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

Transcription

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

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 -2-

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 -3-

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, Lin28-4-

5 Basic Workflow in creating ipscs -5-

6 Basic Workflow in creating ipscs Factors -6-

7 Basic Workflow in creating ipscs Factors Cell types: Fibroblasts Keratinocytes pancreatic beta cells Hepatocytes Any somatic cell? -7-

8 Basic Workflow in creating ipscs Factors Factors: Sox 2, Oct4, KLF4, c-myc, Lin 28, Nanog sirna, small molecules,??? Cell types: Fibrobalsts Pancreatic beta cells Hepatocytes Any somatic cell? -8-

9 Basic Workflow in creating ipscs Factors 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? -9-

10 Basic Workflow in creating ipscs Factors 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? -10-

11 Basic Workflow in creating ipscs Delivery Methods: Infection (lentivirus) Transfection Electroporation Factors 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? How do we validate Pluripotency? -11-

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 -12-

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 -13-

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. -14-

15 Key Considerations in qpcr Assay Design Cycling Conditions Perfect qpcr Assay Design Algorithm Mastermix Functional Validation (Wet-Bench Testing) Ensures all parameters are optimized -15-

16 Key Considerations in qpcr Assay Design Cycling Conditions Design Algorithm Perfect qpcr Assay Mastermix Perfect qpcr Assay Functional Validation (Wet-Bench Testing) Ensures all parameters are optimized Re-validate Assay with New MM -16-

17 qbiomarker PCR Arrays Samples qbiomarker ipsc Colony Screening 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) -17-

18 qbiomarker PCR Arrays Samples qbiomarker ipsc Colony Screening 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) -18-

19 qbiomarker PCR Arrays Samples qbiomarker ipsc Colony Screening 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) -19-

20 qbiomarker PCR Arrays Samples qbiomarker ipsc Colony Screening 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) -20-

21 qbiomarker PCR Arrays Samples qbiomarker ipsc Colony Screening 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) -21-

22 qbiomarker PCR Arrays Samples qbiomarker ipsc Colony Screening 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) -22-

23 qbiomarker PCR Arrays Samples qbiomarker ipsc Colony Screening 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) -23-

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 -24-

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 -25-

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 -26-

27 How do qbiomarker PCR Arrays Work? Isolate Total RNA from Your Sample Isolate RNA using QIAGEN RNeasy. cdna Synthesis 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 -27-

28 How do qbiomarker PCR Arrays Work? Isolate Total RNA from Your Sample Isolate RNA using QIAGEN RNeasy. cdna Synthesis 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 -28-

29 How do qbiomarker PCR Arrays Work? Isolate Total RNA from Your Sample Isolate RNA using QIAGEN RNeasy. cdna Synthesis 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 -29-

30 How do qbiomarker PCR Arrays Work? Isolate Total RNA from Your Sample Isolate RNA using QIAGEN RNeasy. cdna Synthesis 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 -30-

31 Melting Curve Analysis Single Dissociation Peaks for Every Gene Assay Example of QC criteria for every PCR Assay Single peak dissociation curves Single gel bands of predicted size -31-

32 Melting Curve Analysis Single Dissociation Peaks for Every Gene Assay Example of QC criteria for every PCR Assay Single peak dissociation curves Single gel bands of predicted size -32-

33 Melting Curve Analysis Single Dissociation Peaks for Every Gene Assay Example of QC criteria for every PCR Assay Single peak dissociation curves Single gel bands of predicted size -33-

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 Mx3000p, Mx3005p, Mx4000p TP-800 qbiomarker PCR Arrays are guaranteed when using qbiomarker qpcr SYBR Green MasterMix -34-

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? -35-

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 -36-

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 -37-

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. -38-

39 Example of Graphs Ct Value Normalized by GAPDH Ct Value Normalized by GAPDH Ct Fib ipsc 1 ipsc 2 ipsc 3 ipsc 4 ipsc 5 ipsc 6 ESC Ct DNM T3B DPPA4 GDF3 LEFTY1 NANOG PODXL POU5F1 ZFP DNM T3B DPPA4 GDF3 LEFTY1 NANOG PODXL POU5F1 ZFP42 0 Fib ipsc 1 ipsc 2 ipsc 3 ipsc 4 ipsc 5 ipsc 6 ESC Genes Sam ples Fold Change Comparing to Control Sample Fold Change Comparing to Control Sample Fold change (Log10) DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL POU5F1 ZFP42 Genes ipsc 1 ipsc 2 ipsc 3 ipsc 4 ipsc 5 ipsc 6 ESC Fold change (Log10) ipsc 1 ipsc 2 ipsc 3 ipsc 4 ipsc 5 ipsc 6 ESC Samples DNMT3B DPPA4 GDF3 LEFTY1 NANOG PODXL POU5F1 ZFP42-39-

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 -40-

41 ipsc Pluripotency Validation -41-

42 ipsc Pluripotency Validation -42-

43 Summary Reprogramming qbiomarker Expression Lentivirus qbiomarker Expression Plasmids -43-

44 Summary Reprogramming qbiomarker Expression Lentivirus qbiomarker Expression Plasmids ips Cell Induction ipsc colony screening ipsc Pluripotency Validation Yamanaka Factors Thomson Factors Reprogramming Factors Expression -44-

45 Summary Reprogramming qbiomarker Expression Lentivirus qbiomarker 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 -45-

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 -46-

47 Thank you for attending our Webinar! Questions, Comments or Suggestions? -47-