Human mesenchymal stem cells (hMSCs) expressing truncated soluble vascular endothelial growth factor receptor (tsFlk-1) following lentiviral-mediated gene transfer inhibit growth of Burkitt's lymphoma in a murine model

BACKGROUND: Efficient gene transfer to bone marrow derived mesenchymal stem cells (MSC) would provide an important opportunity to express potent anticancer agents in the tumour microenvironment because of their contribution to the tumour stroma. METHODS: HIV-based lentiviral vectors were pseudotyped with four different envelope proteins; amphotropic murine leukaemia virus (ampho), murine leukaemia virus (10A1), feline endogenous virus (RD114), and the vesicular stomatitis virus glycoprotein (VSVG). These pseudotypes were examined for transduction efficiency in human bone marrow derived MSC. The effect of lentiviral expression of truncated soluble vascular endothelial growth factor decoy receptor (tsFlk-1) in MSC on growth of Raji cells was determined, both in vitro and in vivo. RESULTS: All lentiviral vectors produced significant levels of transduction at an multiplicity of infection (MOI) of 1, those bearing the RD114 envelope glycoprotein consistently produced higher transduction levels (mean 70 +/- 6%) compared with the other pseudotyped lentiviral vectors, although there was significant inter-donor variation. Stable transgene expression was achieved after multiple rounds of transduction with VSVG-pseudotyped particles, without alteration in the differentiative capacity of transduced cells. Co-injection of MSC stably expressing tsFlk-1 with Raji Burkitt's lymphoma cells significantly impaired subcutaneous tumour growth in immunodeficient mice when compared to controls where either unmanipulated MSC or GFP-expressing MSC were used. CONCLUSIONS: Human MSC are easily transduced by pseudotyped lentiviral particles but there is inter-donor variation in transduction efficiency. Gene-modified MSC expressing a gene of therapeutic potential can moderate growth of haematological malignancies.     

Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells in-vitro

Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, α6-integrin, β1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells.     

大鼠精原干细胞的微滴培养法

精原干细胞（spermatogonial stem cells,SSCs）作为成体干细胞的一类,既具有自我更新和分化的潜能,又可向子代传递遗传信息。阐明其增殖过程及分化特性对SSCs的进一步应用具有重要意义。小鼠SSCs的微滴培养研究显示,微滴培养技术与常规培养方法相比具有独特的优势。然而其他物种的SSCs能否实现微滴培养尚有待证实。该研究旨在利用微滴培养法建立大鼠SSCs体外培养技术。5、8、10、20、40个大鼠SSCs分别置于20此微滴中培养,用丝裂霉素处理的STO细胞作为滋养层。倒置显微镜观察记录大鼠SSCs的增殖状态。一个月后,对微滴培养的SSCs进行免疫荧光双标记染色鉴定。结果显示,一个微滴内接种5个SSCs就能实现扩增培养;培养一个月后,SSC仍然表达其特异的标记基因分子如CDH1、OCT4、PLZF、Thy1和Gfra1。体外诱导分析显示,微滴培养的大鼠SSCs具有分化为精母细胞的能力。大鼠SSCs微滴培养法的建立,为其他物种SSCs的培养提供了借鉴,也为再生医学和生命科学相关领域的研究提供了技术平台。     

Regeneration of a well-differentiated human airway surface epithelium by spheroid and lentivirus vector-transduced airway cells

BACKGROUND: Following injury to the airway epithelium, rapid regeneration of a functional epithelium is necessary in order to restore the epithelial barrier integrity. In the perspective of airway gene/cell therapy, we analyzed the capacity of human airway epithelial cells cultured as three-dimensional (3-D) spheroid structures to be efficiently transduced on long term by a pseudotyped lentiviral vector. The capacity of the 3-D spheroid structures to repopulate a denuded tracheal basement membrane and regenerate a well-differentiated airway epithelium was also analyzed. METHODS: An HIV-1-derived VSV-G pseudotyped lentiviral vector encoding the enhanced green fluorescent protein (eGFP) was used. Airway epithelial cells were isolated from mature human fetal tracheas and airway xenografts, cultured as 3-D spheroid structures, and either transduced at multiplicity of infection (MOI) 10 and 100 or assayed in an ex vivo and in vivo model to evaluate their regeneration capacity. RESULTS: An in vivo repopulation assay in SCID-hu mice with transduced isolated fetal airway epithelial cells shows that lentiviral transduction does not alter the airway reconstitution. Transduction of the 3-D spheroid structures shows that 12% of cells were eGFP-positive for up to 80 days. In ex vivo and in vivo assays (NUDE-hu mice), the 3-D spheroid structures are able to repopulate denuded basement membrane and reconstitute a well-differentiated human airway surface epithelium. CONCLUSIONS: The efficient and long-term lentiviral transduction of 3-D spheroid structures together with their capacity to regenerate a well-differentiated mucociliary epithelium demonstrate the potential relevance of these 3-D structures in human airway gene/cell therapy.     

