Induced Pluripotent Stem Cells (iPSCs): Frontiers in Reprogramming Michael L. Moeller, MS, PhD Field Application Scientist III Bioscience Division EMD Millipore A Division of Merck KGaA Darmstadt Germany

Epigenetic States Alter Developmental Pathways and Can Be Used to Modify Developmental State A modification of CH Waddington’s Epigenetic landscape model shows cell populations with different develomental potentials (let and their respective epigenetic states (right). Developmental restrictions can be illustrated as marbles rolling down a landscape into one of several valleys (cell fates). Colored marbles correspond to differentiation states purple= totipotent, blue =pluripotent red = multipotent, green= unipotent. Examples of reprogramming processes are shown by dashed arrows . Hochedlinger,K and Plath, K. Development 136, 509-523 (2009) Presentation title in footer | 00 Month 0000

Methods of Factor Delivery Advantages Disadvantages References Moloney-based Retrovirus Silenced in pluripotent cells Self silencing eliminates need for timed factor withdrawal Genomic integration; risk of insertional mutagenesis Limited to dividing cells Expression often maintained in iPSCs; increased tumor incidence in chimeric mice due to transgene reactivation Takahashi and yYamanaka, 2006 HIV based lentivirus Constitutive Transduction of both dividing and nondividing cells Inducible Temporal control over factor expression Lack of silencing in pluripotent state Genomic integration risk of insertional mutagenesis Possibility of leaky expression Grambrink et al., 2008, Blelloch et al., 2007; Yu et al., 2007 Stackfeld et al., 2008b; Brambirink et al 2008 Transient Transfections No viral components Low frequency of genomic integration Technically simple procedure. Multiple rounds of transfection are required Lower levels of expression than when integrated; Delayed kinetics of reprogramming Okita et al., 2008 Adenovirus Repeated infection required for certain cell types Stadtfeld et al., 2008c Nimet Maherali and Konrad Hochedlinger; Cell Stem Cell 3, December 4, 2008 Presentation title in footer | 00 Month 0000

ES-Cell Transcription Factors and Their Role in Reprogramming Activation of pluripotency regulators Repression function Activation of metabolic and proliferative programs Hochedlinger,K and Plath, K. Development 136, 509-523 (2009) Presentation title in footer | 00 Month 0000

Role of 4 Factors During Reprogramming 7. Role of the Four Factors during Reprogramming(A) c-Myc is a major contributor to the downregulation of fibroblast-specific genes at the beginning of the reprogramming process (i). In iPS cells, c-Myc binds and activates many ES cell-specific genes with roles in metabolic regulation, often with the other three factors (iii). For many of these target genes, binding and activation is also found in partially reprogrammed cells (ii). Target genes that are co-bound by Oct4, Sox2, and Klf4 in ES/iPS cells encode some of the most highly expressed regulators of pluripotency (v) and are not bound and activated at the partially reprogrammed state (iv). Our data suggest that the targeting of OSK to these genes represents a barrier to the reprogramming process and that c-Myc-bound genes largely become activated during earlier steps.(B) State of histone H3K4 and K27 trimethylation within promoter regions of genes that become most highly activated during reprogramming and that are co-bound by Oct4, Sox2, and Klf4 in ES/iPS cells but not in partially reprogrammed cells. (A) c-Myc is a major contributor to the downregulation of fibroblast-specific genes at the beginning of the reprogramming process (i). In iPS cells, c-Myc binds and activates many ES cell-specific genes with roles in metabolic regulation, often with the other three factors (iii). For many of these target genes, binding and activation is also found in partially reprogrammed cells (ii). Target genes that are co-bound by Oct4, Sox2, and Klf4 in ES/iPS cells encode some of the most highly expressed regulators of pluripotency (v) and are not bound and activated at the partially reprogrammed state (iv). Our data suggest that the targeting of OSK to these genes represents a barrier to the reprogramming process and that c-Myc-bound genes largely become activated during earlier steps. (B) State of histone H3K4 and K27 trimethylation within promoter regions of genes that become most highly activated during reprogramming and that are co-bound by Oct4, Sox2, and Klf4 in ES/iPS cells but not in partially reprogrammed cells. Rupa Sridharan et al.(2009) :Cell, Volume 136, Issue 2, Pages 364-377 Presentation title in footer | 00 Month 0000

