1. Bch4122: Lecture #6 Stem Cell Treatments for Skeletal Muscle Supplemental Reading: F.D. Price, K. Kuroda, M.A. Rudnicki, Stem cell based therapies to treat muscular dystrophy, BBA 1772: 272-283, 2007.

3. How do you know if stem cell therapy worked? Need to: 1.Show that the stem cell has differentiated into the desired cell type 2.Show that the tissue function has been improved 3.Ensure that the appropriate model system is used 4.Ensure that no negative side-effects occurred

4. Two strategies for cell based therapy to treat muscular dystrophy: Autologous stem cell transfer Allogenic stem cell transfer Muscle stem cells From patient Muscle stem cells with restored dystrophin expression Genetic alteration Re-implantation Muscle stem cells From Donor Transplantation Into patient

5. Two strategies for cell based therapy to treat muscular dystrophy: Autologous stem cell transfer Allogenic stem cell transfer Derived from the patient and therefore less likely to have immune response Genetic alteration can change the stem cells and result in an immune response No genetic alteration Risk of immune rejection Requires immunosuppression and donor compatibility

6. Current Sources of Stem Cells for Muscle Regeneration 3. Mesenchymal stem cells 1. Primary myoblasts from satellite cells 4. Embryonic stem cells 2. Satellite Cells 5. Mesangioblasts

7. Satellite cells are quiescent until they are activated to proliferate and generate cells that either differentiate or remain satellite cells Satellite cell Myoblast Intermediate cell

8. Have been used in clinical trials to treat patients with DMD with limited success Approach: To isolate satellite cells from matched donors and culture them to form primary myoblasts Repetitive intramuscular injections of large quantities of myoblasts 1. Primary myoblasts from satellite cells Muscle fibre

9. Outcome of myoblast transfer Some expression of dystrophin has been restored No substantial physiological correction of the dystrophic phenotype

10. Satellite cell Myoblast Intermediate cell Problems with myoblast transfer 1.Grafted myoblasts have limited migration and repeated local injections are required. Since heart and diaphragm are the biggest problems for DMD patients, it is currently not possible to inject muscle at 1 - 2 mm apart to ensure patient survival. 2.Transplanted myoblasts do not become satellite cells and are limited in long term repair.

11. 2. Satellite Cell transplantation Approach: Isolate a pure population of satellite cells using a surface marker (Note: myoblasts are satellite cells that have been allowed to grow in culture) Problems: Currently difficult because we don’t have good surface markers. Will require a lot of muscle to isolate enough cells

12. Proof of principle experiments performed with Pax3- GFP mice (Buckingham) and with Pax7+/Myf5- mice (Rudnicki): 1. Cells were sorted by fluorescent activated cell sorting (FACS) 2. Cells were injected directly into muscle Found: These cells can restore dystrophin expression in mdx mice (not shown) and contribute to new satellite cells (below). Arrow shows the GFP- labeled satellite cell in a cross section of muscle fibres

13. Bone Hematopoietic stem cell Stromal cells Blood vessel Adipocyte Osteoclast stemcells.nih.gov 3. Mesenchymal Stem cells Bone marrow stromal cells contain mesenchymal stem cells (MSC)

14. Hamada, Hirofumi, Kobune, Masayoshi, Nakamura, Kiminori, Kawano, Yutaka, Kato, Kazunori, Honmou, Osamu, Houkin, Kiyohiro, Matsunaga, Takuya & Niitsu, Yoshiro Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy. Cancer Science 96 (3), 149-156. doi: 10.1111/j.1349-7006.2005.00032.x

15. Mesenchymal Stem Cells (MSCs) Approach: To differentiate Bone Marrow Mesenchymal Stem cells (MSCs) into skeletal muscle and use to fix damaged muscle

16. Mesenchymal Stem cells (MSCs) Outcome: Can differentiate efficiently into skeletal muscle Can be used to repair skeletal muscle when injected intravenously in immunosuppressed rats Satellite cells were formed from the MSCs Rat Human

17. Problems with Mesenchymal Stem Cells The differentiation process required transfection of the Notch intracellular domain. A method of differentiating these cells without transfection of a gene is required for future cell therapy in humans.

18. http://www.geron.com/images/stemcell.jpg 4. Embryonic stem cells

19. Embryonic stem cell differentiation into skeletal muscle Very inefficient but appears to follow in vivo pathways Problems with using in cell therapy: Need to generate large quantities of skeletal muscle Need to avoid forming other cell types Potential advantages: Ability to grow large quantities of cells Easy to genetically manipulate Able to derive immune matched cell lineages for transplant

20. Perlingeiro lab (Nat Med 2008) First example of using mouse embryonic stem cells to fix skeletal muscle: 1.Transfected Pax3 into mouse ES cells 2.Isolated a muscle precursor population by FACS 3.Injected cells into muscle of injured mice 4.Found donor cells created new muscle and enhanced the force generated by new muscle Therefore, embryonic stem cells are a feasible cell source for repairing skeletal muscle.

21. Possible future reality: To have muscle precursor cells frozen and ready for transplant and the same source of immune precursor cells frozen and ready for transplant Therefore there would be no need for “designer” cells since one type of cell would fix everyone’s muscle.

