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Induced Pluripotent Induced Pluripotent Stem Cells (iPSCs): Stem Cells (iPSCs): Frontiers in Frontiers in ReprogrammingReprogrammingMichael L. Moeller, MS, PhDMichael L. Moeller, MS, PhD
Field Application Scientist IIIField Application Scientist III
Bioscience DivisionBioscience Division
EMD MilliporeEMD Millipore
A Division of Merck KGaAA Division of Merck KGaA
Darmstadt GermanyDarmstadt Germany
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Epigenetic States Alter Developmental Pathways and Epigenetic States Alter Developmental Pathways and Can Be Used to Modify Developmental StateCan Be Used to Modify Developmental State
Hochedlinger,K and Plath, K. Development 136, 509-523 (2009)
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Methods of Factor DeliveryMethods of Factor Delivery
Method 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
Genomic integration; risk of insertional mutagenesis
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 Low frequency of genomic integration
Repeated infection required for certain cell types
Delayed kinetics of reprogramming
Stadtfeld et al., 2008c
Nimet Maherali and Konrad Hochedlinger; Cell Stem Cell 3, December 4, 2008
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ES-Cell Transcription Factors and Their ES-Cell Transcription Factors and Their Role in ReprogrammingRole in Reprogramming
Activation of pluripotency regulators
Repression function
Activation of metabolic and proliferative programs
Hochedlinger,K and Plath, K. Development 136, 509-523 (2009)
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Role of 4Role of 4 Factors During ReprogrammingFactors During Reprogramming
Rupa Sridharan et al.(2009) :Cell, Volume 136, Issue 2, Pages 364-377
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The Process of Generation of Induced The Process of Generation of Induced Pluripotent Cells (iPS)Pluripotent Cells (iPS)
Hochedlinger,K and Plath, K. Development 136, 509-523 (2009)
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Methods of iPS Generation: Small Methods of iPS Generation: Small MoleculesMolecules
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.
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Reprogramming ProcessReprogramming Process
Takashi Tada (2008); Cell Stem Cell, Volume 3, Issue 2,Pages 121-122
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All Mycs Are Not Created Equal!!
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•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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Assessment of iPS PotencyAssessment of iPS Potency
Morphology IDMorphology ID– Unlimited self-renewal;
– Visual ID
Molecular IDMolecular 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)Functional ID: (Gold Standards)– Mouse: Tetraploid complementation assay (fused blastocyte+iPS cell gives rise to live pup)
– Human: Teratoma formation
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STEMCCA: Single Vector Delivery of 4 STEMCCA: Single Vector Delivery of 4 Transcription FactorsTranscription Factors
Sommer, C.A.; et al. 2009. Stem Cells 27(3): 543-549.
Sommer, C.A.; et al. 2010. Stem Cells 28(1): 64-74.
TetOn/TetOff Inducible PromoterTetOn/TetOff Inducible Promoter Constitutive Promoter/loxP-Flanked Constitutive Promoter/loxP-Flanked
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Human Reprogramming Requires Human Reprogramming Requires Longer TimeLonger 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
Human
Mouse
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Time Course of Human iPS Colony Time Course of Human iPS Colony FormationFormation
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
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Removal of c-myc Requires Higher MOI and Removal of c-myc Requires Higher MOI and Displays Slower Reprogramming KineticsDisplays Slower Reprogramming Kinetics
Fewer clones (4 vs. 7 – 15)
Higher MOI required (100 vs. 20)
Slower kinetics (24 vs. 14 days)
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OKS/L-Myc STEMCCA: The Latest Polycistronic Vector for hiPSC Generation
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•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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Timecourse of OKS/L-Myc Reprogramming
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hiPSC Boost II
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Oh, The Difference Small Molecules Can Oh, The Difference Small Molecules Can Make!!Make!!
SSEA-4 TRA-1-60
SSEA-4 TRA-1-60
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Healthier, More Robust iPS Production Healthier, More Robust iPS Production Against Different BackgroundsAgainst Different Backgrounds
mTeSR without treatment mTeSR with treatment
StemPro without treatment StemPro with treatment
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iPSC Colonies Generated with OKS/L-Myc and iPS Boost II
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Induction of Sox-2 in Fibroblasts from a Induction of Sox-2 in Fibroblasts from a GFP-Sox-2 Transgenic Mouse ModelGFP-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.
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Expression of Pluripotent Markers in Expression of Pluripotent Markers in STEMCCA-Transduced CellsSTEMCCA-Transduced Cells
Sommer, C.A.; et al. 2009. Stem Cells 27(3): 543-549.
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Following Excision of loxP-Flanked Vector, iPSCs Following Excision of loxP-Flanked Vector, iPSCs Undergo Directed DifferentiationUndergo Directed Differentiation
Sommer, C.A.; et al. 2009. Stem Cells 27(3): 543-549.
Sommer, C.A.; et al. 2010. Stem Cells 28(1): 64-74.
Without excision, STEMCCA is Without excision, STEMCCA is reinduciblereinducible
1. Chimeric embryos generated with STEMCCA-generated iPS cells2. MEFs generated3. TetO promoter activated by doxycycline addition to cell culture
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SSEA-4 and TRA-1-60 As Stage-Specific Markers of SSEA-4 and TRA-1-60 As Stage-Specific Markers of ReprogrammingReprogramming
p3 non-iPS cells
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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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Live Staining to Characterize Fully Live Staining to Characterize Fully Reprogrammed hiPS CellsReprogrammed hiPS Cells
-30 min for SSEA4 and Tra-1-60
20 min for Hoechst
50 minutes total
Antibodies can be washed off without altering morphology and proliferation
Live stain without having to sacrifice colony for analysis
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SSEA-4+ Tra1-60+ Hoechst Dim
Michael L. Moeller, MS, PhDMichael L. Moeller, MS, PhD
Field Application Scientist IIIField Application Scientist III
Bioscience DivisionBioscience Division
EMD MilliporeEMD Millipore
A Division of Merck KGaAA Division of Merck KGaA
Darmstadt Germany Darmstadt Germany