Genomics & Medicine http://biochem158.stanford.edu/ Stem Cells http://biochem158.stanford.edu/Stem%20Cells.html http://biochem158.stanford.edu/ - you may see a videolectures here http://biochem158.stanford.edu/Stem%20Cells.html Doug Brutlag Professor Emeritus of Biochemistry & Medicine Stanford University School of Medicine

Humbio 157/Developmental Biology 257 The Biology of Stem Cells The Role of stem cells in human development and potential for treating disease. Prerequisite Humbio 2A,B or consent of instructor Prof. Margaret Fuller and Roeland Nusse Guest Speakers: Hank Greely, Stefan Heller, David Magnus, Anthony Oro, Theo Palmer, Thomas Rando, Renee Reijo Pera, Irving Weissman, Marius Wernig, Joanna Wysocka Offered next year.

Henry Stewart Talks http://hstalks.com/

Differentiation of Human Tissues ZYGOTE BLASTOCYST GASTRULA Ectoderm (external layer) Mesoderm (middle layer) Endoderm (internal layer) Germ cells Skin cells of epidermis Neuron of brain Pigment cell Cardiac muscle Skeletal cells Tubule cell of the kidney Red blood Smooth (in gut) Pancreatic Thyroid Lung cell (alveolar Cell) Sperm Egg Courtesy Paul Berg

Embryonic Stem Cell Cultures Courtesy Paul Berg

Courtesy Paul Berg

Courtesy Paul Berg

Courtesy Paul Berg

Basic Problems of Stem Cell Therapy HOW TO DIRECT DIFFERENTIATION OF CELLS DOWN SPECIFIC PATHWAYS? e.g. all into muscle or all into nerve; different “cocktails” of growth factors HOW TO OVERCOME IMMUNE REJECTION? e.g. alter histocompatibility genes; therapeutic cloning for “customized” lines HOW TO MAKE AN ORGAN? e.g. combine different cell types in three dimensional arrangements. Courtesy Paul Berg

Fibronectin is a high-molecular weight (~440kDa) extracellular matrix glycoprotein that binds to membrane-spanning receptor proteins called integrins.[1] In addition to integrins, fibronectin also binds extracellular matrix components such as collagen, fibrin and heparan sulfate proteoglycans (e.g. syndecans). Fibronectin exists as a dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds.[1] The fibronectin protein is produced from a single gene, but alternative splicing of its pre-mRNA leads to the creation of several isoforms. Nestin is a type VI intermediate filament (IF) protein.[1][2] These intermediate filament proteins are expressed mostly in nerve cells where they are implicated in the radial growth of the axon. Seven genes encode for the heavy (NF-H), medium (NF-M) and light neurofilament (NF-L) proteins, nestin and α-internexin in nerve cells, synemin α and desmuslin/synemin β (two alternative transcripts of the DMN gene) in muscle cells, and syncoilin (also in muscle cells). Members of this group mostly preferentially coassemble as heteropolymers in tissues. Steinert et al. has shown that nestin forms homodimers and homotetramers but does not form IF by itself in vitro. In mixtures, nestin preferentially co-assembles with purified vimentin or the type IV IF protein -internexin to form heterodimer coiled-coil molecules.[3] Laminins are major proteins in the basal lamina (formerly improperly called "basement membrane"), a protein network foundation for most cells and organs. The laminins are an important and biologically active part of the basal lamina, influencing cell differentiation, migration, adhesion as well as phenotype and survival.[1] Laminins are great trimeric proteins that contain an α-chain, a β-chain, and a γ-chain, found in five, three, and three genetic variants, each. The laminin molecules are named according to their chain composition. Thus, laminin-511 contains α5, β1, and γ1 chains.[2] Fourteen other chain combinations have been identified in vivo. The trimeric proteins form a plus sign with one long arm, giving a structure that can bind to other cell membrane and extracellular matrix molecules.[3] The three shorter arms are particularly good at binding to other laminin molecules, which allows them to form sheets. The long arm is capable of binding to cells, which helps anchor organized tissue cells to the membrane. The laminins are a family of glycoproteins that are an integral part of the structural scaffolding in almost every tissue of an organism. They are secreted and incorporated into cell-associated extracellular matrices. Laminin is vital for the maintenance and survival of tissues. Defective laminins can cause muscles to form improperly, leading to a form of muscular dystrophy, lethal skin blistering disease (junctional epidermolysis bullosa) and defects of the kidney filter Transferrin is a blood plasma protein for iron delivery that, in humans, is encoded by the TF gene.[1] Transferrin is a glycoprotein that binds iron very tightly but reversibly. Although iron bound to transferrin is less than 0.1% (4 mg) of the total body iron, it is the most important iron pool, with the highest rate of turnover (25 mg/24 h). Transferrin has a molecular weight of around 80 kDa and contains 2 specific high-affinity Fe(III) binding sites. The affinity of transferrin for Fe(III) is extremely high (1023 M−1 at pH 7.4)[2] but decreases progressively with decreasing pH below neutrality. Courtesy Paul Berg

