297 A Comparison of Transduction Efficiency and Long Term Repopulating Capacity of Lentiviral Transduced SLAM Versus KSL Enriched HSC Using an Abbreviated Transduction Protocol findings demonstrate mu[.]
findings demonstrate much more efficient gene transfer to skin stem cell populations after early gestational IAGT using lentiviral vector driven by the K5 promoter compared to the CMV promoter The K5 promoter appears to be superior for potential therapeutic applications for congenital skin diseases, particularly those, such as epidermolysis bullosa, that alTect the basal layer CMV K5 (Figure 1) Anti-GFP immunofluorescence images of weeksold mouse injected at E8 with lentiviral vector containing GFP transgene driven by CMV promoter (Left) and K5 promoter (right) 296 Transient Gene Expression, Random Chromosomal Integration and Homologous Recombination in Cynomolgus Monkey Embryonic Stem Cells with Helper-Dependent Adenoviral Vectors Keiichiro Suzuki; Kouichi Hasegawa,' Kaoru Mitsui.' Emi Aizawa,' Haruka Shiiba,' Hirofumi Suemori,' Norio Nakatsuji,' Ko Mitani.' I Research Center for Genomic Medicine Saitama Medical University Hidaka, Saitama, Japan: 'Instinae for Frontier Medical Sciences Kyoto University; Kyoto Japan Considering that a variety of embryonic and adult stem cells have been isolated and characterized, development of a general strategy to obtain efficient gene delivery and homologous recombination (HR) in various cell types would be of paramount importance for biological studies and clinical applications of stem cells HR also can be applied to introduce a gene into safe and transcriptionally active chromosomal sites for gene therapy of inherited diseases However, its application in a variety of cell types, especially in primate embryonic stem (ES) cells, has been hampered by a lack ofa suitable gene delivery method Toward these goals, we examined an ability of helper-dependent adenoviral vector (HO AdV) to transiently and stably express a gene as well as to knockout the hypoxanthine phosphoribosyl transferase (HPRT) locus via HR in male cynomolgus monkey ES cells The efficiencies of transient gene expressionweremeasuredby usingincreasingamountsofGFPencoding HO Advs, and were -10% at a multiplicities of infection (Mal) of 10 GFP-transducing units (gtu; measured on 293 cells) per cell and reached a plateau at -SO% at an Mal of 1000 gtu per cell There was no significant difference in efficiency between the HO AdV with the fiber from human adenovirus type (Ad5) and the Ad35 fiber-pseudotypcd HO AdV The frequency of random chromosomal integration was measured with a HD AdV encoding marker gene G4lS-resistant colonies were obtained at the ~-geo frequencies of 1.7 x 10-5 and 5.0 x 10-1 per infected cell after infection at MOls 000 - 300 and 1000- 3000 gtu per cell, respectively The frequency of chromosomalintegrationvia HR was measured by using a HO AdV encoding a l3-kb homology from introns - S of HPRT, in which exons and were replaced with the IRES-~-geo gene A total of 7.2 x 106 ES cells were infected with the vector at MOls of 100- 300 gtu per cell, and G418-resistant colonies were SII2 obtained Among them, one was 6-thiogunanine-resistant (HPRT knock-out) This colony expressed the alkaline phosphatase at a levelequivalent to that ofparental ES cells, suggesting maintenance ofthe undifferentiatedstate after vector infection CUITCntly, we are analyzing the fidelity of HR as well as dilTerentiation potentials of this HPRT-knockout ES cell clone These results suggest that both efficient transient gene expression as well as HR can be achieved by 1-10AdVs in primate ES cells AdVs can infect almost any cell types efficiently, and therefore, might be a viable option as a tool for chromosomal manipulation ofa variety of stem cells, including human ES cells 297 A Comparison of Transduction Efficiency and Long-Term Repopulating Capacity of Lentiviral Transduced SLAM Versus KSLEnriched HSC Using an Abbreviated Transduction Protocol Pablo Lajc,' Nidal Muvarak,' Philip W Zoltick,' Alan W Flake.l 'The Children s Centerfor Fetal Research Children s Hospital of Philadelphia Philadelphia l'il Background: The SLAM-family receptors have recently been identified as useful markers for the isolation of highly enriched murine hematopoietic stem cells (I-ISC) This HSC enrichment protocol is relatively simple, and results in an enriched I-ISC product that has been shown to have comparable repopulating capacity to c-kit'Sca-l ' Iirr (KSL) HSC The relative capacity to withstand genetic manipulation and, most importantly, maintain long-term repopulating capacity of these highly enriehed HSC populations has not been tested In this study we compare SLAM versus KSLe?riched HSC populations for I) transduction efficiency,and 2) in VIVO long-term repopulating capacity, after lentiviral transduction usinga highly efficient, abbreviated, transductionprotocol Materials and Methods: C57BL6-CD45.1 mice were lethally irradiated and used as recipients Four to eight week old C57BL6-CD45.2 mice were