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.