Difference between revisions of "Stem Cells 2"

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** uses a fluorescent detection of endogenous phosphatase activity in embryonic stem cells
 
** uses a fluorescent detection of endogenous phosphatase activity in embryonic stem cells
  
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[http://www.pnas.org/content/102/23/8239/F5.expansion.html PNAS - Expression of molecular markers characteristic of ES cells in morula-derived cell lines]
  
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===Expression of Zfp42/Rex1 Gene===
  
[http://www.pnas.org/content/102/23/8239/F5.expansion.html PNAS - Expression of molecular markers characteristic of ES cells in morula-derived cell lines]
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* used as a marker of undifferentiated stem cells  
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* regulated by Nanog, Sox2, and Oct4, and by the Wnt pathway
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* also subject to epigenetic regulation by polycomb complexes and DNA methylation
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<pubmed>16714766</pubmed>
  
 
==Stem Differentiation==
 
==Stem Differentiation==

Revision as of 12:26, 25 May 2011

ANAT3231 Cell Biology online lectures from the 2017 course.

Stem cell therapy cartoon.jpg

Embryonic vs Adult Stem Cells

Embryonic Stem Cell Advantages

  • Pluripotency - ability to differentiateinto any cell type.
  • Immortal - one cell can supply endless amounts of cells.
  • Easily available - human embryos from fertility clinics.

Embryonic Stem Cell Disadvantages

  • Unstable - difficult to control differentiation into specific cell type.
  • Immunogenic - potential immune rejection when transplanted into patients.
  • Teratomas - tumor composed of tissues from 3 embryonic germ layers.
  • Ethical Controversy - unethical for those who believes that life begins at conception.


Adult Stem Cell Advantages

  • Already ‘specialised’ - induction of differentiation into specific cell types will be easier.
  • Plasticity - Recent evidences suggest wider than previously thought ranges of tissue types can be derived.
  • No Immune-rejection - if used in autologous transplantations.
  • No Teratomas - unlike ES cells.
  • No Ethical Controversy - sourced from adult tissues.

Adult Stem Cell Disadvantages

  • Minimal quantity - number of isolatable cells may be small.
  • Finite life-span - may have limited lifespan in culture.
  • Ageing - stem cells from aged individuals may have higher chance of genetic damage due to ageing.
  • Immunogenic - potential immune rejection if donor cells are derived from another individual.

Stem Cell Markers

In order to carry out research on stem cells, it is important to be able to identify them. A number of different research groups in the late 90's generated several antibodies which specifically identified undifferentiated, differentiating or differentiated stem cells from a number of different sources and species. Note that the nomenclature in some cases is based upon the antibody used to identify the cell surface marker.

  • Every cell surface has specialized proteins (receptors) that can selectively bind or adhere to other “signalling” molecules (ligands)
  • Different types of receptors differ in structure and affinity for signalling molecules
  • Cells use these receptors and molecules that bind to them as a way of communicating with other cells and to carry out their proper functions in the body
  • Stage-specific embryonic antigen (SSEA)-1, -3 and -4 and tumor-rejection antigen (TRA)-1-60 and -1-81, are expressed in specific combinations by undifferentiated pluripotent cells.
    • embryonic stem cells, induced pluripotent stem cells, embryonal carcinoma cells, primordial germ cells, mesenchymal progenitors in adult murine bone marrow, and embryonic germ cells.
  • Stage-Specific Embryonic Antigen-1 (SSEA-1) cell surface glycan embryonic antigen which has a role in cell adhesion, migration and differentiation and is often differentially expressed during development. Can be identified by Davor Solter monoclonal antibody MC-480 (SSEA-1).
  • Stage-Specific Embryonic Antigen-4 (SSEA-4) cell surface embryonic antigen of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES) which is down-regulated following differentiation of human EC cells. Antigen not expressed on undifferentiated murine EC, ES and EG cells but upregulated on differentiation of murine EC and ES cells. Can be identified by Davor Solter monoclonal antibody MC-813-70 (SSEA-4)
  • Tumor Rejection Antigen (TRA-1-60) Sialylated Keratan Sulfate Proteoglycan expressed on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES).
  • Tumor Rejection Antigen (TRA-1-81) antigen expressed on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES).
    • Both TRA antibodies identify a major polypeptide (Mr 240 kDa) and a minor polypeptide (Mr 415 kDa).
  • Oct-4 (Pou5f1 – Mouse Genome Informatics) gene has an essential role in control of developmental pluripotency (Oct4 knockout embryo blastocysts die at the time of implantation). Oct4 also has a role in maintaining viability of mammalian germline.
  • Stem Cell Antigen 1 (Sca-1) member of the Ly-6 family of GPI-linked surface proteins (Mr 18 kDa) and a major phenotypic marker for mouse hematopoietic progenitor/stem cell subset.
  • CD133, AC133, prominin 5 transmembrane glycoprotein (865 aa) expressed on stem cells with hematopoietic and nonhematopoietic differentiation potential.
  • Alkaline Phosphatase
    • embryonic stem cell is characterized by high level of expression alkaline phosphatase (undifferentiated state) ATCC ELF Phosphatase Detection Kit for Embryonic Stem Cells
    • assay to determine if embryonic stem cells are undifferentiated or are starting to differentiate
    • uses a fluorescent detection of endogenous phosphatase activity in embryonic stem cells

