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STEM CELL BIOLOGY Reprogramming, Pluripotency and Differentiation 1 Outline - Background - Applications A li ti off stem t cell ll research h - Methods for generating stem cells - SBI’s tools for stem cell research What Are Stem Cells? Stem cells are unspecialized cells that are thought to be able to reproduce themselves indefinitely and, under the right conditions conditions, to develop into a wide variety of mature cells with specialized functions. Two important T i t t characteristics: h t i ti - Self-renewal - Pluripotency Human embryonic stem cell colony Types of Stem Cells Embryonic stem cells (also known as ES cells) come from a 4 to 7-day-old embryo. They have the ability to form virtually any type of cell found in the human body. Adult stem cells are more specialized than embryonic stem cells. They are found in the majority of tissues and organs in our body and generate the mature cell types within that tissue or organ. They have a restricted ability to produce different cell types and to self-renew. Types of Stem Cells Stem cells that have the potential to develop into any of the celltypea found in an adult organism are called pluripotent. Embryonic stem cells are pluripotent. Stem cells that only have the potential to make a few cell types in the body are called multipotent. Adult stem cells are multipotent. Applications of Stem Cell Research Study cell differentiation Pluripotent Stem Cells Applications of Stem Cell Research Drug screening & Toxicity testing Study cell differentiation Pluripotent Stem Cells Applications of Stem Cell Research Drug screening & Toxicity testing Study cell differentiation Pluripotent Stem Cells Bone Marrow for leukemia & chemotherapty Nerve cells for neurodegenerative disorders Heart muscle cells for heart disease p py Cell replacement therapy Pancreatic islet cells for diabetes Methods to Generate Stem Cells Methods to Generate Stem Cells Methods to Generate Stem Cells Tools for Stem Cell Research I. Inducing pluripotency with SBI’s iPSC factors Six iPSC factors for stem cell reprogramming Oct4 Nanog Lin28 Sox2 Klf4 c-Myc Protocol of Inducing Pluripotency 4 ttranscription i ti factors f t Human fforeskin H ki fibroblasts Yamanaka S., Cell, 2006 Protocol of Inducing Pluripotency 4 ttranscription i ti factors f t Human fforeskin H ki fibroblasts Replate HFFs on feeder cells Yamanaka S., Cell, 2006 Protocol of Inducing Pluripotency 4 ttranscription i ti factors f t Human fforeskin H ki fibroblasts iPS colony formation Replate HFFs on feeder cells Yamanaka S., Cell, 2006 Protocol of Inducing Pluripotency 4 ttranscription i ti factors f t Expand best colonies 1 2 3 4 5 6 Pick individual colonies Human fforeskin H ki fibroblasts iPS colony formation Replate HFFs on feeder cells Yamanaka S., Cell, 2006 Colony Formation Day 12 Human ES-like Colonies from Retroviral Based Factors Retroviral-Based Teratoma a A AP SSEA--1/4 Nanog Characterization of iPSCs SBI’s Stem Cell Lines Human iPS cell line(SC101A-1) Mouse iPS cell line(SC201A-1) Human or mouse induced pluripotent stem cells were generated by transducing genetically unmodified human or mouse dermal fibroblasts with viruses individually encoding the four transcription factors (Oct4, Sox2, Klf4, and c-Myc) that have been shown to induce the reprogramming of somatic cells to a pluripotent state. The cells were derived using morphological selection criteria and without the use of fluorescent marker or drug selection. When cultured under standard ES cell culture conditions, the morphology of SBI iPSCs are identical to that of ES cells. The cells also express the pluripotency markers SSEA-4/1 Nanog and demonstrate strong endogenous alkaline and Nanog, phosphatase activity. Tools for Stem Cell Research II. Stem cell pluripotency reporters Reporters for human and mouse Oct4 and Nanog allow monitoring, sorting and selecting undifferentiated ES or iPS cells Tools for Stem Cell Research II Stem cell pluripotency reporters II. G GFP Phase Co ontrast H9 hES cells pGreenZeo-CMV pGreenZeo-mOct4 pGreenZeo-mNanog Transduced Transduced Transduced H9 hES cells H9 hES cells H9 hES cells Data courtesy of Drs. Timothy Kamp and Chad H. Koonce Tools for Stem Cell Research III Stem III. St cell ll differentiation diff ti ti reporters t • Available as prepackaged virus or plasmid off the shelf • Custom construction for any reporter gene also offered Protocol for Neural Differentiation hMAP2 hDCX mGAD67 Mouse iPSCs Mouse Neurons Mouse NSCs Mouse Astrocytes mGFAP Tools for Stem Cell Research Stem Cell Differentiation Reporters - Astrocyte reporter construct GFAP: marker for astrocytes Tools for Stem Cell Research Stem Cell Differentiation Reporters - Immature neuron reporter construct DCX: marker for immature neurons Tools for Stem Cell Research Stem Cell Differentiation Reporters - Mature neuron reporter construct MAP2: marker for mature neurons Comparison of SBI Products with Competitors Human iPSC Mouse iPSC iPSC Factors Pluripotency Reporters Differentiation Reporters SBI 9 9 9 9 9 Invitrogen 8 8 8 8 9 8 8 8 8 8 Open Bi Biosystems t 8 8 9 8 8 Stemgent 8 9 9 8 8 Clontech System Biosciences (SBI) www.systembio.com System Biosciences (SBI) 265 North Whisman Rd. Mountain View, CA 94043 Tel: 650-968-2200 Fax: 650-968-2277