1. 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
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Sample & Assay Technologies
3. Why iPSCs? What can they be used for?
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
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
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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
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Sample & Assay Technologies
8. Basic Workflow in creating iPSCs
Factors:
Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog
siRNA, small molecules, ???
Cell types:
Fibrobalsts
Pancreatic beta cells
Hepatocytes
Any somatic cell?
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Sample & Assay Technologies
9. Basic Workflow in creating iPSCs
Factors:
Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog
siRNA, small molecules, ???
Reprogramming (2-3 weeks)
Cell types:
Fibrobalsts
Pancreatic beta cells
Hepatocytes
Any somatic cell?
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Sample & Assay Technologies
10. Basic Workflow in creating iPSCs
Factors:
Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog
siRNA, small molecules, ???
Reprogramming (2-3 weeks)
Cell types:
Fibrobalsts
Pancreatic beta cells
Hepatocytes
Any somatic cell?
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Sample & Assay Technologies
11. Basic Workflow in creating iPSCs
Factors:
Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog
siRNA, small molecules, ???
Delivery Methods:
Infection (lentivirus)
Transfection
Electroporation
Reprogramming (2-3 weeks)
Cell types:
Fibrobalsts
Pancreatic beta cells
Hepatocytes
Any somatic cell?
How do we validate
Pluripotency?
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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
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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
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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 predictive
Biomarker set of genes and data analysis algorithm to clearly differentiate between a
Biological process or define disease.
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Sample & Assay Technologies
24. 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
iPSC Pluripotency Validation
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Sample & Assay Technologies
25. 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
5. Companion Data Analysis Algorithm trained and tested
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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
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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
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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
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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
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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
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Sample & Assay Technologies
31. Melting Curve Analysis
Single Dissociation Peaks for Every Gene Assay
• Example of QC criteria for
every PCR Assay
manufactured by
SABiosciences
• Single peak dissociation
curves
• Single gel bands of
predicted size
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Sample & Assay Technologies
32. Melting Curve Analysis
Single Dissociation Peaks for Every Gene Assay
• Example of QC criteria for
every PCR Assay
manufactured by
SABiosciences
• Single peak dissociation
curves
• Single gel bands of
predicted size
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Sample & Assay Technologies
33. Melting Curve Analysis
Single Dissociation Peaks for Every Gene Assay
• Example of QC criteria for
every PCR Assay
manufactured by
SABiosciences
• Single peak dissociation
curves
• Single gel bands of
predicted size
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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
LightCycler 480
TP-800
Mx3000p, Mx3005p, Mx4000p
qBiomarker PCR Arrays are guaranteed when using
qBiomarker qPCR SYBR Green MasterMix
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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?
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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
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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
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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.
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Sample & Assay Technologies
39. Example of Graphs
∆ Ct Value Norm alized by GAPDH
∆ Ct Value Norm alized by GAPDH
18
18
16
16
Fib
14
DNM T3B
12
iPSC 1
12
DPPA4
iPSC 2
10
iPSC 3
iPSC 4
8
∆ Ct
∆ Ct
14
GDF3
10
LEFTY1
NANOG
8
iPSC 5
6
iPSC 6
4
ESC
PODXL
POU5F1
4
2
6
ZFP42
2
0
0
DNM T3B
DPPA4
GDF3
LEFTY1
NANOG
PODXL
POU5F1
ZFP42
Fib
iPSC 1
iPSC 2
iPSC 3
Genes
iPSC 5
iPSC 6
ESC
Sam ples
Fold Change Comparing to Control Sample
Fold Change Comparing to Control Sample
5.0
5.0
4.5
4.5
4.0
iPSC 1
3.5
iPSC 2
3.0
iPSC 3
2.5
iPSC 4
2.0
iPSC 5
1.5
iPSC 6
1.0
ESC
F o ld ch an g e (L o g 10)
F old ch ang e (L o g10)
iPSC 4
0.5
DNMT3B
4.0
DPPA4
3.5
GDF3
3.0
LEFTY1
2.5
NANOG
2.0
PODXL
1.5
POU5F1
1.0
ZFP42
0.5
0.0
DNMT3B
DPPA4
GDF3
LEFTY1
NANOG
PODXL
POU5F1
0.0
ZFP42
iPSC 1
Genes
iPSC 2
iPSC 3
iPSC 4
iPSC 5
iPSC 6
ESC
Samples
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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.1
iPSC #1
iPSC #2
IMR-90
iPSC Valdation PCR Array
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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
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Sample & Assay Technologies
47. Thank you for attending our Webinar!
Questions, Comments or Suggestions?
Please type all questions into the “Questions” Box.
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Sample & Assay Technologies
