Convenient Generation and Culture of Induced Pluripotent Stem Cells
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1 Pharma&Biotech BioResearch Convenient Generation and Culture of Induced Pluripotent Stem Cells Webinar 24 June 2014 / Speaker: Liz Horton 25 June 2014 / Speaker: Dr. Claudia Schwartz Lonza Walkersville, Inc., Walkersville, MD / June 2014 Lonza
2 Agenda Xeno-free, feeder-free culturing of hpscs L7 hpsc Culture System Generation of induced Pluripotent Stem Cells Reprogramming of PBMCs Adult stem cells An Overview 2
3 Agenda Xeno-free, feeder-free culturing of hpscs L7 hpsc Culture System Generation of induced Pluripotent Stem Cells Reprogramming of PBMCs Adult stem cells An Overview 3
4 Induced Pluripotent Stem Cells (ipscs) Basics Human blastocyst Somatic cells (e.g. fibroblasts or blood cells) Oct4, Klf4, Sox2, c-myc etc. Viral/non-viral transfection Inner Cell mass Culture and selection Human embryonic stem cell (hesc) Induced Pluripotent Stem Cell (ipsc) 4
5 Human PSC Culture What are the potential issues Every day feeding Feeder culture Cell recovery efficient attachment after passaging Differentiation bias Karyotype issues Viability issues Clinical aspirations 5
6 L7 hpsc Culture System: The Components Cell Culture Tools Colony Appearance Colony Expansion Banking L7 hpsc Medium BulletKit L7 hpsc Matrix L7 hpsc Passaging Solution L7 hpsc Cryosolution 6
7 Every-Other-Day Feeding Typical, Healthy Morphology Benefits Daily maintenance of hpscs no longer necessary Similar look and growth characteristics as every-day feeding 7
8 High Levels of Pluripotency Marker Expression Retained Maintained pluripotency Cells cultured for 40 passages in L7 hpsc Medium BulletKit show high level of pluripotency marker expression 8
9 L7 hpsc Culture System Maintains Normal Karyotype After 40 Passages 9
10 L7 hpsc Culture System Supports Embryoid Body Formation hesc-h9 EBs, day 1 EBs, day 7 Ectoderm Endoderm Mesoderm Tuj1 AFP Smal Human pluripotent stem cells cultured with L7 hpsc Culture System Maintain potency and can form embryoid bodies 10
11 And More Importantly, Directed Differentiation hipsc #6.4 Neurospheres Rosettes NSCs 11
12 Characteristic NSC Marker Expression Nestin Sox1 Nestin/DAPI Pax6 DAPI Nestin/Sox1 DAPI Nestin/Pax6 12
13 % Plating Efficiency % Plating Efficiency L7 hpsc Passaging Solution Provides High Re-Plating Efficiency 100% 90% 80% 70% 60% 50% StemPro Media 1 mtesr1 TM 1 Media 2 n=3 40% Dispase 30% 20% 10% 0% L7 hpsc Passaging Solution 13
14 The Time Required to Expand hpscs Using L7 hpsc Passaging Solution is Significantly Reduced L7 hpsc Passaging Solution Reagent L7 hpsc Passaging Solution Post- Dissociation Viability Days to Produce 2 x Cells 97% 27 Dispase 53% 55 Collagenase 51% 56 Scraping 35% 97 14
15 Summary: Advantages of L7 hpsc Culture System Every-other-day feeding High re-plating efficiency Maintainance of pluripotency marker expression Normal karyotype Unaltered directed differentiation capabilities 15
16 Agenda Xeno-free, feeder-free culturing of hpscs L7 hpsc Culture System Generation of induced Pluripotent Stem Cells Reprogramming of PBMCs Adult stem cells An Overview 16
17 Induced Pluripotent Stem Cells (ipscs) Basics Human blastocyst Somatic cells (e.g. fibroblasts or blood cells) Oct4, Klf4, Sox2, c-myc etc. Viral/non-viral transfection Inner Cell mass Culture and selection Human embryonic stem cell (hesc) Induced Pluripotent Stem Cell (ipsc) 17
18 Nucleofector Technology The Principle Notebook Control Unit High transfection efficiency combined with low mortality DNA is directed into the nucleus giving faster gene expression Nucleofection Process Other transfection methods 18
19 Components of Nucleofector Technology Notebook Control Unit A Unique Combination: Nucleofector Device Specific Nucleofector Kits Detailed optimized protocols Enabling excellent transfection performance combined with high functionality! 19