The Process of Generation of Induced Pluripotent Cells (iPS) Hochedlinger,K and Plath, K. Development 136, 509-523 (2009) Presentation title in footer | 00 Month 0000

Methods of iPS Generation: Small Molecules Technology Description Factors to increase efficiency VPA (HDAC inhibitor) efficiency increased 100-fold p53 siRNA; microRNAs (miRNAs); 5-aza-cytodine Small molecule inhibitors Combination of the small molecules BIX-01294 and BayK8644 generate iPS from MEFs that were transfected with only Oct4 and Klf4. RepSox replaces Sox2, need OKM lentivirus 2i + LIF “2i” refers to dual inhibition of MAPK and GSK3 Pushes partially reprogrammed cells “pre-iPS” to “ground state,” in which cells exist free of differentiation and epigenetic restrictions while retaining the ability to self-renew indefinitely. Eliminates Sox2 and c-myc reprogramming factors for converting NSC to iPSC (Oct4 and Klf4 only needed) Induces reactivation of the X chromosome in partially reprogrammed cells. 2i/LIF condition induced stable up-regulation of Oct4 and Nanog, reactivation of the X chromosome, transgene silencing, and competence for somatic and germline chimera Presentation title in footer | 00 Month 0000

Reprogramming Process Figure 1. Generation of iPSCs with Fewer Genetic Modifications Using Chemical Stimuli Increase of chromatin accessibility by open chromatin formation through the Wnt- and c-Myc-mediated pathways or VPA treatment in pre-iPSCs. Reprogramming of adult neural stem cell (NSC) and fetal neural progenitor cell (NPC) to iPSCs by two genes or two genes plus one chemical. AP, Alkaline phosphatase; MEF, mouse embryo fibroblast; CBP, CREB-binding protein; HDAC, histone deacetylase; VPA, valproic acid. Takashi Tada (2008); Cell Stem Cell, Volume 3, Issue 2,Pages 121-122 Presentation title in footer | 00 Month 0000

All Mycs Are Not Created Equal!! •c-Myc, L-Myc, and N-Myc all have the ability to contribute to iPSC generation •c-Myc promotes iPS cell generation, but has significant transformative ability, as well; L-Myc, by contrast, shows very low transformative ability Nakagawa et al. PNAS 107(32): Presentation title in footer | 00 Month 0000

Assessment of iPS Potency Morphology ID Unlimited self-renewal; Visual ID Molecular ID Protein level expression of key pluripotency factors (Oct 4, Nanog) and key specific surface antigens (SSEA-4, Tra-1-60/-81) Functional Telomerase expression Expression of genes involved in retroviral silencing (de novo methyltransferases and Trim28/ transgene independence Epigenetic similarity of ESCs including demethylation at the promoters of pluripotency genes, X chromosome reactivation Bivalent domains of developmental genes consisting of overlapping histone modifications Histone 3 K4 trimethylation/unmethylated Histone 3 K27 = hallmark of iPS and ES cells Histone 3 K4 umethylated/trimethylated Histone 3 K27 =hallmark of differentiated cells Histone 3 K4 trimtehylation + Histone 3 K27 trimethlyation = hallmark of partially reprogrammed iPS cells Functional ID: (Gold Standards) Mouse: Tetraploid complementation assay (fused blastocyte+iPS cell gives rise to live pup) Human: Teratoma formation Presentation title in footer | 00 Month 0000

STEMCCA: Single Vector Delivery of 4 Transcription Factors TetOn/TetOff Inducible Promoter Constitutive Promoter/loxP-Flanked Sommer, C.A.; et al. 2009. Stem Cells 27(3): 543-549. Sommer, C.A.; et al. 2010. Stem Cells 28(1): 64-74. Presentation title in footer | 00 Month 0000

Human Reprogramming Requires Longer Time Slower reprogramming compared to mouse Picking colonies: 18-25 vs. 10-12 days Mouse iPS can be manipulated similarly as ESC after first passage During the first 3 passages, human iPS clones require longer length of time to grow (~18-25 days at p0 and 10-12 days for p1-p3 each passage) to sufficient size for passaging. Approx: 50-60 days to establish Mouse Human Presentation title in footer | 00 Month 0000 12

