Post on 04-Jan-2016
Lecture 12. Stem Cells, Nuclear Transplantation, and Combined Cell &
Gene Therapy Strategies
Gene DefectsCan be Corrected By Homologous Recombination
Embryonic Stem (ES) Cells are Derived from the Inner Cell Mass of the Blastocyst
ES cells are Totipotent in vivo
Totipotent=can become ALLCell Types (Including Germ Cells)
Adult Stem Cells can be Derived from Most Tissues:These Cells are Pluripotent
Pluripotent=Can reconstitute some, but not all, cell types
ES Cells can be Induced to Differentiate into Pluripotent Stem Cells in vitro, given appropriate genetic or hormonal stimuli
Strategy for Today’s Paper:
Step 1:Create ES cells
by Transplanting the Nucleus of aRag2 -/- mouse
Step 2:Repair the
Rag2 gene in theES Cells
Step 3A: Test for Rescue Using Mice Created from theES Cells
Step 3B: DifferentiateES cells to HSC in vitroAnd Place these cells in
Rag2-/- Mice
Step 1A:
Step 1B:
Step 1C: Let Embryo with Nuclear Transplant DevelopTo the Blastocyst Stage and Create ntES cell lines
Step 2: Strategy for Repairing the Mutant Rag2 Allele after Nuclear Transplantation
Southern Blot showing the Repair
Step 3A: Mice Are Made From Repaired ntES Cells
T-cell Receptor Chains are Rearranged in the Rag R/- Mice
Step 3A: Mice Are Made From Repaired ntES Cells
Complete Immune System is Reconstituted in these Mice
Step 3A: Mice Are Made From Repaired ntES Cells
Complete Immune System is Reconstituted when BoneMarrow from these Mice is Transferred into Rag2 -/- Mice
Infect with HoxB4Retrovirus Vector
Step 3B: Reconstitute Mice with Pluripotent Stem CellsDerived from the Repaired ntES cells
Hypothesis: The ntES Derived-Stem Cells Do Not Express Correct Levels of Histocompatibility Type I Antigens and are Eliminated by Natural Killer (NK) cells.
1. Use anti-NK antibody to remove NK cells in Rag-/- Mice2. Use double KO mice: Rag2 -/- and IL-2 Common Cytokine Receptor Chain (C) -/-
As Recipients
Step 3B: First Attempts Were Unsuccessful:No Rescue of Immune System Observed
Results: Modest rescue of B-cells but not of T-cells
Complete RescueOf Myeloid Lineages, but only low level rescue of B-cells and T-Cells
The Repaired AlleleIs present in the Mice
Rearranged T-cell receptors are detected by PCR Analysis of the ntES Engrafted Mice
(~20% of WT)
ntES Engrafted Mice Produce Antibodies
Conclusion: Need to be able to make better HematopoieticStem 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.