Lecture 16

Lecture 16 - BioE10: Lecture 16 Professor Irina Conboy Stem...

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Unformatted text preview: BioE10: Lecture 16 Professor Irina Conboy Stem cell technologies Engineering stem cells for transplantation and gene therapy Objectives: To understand basic principles of directed differentiation of embryonic stem cells and of cell/gene therapy with the help of adult stem cells. Biological fundamentals: hESC, embryoid body, directed differentiation, lineage specific differentiation, DA lineage-specific neuron, PD SCNT, oocyte, polar body, meiosis HSC, SCID, transduction, lentiviral vectors; 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. General strategy for directed differentiation of hESCs Ectoderm Mesoderm Endoderm SCNT and Cloning Spemann (1938): ‘Do nuclei change Do during development?’ Briggs and King (1952) nuclei could direct development to a sexually mature adult. No SCNT with patient’s nuclei was performed; similar or at times identical pictures of already existing hESC colonies (from one cell line) were shown and called different experiments or different patients’-derived cells. patients derived Only in vitro work and no cloning; Both oocytes at metaphase II (MII) and one-cell zygotes have been used as recipients for nuclear transfer. Oocytes at MII that are to be enucleated are cultured in medium containing the microfilament inhibitor cytochalasin D and the DNA-specific fluorochrome Hoechst 3332. Disruption of the microfilaments imparts an elasticity to the cell membranes such that a portion of the oocyte enclosed within a membrane can be aspirated into a pipette. The metaphase plate is removed by aspirating a small amount of cytoplasm from directly beneath the first polar body. Enucleation is confirmed by examining the aspirated cytoplasm under UV for the presence of both the polar body and the metaphase plate. Similarly, zygotes are also incubated in medium containing cytochalasin with the addition of the microtubule inhibitor colchicine. After enucleation, a donor cell (karyoplast) is aspirated into the enucleation pipette, the pipette is inserted through the hole that was created in the zona pellucida and the karyoplast expelled into the perivitelline space. The karyoplast is then placed in contact with the recipient cell or cytoplast. In most situations, cell fusion is induced by application of an electric pulse at 90° to the plane of contact between the two cells, + ionomycin (Ca2+) What is MII oocyte? Maternal determinants In mammals, fertilization typically involves the ovulation of one or a few eggs at one end of the female reproductive tract and the , yp y gg p entry of millions of sperm at the other. Given this disparity in numbers, it might be expected that the more precious commodity— eggs—would be subject to more stringent quality-control mechanisms. However, information from engineered mutations of meiotic genes suggests just the opposite. Specifically, the available mutants demonstrate striking sexual dimorphism in response to meiotic disruption; for example, faced with adversity, male meiosis grinds to a halt, whereas female meiosis soldiers on. This female “robustness” comes with a cost, however, because aneuploidy appears to be increased in the resultant oocytes. (A) Illustration of proximity of major MTOC at the surface; note the perinuclear ring of MTs at this early stage of development. (B) Ethanol-activated ovulated M-2 mouse oocyte in early anaphase that depicts prominent astral array of MTs (green) at the cortical attachment site (CAS) demonstrated by rhodamine phalloidin (red). MT- microtubules; MTOC=microtubule organizing center. During the first cell division following SCNT 1. Nuclear envelope breaks down; g y (spindle) ) 2. Microtubules organize into mitotic arrays ( p 3. DNA replication and mitosis Q: What will happen to the arrays of microtubules of the host cell? What needs to happen to the donor nucleus after SCNT for a successful cell division and thus, for cloning? Other considerations: chromatin status and methylation profile Remodeling of paternal chromatin after fertilization until the first cell division: Sperm DNA is highly compacted d t association with protamine. R t d due to i ti ith t i Removal of protamine i f ll l f t i is followed b bi di of th d by binding f the DNA by acetylated histones that help to maintain the newly formed chromatin in an “open” conformation. Reprogramming of the genome by progressive demethylation of DNA is accompanied by histone modifications, loss of oocyte-specific histone H1, and recruitment of nonhistone proteins t prepare DNA f transcription. hi t t i to for t i ti Methylation levels throughout preimplantation development of normal and i l t ti d l t f l d nuclear transfer-derived embryos. The paternal genome (purple) of normally derived embryos undergoes rapid active demethylation, whereas th maternal d th l ti h the t l genome (yellow) undergoes passive demethylation until the morula stage of pre-implantation development, when de novo methylation commences. Cl th l ti Cloned d embryos (turquoise) undergo a reduced passive demethylation. Directed differentiation Nat Med. 2006 Nov;12(11):1259-68 Goals: G l 1. Differentiate hESCs into dopaminergic neurons p g 2. Assay for the functional properties of these neurons in vitro 3. Assay for functional engraftment of these hESCderived neurons in vivo after transplantation into a rat model of Parkinson’s disease FGF8, Shh (embryonic neurogenesis) + co-culture with human astrocytes that retrovirally express telomerase gene (organ niche) Nat Med. 2006 Nov;12(11):1259-68 ~50% of DA neurons Improving motor skills in living brain by the hESC-based cell replacement 1. 