used as donors HSC were isolated on the basis of the KSL or CD15O+C04S- marker magnetic lineage depletion (AutoMACS Miltenyi) followed by FACS sorting (FACSVantage, BO) Cells were transduced with an HIV-MND2.I-GFP lentiviral vector at an Mal of 100, for a total of 14-16 hours, with no prestimulation, in the presenceof only stem cell factorand thrombopoietin(I OOnglml), in Rctronectin®-coated wells Irradiated mice were transplanted with 5x102 or 5x 103 or 5x IO~ transduced cells, along with x lOs radioprotective syngeneic BM MNCs With every transduction an aliquot ofcells were washed twice to remove all viral particles, ilOd plated in clonogenic media for 72 hours to evaluate the transduction efficiency Mice were analyzed for chimerism at 4, S, 12, and 16 weeks after transplantation The last time point included lineage analysis as well as an evaluation ofchimerism in all hemolymphoid organs Results: We observed consistent transduction effieiency among the multiple experiments, ranging from 60 to 70% after 14 to 16 hours of vector exposure and 72 hours in vector-free culture with no significant difference between the SLAM-HSC and the KSL-HSC groups The was no significant dilTerences in the ability oflransduced cells to repopulate the irradiated mice between both groups, at all time points and cell doses analyzed with chimerism, ranging from to 5%, II to 31% and 30 to 53% GFP-positive PBMC at 16 weeks for the animals transplanted with 500 I 5,000 I 50,000 cells respectively, Multilineage reconstitution was observed for both I-ISC types, and the bone marrow, thymus and spleens contained similar percentages of GFP-positive cells in both HSC groups Conclusions: Our results demonstrate that murine HSC can be efficientlytransduced by lentiviralvector usinga simple protocol that involves minimal in vitro manipulation and no pre-stimulation In addition, we have shown that SLAM enriched HSC are equal to KSL enriched HSC populations with respect to efficiency of Molecular The 4lpy Volume 15 Supplement t• •\ br 2007 Co pyright © "111e American SOI;ic;ty o f Gen e Therapy transduction, and maintenance of long-term repopulating capacity after transductionusing this abbreviatedprotocol.These resultswill be valuable to those interested in genetic manipulation of highly enriched HSC populations 298 Self-Renewal and Differentiation of Human Mesenchymal Stem Cells from Single Cell Clonal Assays C Chang I Lee; Joanne Huang,' Jared E Christensen,' Christine I Mall,' Alyssa C Leapley,' Mervin C Yoder.? Alice F Tarantal.'-' [California National Primate Research Center; University 0/ California, Davis, CA; lHerman B Wells Center/or Pediatric Research, Indiana University School ofMedicine , Indianapolis IN: 'Departmems ofPediatrics and Cell Biology and Human Anatomy, University ofCalifornia, Davis CA Bone marrow-derived mesenchymalstem cells (MSC) from humanand nonhumanprimateshave beenshown to self-renewbeyond the Hayflick limitand differentiatetowardmultiple lineagesofmesenchymalorigin However, it is clear that MSCcultureconsists of a heterogeneouspopulationof cells that may include"true" MSCand lineage-committed progenitors In this study,we sought to identify humanMSC (hMSC)with self-renewal and differentiation potential at a single celllevcl hMSC obtained from bone marrow of healthy donors (N=3) were transduced with an HIV-I-derived lentiviral vector expressing EGFP under the control of the EFIa promoter Single EGFP-positivehMSC were sortedas single cells into24-well plates with and without basement membrane extracellular matrix (ECM) and containingnon-transducedirradiatedautologous hMSC (5xIQ3 cells/em') as feeders in a-MEM supplemented with 20% FBS for 12days Only wells containing single EGFP-positivccells were assessed and counted every three days; wells containing z ~GFP-positive cells were excluded from the study After 12 days III culture, hMSC colonies derived from single sorted cells were trypsinized and expanded EGFP-positive cells were then sorted based on EGFP expression for further experiments To assess differentiation potential ofhMSC expanded from single cell colonies, cells were incubated in adipogenic, chondrogenic, and osteogenic induction medium for 21 days following established protocols Cells were analyzed for gene expression by real-time RT-PCR (Bglap,COMP, LUM, PPARy2, LPL, Lep, BGN,CBFAI, COLIA1, ALPL, SpARC, iBSp, SPPJ) specific for tri-lineagedifferentiation Results indicatedthat 5% of single cells plated on plastic underwent ;:::\0 population doublings during the initial 12·day culture period, whereas 20% of single cells plated on ECM showed comparable growth characteristics, In addition, cells plated on plastic showed a significant reductioningrowth rateand differentiated morphologyin subsequentcultures when compared to cells plated on ECM When differentiation was assessed, 2% of all single cells plated on plastic showed gene expression profiles suggesting multi-differentiation towards all three lineages However, cells plated on ECM showed more robustgrowthand