22. 5. Mesangioblasts Cells associated with vessels that can differentiate into endothelial and muscle lineages Approach: Muscle biopsy taken from a wild type dog donor and mesangioblasts isolated from the blood vessels, amplified in culture and injected back into the muscular dystrophy dog’s vasculature Nature 444, 574-579 (30 November 2006)

23. Find: Amazing recovery of mobility in the dog and expression of dystrophin

24. Mesangioblasts Based on the research in the Dog model, mesangioblasts are now considered good candidates for future cell therapy in humans Exact characteristics of these cells are poorly understood and need to be studied further Some death of some of the animals occurred - not clear if more than in control population

25. Current Sources of Stem Cells for Muscle Regeneration 3. Mesenchymal stem cells 1. Primary myoblasts from satellite cells 4. Embryonic stem cells 2. Satellite Cells 5. Mesangioblasts Jesse Davidson 1980-2009 http://www.jessesjourney.com/

26. Methods used in these papers: Overexpression Overexpression of a gene is used to see the function of that gene. The function can be measured by analysis of mRNA changes (northern blot or PCR) or protein changes (western blot or immunofluorescence). Eg. 1)Myogenic conversion assay (overexpress MyoD and look for muscle by counting myosin heavy chain positive cells (immunofluorescence) or by RT-PCR. 2) Examination of subcellular distribution (nucleus vs. cytoplasm) by immunofluorescence (Paper #2).

27. Loss of Expression/function: You can decrease or eliminate the expression of a protein by: 1.Knocking it out (Paper #4, Fig. 4C) 2.Overexpression of a dominant negative mutant (Paper#3, Figs. 3-5) 3.SiRNA knock-down of expression (Paper #4, Fig. 4)

28. Promoter Assay: A promoter assay examines the ability of a transcription factor to initiate gene expression from a promoter or enhancer region of a gene. All factors are transiently transfected into a chosen cell line and an enzyme assay measures promoter activity (usually luciferase). This was performed in Paper #1, Fig. 3. The weakness of this technique is that it can be viewed as artificial.

29. Chromatin Immunoprecipitation (ChIP) ChIP tells you to which genomic sequences your protein binds to. The protein is cross- linked to DNA, the DNA is sheared, the protein is immunoprecipitated, the associated DNA is analyzed by PCR. Since this technique identifies endogenous genomic interactions occurring in a cell, it is considered to be accurate and powerful (eg. Paper #1, Fig. 2; Paper #3, Fig 4, Paper #4, Fig. 4)

30. Protein-protein interactions can be identified by: 1.Co-immunoprecipitation (a specific antibody pulls out its protein, together with interacting proteins; eg. Paper #1, Fig. 4; Paper #3, Fig. 5; Paper #4, Fig. 2) 2.Creation of a GST-fusion to the protein of interest and purification on glutathione beads. Interacting proteins will co-purify (Paper #1, Fig. 4) 3.Creation of a TAP-fusion and purification by the TAP tag method. Interacting proteins will co- purify (Paper #4, Fig. 2).

31. Chromatin Accessibility Assay: The Chromatin accessibility assay examines the conformation of chromatin and determines if it is open (no nucleasome and therefore a restriction enzyme can cut DNA) or closed (nucleasome present, preventing access of a restriction enzyme; eg. Paper #3, Figs. 1, 3, and 7).

32. Sample questions 2. This figure is from Paper #3 (Embo J.), showing RT-PCR from B22 cells transfected with MyoD A. Explain what this figure shows (worth 4 points).

33. Sample questions 2. This figure is from Paper #3 (Embo J.), showing RT-PCR from B22 cells transfected with MyoD A. Explain what this figure shows (worth 4 points). Answer: This figure shows that: MyoD can induce muscle development to occur (1 point) muscle development is shown by the enhancement of myogenin, Mef2D, MCK, and desmin transcripts. (1 point) MyoD requires functional Brg1 because myogenesis did not occur when Tet was removed, causing a dominant-negative Brg1 to be expressed (2 points)

34. Sample questions 2. This figure is from Paper #3 (Embo J.), showing RT-PCR from B22 cells transfected with MyoD. B. What is missing from this figure? (Worth 1 point).

35. Sample questions 2. This figure is from Paper #3 (Embo J.), showing RT-PCR from B22 cells transfected with MyoD. B. What is missing from this figure? (Worth 1 point). Answer: RT-PCR for 1) MyoD, showing that MyoD was overexpressed or 2) for Brg1, showing dominant negative BRG1 expression (either point is sufficient)

36. Apply knowledge to “new” problem (Bonus question will be of this type) 1. You have identified a novel muscle- specific transcription factor, termed NTX. Design experiments to determine its role in muscle development (10 points).

37. 1.You have identified a novel muscle-specific transcription factor, termed NTX. Design experiments to determine its role in muscle development and state a possible outcome. Potential answer- See if NTX can: convert fibroblasts to muscle in a myogenic conversion assay. Activate skeletal muscle-specific promoters such as MCK-luciferase in a reporter assay. Synergize with /inhibit MyoD in a myogenic conversion assay or a reporter assay. Perform ChIP to determine if it binds skeletal muscle promoters. Create a TAP-NTX fusion protein and identify interacting proteins Note: You should state the outcome: NTX can or can’t - you choose, all the answers should fit together.