Methods to Generate Pluripotent Stem Cells Yamanaka. (2007) Cell Stem Cell Vol 1, pp 39-49. Currently Available Methods to Generate Pluripotent Stem Cells from Adult Somatic or Germ Cells In mouse models, three methods have been reported to generate pluripotent stem cells from somatic cells: nuclear transfer, fusion, and forced expression of defined factors. Also reported is the generation of chimera-competent pluripotent stem cells after the long-term culture of bone marrow cells. In addition, pluripotent stem cells can be established from mouse adult germ cells: multipotent GS cells and parthenogenetic ES cells. Yamanaka. (2007) Cell Stem Cell Vol 1, pp 39-49.

Nanog-Mediated Enhancement of Reprogramming by Fusion Yamanaka. (2007) Cell Stem Cell Vol 1, pp 39-49. Figure 2. Nanog-Mediated Enhancement of Reprogramming by Fusion with ES Cells Nanog-overexpressing mouse ES cells showed a marked increase in reprogramming activity after fusion with neural stem (NS) cells. The forced expression of Nanog in NS cells was found to be less effective. NANOG (pron. nanOg) is a transcription factor critically involved with self-renewal of undifferentiated embryonic stem cells. In humans, this protein is encoded by the NANOG gene.[1][2] Yamanaka. (2007) Cell Stem Cell Vol 1, pp 39-49.

Five Factors Needed to Maintain Pluripotency Yamanaka. (2007) Cell Stem Cell Vol 1, pp 39-49. Oct-3/4 was identified as a novel Oct family protein specifically expressed in EC cells, early embryos, and germ cells (Okamoto et al., 1990; Rosner et al., 1990; Scholer et al., 1990). The Oct family transcription factors contain the POU domain, an 150 amino acid sequence conserved among Pit-l, Oct-1, Oct-2, and uric-86. Oct-3/4 and other POU proteins bind to the octamer sequence (ATTA/TGCAT). Expression of Oct-3/4 is restricted in the blastomeres of the developing mouse embryo, the ICM of blastocysts, the epiblast, and germ cells. It is also expressed in pluripotent stem cells, including ES cells, EG cells, EC cells, and mGS cells Sox2 was identified as a Sox (SRY-related HMG box) protein expressed in EC cells (Yuan et al., 1995). The high mobility group (HMG) domain is a DNA binding domain conserved in abundant chromosomal proteins including HMG1 and HMG2, which bind DNA with little or no sequence specificity, and in sequence-specific transcription factors, including SRY, SOX, and LEF-1 All SOX factors appear to recognize a similar binding motif, A/TA/TCAAA/TG. Like Oct-3/4, Sox2 also marks the pluripotent lineage of the early mouse embryo; expressed in the ICM, epiblast, and germ cells. Unlike Oct-3/4, however, Sox2 is also expressed by the multipotential cells of the extraembryonic ectoderm (Avilion et al., 2003). In addition, Sox2 expression is associated with uncommitted dividing stem and precursor cells of the developing central nervous system (CNS), and it can be used to isolate such cells (Li et al., 1998; Zappone et al., 2000). c-Myc is one of the first proto-oncogenes found in human cancers (Dalla-Favera et al., 1982). The N terminus of Myc binds to several proteins, including TRRAP, which are components of the TIP60 and GCN5 histone acetyltransferase complexes, and TIP48 and TIP49, which contain ATPase domains (Adhikary and Eilers, 2005). The C terminus of the Myc protein contains the basic region/helixloop- helix/leucine zipper (BR/HLH/LZ) domain, through which Myc binds to a partner protein, Max. The Myc- Max dimers bind to a DNA sequence (CACA/GTG), which is a subset of the general E box sequence (CANNTG) that is bound by all bHLH proteins. In addition to binding to DNA, the C terminus of Myc is also involved in transactivation through binding to CBP and p300, which have histone acetylase activities. Yamanaka. (2007) Cell Stem Cell Vol 1, pp 39-49.

