Lecture 15 - BioE10 Lecture 15 Professor Irina Conboy Stem Cell Technologies Key concepts of stem cell engineering Objectives Identifying and using

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Unformatted text preview: BioE10: Lecture 15 Professor Irina Conboy Stem Cell Technologies Key concepts of stem cell engineering Objectives: Identifying and using embryonic gene regulatory networks for directed organogenesis. Cell de-differentiation and production of totipotent stem cells from fibroblasts. Biological fundamentals: PCR, DNA Mircoarray, mRNA, cDNA, ChIP on Chip assay , y, , , p y ICM; Cell fate, self-renewal vs. differentiation, teratoma, transgenic and ko animals Stem cell (adult, embryonic); iPS cell; de-differentiation There are embryonic and adult stem cells and “in-between” stem cells: cord blood, fetal tissues, amniotic fluid Embryonic stem cells Stem cell Asymmetric cell division Symmetric cell division Immortality=ability to self renew and differentiate indefinitely E3.5-5.5 Self-renewed Self renewed Stem cell Somewhat differentiated cell = lineage specific progenitor Differentiated cell Toti- or pluripotenitality:~200 cell-types from single totipotent cell Adult stem cells Where do ESC cultures come from? hESC lines are derived from li d i df ICM of human blastocyst stage embryo SCNT iPS Normally, blastocyst-stage embryos are chosen for derivation of hESCs having well-developed inner cell masses (ICMs) th t are i l t d mechanically. ICMs will f i ll (ICM ) that isolated h i ll ICM ill form rounded cell colonies d d ll l i of small tightly packed cells with a large nucleus to cytoplasmic ratio. It is also possible to derive disease-specific hESCs from embryos with diagnosed mutations by preimplantation g genetic diagnosis. This strategy may result in the identification of new candidate drugs that g gy y g could eventually result in treatments that reduce, or control, these disease conditions. The ‘price’ of this research, however, is destruction of human embryos that have the potential to become new human beings. The fact that these embryos were destined to be discarded and, by implication, implication destroyed because the gamete donors who had lead to their formation no longer wished to continue with their parent programme, was for many not an argument to redirect their fate towards stem cell derivation however noble the cause of curing chronic disability. It is of note, however, that not all societies or religions adopt the same point of view: while Christianbased ethics believe that life begins at fertilization, other beliefs consider 40 days after conception as the crucial time point in determining the moral status of the embryo. Key concepts: Stem cells Embryonic stem (ES) cells are pluripotent — that is, they are still at the base of the differentative tree and have retained their embryonic capacity to give rise to most, if not all, cell types. The potential use for ES cells is their ability to be isolated and grown in large numbers, coupled with their ability to ll i th i bilit t b i l t d d i l b l d ith th i bilit t differentiate into any other cell of the body. This might provide a way to generate specifically predetermined somatic stem cells and precursor cells, which will allow the therapeutic regeneration of damaged adult tissues for which there is no other endogenous or available cell source. The body consists of a large number of specialized, differentiated cells with diverse functions that are organized into specific tissues and organs. During development, and throughout life, many of these tissues can repair and regenerate themselves after tissue damage. This regeneration and repair depend on populations of less-differentiated cells — adult stem cells. The essential property that defines a stem cell population is that, by division, these cells can both that division maintain the reserve pool and provide precursor cells that can develop into the final differentiated state. These stem cell populations are specialized in (that is, committed to) specific directions of differentiation. So, it has been surprising that some studies have shown that stem cells extracted from one tissue have repopulated another. However, in most cases, these studies were not carried out with single purified cell populations, and these results are by no means unambiguous. Nevertheless, highly specialized, tissuespecific stem cells are exactly what is needed for a particular therapy if they could be isolated in sufficient numbers. Criteria f pluripotency/”stemness” of a cell: C for / f 1. Does such cell express genes of “stemness”? 