Biotechnology CIRM Stem Cell Lecture

1. Stem Cells and Regenerative Medicine Notes for Stem Cell Outreach Program presenters: Good morning everyone, in this presentation we’re going to learn about stem cells and regenerative medicine. This presentation was made by the Stem Cell Education Outreach Program (SCEOP) at UC Berkeley. The Stem Cell Education Outreach Program is part of a larger movement in and by our state (the California Stem Cell Education Initiative) to incorporate stem cell topics into high school science classes. This larger initiative is funded by the California Institute for Regenerative Medicine—the ”state stem cell agency”—and our goal is to help you learn about stem cell research. At the SCEOP and CIRM, we are committed to making and delivering unbiased presentations based on scientiﬁc facts and our own observations. The purpose of this presentation is NOT to tell you what to believe about stem cell research. We want to lay out all the information in front of you so you can decide how you feel about it. Also, your opinion is very valuable to us. We are asking students and teachers to ﬁll out our online survey after the presentation. The data gathered from our survey is really important and will inform what materials are included in the state stem cell curriculum. Also, we’ll have a few minutes for questions in the middle and at the end of the presentation (and answer questions on the handouts we gave you).

2. “Glow-in-the-dark” dogs! Here is a recent news story about a research team in South Korea that created puppies that glow red! By the end of the presentation, you’ll learn how they did this and how this relates to stem cell research. CLICK the video. You should hear audio. Since the puppies glow red like rubies, the scientists named them by combining Ruby and Puppy to make Ruppy!

3. “Glow-in-the-dark” dogs! Here is a recent news story about a research team in South Korea that created puppies that glow red! By the end of the presentation, you’ll learn how they did this and how this relates to stem cell research. CLICK the video. You should hear audio. Since the puppies glow red like rubies, the scientists named them by combining Ruby and Puppy to make Ruppy!

4. What is stem cell research? • Understand more about development, aging, disease – Experimental model systems • Prevent or treat diseases and injuries – Cell-based therapies – Pharmaceutical development • Includes testing and drug delivery So, WHAT IS STEM CELL RESEARCH? What is the purpose of studying stem cells? Well, stem cell research helps us understand more about development, aging, and disease. To do this work, scientists look to create experimental model systems which resemble normal processes, in order to learn more about how stem cells change across the lifespan—either in the process of growth in early life or the degeneration of tissues later in life. By using experimental model systems, like studying heart muscle cells beating in a petri dish, scientists can also understand how genetic mutations or speciﬁc chemical messages lead to disrupted stem cell functions in various diseases. Finally, these discoveries can be applied to direct stem cells to do things that will help patients, which is the ultimate goal of regenerative medicine. In this regard, stem cell research may help us prevent or treat diseases and injuries in two ways: cell-based therapies and pharmaceutical development, which includes drug testing and drug delivery.

5. Trachea transplantation: Example of adult stem cell-based tissue regeneration (Click the movie. This ﬁle does not contain audio, so continue with this narration.) Another way we can use stem cells is to grow them into replacement organs. Last year, doctors grew a replacement trachea for a woman whose trachea had collapsed as a result of tuberculosis…They took a trachea from an organ donor, a cadaver…here they’re cutting a segment of windpipe…stripped it of all the donor’s cells that would have been rejected when transplanted into another person…took adult stem cells from the woman who needed the new trachea…grew them up…and grafted them onto the trachea scaffold. They then coaxed those stem cells to repopulate the trachea with mature cells…cut the new trachea to the right size…shaped it to ﬁt…cut out the woman’s damaged trachea…and transplanted it into the woman. She didn’t need drugs to suppress the immune system because the cells came from her own body!

6. Trachea transplantation: Example of adult stem cell-based tissue regeneration (Click the movie. This ﬁle does not contain audio, so continue with this narration.) Another way we can use stem cells is to grow them into replacement organs. Last year, doctors grew a replacement trachea for a woman whose trachea had collapsed as a result of tuberculosis…They took a trachea from an organ donor, a cadaver…here they’re cutting a segment of windpipe…stripped it of all the donor’s cells that would have been rejected when transplanted into another person…took adult stem cells from the woman who needed the new trachea…grew them up…and grafted them onto the trachea scaffold. They then coaxed those stem cells to repopulate the trachea with mature cells…cut the new trachea to the right size…shaped it to ﬁt…cut out the woman’s damaged trachea…and transplanted it into the woman. She didn’t need drugs to suppress the immune system because the cells came from her own body!

7. Outline of Presentation • Fertilization and embryonic development • What makes stem cells unique? • What are the different types of stem cells? • What are examples of stem cell research, therapies, and technologies? • Conclusion and future directions (Go through the outline.)

8. Day 1 In the IVF procedure, sperm and eggs “interact” in a dish leading to insemination. They literally swim up to the egg and burrow toward the nucleus. The first one to get there wins, and all others are blocked out. Male fertility issue: Sometimes sperm cannot latch onto and penetrate the egg. They may choose to have Intra(within)-Cytoplasmic Sperm Injection (ICSI) Sometimes, a couple is unable to conceive the natural way. Do you know what type of clinic they might go to so they can increase the chances of getting pregnant? (wait for student answers…a couple would go to an in vitro fertilization clinic) Does anyone know what in vitro means? (wait for answers) In vitro means within the glass, which is why people produced by in vitro fertilization are sometimes called test tube babies. This is a picture of multiple sperm digesting the outer “shell” of the egg in a race for the center. Sperm use enzymes in their heads and after they get to the egg nucleus, the egg prevents any more sperm from entering. Infertility can stem from many reproductive problems, but in one case where there is a problem with sperm’s ability to latch onto and penetrate the egg, a couple may choose to have Intra-Cytoplasmic Sperm Injection.

9. Intracytoplasmic Sperm Injection places a sperm right inside the egg nucleus so there is a better chance of insemination. Here’s how this procedure works. After ovarian stimulation using hormones, between 10 and 30 eggs are removed from the woman’s ovaries. Sperm is collected, and one sperm is sucked up. (point to right side of picture) On the left, a holding pipette secures the egg in place. Do you see how much bigger the egg is than the sperm? What’s amazing is they both contribute the same amount of genetic material to the developing organism.

10. The sperm, containing the man’s half of the genetic contribution, is injected into the egg, containing the woman’s half of the genes. This is day 1 of fertilization.