PNAS - Expression of molecular markers characteristic of ES cells in morula-derived cell lines

Expression of Zfp42/Rex1 Gene

  • used as a marker of undifferentiated stem cells
  • regulated by Nanog, Sox2, and Oct4, and by the Wnt pathway
  • also subject to epigenetic regulation by polycomb complexes and DNA methylation

<pubmed>16714766</pubmed>

Stem Differentiation

Epithelium

  • each generation at least 1 "immortal" stem cell
    • descendants present in patch in future
  • Other basal cells
    • leave basal layer and differentiate
  • Committed, born different

or may be stem cells equivalent to immortal stem cell in character mortal in sense that their progeny jostled out of basal layer and shed from skin

Amplifying Cells

  • Stem cells in many tissues divide only rarely
  • give rise to transit amplifying cells
  • daughters committed to differentiation that go through a limited series of more rapid divisions before completing the process.
  • each stem cell division gives rise in this way to eight terminally differentiated progeny

Stem Cell Production - Stem Cell Daughter Fates

  • Environmental asymmetry
    • daughters are initially similar
    • different pathways according to environmental influences that act on them after they are born
    • number of stem cells can be increased or reduced to fit niche available
  • Divisional asymmetry
    • stem cell has an internal asymmetry
    • divides in such a way two daughters are already have different determinants at time of their birth

Human embryonic stem cell Differentiation in Culture

Human embryonic stem cell defined conditions.jpg

Characterization of teratomas from H9 cells cultured on an hE-cad-Fc-coated surface. Hematoxylin and eosin staining of paraffin sections through teratomas identified the differentiation huES cells into various tissues:

  • a - immature neuroblastic tissue with neuronal rosettes
  • b - neuroepithelium with pigment
  • c - immature sebaceous tissue
  • d - cartilage
  • e - columnar epithelium
  • f - gut-like epithelial structures
  • g - contains neural tissue (1), cartilage (2), bone parenchyma (3), and epithelial tissue (4).

Bar indicates 100 μm.

Figure from: <pubmed>20525219</pubmed>| BMC Dev Biol.

Current stem cell research

NIH - stem cell cartoon

How to:

  • Isolate
  • Grow
  • Maintain, store
  • Differentiate
  • Therapeutic uses

Growth of Embryonic Stem Cells

  • Mouse blastocyst-derived ES cell line D3
    • from American Type Culture Collection (ATCC)
  • Undifferentiated ES cells
    • maintained on gelatin-coated dishes
    • earlier studies, feeder layer

Growth Media

  • DMEM (dulbecco’s modified essential media)
  • 2 mM glutamine (essential amino acid)
  • 0.001% beta-mercaptoethanol (reducing agent)
  • 1x nonessential amino acids (amino acids for growth)
  • 10% donor horse serum (source of growth factors etc)
  • human recombinant leukemia inhibitory factor (LIF) 2,000 units/ml

Inducible Stem Cell

Human induced pluripotent stem cells 01.jpg

(A) Morphology of human iPS cell clones (2a, 3a and 6a) on surfaces coated with Matrigel or hE-cad-Fc. Scale bar indicates 100 μm. (B) Characterization of teratomas from iPS cells (clone 2a) cultured on an hE-cad-Fc-coated surface. Hematoxylin and eosin staining of teratomas showed the differentiation into various tissues, including immature neuroblastic tissue with neuronal rosettes (a), striated muscle (b), and columnar epithelium (c). Bar indicates 100 μm.

Figure from: <pubmed>20525219</pubmed>| BMC Dev Biol.

Mouse- embryonic stem cell signaling regulation.jpg

Yamanaka Factors

A set of 4 transcription factors when introduced into cells induces stem cell formation. These four transcription factors can be expressed from doxycycline (dox)-inducible lentiviral vectors. The only culture difference in iPS cells and human embryonic stem cell culture is that iPS cell culture require 100ng/ml of bFGF in the culture media.

