Analysis of Gene Tar-

geting and Intrachro-

mosomal Homologous

Recombination Stimu-

lated by Genomic Dou-

ble-Strand

Breaks in Mouse Em-

bryonic Stem

Cells ABSTRACT

To investigate the effects of in vivo ge-nomic DNA double-strand breaks on the efficiency and mechanisms of gene tar-geting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid- to-chromosome) or intrachro-mosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased follow-ing transfection with an I-SceI expres-sion plasmid and a homologous donor plasmid (if needed). The fre-quency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restric-tion site polymorphisms indicates that the frequencies with which donor plas-mid sequences replace the target chro-mosomal sequences decrease with dis-tance from the genomic break site. The frequency of intrachromosomal recombi-nation reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recom-binants exhibit distinct genotypic sector-ing among daughter cells from a single clone, suggesting the existence of het-eroduplex DNA in the original recombi-nation product. GREG DONOHO,1† MARIA JASIN,2 AND PAUL

BERG1*

Department of Biochemistry, Beckman Center for Mo-

lecular and Genetic Medicine, Stanford University Medi-

cal School, Stanford, California 94305,1 and Cell Biology

and Genetics Program, Sloan-Kettering Institute

and Cornell University Graduate School of Medical

Sciences, New York, New York 100212

Stem Cell Edition

Current

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Figure 4. Differentiation of Oct4-GFP clones into embryonal lineages. Three BG01v-derived Oct4-GFP lines (YA06, YA15, and YA18) and one SA002-derived Oct4-GFP line (YB1403) were allowed to form embryoid bodies to characterize the differentiation potential of phiC31 integrase-derived lines. Differentiation into the endodermal (AFP), mesodermal (muscle- �specific actin and Brachyury), and ectodermal ( IIITubulin and Nestin) lineages was analyzed by immunostaining with specific antibodies (red). The cells were counter-stained with 4,6-diamidino- 2- �phenylindole (blue). Abbreviations: AFP, -fetoprotein; SMA, smooth muscle actin.

ABSTRACT It has previously been shown that the phage-derived phiC31 integrase can efficiently target native pseudo-attachment sites in the genome of various species in cultured cells, as well as in vivo. To demonstrate its utility in human embryonic stem cells (hESC), we have created hESC-derived clones containing expression constructs. Variant human embryonic stem cell lines BG01v and SA002 were used to derive lines expressing a green fluorescent protein (GFP) marker under control of either the human Oct4 promoter or �the EF1 promoter. Stable clones were selected by antibi-otic resistance and further characterized. The frequency of integration suggested candidate hot spots in the genome, which were mapped using a plasmid rescue strategy. The pseudo-attP profile in hESC differed from those reported earlier in differentiated cells. Clones derived using this method retained the ability to differentiate into all three germ layers, and fidelity of expression of GFP was verified in differentiation assays. GFP expression driven by the Oct4 promoter recapitulated endogenous Oct4 expression, whereas persistent stable expression of GFP expression �driven by the EF1 promoter was seen. Our results demon-strate the utility of phiC31 integrase to target pseudo-attP sites in hESC and show that integrase-mediated site-specific integration can efficiently create stably expressing engi-neered human embryonic stem cell clones. STEM CELLS

2008;26:119–126

BHASKAR THYAGARAJAN,a YING LIU,a SOOJUNG SHIN,a UMA

LAKSHMIPATHY,a KELLY SCHEYHING,a

HAIPENG XUE,a CATHARINA ELLERSTRO¨M,b RAIMUND STREHL,b

JOHAN HYLLNER,b MAHENDRA S. RAO,a JONATHAN D. CHESNUTa

doi: 10.1634/stemcells.2007-0283 STEMCELLS 2008;26:119–126

Creation of Engineered Human Embryonic Stem Cell Lines Using

phiC31 Integrase

Tissue-specific expression of a BAC transgene tar-

geted to the Hprt locus in mouse

embryonic stem cells Jason D. Heaney, Ashley N. Rettew, and Sarah K. Bronson* Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine H166, 500 University Drive, Hershey, PA 17033-0850, USA