11. Day 1 At 12 hours after fertilization you can see the two bundles of genetic material. These contain the DNA from each parent. By 18-20 hours after fertilization, these bundles fuse and combine genetic material from mom and dad. What starts out as two cells becomes one cell, called the fertilized egg.

12. Day 2 On day 2, that one cell has divided into two cells. How do you guys think these cells are related? (wait for answers) That one cell made a copy of itself, so they are identical.

13. Day 2 Later on day 2, each of those two cells divides, making four identical cells.

14. Day 3 By day 3, each of those four cells divides, making eight identical cells. Each cell is one-eighth the size of the original fertilized egg. Some fertility doctors choose to put the embryos back into the uterus at this stage, and some wait until the embryo is more developed.

15. Day 4 On day 4, the cells have divided several times more and you can’t tell one cell from another cell. How do you think these cells relate to each other? (wait for answers) They’re still identical.

16. Day 5 Day 5 is a big day. Now, ﬂuid builds up inside the ball of cells, making it look hollow like a soccer ball. This structure is called a blastocyst. Are the cells here still identical? (Wait for answers) No, they are not. The outer layer has cells that will become the placenta. There is a clump of cells sitting inside, CLICK! that will become the fetus. However, this is deﬁnitely not a fetus yet. So now, the doctor chooses a few, usually 2-3, healthy-looking 5-day-old blastocysts. He implants those into the woman’s uterus. Hopefully one, and preferably only one of those will successfully burrow into the uterus and develop into a pregnancy. Take home message: EMBRYONIC STEM CELLS MAKE UP THE LITTLE CLUMP OF CELLS INSIDE THE BLASTOCYST. This clump is commonly called the inner cell mass. After the doctor has chosen a few blastocysts to implant, the leftover embryos are frozen. These can later be used by the couple if the pregnancy is unsuccessful or if they want to have more kids. They can be donated to another couple for adoption. They can also be donated with consent to stem cell research. A scientist would harvest the inner cell mass and plate it onto a petri dish to make an embryonic stem cell line. This destruction of the embryo is the main reason why embryonic stem cell research is so controversial.

17. Day 5 Embryonic Stem Cells Day 5 is a big day. Now, ﬂuid builds up inside the ball of cells, making it look hollow like a soccer ball. This structure is called a blastocyst. Are the cells here still identical? (Wait for answers) No, they are not. The outer layer has cells that will become the placenta. There is a clump of cells sitting inside, CLICK! that will become the fetus. However, this is deﬁnitely not a fetus yet. So now, the doctor chooses a few, usually 2-3, healthy-looking 5-day-old blastocysts. He implants those into the woman’s uterus. Hopefully one, and preferably only one of those will successfully burrow into the uterus and develop into a pregnancy. Take home message: EMBRYONIC STEM CELLS MAKE UP THE LITTLE CLUMP OF CELLS INSIDE THE BLASTOCYST. This clump is commonly called the inner cell mass. After the doctor has chosen a few blastocysts to implant, the leftover embryos are frozen. These can later be used by the couple if the pregnancy is unsuccessful or if they want to have more kids. They can be donated to another couple for adoption. They can also be donated with consent to stem cell research. A scientist would harvest the inner cell mass and plate it onto a petri dish to make an embryonic stem cell line. This destruction of the embryo is the main reason why embryonic stem cell research is so controversial.

18. At what point is this a fetus? So remember, natural fertilization happens in the fallopian tube, CLICK! and it takes 7-14 days for the developing embryo to float down to the uterus and implant. When the embryo implants, the “clump” now looks like a disc. CLICK! There are three distinct layers of cells: an outer, middle, and inner layer. Cells in these layers don’t look like mature organs and structures yet, but based on their position they will eventually become specific parts of the body. What parts of the body do you think the outer layer forms? (wait for answers….hint: what’s on the outside of your body?) This layer forms skin and interestingly the nervous system. What do you think the middle layer will form? (wait for answers) It will become the muscles, bones, and heart. What will the inner layer form? (wait for answers) It will become the gut lining and internal organs. CLICK! After this, the nervous system begins to form. The cells in here become the spinal cord and brain. CLICK! Bumps begin to develop near the head-end that will be the future face, neck, mouth, and nose. CLICK! In the next five weeks, parts of the embryo will become specialized into primitive organ systems. The primitive heart starts beating in week 4. CLICK! By week 8, what do we have? (wait) A fetus. Remember, embryonic stem cells used for research are harvested from 5-14 day old embryos, WAY before organ formation starts. The donors ALWAYS have to give their permission to use embryos for research.

19. At what point is this a fetus? • Days 7-14: Uterine implantation So remember, natural fertilization happens in the fallopian tube, CLICK! and it takes 7-14 days for the developing embryo to float down to the uterus and implant. When the embryo implants, the “clump” now looks like a disc. CLICK! There are three distinct layers of cells: an outer, middle, and inner layer. Cells in these layers don’t look like mature organs and structures yet, but based on their position they will eventually become specific parts of the body. What parts of the body do you think the outer layer forms? (wait for answers….hint: what’s on the outside of your body?) This layer forms skin and interestingly the nervous system. What do you think the middle layer will form? (wait for answers) It will become the muscles, bones, and heart. What will the inner layer form? (wait for answers) It will become the gut lining and internal organs. CLICK! After this, the nervous system begins to form. The cells in here become the spinal cord and brain. CLICK! Bumps begin to develop near the head-end that will be the future face, neck, mouth, and nose. CLICK! In the next five weeks, parts of the embryo will become specialized into primitive organ systems. The primitive heart starts beating in week 4. CLICK! By week 8, what do we have? (wait) A fetus. Remember, embryonic stem cells used for research are harvested from 5-14 day old embryos, WAY before organ formation starts. The donors ALWAYS have to give their permission to use embryos for research.

