1. 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

2. 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, (2009)
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3. 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
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4. 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, (2009)
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5. 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
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6. The Process of Generation of Induced Pluripotent Cells (iPS)
Hochedlinger,K and Plath, K. Development 136, (2009)
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7. 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 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
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8. 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
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9. 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):
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10. 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
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11. STEMCCA: Single Vector Delivery of 4 Transcription Factors
TetOn/TetOff Inducible Promoter Constitutive Promoter/loxP-Flanked
Sommer, C.A.; et al Stem Cells 27(3):
Sommer, C.A.; et al Stem Cells 28(1):
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12. Human Reprogramming Requires Longer Time
Slower reprogramming compared to mouse
Picking colonies: vs 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 (~ days at p0 and days for p1-p3 each passage) to sufficient size for passaging. Approx: days to establish
Mouse
Human
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13. 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): days
p0 to p3: days for each passage; days total
p3 to p4: 7 days
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14. 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 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.
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15. 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
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16. Timecourse of OKS/L-Myc Reprogramming
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17. hiPSC Boost II
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18. Oh, The Difference Small Molecules Can Make!!
SSEA TRA-1-60
SSEA TRA-1-60
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19. Healthier, More Robust iPS Production Against Different Backgrounds
mTeSR without treatment mTeSR with treatment
StemPro without treatment StemPro with treatment
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20. iPSC Colonies Generated with OKS/L-Myc and iPS Boost II
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21. 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 Stem Cells 27(3):
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22. Expression of Pluripotent Markers in STEMCCA-Transduced Cells
Sommer, C.A.; et al Stem Cells 27(3):
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23. 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 Stem Cells 27(3):
Sommer, C.A.; et al Stem Cells 28(1):
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24. 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.
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25. 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
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26. michael.moeller@merckgroup.com Michael L. Moeller, MS, PhD
Field Application Scientist III
Bioscience Division
EMD Millipore
A Division of Merck KGaA
Darmstadt Germany