Induced Pluripotent Stem Cells (iPSCs): Frontiers in Reprogramming Michael L. Moeller, MS, PhD Field...

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

michael.moeller@merckgroup.com