withnormalhMSC morphology The multilineage differentiationpotential of cells plated on ECM is currently under investigation Current findings suggest: (I) the existence of "true" hMSC that can both self-renew at a single cell level and differentiate toward multiple lineages of mesenchymal origin; (2) the frequency of such hMSC in routine culture is very low; and (3) that culturinghMSC with ECMsignificantly enhancedproliferation of cel.ls derived from single cells Further studies in progress are focu~mg on karyotype,gene expressionprofiles,surface phenotype, and lineage mapping of individual colonies ofhMSC 299 Hepatocyte Propagation In Vivo for Liver Tissue Engineering Kazuo Ohashi,' Kohei Tatsumi ' Miho Kataoka,' Chise Tatcno, ' Katsutoshi Yoshizato,' Akira Yoshioka.' Teruo Okano,' Yoshiyuki Nakajima.' 'Department ofSurgery, Nara Medical University, Kashihara, Nara, Japan: lDepartment ofPediatrics, Nara Medical University, Kashihara, Nara, Japan; J}'oshizato Project, Hiroshima Prefectural Institute ofIndustrial Science and Technology, Higashihlroshima, Hiroshima, Japan : 'Department 0/Biological Science, Hiroshima University, Higashihiroshima, Hiroshima, Japan: Sinstitllte 0/Advanced Biomedical Engineering and Science, TOkyo Women s Medical University, Shinjyuku, TOkyo Japan Background: Recent success in hepatocyte transplantation toward the treatment of liver diseases, including metabolic defects has encouraged further investigation into bioengineering hepatic tissues as a cell-based therapy Our group has recently developed an approachto engineerstable liver tissues under the kidneycapsule while retainingfull hepatocyte functionality Consideringthe serious donor livershortagefor hepatocyte isolation, itwould be importantto establish a newsystem to expandfunctional hepatocytes Researchers, includingour group, have recently shown that allogenicas well as xenogenic hepatocytes could be propagated in the liver of small animals if the host livers had been placed under certain conditions Herewe demonstratea novel strategyto engineer liver tissues under the kidney capsule using hepatocytes that have been propagated in mouse livers Methods: Donor hepatocytes were isolated from human alpha-I antitrypsin (A1AT) transgenic mice (provided by Dr Bumgardner, Ohio State Univ.) with the FVBIN genetic background Hcpatocytes were separated from non-parenchymal cells using low-speed centrifugation (hepatocyte purity >99%) Viable hepatocytes (5 x lOS) were transplanted into the liver of urokinasetype plasminogen activator transgenic SCID (uPA/SCID) mice The viability and the propagation status of the transplanted A IAT hepatocytes within the uPA/SCID Iivcrs were determined by serum A IAT levels and histological examination of the liver tissues The propagated A IAThepatocytes were subsequently isolated from the livers of the recipient upA/SCID mouse Liver tissue engineering was performed by transplanting the propagatedA1AT hepatocytes under the kidney capsule space of wild type of rVBIN mice Results: Serum A IAT levels of recipient uPA/SCID mouse showed rapid increase and attained the original donor AIAT mouse levels within weeks, which demonstrated the progressive proliferation and propagationof the donor hepatoytcs Histological examination showed propagatedA lAT hcpatocytes occupied more than 98% of the uPNSCID livers Then we performed liver tissue engineering methodsin the kidneycapsuleusingA t AThepatocytes that had been propagated within upA/SCID livers The engineered liver tissues stably persistedforover 10weeksand functional livercharacteristics were confirmedby histological analyses, Conclusions: The present cell-based strategy using propagated hepatocytes to engineer new liver tissue in vivo could represent a novel therapeutic method in the treatment of liver diseases, 300 Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders Liesbeth De Waele,1 Kathleen Preson,' VeerleGillijns,' Chantal Thys.! Chris Van Geet.! Desire Collen, I Thierry VandenDriessche ,' Marinee Chuah.' 'Center for Transgene Technology and Gene Therapy, VIB-University 0/Leuven, Leuven, Belgium; /Center for Molecular and Vascular Biology, University 0/Leuven, Leuven, Belgium Introduction: The prevalenceof congenitalplateletdisordershas not beenestablished, butfor some life-threatening bleedingdisorders Molecular Therapy Volume 15.Supplem ent I M;'l"2007 Coprright © The American Society o f Gene Th erapy SI13 .. .transduction, and maintenance of long- term repopulating capacity after transductionusing this abbreviatedprotocol.These resultswill be valuable to those interested in genetic manipulation of. .. Kataoka,'' Chise Tatcno, '' Katsutoshi Yoshizato,'' Akira Yoshioka.'' Teruo Okano,'' Yoshiyuki Nakajima.'' ''Department ofSurgery, Nara Medical University, Kashihara, Nara, Japan: lDepartment ofPediatrics,... Research, Indiana University School ofMedicine , Indianapolis IN: ''Departmems ofPediatrics and Cell Biology and Human Anatomy, University ofCalifornia, Davis CA Bone marrow-derived mesenchymalstem cells