20. At what point is this a fetus? • Days 7-14: Uterine implantation • Day 14: Three distinct layers begin to form (no more pluripotent stem cells) So remember, natural fertilization happens in the fallopian tube, CLICK! and it takes 7-14 days for the developing embryo to float down to the uterus and implant. When the embryo implants, the “clump” now looks like a disc. CLICK! There are three distinct layers of cells: an outer, middle, and inner layer. Cells in these layers don’t look like mature organs and structures yet, but based on their position they will eventually become specific parts of the body. What parts of the body do you think the outer layer forms? (wait for answers….hint: what’s on the outside of your body?) This layer forms skin and interestingly the nervous system. What do you think the middle layer will form? (wait for answers) It will become the muscles, bones, and heart. What will the inner layer form? (wait for answers) It will become the gut lining and internal organs. CLICK! After this, the nervous system begins to form. The cells in here become the spinal cord and brain. CLICK! Bumps begin to develop near the head-end that will be the future face, neck, mouth, and nose. CLICK! In the next five weeks, parts of the embryo will become specialized into primitive organ systems. The primitive heart starts beating in week 4. CLICK! By week 8, what do we have? (wait) A fetus. Remember, embryonic stem cells used for research are harvested from 5-14 day old embryos, WAY before organ formation starts. The donors ALWAYS have to give their permission to use embryos for research.

21. At what point is this a fetus? • Days 7-14: Uterine implantation • Day 14: Three distinct layers begin to form (no more pluripotent stem cells) • Days 14-21: Beginning of future nervous system So remember, natural fertilization happens in the fallopian tube, CLICK! and it takes 7-14 days for the developing embryo to float down to the uterus and implant. When the embryo implants, the “clump” now looks like a disc. CLICK! There are three distinct layers of cells: an outer, middle, and inner layer. Cells in these layers don’t look like mature organs and structures yet, but based on their position they will eventually become specific parts of the body. What parts of the body do you think the outer layer forms? (wait for answers….hint: what’s on the outside of your body?) This layer forms skin and interestingly the nervous system. What do you think the middle layer will form? (wait for answers) It will become the muscles, bones, and heart. What will the inner layer form? (wait for answers) It will become the gut lining and internal organs. CLICK! After this, the nervous system begins to form. The cells in here become the spinal cord and brain. CLICK! Bumps begin to develop near the head-end that will be the future face, neck, mouth, and nose. CLICK! In the next five weeks, parts of the embryo will become specialized into primitive organ systems. The primitive heart starts beating in week 4. CLICK! By week 8, what do we have? (wait) A fetus. Remember, embryonic stem cells used for research are harvested from 5-14 day old embryos, WAY before organ formation starts. The donors ALWAYS have to give their permission to use embryos for research.

22. At what point is this a fetus? • Days 7-14: Uterine implantation • Day 14: Three distinct layers begin to form (no more pluripotent stem cells) • Days 14-21: Beginning of future nervous system • Days 21-24: Beginning of future face, neck, mouth, and nose So remember, natural fertilization happens in the fallopian tube, CLICK! and it takes 7-14 days for the developing embryo to float down to the uterus and implant. When the embryo implants, the “clump” now looks like a disc. CLICK! There are three distinct layers of cells: an outer, middle, and inner layer. Cells in these layers don’t look like mature organs and structures yet, but based on their position they will eventually become specific parts of the body. What parts of the body do you think the outer layer forms? (wait for answers….hint: what’s on the outside of your body?) This layer forms skin and interestingly the nervous system. What do you think the middle layer will form? (wait for answers) It will become the muscles, bones, and heart. What will the inner layer form? (wait for answers) It will become the gut lining and internal organs. CLICK! After this, the nervous system begins to form. The cells in here become the spinal cord and brain. CLICK! Bumps begin to develop near the head-end that will be the future face, neck, mouth, and nose. CLICK! In the next five weeks, parts of the embryo will become specialized into primitive organ systems. The primitive heart starts beating in week 4. CLICK! By week 8, what do we have? (wait) A fetus. Remember, embryonic stem cells used for research are harvested from 5-14 day old embryos, WAY before organ formation starts. The donors ALWAYS have to give their permission to use embryos for research.

23. At what point is this a fetus? • Days 7-14: Uterine implantation • Day 14: Three distinct layers begin to form (no more pluripotent stem cells) • Days 14-21: Beginning of future nervous system • Days 21-24: Beginning of future face, neck, mouth, and nose • Weeks 3-8: Beginning of organ formation So remember, natural fertilization happens in the fallopian tube, CLICK! and it takes 7-14 days for the developing embryo to float down to the uterus and implant. When the embryo implants, the “clump” now looks like a disc. CLICK! There are three distinct layers of cells: an outer, middle, and inner layer. Cells in these layers don’t look like mature organs and structures yet, but based on their position they will eventually become specific parts of the body. What parts of the body do you think the outer layer forms? (wait for answers….hint: what’s on the outside of your body?) This layer forms skin and interestingly the nervous system. What do you think the middle layer will form? (wait for answers) It will become the muscles, bones, and heart. What will the inner layer form? (wait for answers) It will become the gut lining and internal organs. CLICK! After this, the nervous system begins to form. The cells in here become the spinal cord and brain. CLICK! Bumps begin to develop near the head-end that will be the future face, neck, mouth, and nose. CLICK! In the next five weeks, parts of the embryo will become specialized into primitive organ systems. The primitive heart starts beating in week 4. CLICK! By week 8, what do we have? (wait) A fetus. Remember, embryonic stem cells used for research are harvested from 5-14 day old embryos, WAY before organ formation starts. The donors ALWAYS have to give their permission to use embryos for research.

24. Embryonic Development: Fish embryo Keller et al. 2008 This is going to be a video of the development of a ﬁsh embryo. We thought this would help you visualize how an embryo develops, because every vertebrate—or animal with a spine—pretty much starts out like this. This movie begins with the ﬁsh having 64 cells. It proceeds very much like human embryonic development. On the left we’re seeing one side of the embryo. This is a view of the ANIMAL pole, which is the side of the embryo that has actively dividing cells. On the right is the opposite side of the embryo, the VEGETAL pole, which has “yolk” cells that divide less rapidly and give nutrients to the cells of the embryo. CLICK the video. On the left you can see cells rapidly dividing or proliferating…You can see cells migrating around the embryo to form the hollow ball…Then, the clump of embryonic stem cells forms at the future dorsal side of embryo (point to your back)…The clump reshapes and starts to form the spinal cord and future brain. You can see that the brain is the heart-shaped formation on left, with the beginnings of two hemispheres, and on the right is the beginnings of a segmented spinal cord.