Comparative Analysis of Transduced Primary Human Dendritic Cells Generated by the Use of Three Different Lentiviral Vector Systems

Lentiviral gene transfer vectors are suitable for genetically modifying non-cycling primary human cells. In this study, we analyzed transduced human dendritic cells (DC) generated by the use of three different GFP-encoding lentiviral vectors, HIV-2 ROD A Δenv-GFP (ROD A), SIVsmm PBj ΔE EGFP (PBj), and SIVmac ΔE EGFP (SIVmac). CD14⁺ monocytes were isolated from buffy coat, transduced, and differentiated to immature and mature DC. Cytofluometric analysis of DC revealed high transduction efficiencies at MOI 1 for simian immunodeficiency virus (SIV)-derived vectors PBj and SIVmac ranging between 80–90 and 70–90%, respectively. In contrast, transduction with ROD A resulted only in approximately 30%-positive DC at the same MOI. Of note, none of the analyzed vectors affected expression of maturation and/or activation markers. Moreover, transduction with PBj or SIVmac did not induce significant cytokine responses whereas ROD A transduction stimulated weak interferon-alpha responses. SIVmac transduced DC showed normal phagocytosis of antigen and normal allo T cell stimulatory capacity when compared with untreated DC. Thus, the SIVmac lentiviral transduction vector is suitable for efficient genetic modification of human DC without affecting phenotype or function and thus qualifies this vector as a versatile tool for use in basic research.     

食蟹猴精原干细胞体外培养体系的初步建立

为了掌握食蟹猴(Macaca fascicularis)精原于细胞(spermatogonial stem cells,SSCs)体外培养生长特性,并建立其培养体系.手术法获得幼年期食蟹猴单侧睾丸,改良的两步酶消化法获得其细胞悬液,添加特定培养液进行体外培养,以碱性磷酸酶(AKP)染色鉴定培养细胞,并评价不同饲养层细胞...     

Lentiviral vector-mediated gene transfer to endotherial cells compared with adenoviral and retroviral vectors

Human immunodeficiency virus (HIV, lentivirus) vector has attractive features for gene therapy, including the ability to transduce non-dividing cells and long-term transgene expression. We have already reported that lentivirus vector can transduce well-differentiated rat cardiac myocytes. Endothelial cells (EC) are an attractive target for gene therapy, both for the treatment of cardiovascular disease and for the systemic delivery of recombinant gene products directly into the circulation. There are several reports regarding application of adenovirus and retrovirus based vectors to EC. However, there have been few reports which show the effect to lentivirus-mediated gene transfer efficiency, compared with adenovirus and retrovirus. In this study, bovine aortic endothelial cells (BAECs) were infected, in vitro, with these virus vectors. Transduction efficiency (TE) of beta-Gal gene transfer in BAECs by adenovirus, lentivirus, or retrovirus at MOI10 (Multiplicity of infection) (determined on Hela cells) is 69+/-11, 33+/-8, or 22+/-6% respectively. In adenovirus and lentivirus, almost 100% of BAECs were transduced at MOI 50. However, in retrovirus, TE showed only 48+/-6% at MOI 50 and no increase at MOI 100. The percentage of beta-Gal positive cells was decreased rapidly at longer passage of cells after being transduced by adenovirus. However, lentivirus and retrovirus showed sustained higher percentage of positive cells. Furthermore, transduction by lentiviral vectors had no significant effect on viability of BAECs. Our results indicate that lentivirus showed high-level and long term gene expression in BAECs. Lentivirus can be an effective vector for the ex vivo, genetically modified EC implantation and in vivo gene therapy.     

