Stem Cells 2

From CellBiology

ANAT3231 Cell Biology online lectures from the 2017 course.

Embryonic Stem Cell Signaling Regulation

Mouse- embryonic stem cell signaling regulation.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-1 (SSEA-1) cell surface 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

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



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

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.

  • 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

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.

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