25. Embryonic Development: Fish embryo Keller et al. 2008 This is going to be a video of the development of a ﬁsh embryo. We thought this would help you visualize how an embryo develops, because every vertebrate—or animal with a spine—pretty much starts out like this. This movie begins with the ﬁsh having 64 cells. It proceeds very much like human embryonic development. On the left we’re seeing one side of the embryo. This is a view of the ANIMAL pole, which is the side of the embryo that has actively dividing cells. On the right is the opposite side of the embryo, the VEGETAL pole, which has “yolk” cells that divide less rapidly and give nutrients to the cells of the embryo. CLICK the video. On the left you can see cells rapidly dividing or proliferating…You can see cells migrating around the embryo to form the hollow ball…Then, the clump of embryonic stem cells forms at the future dorsal side of embryo (point to your back)…The clump reshapes and starts to form the spinal cord and future brain. You can see that the brain is the heart-shaped formation on left, with the beginnings of two hemispheres, and on the right is the beginnings of a segmented spinal cord.

26. Outline of Presentation • Fertilization and embryonic development • What makes stem cells unique? • What are the different types of stem cells? • What are examples of stem cell research, therapies, and technologies? • Conclusion and future directions What makes stem cells unique?

27. Symmetric cell division At the beginning of embryonic development, stem cells undergo symmetric cell division. They divide symmetrically, where one cell splits and gives rise to two identical cells that have the same potential. This is why we said the stem cells in the early embryo remain the same and are identical. Interestingly, this is also how fully mature cells in your body divide. What’s the technical term for this that you guys probably learned in biology? (wait) MITOSIS!

28. Asymmetric cell division Progenitor cell Stem cell Stem cell Then at blastocyst formation and gastrulation the stem cells start to divide asymmetrically. When these stem cells divide, they give rise to two cells that are different from each other. One of the cells remains a stem cell—the yellow one—and the other changes into a progenitor cell—the green square. That progenitor cell is a young cell that will change into a mature cell type, like the epithelial cell in green. Asymmetric cell division is when a stem cell divides to produce two cells that are different from each other. CLICK! One of the cells remains a stem cell, demonstrating self-renewal, and the other CLICK! differentiates into a progenitor cell.

29. Asymmetric cell division 1. Self-renews Progenitor cell Stem cell Stem cell Then at blastocyst formation and gastrulation the stem cells start to divide asymmetrically. When these stem cells divide, they give rise to two cells that are different from each other. One of the cells remains a stem cell—the yellow one—and the other changes into a progenitor cell—the green square. That progenitor cell is a young cell that will change into a mature cell type, like the epithelial cell in green. Asymmetric cell division is when a stem cell divides to produce two cells that are different from each other. CLICK! One of the cells remains a stem cell, demonstrating self-renewal, and the other CLICK! differentiates into a progenitor cell.

30. Asymmetric cell division 1. Self-renews 2. Differentiates Progenitor cell Stem cell Stem cell Then at blastocyst formation and gastrulation the stem cells start to divide asymmetrically. When these stem cells divide, they give rise to two cells that are different from each other. One of the cells remains a stem cell—the yellow one—and the other changes into a progenitor cell—the green square. That progenitor cell is a young cell that will change into a mature cell type, like the epithelial cell in green. Asymmetric cell division is when a stem cell divides to produce two cells that are different from each other. CLICK! One of the cells remains a stem cell, demonstrating self-renewal, and the other CLICK! differentiates into a progenitor cell.

31. D FE IF EN R N IO IAT T D FE IF EN R N IO IAT T  SELF – RENEWAL  Ok so that stem cell divided to produce a green progenitor cell which differentiates into a green skin cell (CLICK!). That stem cell can divide again, this time producing a different type of progenitor cell CLICK! which matures into yet another cell type, CLICK! like this brain cell. OK so what makes a stem cell unique? (wait for answers) FIRST: it maintains its own population—self-renewal—and SECOND: its capacity to give rise to different progenitors that change into a mature cell—differentiation.

36. Outline of Presentation • Fertilization and embryonic development • What makes stem cells unique? • What are the different types of stem cells? • What are examples of stem cell research, therapies, and technologies? • Conclusion and future directions

37. CLICK! This diagram will eventually show the entire range of development, from fertilized egg to mature cell types in the body. Each cell in the 8-cell embryo, here in red, can generate every cell in the embryo as well as the placenta and extra-embryonic tissues. These cells are called CLICK! TOTIPOTENT stem cells. Why are they called totipotent? (wait for answers) Because one red cell can potentially make all necessary tissues for development. CLICK! During In Vitro Fertilization, can parents choose whether their baby is going to be a boy or a girl? (wait) Yes, there is a widely-practiced procedure called pre-implantation genetic diagnosis, where one cell is removed from the 8-cell embryo and its DNA is examined. What might you look for when trying to identify the embryo’s sex? (wait) If there’s an X and Y chromosome it’s a boy and if there are two X’s it’s a girl. The parents can decide whether to implant it. Also parents with a genetic disease might want to see if their baby has any identiﬁable genetic disorders and decide whether to implant based on this information. Pre-implantation genetic diagnosis doesn’t destroy the embryo. Scientists are attempting to adapt this pre-implantation genetic diagnosis procedure and use it to create a stem cell line from one single TOTIPOTENT cell, without destroying the embryo. The embryonic stem cells inside the blastocyst, here in purple, can generate every cell in the body except placenta and extra-embryonic tissues. These are called CLICK! PLURIPOTENT stem cells…why? (wait for answers) Because they can differentiate into all the 200+ cell types in the body, but they do not form the placenta. CLICK! Pluripotent stem cells can be isolated and grown in culture, or left to develop into more specialized cells in the body. CLICK! Adult stem cells or tissue-speciﬁc stem cells have restricted lineages. Adult stem cells show up when the three distinct layers form in the 14-day-old embryo, and are present in the fetus, baby, child, and so forth. Adult just means they’ve gone further down their lineage pathway than the initial stem cells in the embryo. They are called CLICK! MULTIPOTENT stem cells because they will only become mature cells from the tissue in which they reside. Adult stem cells are present throughout your life and replace fully mature CLICK!, yet damaged and dying cells.

