Reprogramming, Screening and Validation of ipscs and Terminally Differentiated Cells using the qbiomarker PCR Array System

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Reprogramming, Screening and Validation of iPSCs and

Terminally Differentiated Cells using the

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Outline of Webinar

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What are induced pluripotent Stem Cells (iPS Cells or iPSCs)?

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Methods for creating iPSCs

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Screening and Validation iPSCs using qPCR

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The qBiomarker PCR Array System

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Application Data

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Screening of iPSC for Pluripotency

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Why iPSCs? What can they be used for?

Uses of iPSCs in Biomedical Research:

Basic Research into pluripotency and differentiation

Applied Research into Disease Specific model systems

Translational Research Uses in regenerative medicine

iPSCs are derived from somatic cells and are not subjected to moral/policy issues that surround embryonic stem cells.

Not necessary to destroy or create embryos

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What are inducible pluripotent Stem Cells (iPS or iPSCs?)

Most common technique for iPSCs Creation

Reprogram a somatic cell by the over expression of key regulatory factors: Yamanaka Factors:

Oct4, Sox2, KLF4, c-MYC Thomson Factors:

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Basic Workflow in creating iPSCs

F ac_to

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Basic Workflow in creating iPSCs

F ac_to rs Cell types: Fibroblasts Keratinocytes

pancreatic beta cells Hepatocytes

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Basic Workflow in creating iPSCs

F ac_to rs Cell types: Fibrobalsts

Pancreatic beta cells Hepatocytes

Any somatic cell?

Factors:

Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog siRNA, small molecules, ???

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Basic Workflow in creating iPSCs

F ac_to rs Reprogramming (2-3 weeks) Factors:

Cell types:

Fibrobalsts

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Basic Workflow in creating iPSCs

Cell types:

Fibrobalsts

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Basic Workflow in creating iPSCs

Delivery Methods: Infection (lentivirus) Transfection Electroporation

How do we validate

Pluripotency?

Cell types: Fibrobalsts

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Pluripotency Validation

How Can we validate the pluripotency of our iPSCs? Chimeria Formation

Teratoma Formation Biochemical Assays

Differentiation and Morphology (Microscopy) Biomarkers using qPCR

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Pluripotency Validation

How Can we validate the pluripotency of our iPSCs? Chimeria Formation

Teratoma Formation Biochemical Assays

Differentiation and Morphology (Microscopy)

Biomarkers using qPCR

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Using qPCR As a Screening and Validation Technology

qPCR: (Real-Time PCR)

Able to accurately identify and provide quantitation for nucleic acids

Small amounts of sample needed

Can look at multiple genes simultaneously SABiosciences’ Solution to qPCR is the PCR Array

Collection of Wet-Bench Validated SYBR Green qPCR Assays

Biological Content for understanding biological pathway or defining mechanism of action

Standardized for any qPCR instrument

Data Analysis and Biological Interpretation

qBiomarker PCR Arrays use laboratory validated samples to define both a predictive Biomarker set of genes and data analysis algorithm to clearly differentiate between a Biological process or define disease.

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Key Considerations in qPCR Assay Design

Perfect qPCR Assay Mastermix Cycling Conditions Design Algorithm

Functional Validation (Wet-Bench Testing) Ensures all parameters are optimized

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Key Considerations in qPCR Assay Design

Perfect qPCR Assay Mastermix Cycling Conditions Design Algorithm

Functional Validation (Wet-Bench Testing) Ensures all parameters are optimized

Perfect qPCR

Assay

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qBiomarker PCR Arrays

qBiomarker Screening PCR Arrays 8 samples per Plate

8 predictive Biomarkers 1 Normalization Gene

GDC (Genomic DNA Control)

RTC (Reverse Transcription Control) PPC (Positive PCR Control) Samples

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DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC

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qBiomarker PCR Arrays

qBiomarker Screening PCR Arrays

8 samples per Plate

8 predictive Biomarkers 1 Normalization Gene

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qBiomarker PCR Arrays

8 predictive Biomarkers

1 Normalization Gene

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qBiomarker PCR Arrays

8 predictive Biomarkers 1 Normalization Gene

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qBiomarker PCR Arrays

8 predictive Biomarkers

GDC (Genomic DNA Control)

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qBiomarker PCR Arrays

RTC (Reverse Transcription Control)

PPC (Positive PCR Control) Samples

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qBiomarker PCR Arrays

RTC (Reverse Transcription Control)

PPC (Positive PCR Control) Samples

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qBiomarker Screening and Validation PCR Arrays

2 different formats depending on number of Biomarkers

qBiomarker Screening PCR Arrays 8 samples/plate

iPSC Colony Screening Yamanaka Factors

Thomson Factors

qBiomarker Validation PCR Arrays 4 samples/plate

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qBiomarker PCR Arrays Biological Content

Biomarkers are selected using multi-step process 1. Text mining and literature research 2. Large gene list developed

3. Biological Samples assayed through collaboration 4. Predicative Gene Signature based on Results

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How do qBiomarker PCR Arrays Work?