Outline of the MEF reprogramming protocol 1 Outline of the MEF reprogramming protocol 2 | stained with anti-Rex1, Sox2 and SSEA1 antibodies


  • OCT4 Transcription factors containing the POU homeodomain
  • MYC The MYC protooncogene encodes a DNA-binding factor that can activate and repress transcription. Ectopic expression of c-Myc can also cause tumorigenicity in offspring.
  • SOX2 SRY-RELATED HMG-BOX GENE 2
  • KLF4


<pubmed>16904174</pubmed>


Links: Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

More recently shown that Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells.

<pubmed>18594515</pubmed>


Thompson Factor

Neural Therapeutic Uses?

Stem cell therapy cartoon

NIH - Use of Genetically Modified Stem Cells in Experimental Gene Therapies


Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model

  • Implantation of fetal dopamine (DA) neurons can reduce parkinsonism in patients
  • current methods are rudimentary
  • lacking a reliable donor cell source

Transplanted ES cells can develop spontaneously into dopamine (DA) neurons

  • Such DA neurons can restore cerebral function and behavior in an animal model of Parkinson's disease
  • Björklund et al Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 4, 2344-2349, February 19, 2002

Parkinson Rat Model

Embryonic stem cell Transplant

  • transplanting low doses of undifferentiated mouse embryonic stem (ES) cells into rat striatum
  • results in a proliferation of ES cells into fully differentiated DA neurons
  • ES cell-derived DA neurons caused gradual and sustained behavioral restoration of DA-mediated motor asymmetry


Staining of a Graft

  • 16 weeks after implantation of D3 ES cells into adult 6-OHDA lesioned striatum
    • TH-positive neurons were found within the graft (A and B, green)
    • All TH-positive profiles coexpressed the neuronal marker NeuN (A, red)
    • TH (B) also was coexpressed with DAT (C, red) and AADC (D, blue), shown by white triple labelling (E)

Rotation response to Amphetamine

  • 6-OHDA-lesioned animals were selected for transplantation by quantification of rotational behaviour in response to amphetamine
  • response was examined post-transplantation at 5, 7, and 9 weeks
  • Animals with ES cell-derived DA neurons showed recovery over time from amphetamine-induced turning behavior

Seizure suppression in amygdala-kindled mice by transplantation of neural stem/progenitor cells derived from mouse embryonic stem cells

Neurol Med Chir (Tokyo). 2010;50(2):98-105; disucussion 105-6.

Shindo A, Nakamura T, Matsumoto Y, Kawai N, Okano H, Nagao S, Itano T, Tamiya T. Source Department of Neurological Surgery, Kagawa University Faculty of Medicine, Kagawa.

Abstract

Embryonic stem cells (ES cells) differentiate into multiple cell lineages including neural cells. The present study optimized the method to induce differentiation of gamma-aminobutyric acid-producing neurons (GABAergic neurons) from ES cell-derived neural stem/progenitor cells (NS/PCs), and transplanted these ES cell-derived GABAergic neurons producing neural progenitors into kindled epileptic mice, and analyzed the morphological and functional recovery from epilepsy. The response of kindling was evaluated by the modified Racine scale. Following stage 5 kindling, the mice were divided into two groups. Group 1 received NS/PCs derived from the ES cells ubiquitously expressing green fluorescent protein transplanted into the dorsal hippocampal area. Group 2 received microinjections of only the medium. After transplantation, the recovery of seizures was evaluated by the modified Racine scale again. All mice were perfused and fixed for immunohistochemical analysis after finishing the kindling experiment. In Group 1, one mouse was classified as stage 0, five as stage 3, and one as stage 4 recovering from stage 5 at 6 weeks after transplantation. In Group 2, all mice remained in stage 5. The transplanted cells were examined immunohistochemically using neuronal and GABAergic markers. In the transplanted mice, substantial hippocampal GABAergic re-innervation and seizure-suppressing effects were observed. NS/PCs derived from ES cells have high potential for use in transplantation therapy for clinically intractable epilepsies.

PMID: 20185872 http://www.ncbi.nlm.nih.gov/pubmed/20185872 Neurol Med Chir (Tokyo)

Cytoskeleton Disease?

Targeted Gene Correction of Laminopathy-Associated LMNA Mutations in Patient-Specific iPSCs

Cell Stem Cell. 2011 May 18.

Liu GH, Suzuki K, Qu J, Sancho-Martinez I, Yi F, Li M, Kumar S, Nivet E, Kim J, Soligalla RD, Dubova I, Goebl A, Plongthongkum N, Fung HL, Zhang K, Loring JF, Laurent LC, Izpisua Belmonte JC. Source Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.