40. This cell Can form the Embryo and placenta CLICK! This diagram will eventually show the entire range of development, from fertilized egg to mature cell types in the body. Each cell in the 8-cell embryo, here in red, can generate every cell in the embryo as well as the placenta and extra-embryonic tissues. These cells are called CLICK! TOTIPOTENT stem cells. Why are they called totipotent? (wait for answers) Because one red cell can potentially make all necessary tissues for development. CLICK! During In Vitro Fertilization, can parents choose whether their baby is going to be a boy or a girl? (wait) Yes, there is a widely-practiced procedure called pre-implantation genetic diagnosis, where one cell is removed from the 8-cell embryo and its DNA is examined. What might you look for when trying to identify the embryo’s sex? (wait) If there’s an X and Y chromosome it’s a boy and if there are two X’s it’s a girl. The parents can decide whether to implant it. Also parents with a genetic disease might want to see if their baby has any identiﬁable genetic disorders and decide whether to implant based on this information. Pre-implantation genetic diagnosis doesn’t destroy the embryo. Scientists are attempting to adapt this pre-implantation genetic diagnosis procedure and use it to create a stem cell line from one single TOTIPOTENT cell, without destroying the embryo. The embryonic stem cells inside the blastocyst, here in purple, can generate every cell in the body except placenta and extra-embryonic tissues. These are called CLICK! PLURIPOTENT stem cells…why? (wait for answers) Because they can differentiate into all the 200+ cell types in the body, but they do not form the placenta. CLICK! Pluripotent stem cells can be isolated and grown in culture, or left to develop into more specialized cells in the body. CLICK! Adult stem cells or tissue-speciﬁc stem cells have restricted lineages. Adult stem cells show up when the three distinct layers form in the 14-day-old embryo, and are present in the fetus, baby, child, and so forth. Adult just means they’ve gone further down their lineage pathway than the initial stem cells in the embryo. They are called CLICK! MULTIPOTENT stem cells because they will only become mature cells from the tissue in which they reside. Adult stem cells are present throughout your life and replace fully mature CLICK!, yet damaged and dying cells.

41. This cell Can form the Embryo and placenta CLICK! This diagram will eventually show the entire range of development, from fertilized egg to mature cell types in the body. Each cell in the 8-cell embryo, here in red, can generate every cell in the embryo as well as the placenta and extra-embryonic tissues. These cells are called CLICK! TOTIPOTENT stem cells. Why are they called totipotent? (wait for answers) Because one red cell can potentially make all necessary tissues for development. CLICK! During In Vitro Fertilization, can parents choose whether their baby is going to be a boy or a girl? (wait) Yes, there is a widely-practiced procedure called pre-implantation genetic diagnosis, where one cell is removed from the 8-cell embryo and its DNA is examined. What might you look for when trying to identify the embryo’s sex? (wait) If there’s an X and Y chromosome it’s a boy and if there are two X’s it’s a girl. The parents can decide whether to implant it. Also parents with a genetic disease might want to see if their baby has any identiﬁable genetic disorders and decide whether to implant based on this information. Pre-implantation genetic diagnosis doesn’t destroy the embryo. Scientists are attempting to adapt this pre-implantation genetic diagnosis procedure and use it to create a stem cell line from one single TOTIPOTENT cell, without destroying the embryo. The embryonic stem cells inside the blastocyst, here in purple, can generate every cell in the body except placenta and extra-embryonic tissues. These are called CLICK! PLURIPOTENT stem cells…why? (wait for answers) Because they can differentiate into all the 200+ cell types in the body, but they do not form the placenta. CLICK! Pluripotent stem cells can be isolated and grown in culture, or left to develop into more specialized cells in the body. CLICK! Adult stem cells or tissue-speciﬁc stem cells have restricted lineages. Adult stem cells show up when the three distinct layers form in the 14-day-old embryo, and are present in the fetus, baby, child, and so forth. Adult just means they’ve gone further down their lineage pathway than the initial stem cells in the embryo. They are called CLICK! MULTIPOTENT stem cells because they will only become mature cells from the tissue in which they reside. Adult stem cells are present throughout your life and replace fully mature CLICK!, yet damaged and dying cells.

42. This cell Can form the Embryo and placenta This cell Can form the Embryo CLICK! This diagram will eventually show the entire range of development, from fertilized egg to mature cell types in the body. Each cell in the 8-cell embryo, here in red, can generate every cell in the embryo as well as the placenta and extra-embryonic tissues. These cells are called CLICK! TOTIPOTENT stem cells. Why are they called totipotent? (wait for answers) Because one red cell can potentially make all necessary tissues for development. CLICK! During In Vitro Fertilization, can parents choose whether their baby is going to be a boy or a girl? (wait) Yes, there is a widely-practiced procedure called pre-implantation genetic diagnosis, where one cell is removed from the 8-cell embryo and its DNA is examined. What might you look for when trying to identify the embryo’s sex? (wait) If there’s an X and Y chromosome it’s a boy and if there are two X’s it’s a girl. The parents can decide whether to implant it. Also parents with a genetic disease might want to see if their baby has any identiﬁable genetic disorders and decide whether to implant based on this information. Pre-implantation genetic diagnosis doesn’t destroy the embryo. Scientists are attempting to adapt this pre-implantation genetic diagnosis procedure and use it to create a stem cell line from one single TOTIPOTENT cell, without destroying the embryo. The embryonic stem cells inside the blastocyst, here in purple, can generate every cell in the body except placenta and extra-embryonic tissues. These are called CLICK! PLURIPOTENT stem cells…why? (wait for answers) Because they can differentiate into all the 200+ cell types in the body, but they do not form the placenta. CLICK! Pluripotent stem cells can be isolated and grown in culture, or left to develop into more specialized cells in the body. CLICK! Adult stem cells or tissue-speciﬁc stem cells have restricted lineages. Adult stem cells show up when the three distinct layers form in the 14-day-old embryo, and are present in the fetus, baby, child, and so forth. Adult just means they’ve gone further down their lineage pathway than the initial stem cells in the embryo. They are called CLICK! MULTIPOTENT stem cells because they will only become mature cells from the tissue in which they reside. Adult stem cells are present throughout your life and replace fully mature CLICK!, yet damaged and dying cells.