1 Rats are lesioned on the right side of their brains with 6OHDA injections 2. Rats will turn contralateral (away from lesion) left upon stimulation of DA neurons with apomorphine 3. 3 hESC-derived DA neurons (or control saline) are injected into the same brains. 4. Test for functional recovery Q: Knowing that property of hESC is to produce teratoma, what undesired outcome can follow the transplantation of these DA neurons? Nat Med. 2006 Nov;12(11):1259-68 Bioengineering methods that can greatly improve directed differentiation of hESC- or iPS-cells 1. Synthetic biomaterials could be used to produce 3D differentiation matrix (mimics physiological organogenesis much better); 2. Stages of development (directed differentiation) of hESCs or iPSCs could be orchestrated by controlled release of FGF8, Shh, etc. from such matrix (combination of exogenous and endogenous factors is beneficial) 3. physiological matrix stiffness and electrical stimuli could be matched to those of developing brain (generally speaking). 4. Co cultures 4 Co-cultures with astrocytes and other cells present in neuronal niche could be set-up in 3D synthetic matrices. 5. Micro-sorting of single differentiated cells could prevent teratoma formation. f ti 6. Neuronal networks (synaptic connections between many neurons) could be patterned and coordinated in synthetic biomaterial (less danger of inadvertently “reprogramming” patient’s brain by the transplant). Stem cell and gene therapy Passegue, at. al, 2003 Hematopoietic sc and blood cell lineages. HSCs can be divided into LT-HSCs, highly self-renewing cells that reconstitute an animal for its entire life span, or ST-HSCs, which reconstitute the animal for a limited period. ST-HSCs differentiate into MPPs, which do not or briefly self-renew, and have the ability to differentiate into oligolineage-restricted progenitors that ultimately give rise to differentiated progeny through functionally irreversible maturation steps. The CLPs give rise to T steps lymphocytes, B lymphocytes, and natural killer (NK) cells. The CMPs give rise to GMPs, which then differentiate into monocytes macrophages and granulocytes, and to megakaryoticerythroid progenitors (MEP), which produce megakaryocytesplatelets and erythrocytes. Both CMPs and CLPs can give rise to dendritic cells. All of these stem and progenitor populations are separable as pure populations by using cell surface markers. BM-based gene corrections and cell transplantation therapy CD34=cell surface protein; Fibronectin+cytokines=ECM substrate+growth factors Summary of current gene-therapy protocols in humans. Harvested or mobilized HSCs are CD34 enriched and cultured with fibronectin, cytokines and retroviral supernatant for one to two days (lentiviral vectors) or three to four days [murine leukemia virus (MLV)-based vectors (retroviral) ]. Autologously transplanted cells migrate to the marrow and initiate hematopoiesis. De-differentiation is not Trans-differentiation Q: Cell A (BM cell) expresses GFP protein; and after BM transplantation muscle cells (or neurons) are green. A. Does this mean that BM cell trans-differentiated into muscle ff or brain cells? B. Provide an alternative explanation. C. How would you test if cell C H ld ll fusion occurred? Conlusions: Current ex-vivo approaches for organ and tissue manufacturing recapitulate the key aspects of embryonic development Current caveats include teratoma formation (purity of transplanted (p y p cells needs to be increased); techniques for robust expansion of progenitor cells remain to be established; integration of engineered tissues into physiologic organs (especially pathological) needs to be developed. Cell and gene therapies work well for the best characterized subsets of stem cells (such as HSC); thus, understanding of stem cell biology will t ll ( h HSC) th d t di f t ll bi l ill yield better strategies for tissue engineering. Check your understanding: What is the main principle of directed differentiation of hESC or iPSC into DA neurons? How to test function of these neurons? What are the caveats of this method? You h ld b bl t d i t h Y should be able to depict schematically gene therapy approach using HSCs. ti ll th h i HSC You should be able to depict schematically SCNT, listing key events and difficulties. What is MII oocyte? What is polar body? What is maternal determinant? ...
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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 Berkeley.

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