i psc and stem cell 2013
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TRANSCRIPT
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
Sample & Assay Technologies- 3 -
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
Sample & Assay Technologies- 4 -
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
Sample & Assay Technologies- 7 -
Basic Workflow in creating iPSCs
Cell types: FibroblastsKeratinocytespancreatic beta cellsHepatocytesAny somatic cell?
Sample & Assay Technologies- 8 -
Basic Workflow in creating iPSCs
Cell types: FibrobalstsPancreatic beta cellsHepatocytesAny somatic cell?
Factors:Sox 2, Oct4, KLF4, c-Myc, Lin 28, NanogsiRNA, small molecules, ???
Sample & Assay Technologies- 9 -
Basic Workflow in creating iPSCs
Reprogramming (2-3 weeks)
Factors:Sox 2, Oct4, KLF4, c-Myc, Lin 28, NanogsiRNA, small molecules, ???
Cell types: FibrobalstsPancreatic beta cellsHepatocytesAny somatic cell?
Sample & Assay Technologies- 10 -
Basic Workflow in creating iPSCs
Reprogramming (2-3 weeks)
Factors:Sox 2, Oct4, KLF4, c-Myc, Lin 28, NanogsiRNA, small molecules, ???
Cell types: FibrobalstsPancreatic beta cellsHepatocytesAny somatic cell?
Sample & Assay Technologies- 11 -
Basic Workflow in creating iPSCs
Reprogramming (2-3 weeks)
Factors:Sox 2, Oct4, KLF4, c-Myc, Lin 28, NanogsiRNA, small molecules, ???
Delivery Methods:Infection (lentivirus)TransfectionElectroporation
How do we validate Pluripotency?
Cell types: FibrobalstsPancreatic beta cellsHepatocytesAny somatic cell?
Sample & Assay Technologies- 12 -
Pluripotency Validation
How Can we validate the pluripotency of our iPSCs?
Chimeria Formation
Teratoma Formation
Biochemical Assays
Differentiation and Morphology (Microscopy)
Biomarkers using qPCR
? ??
Sample & Assay Technologies- 13 -
Pluripotency Validation
How Can we validate the pluripotency of our iPSCs?
Chimeria Formation
Teratoma Formation
Biochemical Assays
Differentiation and Morphology (Microscopy)
Biomarkers using qPCR
? ??
Sample & Assay Technologies- 14 -
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.
Sample & Assay Technologies- 15 -
Key Considerations in qPCR Assay Design
Perfect qPCR Assay
Mastermix
Cycling Conditions
Design Algorithm
Functional Validation (Wet-Bench Testing) Ensures all parameters are optimized
Sample & Assay Technologies- 16 -
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
Sample & Assay Technologies- 17 -
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
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
qBiomarker iPSC Colony Screening
Sample & Assay Technologies- 18 -
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
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
qBiomarker iPSC Colony Screening
Sample & Assay Technologies- 19 -
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
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
qBiomarker iPSC Colony Screening
Sample & Assay Technologies- 20 -
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
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
qBiomarker iPSC Colony Screening
Sample & Assay Technologies- 21 -
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
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
qBiomarker iPSC Colony Screening
Sample & Assay Technologies- 22 -
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
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
qBiomarker iPSC Colony Screening
Sample & Assay Technologies- 23 -
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
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL OCT4 ZFP42 GAPDH GDC RTC PPC
qBiomarker iPSC Colony Screening
Sample & Assay Technologies- 24 -
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
Sample & Assay Technologies- 25 -
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
Sample & Assay Technologies- 26 -
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
Sample & Assay Technologies- 27 -
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
Sample & Assay Technologies- 28 -
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
Sample & Assay Technologies- 29 -
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
Sample & Assay Technologies- 30 -
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
Sample & Assay Technologies- 31 -
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
Sample & Assay Technologies- 32 -
Single Dissociation Peaks for Every Gene Assay
Melting Curve Analysis
• Example of QC criteria for every PCR Assay manufactured by SABiosciences
• Single peak dissociation curves
• Single gel bands of predicted size
Sample & Assay Technologies- 33 -
Single Dissociation Peaks for Every Gene Assay
Melting Curve Analysis
• Example of QC criteria for every PCR Assay manufactured by SABiosciences
• Single peak dissociation curves
• Single gel bands of predicted size
Sample & Assay Technologies- 34 -
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
Sample & Assay Technologies- 35 -
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?
Sample & Assay Technologies- 36 -
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
Sample & Assay Technologies- 37 -
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
Sample & Assay Technologies- 38 -
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.
Sample & Assay Technologies- 39 -
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
Sample & Assay Technologies- 40 -
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
Sample & Assay Technologies- 43 -
Summary
Reprogramming
qBiomarker Expression LentivirusqBiomarker Expression Plasmids
Sample & Assay Technologies- 44 -
Summary
Reprogramming
qBiomarker Expression LentivirusqBiomarker Expression Plasmids
iPS Cell Induction•iPSC colony screening•iPSC Pluripotency Validation•Yamanaka Factors•Thomson Factors•Reprogramming Factors Expression
Sample & Assay Technologies- 45 -
Summary
Reprogramming
qBiomarker Expression LentivirusqBiomarker Expression Plasmids
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
Sample & Assay Technologies- 46 -
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