Induction of Pluripotent Stem Cells (iPS) from Somatic Stem Cells Yamanaka. (2007) Cell Stem Cell Vol 1, pp 39-49. Figure 3. Putative Roles of the Four Factors in the Induction of iPS Cells Pluripotent stem cells are immortal and have open and active chromatin structure. It is likely that c-Myc induces these two important properties. However, c-Myc also induces apoptosis and senescence, which are probably suppressed by KLF4. Oct-3/4 probably changes the cell fate from tumor cells to ES-like cells. To establish pluripotency, Sox2 is also required. Yamanaka. (2007) Cell Stem Cell Vol 1, pp 39-49.

Adipose Tissue Provides iPSC Efficiently Sun et al, Proc Natl Acad Sci U S A. 2009 Sep 15;106(37):15720-5. Don’t need fibroblast feeder cells to grow Two times faster growth and 20 more efficient. Sun et al, Proc Natl Acad Sci U S A. 2009 Sep 15;106(37):15720-5.

Using CRE – Recombinase to Remove Viral Transforming DNA from iPSCs Soldner et al. Cell. 2009 Mar 6;136(5):964-77. Soldner et al. Cell. 2009 Mar 6;136(5):964-77.

Cre-Lox Recombination to Remove Viral DNA In the early 1990's a new method was developed to delete a specific portion of DNA. The procedure took advantage of the basic research performed on the bacteriophage called P1. In this virus, there is an enzyme called cre and particular DNA sequences called lox P sites. The lox P sites work in pairs and they flank a segment of DNA called a target (figure 1).

Inducing iPSCs using Transcription Factor Proteins

Alternate Stem Cell Fates Embryonic Stem Cells Adult Courtesy Minx Fuller

signals from niches maintain adult stem cells and tissues Central elements niche model Limited availability and range of the niche signals stem cells compete for niche signals niches and signals maintain tissues Courtesy Roel Nusse

In the absence of niche signals, adult stem cells will differentiate, by default Outside the niche, stem cells differentiate because the absence of signals lead to differentiation, stem cells hard to propagate what are the signals how is range controled the intrinsic state of the cell (stem-ness) outside signals Self-renewing signals: very complex 1. inhibit differentiation 2. control proliferation 3. are local and niches have a limited capacity 4. include the main developmental pathways: Notch, Hedgehog, BMP, FGF and Wnt 1. Self-renewal is proliferation coupled to blocking differentiation, controlled by signals. 2.Signals are local; niches have a limited capacity and cells compete for the signals 3. The signals control tissue homeostasis, also after damage Courtesy Roel Nusse

Oocyte Niche in the Drosophila Germarium Li and Xie, Ann. Rev. Dev. Biol. 2005, 605-663 Drosophila germarium cross section showing the locations of germ line stem cells (GSCs), somatic stem cells (SSCs), and their niches. Two or three GSCs (red cells, left) are situated in their niche, composed of cap cells (green cells, left) and terminal filament cells (light blue cells, left tip), whereas their differentiated progeny, including cystoblasts and differentiated cysts (yellow cells, middle), are surrounded by inner sheath cells (purple cells and green cells, bottom and top). Two or three SSCs (red cells, bottom and top) directly contact the posterior group of inner sheath cells (green cells, bottom and top) forming their niche, whereas their differentiated progeny, also known as follicle progenitor cells (orange cells on right), further proliferate and generate differentiated follicle cells. Two inserts depict major signaling pathways controlling GSC (top and left) and SSC (top and right) self-renewal and proliferation; these inserts also depict niche cells (green) and stem cells (pink). Li and Xie, Ann. Rev. Dev. Biol. 2005, 605-663

Cell-Cell Interactions at Oocyte Niche Li and Xie, Ann. Rev. Dev. Biol. 2005, 605-663 Drosophila germarium cross section showing the locations of germ line stem cells (GSCs), somatic stem cells (SSCs), and their niches. Two or three GSCs (red cells, left) are situated in their niche, composed of cap cells (green cells, left) and terminal filament cells (light blue cells, left tip), whereas their differentiated progeny, including cystoblasts and differentiated cysts (yellow cells, middle), are surrounded by inner sheath cells (purple cells and green cells, bottom and top). Two or three SSCs (red cells, bottom and top) directly contact the posterior group of inner sheath cells (green cells, bottom and top) forming their niche, whereas their differentiated progeny, also known as follicle progenitor cells (orange cells on right), further proliferate and generate differentiated follicle cells. Two inserts depict major signaling pathways controlling GSC (top and left) and SSC (top and right) self-renewal and proliferation; these inserts also depict niche cells (green) and stem cells (pink). Gbb glass bottom boat = TGF-beata at 60A ZPG = zero population growth = adhesion Innexin 4 similar to mamalian Connexin Also ARM + cadherin Li and Xie, Ann. Rev. Dev. Biol. 2005, 605-663