The hypoxanthine phosphoribosyltransferase (Hprt) locus has been shown to have mini-mal influence on transgene expression when used as a surrogate site in the mouse ge-nome. We have developed a method to transfer bacterial artificial chromosomes (BACs) as a single copy into the partially deleted Hprt locus of embryonic stem cells. BACs were modified by Cre/loxP recombination to contain the sequences necessary for homologous recombination into and complementation of the partially deleted Hprt locus. Modified BACs were shown to undergo homologous recombination into the genome intact, to be stably transmitted through the germ line of transgenic mice, and to be expressed in the proper tissue-specific manner. This technology will facilitate many studies in which correct inter-pretation of data depends on developmentally appropriate transgene expression in the absence of rearrangements or deletions of endogenous DNA. doi:10.1016/j.ygeno.2003.12.015

Fig. 4. Localization of the BAC transgene to the X chromosome of clones (A) 2338M9[ES]-4 and (B) 2338M9[ES]-9 by FISH. Metaphase chromosome spreads were hybridized with labeled X-chromosome centromeric repeat probe DXwas70 (green) and labeled mo__│dified or unmodified BAC DNA (red). Chromosomes were counterstained with DAPI (blue). Arrows indicate chromosomes with detectable signals from the probes.

Instant MR Labeling of Stem Cells Using

Magnetoelectroporation

ABSTRACT

For cellular MR imaging, conventional approaches to intracellular magnetic labeling of nonphagocytic cells rely on the use of secondary compounds such as transfection agents and prolonged incubation of cells.

Magnetoelectroporation (MEP) was investigated as an alternative method to achieve instant (<1 s) en-

dosomal labeling with the FDA-approved formulation Feridex, without the need for adjunct agents or initi-ating cell cultures. While MEP was harmful at higher voltages or pulse durations, the procedure could be

properly calibrated using a pulse of 130 V and 17 ms. Labeling was demonstrated for stem cells from mice,

rats, and humans; the uptake of iron was in the picogram range and comparable to values obtained using transfection agents. MEP-labeled stem cells exhibited an unaltered viability, proliferation, and mitochon-

drial metabolic rate. Labeled mesenchymal stem cells (MSCs) and neural stem cells (NSCs) differentiated

into adipogenic, osteogenic, and neural lineages in an identical fashion as unlabeled cells, while containing Feridex particles as demonstrated by double immunofluorescent staining. MEP-labeled NSCs proliferated

normally following intrastriatal transplantation and could be readily detected by MR imaging in vivo. As

MEP circumvents the use of secondary agents, obviating the need for clinical approval, MEP labeling may be ideally suitable for bedside implementation. Magn Reson Med 54:769–774, 2005.

P. Walczak, D. A. Kedziorek, A. A. Gilad, S. Lin, and J. W. M. Bulte*

FIG. 4. Normal stem cell differentiation following MEP treatment (2 mg Fe/mL). (a– d) Adipogenic differentiation of MEP-labeled MSCs (a, c), as mani-fested by prominent vacuolization and accumulation of neutral lipids stained with Oil red O, is unaltered compared to unlabeled controls (b, d). Colocaliza-tion of immunostained dextran-positive granules (green) with fat vacuoles (c) demonstrates normal cell functionality in the presence of intracellular Feridex. Similar vacuoles without dextran can be seen in unlabeled control cells (d). The osteogenic differentiation of MEP-labeled MSCs (f), as visual-ized by Von Kossa staining of calcium precipitates, is also unaltered compared to controls (f). NSCs develop typical neuronal morphology and express �neuronal protein III-tubulin (red) for both MEP-labeled (g) and unlabeled (h) cells. Note the low density of dextran- (Feridex, green granules) con-taining endosomes in (g), due to the high prolifera-tion of NSCs and dilution of Feridex before terminal differentiation

FIG 4

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Activin A expression regulates multipotency of mesenchymal progenitor

cells

ABSTRACT

Introduction: Bone marrow (BM) stroma currently represents the most common and investigated source of mesenchymal progenitor cells (MPCs); how-ever, comparable adult progenitor or stem cells have also been isolated from a wide variety of tissues. This study aims to assess the functional similarities of MPCs from different tissues and to identify specific factor(s) related to their multipotency.