Isolate Total RNA from Your Sample _{Isolate RNA using QIAGEN RNeasy}

. cDNA Synthesis (C-03 kit)

– Genomic DNA Removal Step (5 min.)

– Reverse Transcription Step (20 min.)

. Load Plates

 Multiple Samples per PCR Array

 2 minutes with multi-channel pipet

. Run 40 cycle qPCR Program

 Standard cycling conditions for all Real Time PCR Instruments

 2 hours

. Upload and Analyze Data (FREE)

 10 minutes from Raw Ct values to interpretation of Biology and Fold

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How do qBiomarker PCR Arrays Work?

. cDNA Synthesis

– Genomic DNA Removal Step (5 min.)

– Reverse Transcription Step (20 min.)

. Load Plates

 2 hours

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How do qBiomarker PCR Arrays Work?

. cDNA Synthesis

– Genomic DNA Removal Step (5 min.) – Reverse Transcription Step (20 min.)

. Load Plates

 2 hours

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How do qBiomarker PCR Arrays Work?

. cDNA Synthesis

. Load Plates

 2 hours

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How do qBiomarker PCR Arrays Work?

. cDNA Synthesis

. Load Plates

 2 hours

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Melting Curve Analysis

• Example of QC criteria for every PCR Assay

• Single peak dissociation curves

• Single gel bands of predicted size

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Single Dissociation Peaks for Every Gene Assay

Melting Curve Analysis

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Single Dissociation Peaks for Every Gene Assay

Melting Curve Analysis

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Compatible Instrumentation: 96- & 384-Well Formats

 96-Well Blocks: 7000, 7300, 7500, 7700  FAST 96-Well Blocks: 7500, 7900HT  FAST 384-Well Block: 7900HT

 StepOnePlus

 iCycler, MyiQ, MyiQ2, iQ5, CFX96, CFX384  Opticon, Opticon 2, Chromo 4

 Mastercycler ep realplex 2/2S/4/4S

 Mx3000p, Mx3005p, Mx4000p

 LightCycler 480

 TP-800

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Data Analysis is Simple, Quick and Predictive

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An integral part of ALL qBiomarker PCR Arrays

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Each Pathway has a specific Data Analysis Template

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Interprets Gene Expression Changes into Biological Process

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Are my iPSCs Pluripotent?

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What is the expression level of endogenous reprogramming

Factors? Exogenous?

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Have my iPSCs undergone spontaneous differentiation?

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Screening iPS Cells for Pluripotency

Experiment:

Created 6 different iPS cell lines from fibroblasts following 3 weeks of reprogramming. Which cell lines are totally pluripotent and worth using?

iPS Colony Screening

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Screening iPS Cells for Pluripotency

Experiment:

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Screening iPS Cells for Pluripotency

Experiment:

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Example of Graphs

Ct Value Norm alized by GAPDH

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DNM T3B DPPA4 GDF3 LEFTY1 NANOG PODXL POU5F1 ZFP42

Genes  Ct Fib iPSC 1 iPSC 2 iPSC 3 iPSC 4 iPSC 5 iPSC 6 ESC

Ct Value Norm alized by GAPDH

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Fib iPSC 1 iPSC 2 iPSC 3 iPSC 4 iPSC 5 iPSC 6 ESC

Sam ples  Ct DNM T3B DPPA4 GDF3 LEFTY1 NANOG PODXL POU5F1 ZFP42

Fold Change Comparing to Control Sample

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Fol d c h a ng e ( L og1 0 ) iPSC 1 iPSC 2 iPSC 3 iPSC 4 iPSC 5 iPSC 6 ESC

Fold Change Comparing to Control Sample

1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 F ol d c ha nge ( Log 1 0 ) DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL POU5F1

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Application Data

. Determining The Pluripotent Status of iPSCs:

One of the problems facing iPSC researchers is spontaneous differentiation and loss of

pluripotency during culture and passage conditions.

Are my IPSCs still pluripotent?

ESC A.1 iPSC #1 iPSC #2 IMR-90

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Summary

Reprogramming

qBiomarker Expression Lentivirus qBiomarker Expression Plasmids

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Summary

Reprogramming

iPS Cell Induction

•iPSC colony screening

•iPSC Pluripotency Validation

•Yamanaka Factors

•Thomson Factors

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Summary

Reprogramming

iPS Cell Induction

•iPSC colony screening

•iPSC Pluripotency Validation

•Yamanaka Factors

•Thomson Factors

•Reprogramming Factors Expression iPS Cell Differentiation

•Embryoid Body

•Neuronal Differentiation

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Summary

qBiomarker PCR Arrays

 Laboratory validated performance and biological content

 Available for any qPCR instrument

 Companion Data Analysis for interpretation of results

Applications:

 Screening or Validation of iPS Cell inductions

 Validation of Cardiomyocyte and Neuronal Differentiation

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