2. Does it produce teratoma? 3. Does it produce chimeric embryos and animals? Q: How to test if a cell (hMSC (fibroblast), cord blood-derived, etc.) is a stem cell, e.g. is pluripotent and can self-renew? Teratoma formation is a property of totipotent stem cells Embryonic stem cells are E b i t ll totipotent= can give rise to all organs and tissues; and can selfrenew indefinitely. Formations of teratomas and serial transplantation of dissociated teratomas proves both i t b th points. Simplified scheme of Transgenic/KO mice production Cultured ES cells from Brown mouse Transfect with g gene of interest Neomycin selection White mouse Only stem cells that are pluripotent and self-renew will give rise to all tissues and organs in an embryo and newborn animal Q: How to use the GFPreporter approach for testing if cell of interest (e.g. hMSC) is a true pluripotent selfrenewing stem cell? Cell. 2007 Nov 30;131(5):861-72. Creating autologous (syngeneic) tissues: De-differentiation De-differentiation i t embryonic-like stem cell D diff ti ti into b i lik t ll Induced pluripotency state (iPS) cells: Are now produced from mouse and human somatic cells by viral and plasmid vector delivery of the master-regulatory genes of stemness (Oct4 and Sox2, plus cMyc (cell proliferation) and Klf4. This work has several important goals and outcomes: 1. Scientific: What mechanisms make cell a stem cell vs. differentiated cell. 2. Clinical: Autologous (syngeneic) pluripotent cell derivation for tissue replacement, studies of genetic and acquired disorders, etc. 3. Ethical: circumvents the issue of embryo destruction. y 4. Philosophical: redefines the concept of stem cell and of cloning. cloning Cell, 2007. Induction of Pluripotent Stem cells from Adult Human Fibroblasts by Defined factors Kazutoshi Takahashi, Koji Tanabe Mari Ohnuki Megumi Narita Takahashi Tanabe, Ohnuki, Narita, Tomoko Ichisaka, Kiichiro Tomoda, and Shinya Yamanaka Candidate gene approach: what are the “genes of stemness”? 1. Expressed in embryonic stem cells, but not in differentiated cells. 2. Lack of expression causes the loss of “stemness”. 3. Over-expression induces “stemness” Sox-2 S 2 The phenotypic effects of gene-targeting experiments in mice. The phenotypic effects of gene-targeting experiments in mice provided insights into the pluripotency of embryos and embryonic stem cells. Oct4–/– (octamer-binding transcription factor-4), Nanog–/–, Stat3–/– (signal transducer and activator of transcription-3), gp gp130–/– (glycoprotein-130), Lif –/– (leukaemia inhibitory factor) and Lifr –/– (LIF receptor) embryos can (g y p ), ( y ) ( p ) y develop to various stages in vivo— as highlighted by the outer ring in the figure, but they all fail (truncated line from the ring to the centre) to yield ESCs (embryonic stem cells) in vitro (see centre of the figure). ESCs might only represent some aspects of the natural embryo, and studying cells and phenomena in vivo is indispensable. E1.5, E3.5, E8.5 and E18.5 represent the number of days of embryonic (E) development although developmental stages following E10 5 should properly be referred to as development, E10.5 fetal. Q: place ES regulatory genes in temporal hierarchy from most to least important for p g yg p y p pluripotentiality. ChIP on Chip assay or how the “genes of stemness” were identified ChIP (Chromatin immuno-precipitation) on Chip assay Bead Introducing few copies of Oct4 and Sox2 genes (proteins) will activate a selfreinforcing gene regulatory embryonic stem cell circuitry Cell. 2005 Sep 23;122(6):947-56 The main functions of this regulatory network Transcriptional activation of embryonic genes Chromatin remodeling Transcriptional ( (histone acetylation, y , silencing of differentiated genes methylation) to more permanently silence differentiated genes and activate embryonic gene Cell. 2005 Sep 23;122(6):947-56 Experimental approach of de-differentiation (general scheme): Several genes of “stemness” were expressed in fibroblasts (otherwise known as “mesenchymal stem cells” and turned fibroblasts into embryonic-like stem cells (iPS= induced pluripotent state) Fibroblasts (MSC) differentiate into cells of above lineages in specific liquid media (known for many years); for each cell-fate a combination of chemical factors from media with a particular rigidity of the adhesion substrate works the best; differentiation into cells expressing muscle and neuronal markers was also tested. (Discher, (Discher Cell 2006). 