Lecture 12. Stem Cells, Nuclear Transplantation, and Combined Cell

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Lecture 12. Stem Cells, Nuclear Transplantation,
and Combined Cell Gene Therapy Strategies
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Embryonic Stem (ES) Cells are Derived from the
Inner Cell Mass of the Blastocyst ES cells are
Totipotent in vivo
Totipotentcan become ALL Cell Types (Including
Germ Cells)
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Adult Stem Cells can be Derived from Most
Tissues These Cells are Pluripotent
PluripotentCan reconstitute some, but not all,
cell types
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ES Cells can be Induced to Differentiate into
Pluripotent Stem Cells in vitro, given
appropriate genetic or hormonal stimuli
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Strategy for Todays Paper
Step 3A Test for Rescue Using Mice Created from
the ES Cells
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Step 1A
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Step 1B
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Step 1C Let Embryo with Nuclear Transplant
Develop To the Blastocyst Stage and Create ntES
cell lines
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Step 2 Strategy for Repairing the Mutant Rag2
Allele after Nuclear Transplantation
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Step 3A Mice Are Made From Repaired ntES Cells
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Step 3A Mice Are Made From Repaired ntES Cells
Complete Immune System is Reconstituted in these
Mice
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Step 3A Mice Are Made From Repaired ntES Cells
Complete Immune System is Reconstituted when
Bone Marrow from these Mice is Transferred into
Rag2 -/- Mice
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Step 3B Reconstitute Mice with Pluripotent Stem
Cells Derived from the Repaired ntES cells
Infect with HoxB4 Retrovirus Vector
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Step 3B First Attempts Were Unsuccessful No
Rescue of Immune System Observed

• Hypothesis The ntES Derived-Stem Cells Do Not
  Express
• Correct Levels of Histocompatibility Type I
  Antigens
• and are Eliminated by Natural Killer (NK) cells.
• Use anti-NK antibody to remove NK cells in Rag-/-
  Mice
• Use double KO mice Rag2 -/- and
• IL-2 Common Cytokine Receptor g Chain (gC) -/-
• As Recipients

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Results
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Rearranged T-cell receptors are detected by PCR
Analysis of the ntES Engrafted Mice (20 of WT)
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ntES Engrafted Mice Produce Antibodies
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Conclusion Need to be able to make better
Hematopoietic Stem Cells from the ntES
cells Simply overexpressing Hox4B with the
Retrovirus Vector Favors Myeloid vs. Lymphoid
Differentiation Retrovirus Gene Transfer is
ineffective for Gene Therapy.