Nucleofector Technology in. Somatic Stem Cell Research. gene transfer begins here

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1 Nucleofector Technology in Somatic Stem Cell Research

2 The Nucleofector technology The first highly efficient method for non-viral gene transfer into primary cells and difficult-to-transfect cell lines Nucleofector Device Nucleofector Kits

3 Nucleofector II Cell specific Programs Cell specific Solutions Certified Cuvettes

4 Conventional Electroporation Technology Gene of interest Gene transfer into + PULSE - the cytoplasm Cell of interest Gene in the nucleus Only after cell division

5 amaxa s Nucleofector Technology + Cell Specific Program - Gene of interest Cell of interest Gene transfer directly into the nucleus! Cell type specific Solution

6 Nucleofection and Stem Cells Nucleofection is the ideal transient transfection method for stem cells.

7 Nucleofection of CD34+ Hematopoietic Progenitor Cells with 54% Efficiency Transfection of normal CD34+ progenitors by nucleofection Normal human CD34+ progenitor cells were nucleofected with an Akt-GFP fusion plasmid and expression assessed by fluorescence microscopy. Overall transfection efficiency of Akt-GFP in CD34+ cells was 54+/-9%. (Data kindly provided by Prof. A. Khwaja, Dept. of Haematology, University College London).

8 Nucleofection of CD34+ Long-term transgene expression Up to 200 hours of deltalngfr expression after nucleofection. Peripheral Blood Stem Cells Bone Marrow Stem Cells Kinetics of deltalngfr expression were determined by flow cytometric analysis. 39 ± 5.9% of Peripheral Blood Stem Cells showed deltalngfr staining 4 h after transfection with a continuous decrease (n = 3, 3 patients). Bone Marrow Stem Cells showed maximal deltalngfr expression with 26 ± 9.7% 84 h after transfection, which then decreased in the proliferating culture (n = 3,single patient).

9 Nucleofection of Mesenchymal Stem Cells Maintained differentiation ability Differentiation of nucleofected MSC into adipocytes and osteoblasts. phase contrast fluorescence fixed/stained adipocytic (C) differentiation osteoblastic (F) differentiation Reprinted with permission of Data courtesy of Aluigi M, Fogli M, Curti A, Isidori A, Gruppioni E, Chiodoni C, Colombo MP, Versura P, D Errico-Grigioni A, Ferri E, Baccarani M and Lemoli RM,, Institute of Hematology and Medical Oncology, Italy.)

10 Nucleofection of Mesenchymal Stem Cells Nucleofection superior to lipofection MSC were transfected with 2 μg pcdna3/nt-gfp using either nucleofection (Human MSC Nucleofector Kit and programme U-023) or the lipidbased reagents Fugene 6 or DOTAP (both Roche Applied Science). Nucleofected MSC were analzyed for transfection efficiency roughly 60 hours post nucleofection, cells transfected with Fugene 6 or DOTAP were analyzed after 72 hours. Transfection efficiency was scored by flow cytometric analysis and reported as percentage of GFP+ cells. The percentage of viable cells was estimated by trypan blue exclusion. (Data courtesy of AluigiM, FogliM, CurtiA, IsidoriA, Gruppioni E, Chiodoni C, Colombo MP, Versura P, D Errico- Grigioni A, Ferri E, Baccarani M and Lemoli RM, Institute of Hematology and Medical Oncology, Bologne, Italy.)

11 Nucleofection of Mouse Embryonic Cells Nucleofection superior to electroporation Comparison of nucleofection and electroporation for transfection of mouse ES cells. Mouse ES cells were nucleofected using program A-13 and compared to mock-transfected (no DNA) and electroporated [EP] ES cells using Biorad Gene Pulser. Cells were stained 48h after transfection for transient lacz expression. Nucleofection of mouse embryonic fibroblasts Spontaneously immortalized mouse embryonic fibroblasts (strain: C57BL/6 x 129Sv) were nucleofected using the MEF Nucleofector Kit 1 and a plasmid encoding the enhanced green fluorescent protein egfp. 24h post nucleofection, the cells were analyzed by fluorescence microscopy. (Photograph courtesy of Dr. H. Hermanns and Prof. P.H. Heinrich, University of Aachen, Germany)

12 Nucleofection of Neural Stem Cells (NSC) Rat NSC showing unaltered cell differentiation potential Primary rat neural stem cells isolated from rat embryos (E14) were nucleofected using the Rat NSC Nucleofector Kit, program A-31 and a plasmid encoding the enhanced green fluorescent protein egfp under control of an EF1a promoter. After nucleofection, the cells were cultured with bfgf for 2 days, then for 5 days without bfgf to differentiate NSC into neurons and astrocytes. Cells were analyzed by fluorescence microcopy 2 days (A) and 7 days post nucleofection (B). (Photograph courtesy of Dr. S. H. Lee, College of Medicine, Dept. of Biochemistry, Hanyang University, Seoul, South Korea). 2 days 7 days post nucleofection

13 Nucleofection of Neural Stem Cells (NSC) Mouse NSC showing unaltered cell differentiation potential Differentiation of mouse embryonic brain NSCs nucleofected with Olig2. (A): Nucleofected NSCs, stained by Hoechst nuclear staining (blue), show intense intranuclear immunostaining for Olig2 (red) 24 hours after Olig2 gene nucleofection. Only apoptotic ells with condensed nuclei (arrowhead) do not express Olig2. (B): Nontransfected NSCs cultured for 4 days in SATO medium differentiate, as a major default, into astrocytes (GFAP- immunostaining: green), whereas only a small minority (approx. 10%) differentiate into an oligodendrocyte cell lineage as demonstrated by O4- immunostaining (red). (C): In contrast, over 25% of NSCs transfected with Olig2 differentiate into O4 positive (red), extensively branched oligodendrocytes (green=gfap immunostaining). A B C GFAP = glial fibrillary acidic protein

14 Nucleofection of Neural Stem Cells (NSC) Mouse NSC showing unaltered cell differentiation potential Differentiation of mouse embryonic brain NSCs nucleofected with Olig2. (D) Olig2-nucleofected NSC-derived oligodendrocytes (arrowheads) express the oligodendrocytic specific protein MBP (green). (D ) as well as intranuclearly the transcription factor Nkx2.2 (red); overlay of D and D in D. (D ) overlay of D and D D D D MBP = myelin basic protein

15 Nucleofection of Neural Stem Cells (NSC) Mouse NSC showing unaltered cell differentiation potential Olig2-nucleofected NSC-derived oligodendrocytes show major features of fully differentiated in-vitro oligodendrocytes including extensive arborisations positive for O4 (E) and GalC-positive myelin flaps (F). GALc = galactosidase C

16 Benefit from Non-viral technology No biosafety concerns High efficiencies Over 70% transfection efficiency in stem cells with high cell viabilities Versatility Identical transfection parameters for transfection of DNA,RNA and sirna Functionality Unaltered cell differentiation potential after nucleofection demonstrated for numerous cell types Optimized protocols Individually optimized Nucleofector Kits for each cell type Easy procedure Comprehensive handling and easy optimization schemes

17 Europe/World USA amaxa GmbH Nattermannallee Köln, Germany amaxa Inc. 205 Perryparkway Suite 7 Gaithersburg, MD Scientific Support Team phone +49 (0) fax +49 (0) scientific-support@amaxa.com Scientific Support Team phone fax scientific-support.us@amaxa.com Thank you very much!