Time Course of Human iPS Colony Formation Human iPS cells in these images were generated using mouse STEMCCA lentivirus Timing: Infection to colony formation (p0): 18-25 days p0 to p3: 10-12 days for each passage; 50-60 days total p3 to p4: 7 days Presentation title in footer | 00 Month 0000 13

Removal of c-myc Requires Higher MOI and Displays Slower Reprogramming Kinetics Fewer clones (4 vs. 7 – 15) Higher MOI required (100 vs. 20) Slower kinetics (24 vs. 14 days) Absence of c-myc resulted in a lower number of iPS clones with significantly higher MOI required and a delayed onset of iPS clones. Clone 100.1 displayed a visual absence of GFP expression, but could be expanded as iPS cells, suggesting that a low level of transgene expression (i.e. possibly minimal viral integration(s) is sufficient for reprogramming but insufficient for selection of cre-excised mouse iPS cells via FACS. Presentation title in footer | 00 Month 0000 14 14

OKS/L-Myc STEMCCA: The Latest Polycistronic Vector for hiPSC Generation •Human forms of Oct-4, Klf4, and Sox2 •L-Myc in place of c-Myc •loxP-flanked, so can be removed with Cre recombinase Presentation title in footer | 00 Month 0000

Timecourse of OKS/L-Myc Reprogramming Presentation title in footer | 00 Month 0000

hiPSC Boost II Presentation title in footer | 00 Month 0000

Oh, The Difference Small Molecules Can Make!! SSEA-4 TRA-1-60 SSEA-4 TRA-1-60 Presentation title in footer | 00 Month 0000

Healthier, More Robust iPS Production Against Different Backgrounds mTeSR without treatment mTeSR with treatment StemPro without treatment StemPro with treatment Presentation title in footer | 00 Month 0000

iPSC Colonies Generated with OKS/L-Myc and iPS Boost II Presentation title in footer | 00 Month 0000

Induction of Sox-2 in Fibroblasts from a GFP-Sox-2 Transgenic Mouse Model GFP-Sox-2 transgenic animals Fibroblasts harvested and cultured Cultures transduced with STEMCCA Cells assayed via FACS against GFP expression Sommer, C.A.; et al. 2009. Stem Cells 27(3): 543-549. Presentation title in footer | 00 Month 0000

Expression of Pluripotent Markers in STEMCCA-Transduced Cells Sommer, C.A.; et al. 2009. Stem Cells 27(3): 543-549. Presentation title in footer | 00 Month 0000

Following Excision of loxP-Flanked Vector, iPSCs Undergo Directed Differentiation Without excision, STEMCCA is reinducible 1. Chimeric embryos generated with STEMCCA-generated iPS cells 2. MEFs generated 3. TetO promoter activated by doxycycline addition to cell culture Sommer, C.A.; et al. 2009. Stem Cells 27(3): 543-549. Sommer, C.A.; et al. 2010. Stem Cells 28(1): 64-74. Presentation title in footer | 00 Month 0000

SSEA-4 and TRA-1-60 As Stage-Specific Markers of Reprogramming p3 non-iPS cells At this stage, can use Human iPS Selection Kit to exclude SSEA-4 negative colonies and focus on SSEA-4 positive colonies. Presentation title in footer | 00 Month 0000 24

Live Staining to Characterize Fully Reprogrammed hiPS Cells Live stain without having to sacrifice colony for analysis -30 min for SSEA4 and Tra-1-60 20 min for Hoechst 50 minutes total SSEA-4+ Tra1-60+ Hoechst Dim Antibodies can be washed off without altering morphology and proliferation Human iPS cells in these images were generated using mouse STEMCCA lentivirus Presentation title in footer | 00 Month 0000 25 25

michael.moeller@merckgroup.com Michael L. Moeller, MS, PhD Field Application Scientist III Bioscience Division EMD Millipore A Division of Merck KGaA Darmstadt Germany michael.moeller@merckgroup.com