46. This cell Can form the Embryo and placenta This cell Can form the Embryo Fully mature CLICK! This diagram will eventually show the entire range of development, from fertilized egg to mature cell types in the body. Each cell in the 8-cell embryo, here in red, can generate every cell in the embryo as well as the placenta and extra-embryonic tissues. These cells are called CLICK! TOTIPOTENT stem cells. Why are they called totipotent? (wait for answers) Because one red cell can potentially make all necessary tissues for development. CLICK! During In Vitro Fertilization, can parents choose whether their baby is going to be a boy or a girl? (wait) Yes, there is a widely-practiced procedure called pre-implantation genetic diagnosis, where one cell is removed from the 8-cell embryo and its DNA is examined. What might you look for when trying to identify the embryo’s sex? (wait) If there’s an X and Y chromosome it’s a boy and if there are two X’s it’s a girl. The parents can decide whether to implant it. Also parents with a genetic disease might want to see if their baby has any identiﬁable genetic disorders and decide whether to implant based on this information. Pre-implantation genetic diagnosis doesn’t destroy the embryo. Scientists are attempting to adapt this pre-implantation genetic diagnosis procedure and use it to create a stem cell line from one single TOTIPOTENT cell, without destroying the embryo. The embryonic stem cells inside the blastocyst, here in purple, can generate every cell in the body except placenta and extra-embryonic tissues. These are called CLICK! PLURIPOTENT stem cells…why? (wait for answers) Because they can differentiate into all the 200+ cell types in the body, but they do not form the placenta. CLICK! Pluripotent stem cells can be isolated and grown in culture, or left to develop into more specialized cells in the body. CLICK! Adult stem cells or tissue-speciﬁc stem cells have restricted lineages. Adult stem cells show up when the three distinct layers form in the 14-day-old embryo, and are present in the fetus, baby, child, and so forth. Adult just means they’ve gone further down their lineage pathway than the initial stem cells in the embryo. They are called CLICK! MULTIPOTENT stem cells because they will only become mature cells from the tissue in which they reside. Adult stem cells are present throughout your life and replace fully mature CLICK!, yet damaged and dying cells.

47. Induced Pluripotent Stem (iPS) Cells Skin cells iPS cells There is another type of stem cell. One way to obtain pluripotent stem cells without destroying an embryo would be to just make them, simply engineer them…. This is like turning back the clock in a subset of cells to create pluripotency, rather than directly harvesting or cloning embryonic stem cells. In induced Pluripotent Stem Cell technology, you FIRST isolate and culture skin cells from a patient. SECOND, you introduce three or four pluripotency genes into the skin cells by using an engineered virus carrier. THE EXPRESSION OF THESE GENES REGENERATES THE STEM CELL PHENOTYPE. The viruses simply deliver the genes of interest and are themselves engineered not be harmful. Here, the red cells indicate the cells actively expressing the four essential, stem cell or pluripotency, genes. In the THIRD image, you harvest and culture the cells according to the method for embryonic stem cell culture. FINALLY, through this process, a subset of the cells generates embryonic-stem-cell-like colonies called induced Pluripotent Stem cells. Already, scientists at Harvard and other institutions have used this process to create over twenty stem cell lines that model different genetic diseases. They do this by starting with skin cells from patients with the diseases.

48. Pros and Cons to iPS cell technology What are the pros and cons to iPS cell technology? The PROS (CLICK!) are that the induced Pluripotent Stem cells would be genetically identical to the person who donated the skin cells, so any cells derived from these iPS cells would not be rejected by the patient’s immune system. Ultimately, this would be a move toward personal, cell-based therapies. Another advantage is that you do not need to use an embryo to get pluripotent cells. So what about the CON’s…. (CLICK!) Any cell created from a patient with a genetic disease, as opposed to an environmentally-caused disease, would carry the same defective genes. So if a Parkinson’s patient needed new motor neurons, iPS would not work because the stem cells would display the same symptoms. Another disadvantage is that one of the pluripotency genes is also a cancer gene. This gene is used to stimulate the cells to start dividing again, and it must be controlled. If some of the iPS cells turn into cancer cells, then patients might develop tumors as a result of the transplant. Also, since viruses insert these pluripotency genes randomly into the genome, you might be interrupting the normal sequence of an important gene and cause an undesireable mutation. Scientists have already found substitutes for this cancer gene as well as alternative methods of delivering and expressing these genes, which could eventually make this process safer for transplantation. For now, these cells are perfect for making and studying diseases in a dish to promote basic research. But many important details need to be worked out before this tool is used as an actual technology in the clinic to treat patients.

49. Pros and Cons to iPS cell technology • Pros: – Cells would be genetically identical to patient or donor of skin cells (no immune rejection!) – Do not need to use an embryo What are the pros and cons to iPS cell technology? The PROS (CLICK!) are that the induced Pluripotent Stem cells would be genetically identical to the person who donated the skin cells, so any cells derived from these iPS cells would not be rejected by the patient’s immune system. Ultimately, this would be a move toward personal, cell-based therapies. Another advantage is that you do not need to use an embryo to get pluripotent cells. So what about the CON’s…. (CLICK!) Any cell created from a patient with a genetic disease, as opposed to an environmentally-caused disease, would carry the same defective genes. So if a Parkinson’s patient needed new motor neurons, iPS would not work because the stem cells would display the same symptoms. Another disadvantage is that one of the pluripotency genes is also a cancer gene. This gene is used to stimulate the cells to start dividing again, and it must be controlled. If some of the iPS cells turn into cancer cells, then patients might develop tumors as a result of the transplant. Also, since viruses insert these pluripotency genes randomly into the genome, you might be interrupting the normal sequence of an important gene and cause an undesireable mutation. Scientists have already found substitutes for this cancer gene as well as alternative methods of delivering and expressing these genes, which could eventually make this process safer for transplantation. For now, these cells are perfect for making and studying diseases in a dish to promote basic research. But many important details need to be worked out before this tool is used as an actual technology in the clinic to treat patients.