Drosophila Spermatogonial Niche Li and Xie, Ann. Rev. Dev. Biol. 2005, 605-663 Cross section of the apical tip of the Drosophila testis, showing the locations of germ line stem cells (GSCs), somatic stem cells (SSCs), and their niches. Hub cells (green) at the apical tip of the testis form niches for both GSCs (red) and SSCs (gray, left), which generate, respectively, spermatogonial cells (yellow) and somatic cyst cells (light gray) encapsulating differentiated spermatogonial cells. The insert on top describes major signaling pathways involved in communication between GSCs and the niche cells for controlling self-renewal and proliferation. Li and Xie, Ann. Rev. Dev. Biol. 2005, 605-663

Cell-Cell Interactions at the Spermatogonial Niche Li and Xie, Ann. Rev. Dev. Biol. 2005, 605-663 Upd (unpaired) from the hub activates the JAK-STAT pathway in GSCs and promotes their self- renewal (Kiger et al. 2001, Tulina & Matsunis 2001). Additionally, the activation of JAK- STAT signaling can reprogram mitotic germ cysts into GSCs (Brawley & Matunis 2004). As in the ovary, BMP signaling is required for controlling GSC self-renewal in the testis (Kawase et al. 2004, Schulz et al. 2004, Shivdasani & Ingham 2003). Hub cells and somatic cyst cells express gbb at high levels and dpp at much lower levels; consequently, BMP downstream components are essential for controlling testicular GSC self-renewal (Kawase et al. 2004). Because dpp overexpres- sion fails to suppress completely spermatogo- nial cell differentiation, BMP signaling likely plays a permissive role in controlling male GSC self-renewal. BMP and JAK-STAT sig- naling pathways are required for controlling male GSC self-renewal; thus, they must some- how interact with each other. The integra- tion between these two pathways in male GSCs is an important area in need of future exploration. Li and Xie, Ann. Rev. Dev. Biol. 2005, 605-663

Hair Follicle Niche Li and Xie, Ann. Rev. Dev. Biol. 2005, 605-663 Illustration of the epidermal stem cells. Stem cells are located in the bulge region of the hair follicle beneath the sebaceous gland. Upon activation, stem cells undergo division; the daughter cells retained in the bulge remain as stem cells while other daughter cells migrate down to become hair-matrix progenitors responsible for hair regeneration. In neonatal mice or in damaged skin, stem cells can also migrate upward and convert to epidermal progenitors that replenish lost or damaged epidermis. The bulge area is an environment that restricts cell growth and differentiation by expressing Wnt inhibitors, including DKK,Wif, and sFRP as well as BMPs. During the early anagen phase, Wnts from dermal papilla (DP) and Noggin, which is derived from both DP and bulge (J. Zhang & L. Li, unpublished data), coordinate to overcome the restriction signals imposed by both BMPs and Wnt inhibitors; this leads to stem cell activation and subsequent hair regeneration. The FGF and Notch pathways are also involved in DP function for hair-matrix cell proliferation and lineage fate determination, but their influence on stem cells is not clear. Li and Xie, Ann. Rev. Dev. Biol. 2005, 605-663

Intestinal Stem Cells in the Crypts Hans Clever’s annimations

Asymmetric stem cell divisions Extrinsic factor(s) Niche Courtesy Minx Fuller

John Cairns: The Immortal Parental Strands Cairns (1975) Nature 255, 197 Cairns (1975) Nature 255, 197

Motivation for Asymmetric Strand Segregation Adult rat contains 6x1010 cells In its small intestine, a rat sheds over 1013 epithelial cells during its lifetime. Requires 103 symmetric cell doublings from embryo to adult followed by 1013 asymmetric cell doublings during its lifetime How do epithelial cells minimize mutations that lead to cancer? Cairns (1975) Nature 255, 197 Cairns (1975) Nature 255, 197