20 Generation of ips Cells via Transfection of PBMCs Using Nucleofector Technology Reviving & Priming Nucleofection Recovery Colony appearance Colony selection & expansion Banking & Characterization Day -9 Day 0 Day 0-4 Day 9 Day (P0) Day 76 (P5) L7 PBMC Reprogramming Protocol (coming soon) L7 hpsc Culture System 20
21 PBMC-Derived ipsc Characterization Attachment and Pluripotency Markers Morphology (P15, D3) Alkaline Phosphatase (P15, D4) DAPI TRA-1-60/Oct4 Lonza LiPSC-34B 21
22 PBMC-Derived ipsc Characterization Pluripotency Markers LiPSC34B (P13, D4) SSEA4-PE TRA-1-60-PE TRA-1-81-PE 22
23 PBMC-derived ipsc Characterization Embryoid Body Formation ipcs can be differentiated into cells representative of all 3 germ layers EB Colonies SMA, DAPI AFP, DAPI Tuj1, DAPI LiPSC34B P15 23
24 PBMC-Derived ipsc Characterization Karyotype and Plasmid Clearance Clear of Plasmid: LiPSC-34B (P5) Undetectable level of plasmid confirmed by a qpcr based assay based on the level of CT value Normal Karyotype: LiPSC-34B (P11) 24
25 ips Generation Typically Used Human Cells Types Human Cells* Efficiency (pmaxgfp Vector) References Dermal fibroblasts 40-95% Yu J et al. (2009) Science, 324(5928): Mehta A et al. (2011) Cardiovasc Res 91: Keratinocytes 40-80% Bone marrow CD34+ cells 66% Chou BK et al. (2011) Cell Research, 21: Cord blood CD34+ cells 82% Chou BK et al. (2011) Cell Research, 21: Mack A et al. (2011) PlosOne 6 (11) Bone marrow mononuclear cells Peripheral blood mononuclear cells Neural progenitor cells up to 90% Hu K et al. (2011) Blood, 117(14): e109-e119 Chou BK et al. (2011) Cell Research, 21: % Hu K et al. (2011) Blood, 117(14): e109-e119 Chou BK et al. (2011) Cell Research, 21: Adipose-derived stem cells 65-90% Jia et al. (2010) Nature Methods, 7: *Also available from Lonza 25
26 Agenda Xeno-free, feeder-free culturing of hpscs L7 hpsc Culture System Generation of induced Pluripotent Stem Cells Reprogramming of PBMCs Adult stem cells An Overview 26
27 Stem Cell Sources and Relevance ESCs Adult SCs IPSCs Origin Blastocyst of embryo Bone marrow, circulation or resident issue Strengths - Pluriotent (3 germ layers) - Self-renewal and high replicative capacity Weaknesses - Immunological concerns - Subject to ethical debate - Potential for teratoma and teratocarcinoma - Currently no clinical trial data - Autologous - Clinical safety and efficacy data - Typically lineage committed - Limited number - Limited replicative capacity - Lineage restricted Reprogramming of somatic cells - Totipotent (3 germ layers and trophoblast) - Autologous - Large reservoir of cells - Potential for teratoma and teratocarcinoma - No clinical data Leeper N J et al. Circulation. 2010;122:
28 Overview Adult Stem Cell and Precursors Real Adult Stem Cells Human ADSC Human MSC Rat MSC Precursors Human CD34+ cells Human pre-adipocytes Human osteoclast precursors Human neural progenitors Human motor neuron progenitors 28
29 Adult Stem Cells MSCs MSC studies: General Clinical trails Relevance: No ethical issues Immunoprivileged Tissue engineering 29
30 Efficient Transfection of Various Adult Stem Cell Types Using Nucleofector Technology 100% 80% 60% 40% 20% Efficiency Viability 0% For more data see: 30
31 Products and Services for Stem Cell Research Adult Stem Cells Precursors Pluripotent SC ipsc Cell Sourcing Human ADSC* Human MSC Rat MSC Human CD34+ cells Human pre-adipocytes Human osteoclast precursors Human neural progenitors Human motor neuron progenitors Human fibroblasts Human CD34+ cells Human MNCs MEFs Human keratinocytes Human ADSC Cell Culture L7 TM hpsc Culture System Transfection Nucleofector Technology Differentiation Differentiation media Services Characterization Services Expansion Services *normal and disease 31
32 Support Tools and Contact Details Our online databases Cell Database: Citations: Primary cell culture experts Scientific Support Team US: or Scientific Support Team EU + ROW: scientific.support.eu@lonza.com or
33 Interested in Learning More? Join our upcoming webinar in October: Gene Editing Transfecting ZFNs, TALENs or CRISPR/Cas Using Nucleofector Technology Register at: 33
34 Thank You!
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