50. Pros and Cons to iPS cell technology • Pros: – Cells would be genetically identical to patient or donor of skin cells (no immune rejection!) – Do not need to use an embryo • Cons: – Cells would still have genetic defects – One of the pluripotency genes is a cancer gene – Viruses might insert genes in places we What are the pros and cons to iPS cell technology? The PROS (CLICK!) are that the induced Pluripotent Stem cells would be genetically identical to the person who donated the skin cells, so any cells derived from these iPS cells would not be rejected by the patient’s immune system. Ultimately, this would be a move toward personal, cell-based therapies. Another advantage is that you do not need to use an embryo to get pluripotent cells. So what about the CON’s…. (CLICK!) Any cell created from a patient with a genetic disease, as opposed to an environmentally-caused disease, would carry the same defective genes. So if a Parkinson’s patient needed new motor neurons, iPS would not work because the stem cells would display the same symptoms. Another disadvantage is that one of the pluripotency genes is also a cancer gene. This gene is used to stimulate the cells to start dividing again, and it must be controlled. If some of the iPS cells turn into cancer cells, then patients might develop tumors as a result of the transplant. Also, since viruses insert these pluripotency genes randomly into the genome, you might be interrupting the normal sequence of an important gene and cause an undesireable mutation. Scientists have already found substitutes for this cancer gene as well as alternative methods of delivering and expressing these genes, which could eventually make this process safer for transplantation. For now, these cells are perfect for making and studying diseases in a dish to promote basic research. But many important details need to be worked out before this tool is used as an actual technology in the clinic to treat patients.

51. How do cells know what to All cells in a person have the same DNA Yet eye cells differ from nose cells Central dogma of biology Genetic engineeri ng Tissue therapy With the case of iPS cells, scientists use genetic engineering to add genes into the cells’ genomes, leading to the production of proteins that change the cell phenotype. These cells may be useful for personalized tissue therapies. But normally, in the body, how do cells know what to become? All cells in a person share the same genetic code, or genotype. Given that their DNA sequences are the same, why are your eye cells different from your nose cells? If you cut your hand, how do the cells know to make more hand cells, not eye cells? Well, that environment outside the cells, also called the niche (neesh), inﬂuences the switching on and off of different genes. The change in gene expression alters the physical characteristics, or phenotype, of the cells. This should sound familiar to everyone since this is the central dogma of biology—that is, gene expression drives the presence and levels of certain proteins which dictate the cell type. These cells can be used for tissue therapy. Genetic engineering alters DNA sequences which can eventually alter the cell type.

52. Signals to Stem Cells Matrix Molecules Self-Renewal Soluble Factors Other Cells Differentiation Little, et al. Chemical Reviews (2008). Cells stick and respond to molecules embedded in their extracellular environment (top left). They also respond to chemicals or molecules ﬂoating around in the liquid surrounding them (middle left). Cells can feel and communicate with each other (bottom left), and also can respond to forces. How might an embryonic stem cell respond if it touches a bunch of muscle cells? (wait for answers) It might differentiate into a muscle ﬁber. How do you think that same stem cell would respond to culture with a bunch of neurons? (wait) It’ll turn into a neuron. Here, this stem cell is going to make a decision to self-renew or differentiate based on the individual components and combinations of these factors in the extracellular environment.

53. Factors known to affect stem cells • • • • • • Low stress levels Regular exercise Enriching experiences Learning new information Healthy diets: rich in antioxidants Avoid excessive drinking Helping you help yourself So if growth factors and hormones affect stem cell functions, then our lifestyles…. our experiences and behaviors…. are also likely to inﬂuence homeostatic stem-cell related processes—for example, regular cell turnover in blood and skin, wound healing throughout the body, and even our sensory and cognitive abilities when it comes to adult stem cells in the brain. So not surprisingly, what is good for your body (point to bullets) is good for your stem cells…. and so, what’s good for your stem cells is also good for your body. Why? Because the regulation of stem cells not only plays a really important role in various disease states but also these proliferative cells actively participate in maintaining our overall health. If you’re good to your stem cells, they’ll be good to you.

54. Outline of Presentation • Fertilization and embryonic development • What makes stem cells unique? • What are the different types of stem cells? • What are examples of stem cell research, therapies, and technologies? • Conclusion and future directions So now that you know a bit about where embryonic stem cells come from, and that several tissues in the body retain populations of stem cells into adulthood; and that the local stem cell niche, chemical messengers and even our own lifestyles affect stem cell functions…. Let’s talk a bit about what’s going on right now in terms of stem cell therapies and technologies to obtain or create stem cells for research and future cures.

55. Experimental model system Heart muscle cells beating in a petri dish! Videos by The Exploratorium Here is an example of using stem cells to create experimental model systems. These cells are heart muscle cells. They were derived from mouse embryonic stem cells, and they beat spontaneously in the petri dish. CLICK THE LEFT VIDEO. The ones on the left have one epicenter of beating. CLICK THE RIGHT VIDEO. The ones on the right have three epicenters. These are experimental model systems that give scientists a way to study how the heart works. Scientists can also genetically manipulate these cells so that they might have a defect or disease, then study how the disease develops. They can also be used as a tool for drug screening in early stages of pharmaceutical development instead of having to use animals or humans.

58. Bone marrow transplant: Example of adult stem cell-based therapy Here is an example of an existing stem-cell-based therapy. Although they are more difficult to obtain in pure form than embryonic stem cells, adult stem cells do have therapeutic potential. A well-established adult stem cell therapy is a bone marrow transplant, which is usually a mix of several types of cells including adult stem cells. Bone marrow transplants have been practiced for 40 years as a treatment for diseases of the blood and certain types of cancer like leukemia. FIRST, a donor’s tissue type is matched with the patient’s tissue type to make sure the patient won’t reject the transplant. NEXT, bone marrow containing hematopoietic (hee-mat-oh-poetic) stem cells, or blood-forming cells, is taken from the donor’s pelvis. THEN, right before the transplant, the recipient patient receives chemotherapy to destroy all of their malignant blood cells. FINALLY, the donor’s marrow is ﬁltered to increase the ratio of stem cells and then given in a transfusion to the patient. The stem cells will ﬁnd their way to the bone marrow and eventually repopulate the patient’s blood system. Sometimes instead of receiving stem cells from a donor, the patient can receive their own stem cells. Umbilical cord, the tissue connecting baby to mother before birth, is a rich source of hematopoietic (hee-mat-oh-poetic) stem cells. The umbilical cord is usually thrown away after a baby is born, but some people choose to “bank” the umbilical cord blood cells in case the child needs to use those stem cells later on. Hematopoietic stem cells from umbilical cord do not have the same immune-rejection issues as hematopoietic stem cells from bone marrow, which makes them ideal for therapies.