Methods: For this purpose, we directly compared MPCs isolated from different adult tissues, including bone marrow, tonsil, muscle, and dental pulp. We first examined and compared proliferation rates, immunomodulatory properties, and multidifferentiation potential of these MPCs in vitro. Next, we specifically evaluated activin A expression profile and activin A:follistatin ratio in MPCs from the four sources.

Results: The multidifferentiation potential of the MPCs is correlated with activin A level and/or the activin A:follistatin ratio. Interestingly, by siRNA-mediated activin A knockdown, activin A was shown to be required for the chondrogenic and osteogenic differentiation of MPCs. These findings strongly suggest that activin A has a pivotal differentiation-related role in the early stages of chondrogenesis and osteogenesis while inhibiting adipogenesis of MPCs.

Conclusions: This comparative analysis of MPCs from different tissue sources also identifies bone marrow-derived MPCs as the most potent MPCs in terms of multilineage differentiation and immunosuppression, two key requirements in cell-based regenerative medicine. In addition, this study implicates the significance of activin A as a functional marker of MPC identity.

Figure 2. In vitro-induced differentiation of MPCs from different tissue sources. (a-d) Chondrogenic differentiation evaluated after 21 days in micropellet culture. (a) Im-munohistochemical analysis of cartilage markers (COL2 and AGN) and alcian blue (AB) and picrosirius (PS) staining in pellet cultures of MPCs. (d) Expression of COL2, a specific marker for chondrogenesis, meas-ured in differentiated MPC cultures com-pared with day 0 (D0) cultures, determined by quantitative RT-PCR with GAPDH as an internal control. (b-e) Osteogenic differentia-tion of MPCs assessed after 21-day culture in osteogenic medium. (b) All MPCs tested exhibited ALP activity and produced a miner-alized matrix stained with alizarin red. T-MPCs displayed less alizarin red staining compared with BM-MPCs, M-MPCs, and DP-MPCs. (e) Significant upregulation of the expression of osteocalcin (OC), an os-teoblast-specific gene, compared with undif-ferentiated MPCs was observed after osteo-genic induction of BM-MPCs, T-MPCs, M-MPCs, and DP-MPCs compared with D0 cultures. (c-f) Adipogenic differentiation of MPCs cultured in adipogenic medium for 21 days. (c) All MPCs tested exhibited lipid-containing intracellular vacuoles that stained with oil red O at the end of the differentiation period. (f) MPCs cultured in adipogenic me-dium upregulated the expression of adipo-cyte-specific genes (PPAR-γ and LPL) com-pared with MPCs at D0. Quantitative RT-PCR data represent the mean ± SD in three independent experiments. *P ≤ 0.05, versus D0; **P ≤ 0.05, versus BM-MPCs.

Electro-Molecular Therapy using Adult Mesenchymal Stem Cells Abstract— Clinically chemo-refractive types of cancers do not respond well to conventional therapies. To treat and enhance the efficacy of drug delivery for these cancers, we have developed an in vitro model of a combination ther-apy using adult Mesenchymal stem cells. Adult Mesenchymal stem cells have been used for this study primarily be-cause of their ability to home towards tumor cells, making the possibility to practice targeted tumor therapy more re-alistic. These cells, derived from Human adult bone marrow were subjected to high intensity, short duration (1200V/cm, 100µs), and low intensity, long duration (200V/cm, 40ms and 450V/cm, 25ms) pulses. The effect of these volt-ages on the viability and proliferation ability of these cells in the presence and absence of Bleomycin (FDA approved chemodrug used for treating various cancers) indicate the possibility of transfer of this technique to clinical practice for effective electro-molecular targeted stem cell therapy. An analysis of the electrical energy applied vs. the viability illustrates a linear relationship. The dose curve exhibits a non-linear relationship. These results indicate that the high efficacy of MSC targeted combination therapy would provide efficient, economical, and enhanced clinical benefit for many types of cancers which need alternate treatments.

Figure 1: Cancer stem cells (CSCs) are cancer cells (found within tumors or hematological cancers) that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. CSCs are therefore tumorigenic (tumor-forming), perhaps in contrast to other non-tumorigenic cancer cells.

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