Asymmetric Segregation of Parental DNA Strands Rando (2007) Cell 129 1239 Rando (2007) Cell 129 1239

Asymmetric Stem Cell Growth with Asymmetric Parental Strand Segregation Rando (2007) Cell 129 1239 Rando (2007) Cell 129 1239

Asymmetric DNA Labeling Patterns Rando (2007) Cell 129 1239 Rando (2007) Cell 129 1239

Duplicating Muscle Cell Pairs Display Asymmetric DNA Labeling Patterns Conboy et al, PLOS Biology (2007) Conboy et al, PLOS Biology (2007)

Asymmetric Stem Cell Growth with Asymmetric Parental Strand Segregation Rando (2007) Cell 129 1239 Rando (2007) Cell 129 1239

Wnt signaling TCF APC GSK3 Axin1 Wnt LRP Frizzled Courtesy Roel Nusse

Wnt Signaling Pathway © BioCarta Wnt Signaling Pathway Wnt family members are secreted glycoproteins who bind to cell surface receptors such as Frizzled. Wnt members can play a role in the expression of many genes by interacting with multiple disparate signaling pathways. Shown is the Wnt/beta-catenin pathway. © BioCarta

The Hedgehog pathway Shh Patched Smoothened SuFu Gli Ligand Receptor 7-pass membrane transducer I study hedgehog signal transduction for two main reasons: Biochemical steps in the pathway are not understood. Biochemical activities of the components are not known. As a consequence, we don’t know how the Smo inhibitors work– how will we deal with resistance? No tools to assay activity in patient samples. One of the major breakthroughs in the field has been the finding that Hh signaling is dependant on a very specialized structure called the primary cilium.. Cytoplasmic Negative Regulator SuFu Transcription Factor Gli Target Genes : Gli1 and Ptc1 Courtesy Raj Rohatgi

The primary cilium: A specialized compartment for signal transduction Nucleus Cilium NIH 3T3 Cells Vesicle Cilia-targeted Protein Transport Complex AXONEME Rosenbaum and Witman, Nature Reviews Mol Cell Biol (2002) Introduce the structure: Solitary projection; basal body; size Why the recent interest-- Polycystic Kidney Disease; Bardet Biedl Syndrome --General Components of Signaling. Kathryn Anderson: Mutants in IFT; Kinesin; Dyenin all have Hh phenotypes Studying signaling in this structure is a technical challenge. It has required the development of new way to study signaling using high resolution microscopy and new optical probes to follow protains, We have started to unravel the cell biology of how Hh signaling depends on primary cilia. Plasma Membrane Basal Body Cytoplasm Rosenbaum and Witman, Nature Reviews Mol Cell Biol (2002) Courtesy Raj Rohatgi

Cilia as sensors for Shh: Shh binds to its receptor Patched1 at primary cilia in live cells Shh-A594 Ptc1-YFP Ptc1-YFP Shh-A594 Shh Ptc1 Smo If we start at the top of the pathway, we have found that the receptor is not distributed all over the cell but rather concentrated within cilia. This is an image taken w/ a spinning disc confocal to give a thin optical section w/o causing photobleacing. Next I synthesized a version of the ligand modified by a fluorophore. Binding is not static but causes a change in localization– measure patched fluorescence from pictures like this Ptc1 is dislocated from cilia. SuFu Gli Courtesy Raj Rohatgi

Smo moves to cilia and when the Hedgehog pathway is activated 3T3 Cells Shh Ptc1 Smo What happens next? The dissapearance of Ptc1 is followed by the marked concentration of Smo into cilia. We think that Ptc1 works by excluding Smo from cilia because when we look at cells lacking Ptc1 Smo is constitutively in cilia. Thus localization of Smo at cilia causes pathway activation. SuFu Gli Courtesy Raj Rohatgi

Smo activates downstream signaling components in cilia SuFu Cilia Gli2-YFP Cilia Ptc1 Smo Shh SuFu Gli What happens when Smo gets to cilia: The downstream components are localized in cilia and so the thought is that SMo can engage downstream machinery. Courtesy Raj Rohatgi

Smo as a model for signal-regulated protein transport at primary cilia Smo Cilia Nucleus +Shh Courtesy Raj Rohatgi

Models for ciliary protein transport Post Golgi Vesicle Smo C N 2a: Lateral Transport Plasma Membrane Cilium 1: Direct Trafficking Endosome 2b: Recycling Mediated 2: Trafficking to plasma membrane Courtesy Raj Rohatgi