59. Spinal cord injury: Example of embryonic stem cell-based therapy Geron video: http://www.geron.com/ This is also an example of a cell-based-therapy, although it is still in the early stages of development. One way that we can use embryonic stem cells is to coax them to differentiate into progenitor cells for a certain type of tissue, such as nervous. Those progenitor cells have a healing effect when transplanted into the injured spinal cord. Geron, a Bay Area biotech company, is sponsoring the ﬁrst FDA-approved HUMAN embryonic stem cell trial to try to bridge the gap from research to therapeutics. Let’s check out a video describing the work in rats that lead to this trial in humans. CLICK the video. You should hear audio. Note that embryonic stem cells cannot be transplanted “as-is” for therapies. If you inject embryonic stem cells into a patient, those cells often turn into nasty tumors called teratomas. Teratomas contain many differentiated tissues because the extracellular environment has given them abnormal differentiation instructions. In order to be used for therapies, embryonic stem cells must ﬁrst be coaxed to differentiate into adult stem cells, progenitor cells, or fully mature cells.

60. Spinal cord injury: Example of embryonic stem cell-based therapy Geron video: http://www.geron.com/ This is also an example of a cell-based-therapy, although it is still in the early stages of development. One way that we can use embryonic stem cells is to coax them to differentiate into progenitor cells for a certain type of tissue, such as nervous. Those progenitor cells have a healing effect when transplanted into the injured spinal cord. Geron, a Bay Area biotech company, is sponsoring the ﬁrst FDA-approved HUMAN embryonic stem cell trial to try to bridge the gap from research to therapeutics. Let’s check out a video describing the work in rats that lead to this trial in humans. CLICK the video. You should hear audio. Note that embryonic stem cells cannot be transplanted “as-is” for therapies. If you inject embryonic stem cells into a patient, those cells often turn into nasty tumors called teratomas. Teratomas contain many differentiated tissues because the extracellular environment has given them abnormal differentiation instructions. In order to be used for therapies, embryonic stem cells must ﬁrst be coaxed to differentiate into adult stem cells, progenitor cells, or fully mature cells.

61. Outline of Presentation Fertilization and embryonic development What makes stem cells unique? What do stem cells look like? What are the different types of stem cells? • What are examples of stem cell research, therapies, and technologies? • Conclusion and future directions • • • • So let’s put this all together.

62. Why do researchers want to use embryonic stem cells along with other technologies? • Pluripotent – Expanded developmental potential allows them to be used in ways that adult stem cells cannot • Can proliferate indeﬁnitely in culture • Easier to obtain than adult stem cells Why are people so interested in using human embryonic stem cells? Why not just use adult stem cells or induced Pluripotent Stem cells for research and therapies? Embryonic stem cells are pluripotent, and their expanded developmental potential allows us to study development and model disease in ways we can’t do with adult stem cells. Embryonic stem cells can proliferate, or divide symmetrically, forever. Some people call them immortal cells. Adult stem cells stop proliferating after a certain number of cell divisions. Embryonic stem cells are easier to obtain than most adult stem cells. This is also why some people do not want to use embryonic stem cells. The ethical framework for obtaining embryonic stem cells by destroying a blastocyst is debatable. Some believe induced Pluripotent Stem cells will replace embryonic stem cells for use in therapies, however this remains to be seen.

63. Science is discovering the unknown • Stem cell field is still in its infancy • Human embryonic stem cell research is a decade old, adult stem cell research has 30year head start • Holds hope for curing or improving treatments for 70+ diseases How can you help to shape the direction of this New scientific disciplines, like stem cell research, are all about posing questions and finding answers to life’s mysteries. We have already learned a lot in the ten years we’ve been doing human embryonic stem cell research and in the 30 years before that doing adult stem cell research. The knowledge we have now, including the information in this presentation, is subject to change because science is always evaluating the existing body of work and filling in the gaps. Because scientific inquiry and the need for more advanced medical technology guides the questions researchers ask, we can’t predict exactly where the stem cell field is going and what cures may or may not exist ten years from now. But we do know that stem cell research and regenerative medicine hold hope for curing or improving treatments for more than seventy diseases.

64. Somatic Cell Nuclear Transfer (SCNT) Udder cell Egg cell Here we’re going to demonstrate cloning using Dolly the Sheep. In Somatic Cell Nuclear Transfer, you FIRST harvest an egg cell from one sheep, then remove the egg’s genetic material, leaving an egg that still has all its internal chemical signals but no nucleus and so none of its own DNA. NEXT, you take an udder cell from the sheep you want to clone— in the case of Dolly it was her mom—and transfer this nucleus into the enucleated egg. In the case of the “Ruppies,” researchers inserted ﬂuorescent-encoding genes into the skin cells before transferring their nuclei into egg cells. FINALLY, applying a small jolt of electricity will start the process of cell division and trigger development that you saw early in this presentation. But this alone isn’t enough to produce a cloned sheep OR puppy.

65. Types of Cloning For the case of REPRODUCTIVE CLONING, when the embryo reaches the blastocyst stage, you HAVE to inject it back into the egg donor, who later gives birth to the cloned sheep. That’s how Dolly was cloned—a perfect GENETIC replica of her mother. So what’s different about cloning embryonic stem cells? So for research purposes, CLICK! you harvest the cloned embryonic stem cells and plate them on a petri dish for a scientist to use in her research. Rather than create a whole animal, scientists use these cells to study disease and eventually find cures. This is called RESEARCH CLONING. If her work is particularly promising, she can move to more clinical experiments. In THERAPEUTIC CLONING, CLICK! she can take those plated stem cells from her research, make them into the cell type needed by the sick sheep who donated the skin cell, and transplant them into the sheep to repair specific tissue, not create a whole animal. In humans this would be an ideal use of cloned embryonic stem cells. However, scientists haven’t YET figured out how to get the cells to divide past the first few divisions. If we could get somatic cell nuclear transfer to work, what would be the advantage to using cloned cells? (They are genetically identical to the patient and would not be rejected as foreign by the immune system.) And remember these cells CANNOT be used to generate new organisms outside of the proper niche, the uterus. REFER TO HANDOUT: The Facts about Stem Cell Research question 6. “What was unique about the dogs and how did they make them? Ask students to describe how the Ruppies were made. They can draw a picture if it is easier for them. Scientists introduced a gene that produces Red Fluorescent Protein into the DNA of skin cells cultured from a donor dog. They then transferred the identical nuclei of those cells into egg cells without nuclei, continued with the cloning

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