1. Lecture 12. Stem Cells, Nuclear Transplantation, and Combined Cell & Gene Therapy Strategies

2. Gene Defects Can be Corrected By Homologous Recombination

3. Embryonic Stem (ES) Cells are Derived from the Inner Cell Mass of the Blastocyst ES cells are Totipotent in vivo Totipotent=can become ALL Cell Types (Including Germ Cells)

4. Adult Stem Cells can be Derived from Most Tissues: These Cells are Pluripotent Pluripotent=Can reconstitute some, but not all, cell types

5. ES Cells can be Induced to Differentiate into Pluripotent Stem Cells in vitro, given appropriate genetic or hormonal stimuli

6. Strategy for Today’s Paper: Step 1: Create ES cells by Transplanting the Nucleus of a Rag2 -/- mouse Step 2: Repair the Rag2 gene in the ES Cells Step 3A: Test for Rescue Using Mice Created from the ES Cells Step 3B: Differentiate ES cells to HSC in vitro And Place these cells in Rag2-/- Mice

7. Step 1A:

8. Step 1B:

9. Step 1C: Let Embryo with Nuclear Transplant Develop To the Blastocyst Stage and Create ntES cell lines

10. Step 2: Strategy for Repairing the Mutant Rag2 Allele after Nuclear Transplantation Southern Blot showing the Repair

11. Step 3A: Mice Are Made From Repaired ntES Cells T-cell Receptor Chains are Rearranged in the Rag R/- Mice

12. Step 3A: Mice Are Made From Repaired ntES Cells Complete Immune System is Reconstituted in these Mice

13. 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

14. Infect with HoxB4 Retrovirus Vector Step 3B: Reconstitute Mice with Pluripotent Stem Cells Derived from the Repaired ntES cells

15. 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-/- Mice 2.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

16. Results: Modest rescue of B-cells but not of T-cells Complete Rescue Of Myeloid Lineages, but only low level rescue of B-cells and T-Cells The Repaired Allele Is present in the Mice

17. Rearranged T-cell receptors are detected by PCR Analysis of the ntES Engrafted Mice (~20% of WT)

18. ntES Engrafted Mice Produce Antibodies

19. 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.