Stem cell and gene therapy

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Stem cell and gene therapy

• Dr. Kuang-Wen Liao
• NCTU

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Stem cell

• The classical definition of a stem cell requires
  that it possess two properties
• Self-renewal - the ability to go through numerous
  cycles of cell division while maintaining the
  undifferentiated state.
• Potency - the capacity to differentiate into
  specialized cell types. In the strictest sense,
  this requires stem cells to be either totipotent
  or pluripotent - to be able to give rise to any
  mature cell type, although multipotent or
  unipotent progenitor cells are sometimes referred
  to as stem cells.

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• The two broad types of mammalian stem cells are
  embryonic stem cells that are found in
  blastocysts, and adult stem cells that are found
  in adult tissues.
• In a developing embryo, stem cells can
  differentiate into all of the specialized
  embryonic tissues.
• In adult organisms, stem cells and progenitor
  cells act as a repair system for the body,
  replenishing specialized cells, but also maintain
  the normal turnover of regenerative organs, such
  as blood, skin or intestinal tissues.

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• Potency specifies the differentiation potential
  (the potential to differentiate into different
  cell types) of the stem cell.
• Totipotent stem cells are produced from the
  fusion of an egg and sperm cell. Cells produced
  by the first few divisions of the fertilized egg
  are also totipotent. These cells can
  differentiate into embryonic and extraembryonic
  cell types.
• Pluripotent stem cells are the descendants of
  totipotent cells and can differentiate into cells
  derived from any of the three germ layers.
• Multipotent stem cells can produce only cells of
  a closely related family of cells (e.g.
  hematopoietic stem cells differentiate into red
  blood cells, white blood cells, platelets, etc.).
  
• Unipotent cells can produce only one cell type,
  but have the property of self-renewal which
  distinguishes them from non-stem cells (e.g.
  muscle stem cells).

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• Embryonic stem cell lines (ES cell lines) are
  cultures of cells derived from the epiblast
  tissue of the inner cell mass (ICM) of a
  blastocyst or earlier morula stage embryos.
• A blastocyst is an early stage
  embryoapproximately four to five days old in
  humans and consisting of 50150 cells.
• ES cells require very different environments in
  order to maintain an undifferentiated state.
  Mouse ES cells are grown on a layer of gelatin
  and require the presence of Leukemia Inhibitory
  Factor (LIF).
• Human ES cells are grown on a feeder layer of
  mouse embryonic fibroblasts (MEFs) and require
  the presence of basic Fibroblast Growth Factor
  (bFGF or FGF-2).
• Without optimal culture conditions or genetic
  manipulation, embryonic stem cells will rapidly
  differentiate.

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• The term adult stem cell refers to any cell which
  is found in a developed organism that has two
  properties the ability to divide and create
  another cell like itself and also divide and
  create a cell more differentiated than itself.
• Pluripotent adult stem cells are rare and
  generally small in number but can be found in a
  number of tissues including umbilical cord blood.
• Most adult stem cells are lineage-restricted
  (multipotent) and are generally referred to by
  their tissue origin (mesenchymal stem cell,
  adipose-derived stem cell, endothelial stem cell,
  etc.).

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Bone marrow transplantation

• The BMT can rebuilt up immunity and blood system.

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Highly efficient and large-scale generation of
functional dopamine neurons from human embryonic
stem cells.

• When grafted into a parkinsonian rat model, the
  hESC-derived DA neurons elicited clear behavioral
  recovery in three behavioral tests. In summary,
  our study paves the way for the large-scale
  generation of purer and functional DA neurons for
  future clinical applications (Proc Natl Acad Sci
  U S A. 2008 Mar 4105(9)3392-7) .

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Transplantation of magnetically labeled
mesenchymal stem cells improves cardiac function
in a swine myocardial infarction model.

• Transplanted MR-MSCs can regenerate new
  myocardium and prevent remolding in an MI model
  at 2-month follow-up and represent a preferred
  method to better understand the mechanisms of
  stem cell therapy in future clinical studies
  (Chin Med J (Engl). 2008 Mar 20121(6)544-50) .

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Stem cells for liver tissue repair Current
knowledge and perspectives

• The current research on the hepatic fate of stem
  cells is still facing difficulties to demonstrate
  the acquisition of a full mature hepatocyte
  phenotype, both in vitro and in vivo.
• Furthermore, the lack of obvious demonstration of
  in vivo hepatocyte-like cell functionality
  remains associated to low repopulation rates
  obtained after current transplantation procedures
  (World J Gastroenterol  2008 February 14 14(6)
  864-875).

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• The problems of stem cells

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• Tissue regeneration

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Definition of gene therapy

• The treatment of certain disorders, especially
  those caused by genetic anomalies or
  deficiencies, by introducing specific engineered
  genes into a patient's cells.

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Regulation of gene

• Continuous expression
• CMV
• LTR
• SV40
• - Mammalial constant expressive promoter b-actin
  promoter
• Regulatory expression
• Tetracycline regulation system

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Gene delivery

• Viral vector
• Non-viral technique

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Viral vector

• Retrovirus
• Adenovirus
• Adeno-associated virus

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Retrovirus

• Element
• LTR
• Packaged signal ?
• Package cell line
• Advantages
• Integrate into genome
• Permanent and stable expression
• Disadvantages
• Low safty
• Low titer

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Adenovirus

• Adenoviruses (AV) infect a wide range of
  non-dividing cells, and have been used
  extensively for live vaccines against respiratory
  infections without side effects (low
  pathogenicity to man).
• These features make AV a likely prospect for
  delivering genes to target cells.

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• High titer
• Induction of high immunity against AV

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Adeno associated virus

• Single stand DNA virus
• High safety
• Weak expression

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Non-viral technique

• Cationic liposome
• Calcium phosphate coprecipitation
• Directly muscular injection
• Electroporation
• PEI (polyrthylenimine)
• Gene gun

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Calcium phosphate coprecipitation
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Directly muscular injection

• Naked DNA
• Muscular cell
• Epidermis

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PEI
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Electroporation
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Gene Gun
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Clinical trial for gene therapy

• Dr. Flake was able to cure a child of a rare
  immune disease, X-linked severe combined
  immunodeficiency disorder (X-SCID), by using a
  prenatal stem cell transplant in 1996 that
  rebuilt his immune system.

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Gene Therapy for Aspartoacylase (ASPA)-deficient
patients Canavan disease
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