2006) Q: is the rigidity of adhesion substrate (1) sufficient, (2) necessary or (3) limiting factor in the cell-fate determination? More details on the experimental approach Fibroblasts are engineered to express a selection marker, e.g. Nanog promoter that drives antibiotic resistance gene under a positive transcriptional control by Oct4. ATG Neomycin or Puromycin resistance Nanog promoter Oct4 Retroviral transduction Oct-4; Sox2 (nanog); cMyc, KLF4 Retroviral (Lyn28) transduction These cells express the selection l ti marker Cell. 2006 Aug 25;126(4):663-76. ~0.2% survival Neo, Puro selection Q: Will both endogenous Nanog and synthetic Nanog promoter driven antibiotic resistance genes be expressed in dedifferentiated fibroblasts? Q: How many cells one should start with in order to obtain 50 de-differentiated fibroblast-derived colonies? 1colony=1cell Only fibroblasts that reactivated the embryonic gene expression profile, profile e.g. nanog, will survive Cell. 2007 Nov 30;131(5):861-72. Even more details on this experimental approach One of the key approaches for successfully de-differentiating HUMAN fibroblasts (as compared to MOUSE fibroblasts) fib bl t ) was engineering of i i f human cells with synthetic mouse-derived Sic7a1receptor. This receptor is readily used by the retroviruses which deliver genes of “stemness” to HUMAN fibroblasts fibroblasts. Expression of a reporter gene delivered by retroviral vector is shown as a proof of the aforementioned point. Q: How many different retroviral vectors were introduced into human fibroblasts (if 2 genes maximum could be delivered from one retrovirus)? MEF=mouse embryonic fibroblasts; HDF=human derived fibroblasts. Cell. 2007 Nov 30;131(5):861-72. The main functions of this regulatory network Q: Is continuous expression of retrovirally delivered genes (Oct4, Sox2, etc.) necessary, or would endogenous genes maintain “stemness” after reactivation by the exogenously expressed Oct4, Sox2, etc.? Transcriptional activation of embryonic genes Chromatin remodeling Transcriptional ( (histone acetylation, y , silencing of differentiated genes methylation) to more permanently silence differentiated genes and activate embryonic gene Cell. 2005 Sep 23;122(6):947-56 Q: What chromatin confirmation will be at Nanog genetic locus in (a) Neuron (b) iPS cell (c) embryonic stem cell? Q: for female cells how many X chromosomes do you expect to be inactivated in (a) fibroblast, (b) iPS cell derived from such fibroblast and (c) neuron produced by such iPS cell? Potential applications and Caveats Cells positive for beta III tubulin and tyrosine hydroxylate (TH=marker of mature Dopaminergic neurons) -each clone has unique pattern each of viral integration sites of c-myc 1. Strong retroviral promoters might localize next to oncogenes; 2. Unknown number of copies of exogenous genes and retroviral genomes are 2 U k b f i f d t i l introduced; Cell. 2007 Nov 30;131(5):861-72. Conlusions: Embryonic stem cells or induced pluripotency cells are ideal candidates for tissue engineering and regenerative medicine applications; Not every cell can be called “stem cell”, specific criteria of self-renewal and pluripotency or dedicated differentiation do apply Candidate gene approach was successfully used to identify “genes of stemness” Recombinant DNA t h l R bi t technology was used for negative and positive df ti d iti selection of iPS cells There are certain caveats in current generation of iPS cells, e g use or cells e.g. retroviral vectors. Check your understanding: You should be able to compare the properties of adult and embryonic stem cells You should be able to propose strategies for identifying whether a cell is a stem cell or not You should understand the main experimental approaches of iPS cell production; and know the difference between de-differentiation and transdifferentiaiton. You should be able to depict schematically ChIP on Chip assay. ...
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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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