I psc and stem cell 2013

Sample & Assay Technologies

Reprogramming, Screening and Validation of iPSCs and Terminally Differentiated Cells using the

qBiomarker PCR Array System

Sample & Assay Technologies- 2 -

Outline of Webinar

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

�Methods for creating iPSCs

�Screening and Validation iPSCs using qPCR

�The qBiomarker PCR Array System

�Application Data� Screening of iPSC for Pluripotency

�Summary, Questions and Answers


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 �Not subjected to Federal/State/Political funding issues


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:Oct4, Sox2, Nanog, Lin28


Basic Workflow in creating iPSCs





Cell types: FibroblastsKeratinocytespancreatic beta cellsHepatocytesAny somatic cell?



Cell types: FibrobalstsPancreatic beta cellsHepatocytesAny somatic cell?

Factors:Sox 2, Oct4, KLF4, c-Myc, Lin 28, NanogsiRNA, small molecules, ???



Reprogramming (2-3 weeks)












Delivery Methods:Infection (lentivirus)TransfectionElectroporation

How do we validate Pluripotency?



Pluripotency Validation

How Can we validate the pluripotency of our iPSCs?

Chimeria Formation

Teratoma Formation

Biochemical Assays

Differentiation and Morphology (Microscopy)

Biomarkers using qPCR

? ??


Pluripotency Validation

How Can we validate the pluripotency of our iPSCs?

Chimeria Formation

Teratoma Formation

Biochemical Assays

Differentiation and Morphology (Microscopy)

Biomarkers using qPCR

? ??


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 predictiveBiomarker set of genes and data analysis algorithm to clearly differentiate between a Biological process or define disease.


Key Considerations in qPCR Assay Design

Perfect qPCR Assay

Mastermix

Cycling Conditions

Design Algorithm

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


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

Re-validate Assay with New MM


qBiomarker PCR Arrays

qBiomarker Screening PCR Arrays

8 samples per Plate

8 predictive Biomarkers1 Normalization Gene

GDC (Genomic DNA Control)RTC (Reverse Transcription Control)PPC (Positive PCR Control)

Samples

12345678

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








qBiomarker iPSC Colony Screening




8 samples per Plate



Samples

12345678













8 samples per Plate



Samples

12345678













8 samples per Plate



Samples

12345678













8 samples per Plate



Samples

12345678













8 samples per Plate



Samples

12345678













8 samples per Plate



Samples

12345678











qBiomarker Screening and Validation PCR Arrays2 different formats depending on number of Biomarkers

qBiomarker Screening PCR Arrays8 samples/plateiPSC Colony ScreeningYamanaka FactorsThomson Factors

qBiomarker Validation PCR Arrays4 samples/plateiPSC Pluripotency Validation


qBiomarker PCR Arrays Biological Content

Biomarkers are selected using multi-step process1. Text mining and literature research2. Large gene list developed 3. Biological Samples assayed through collaboration4. Predicative Gene Signature based on Results5. Companion Data Analysis Algorithm trained and tested


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 Change results






































Melting Curve Analysis

• Example of QC criteria for every PCR Assay manufactured by SABiosciences

• Single peak dissociation curves

• Single gel bands of predicted size

Single Dissociation Peaks for Every Gene Assay














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

qBiomarker PCR Arrays are guaranteed when using qBiomarker qPCR SYBR Green MasterMix


Data Analysis is Simple, Quick and Predictive

.An integral part of ALL qBiomarker PCR Arrays� Each Pathway has a specific Data Analysis Template� Interprets Gene Expression Changes into Biological Process

� Are my iPSCs Pluripotent?� What is the expression level of endogenous reprogramming

Factors? Exogenous?� Have my iPSCs undergone spontaneous differentiation?� Are my cardiomyocytes or neurons terminally differentiated?


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

Delta Ct Values are interpreted













Drop iPSC 1 and iPSC 2 from further experiments.


Example of Graphs

∆∆∆∆Ct Value Normalized by GAPDH

0

2

4

6

8

10

12

14

16

18

DNM T3B DPPA4 GDF3 LEFTY1 NANOG PODXL POU5F1 ZFP42

Genes

∆∆ ∆∆C

t

Fib

iPSC 1

iPSC 2

iPSC 3

iPSC 4

iPSC 5

iPSC 6

ESC

∆∆∆∆Ct Value Normalized by GAPDH

0

2

4

6

8

10

12

14

16

18

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

Samples

∆∆ ∆∆C

t

DNM T3B

DPPA4

GDF3

LEFTY1

NANOG

PODXL

POU5F1

ZFP42

Fold Change Comparing to Control Sample

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL POU5F1 ZFP42

Genes

Fo

ld c

han

ge

(Lo

g10

) iPSC 1

iPSC 2

iPSC 3

iPSC 4

iPSC 5

iPSC 6

ESC

Fold Change Comparing to Control Sample

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

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

Samples

Fo

ld c

han

ge

(Lo

g10

)

DNMT3B

DPPA4

GDF3

LEFTY1

NANOG

PODXL

POU5F1

ZFP42


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.1iPSC #1iPSC #2IMR-90

iPSC Valdation PCR Array


iPSC Pluripotency Validation


iPSC Pluripotency Validation


Summary

Reprogramming

qBiomarker Expression LentivirusqBiomarker Expression Plasmids


Summary

Reprogramming


iPS Cell Induction•iPSC colony screening•iPSC Pluripotency Validation•Yamanaka Factors•Thomson Factors•Reprogramming Factors Expression


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•Cardiomyocytes Differentiation


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

�Complete System for Reprogramming, Screening and Validating iPS Cells


Thank you for attending our Webinar!

Questions, Comments or Suggestions?

Please type all questions into the “Questions” Box.

Documents

I psc and stem cell 2013