Abstract

Combination of stem cell-based approaches with gene-editing technologies represents an attractive strategy for studying human disease and developing therapies. However, gene-editing methodologies described to date for human cells suffer from technical limitations including limited target gene size, low targeting efficiency at transcriptionally inactive loci, and off-target genetic effects that could hamper broad clinical application. To address these limitations, and as a proof of principle, we focused on homologous recombination-based gene correction of multiple mutations on lamin A (LMNA), which are associated with various degenerative diseases. We show that helper-dependent adenoviral vectors (HDAdVs) provide a highly efficient and safe method for correcting mutations in large genomic regions in human induced pluripotent stem cells and can also be effective in adult human mesenchymal stem cells. This type of approach could be used to generate genotype-matched cell lines for disease modeling and drug discovery and potentially also in therapeutics.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID: 21596650 | Cell Stem Cell.

Human embryonic stem cells (hESCs) without the use of additional human embryos

  1. Reprogramming of adult cell nucleus - use existing hESCs to fuse with an adult somatic cell, generating a cell line that retains ESC specific properties and yet has the genotype of the somatic cell donor. However, there is no technology available to selectively remove all the ESC chromosomes while retaining the somatic cell chromosomes.
  2. ESCs from embryo like entities - use of somatic cell nuclear transfer (SCNT) to produce developmentally compromised embryo-like structures, with the help of genetically premodified deficient nuclei which cannot support development. The zygote produced by such nuclear transfer undergoes cleavage in-vitro and produce ICM cells, which would be used for deriving ESCs, but would not proceed further in development. A proof of principle to this was accomplished by generating mouse ESCs, using a donor nucleus which was silenced for Cdx2 gene. This is ethically correct for those who believe that fetal life begins only after the embryo implants. However, one need not go for creating a mutation to achieve this target, as a blastocyst cannot develop into a complete human life in vitro, irrespective of the presence or absence of any kind of genetic alterations.
  3. ESC lines from single blastomeres - a single cell can be isolated from the cleavage stage embryo, a technique well established for preimplantation genetic diagnosis (PGDs), and used to create a cell line from it; the rest of the embryo can be transferred back to the uterus to give rise to a fetus. Robert Lanza's group has shown that ESC lines could be established from single cell biopsies of the mouse and human embryos. However, this technique is very difficult to translate to human being. Also, the fate of the residual embryos if they are transferred is largely unknown, as there is a lack of long term studies supporting the health of babies born following PGD.
  4. ESC lines from induced somatic cell dedifferentiation - adult somatic cells are genetically modified and reprogrammed to undergo a process of dedifferentiation, by inducing the expression of pluripotency related genes. Recently, induced pluripotent stem cell lines have been derived by allowing trans-acting factors present in the mammalian oocytes to reprogram somatic cell nuclei to an undifferentiated state. They have demonstrated that four factors OCT-4, SOX-2, Nanog and LIN28 are sufficient to reprogram human somatic stem cells to pluripotent stem cells. Whereas, Takahashi and Yamanaka (2006) induced somatic cells into pluripotent stem cells by introducing four factors OCT-4, SOX-2, c-Myc and KLF-4. These cells designated as induced pluripotent stem cells (iPS) exhibit morphology of embryonic stem cells and express ES cell markers. Several technical limitations such as use of retrovirus or lentiviruses for transfecting OCT-4, Nanog, SOX-2, C-MYC, LIN28 or KLF4 restrict the use of such cell lines for clinical applications (Hanna et al., 2007).
  5. Embryonic like stem cells from alternative sources - adult stem cells similar to blastomeres of the preimplantation stage embryos have been identified and isolated by Henry Young and coworkers. These cells called the blastomere-like stem cells (BLSCs) are found to be totipotent due to their potential to give rise to all tissue types including the gametes. These BLSCs can be induced to differentiate in a unidirectional manner to form pluripotent embryonic-like stem cells (ELSCs). It is also claimed that these cells do not express the MHC class-I or HLA DR-II cell surface markers. More recently Meng et al., (2007) have discovered a population of stem cells in the menstrual blood. These cells named as the "Emdometrial Regenerative Cells" are shown to be capable of differentiating into 9 tissue lineages namely: cadiomyocytic, respiratory epithelial, neurocytic, myocytic, endothelial, pancreatic, hepatic, adipocytic, and osteogenic.

(Text modified from <pubmed>18230169</pubmed>| J Transl Med.)

2012 Course Content

Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton - Intermediate Filaments | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | 2012 Revision | Development


Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Microarray Visit | Tissue Culture 1 | Tissue Culture 2 | Stem Cells Lab | Stem Cells Analysis



2012 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7 | Group 8 | Group 9

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G