controlling variable cell fate determination on a single substrate through gradient-based asymmetry in Notch signaling strength. How to deliver Gene X and its functionality to the wounded tissue? Engineered cells of future tissue Biomaterial with a combination of protein factors, plasmid, viral vectors, etc. t t Orchestrated tissue repair and maintenance, Neo-organogenesis. Synthetic biomaterials are typically: N-(2N (2 hydroxypropyl)-methacrylamide (HPMA) or PEG or gel-like interpenetrating polymeric network (IPN) of poly(acrylamide) and poly(ethylene glycol general name is hydrogels, hydrogels since they have high and variable water content) . [–CH2–CH2–O–]–monomer for PEG; Natural structural biomaterial are ECM proteins, which surround cells and are secreted by cells, e.g.: laminin, collagen, y g g fibronectin. Bioreactor Liquid N2 Considerations: immune-rejection, inflammation, blood-clotting (structural components and cells); Syngeneic engineered cells gene X timing/duration and place or the response; (need to could b generated and b k d f ld be t d d banked for overlap with tissue regeneration in space and time) everyone (and used as needed) Conclusions: •Early embryonic development starts after fertilization and results in g y formation of germ layers: ecto-;meso-;endoderm •Sperm entry site (Ca2+ flux) and maternal mRNA localization create asymmetry. Networks of regulatory genes can be orchestrated by initial polarity=asymmetry Cells instruct each other with respect to specific fate. Cell fate is regulated not only by “on”/”off” switches, but also by timing and dosage of gene expression •Cell fate is regulated by distinct time-space domains of morphogenic factors During embryogenesis, cells migrate and encounter changing signaling environments. environments Epigenetic pattern differs in normal and induced embryogenesis Knowing physiological organogenesis enables bioengineering tools for regenerative medicine ...
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This note was uploaded on 04/21/2010 for the course BIOE 10 taught by Professor Conboy during the Fall '09 term at University of California, Berkeley.

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