In vitro survival and proliferation of porcine primordial germ cells

Primordial germ cells (PGC) collected from the genital ridge of Day 25 porcine embryos were cultured on STO feeder cells in medium with or without supplemented growth factors. The effects on porcine PGC proliferation of leukemia inhibitory factor (LIF), LIF + stem cell factor (SCF) or LIF + SCF + basic fibroblast growth factor (bFGF), growth factors shown to be essential for in vitro survival and proliferation of murine PGC, were tested. After histochemical staining, both freshly collected and cultured PGC expressed alkaline phosphatase activity. With or without supplemented growth factors, porcine PGC survived and proliferated in culture for at least 5 d. None of the growth factors tested markedly enhanced in vitro growth of porcine PGC. These results suggest that growth factors provided by either the STO feeder layer or the cultured PGC themselves are sufficient to support in vitro survival and proliferation of porcine PGC. With the support of STO cells, addition of growth factors shown to be essential for the in vitro growth of murine PGC is not required for survival and proliferation of cultured porcine PGC.     

Production of transgenic rats via lentiviral transduction and xenogeneic transplantation of spermatogonial stem cells

Spermatogonial stem cells (SSCs) continue to proliferate in the testis to support spermatogenesis throughout life, which makes them ideal targets for germline modification. Although recent success in the production of transgenic and knockout animals using SSCs has opened up new experimental possibilities, several problems, including the low efficiency of germ cell transplantation and poor fertility rates, remain to be resolved. In the present study, we took advantage of the xenogeneic transplantation to resolve these problems. Rat SSCs were transduced in vitro with a lentiviral vector that expressed enhanced green fluorescent protein (EGFP), and then transplanted into the testes of immunodeficient mice. The transduced rat SSCs produced EGFP-expressing spermatogenic cells, and microinsemination using these cells was used to produce transgenic rats, which stably transmitted the transgene to the next generation. Thus, xenogeneic transplantation is a powerful strategy for transgenesis, and smaller xenogeneic surrogates can be used for male germline modification using SSCs.     

Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors: assessment of immunophenotype and differentiation capacity in vitro

Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-016-9945-6) contains supplementary material, which is available to authorized users.     

Human amniotic epithelial cells maintain mouse spermatogonial stem cells in an undifferentiated state due to high leukemia inhibitor factor (LIF) expression

Spermatogonial stem cells (SSCs), like other stem cells, have unique properties: prolonged proliferation, self-renewal, generation of differentiated progeny, and maintenance of developmental potential. Long-term cultivation of normal SSCs into stable cell lines, and maintaining SSCs in an undifferentiated state capable of self-renewal, is a major challenge. Here, we compare the effect of leukemia inhibitory factor (LIF) expression on mouse SSCs isolated from testicular tissue cultured under different conditions. We found that human amniotic epithelial cells (hAECs) with high LIF expression (LIF(high)) feeder cells allowed mouse SSCs to maintain a high level of AP activity when cultured long term. Expression of some important stem cell markers was higher in mouse SSCs cultured on hAECs (LIF(high)) compared to those cultured on hAECs (LIF(low)). Taken together, these results suggest that LIF expression could be a crucial component for feeder cells to maintain mouse SSCs in an undifferentiated, proliferative state capable of self-renewal.     

Use of human amniotic epithelial cells as a feeder layer to support undifferentiated growth of mouse spermatogonial stem cells via epigenetic regulation of the Nanog and Oct-4 promoters

Spermatogonial stem cells (SSCs) are defined by unique properties like other stem cells. However, there are two major challenges: long-term cultivation of normal SSCs into stable cell lines and maintaining the SSCs as undifferentiated and capable of self-renewal. Here, we compared different culture methods for mouse SSCs isolated and cultured from testicular tissue. We found that human amniotic epithelial cells (hAECs) can behave as feeder cells, allowing mouse SSCs to maintain a high level of alkaline phosphatase (AP) activity when cultured long-term. Also, we observed that expression of Nanog, Oct-4 and other important stem cells markers were higher in mouse SSCs cultured on hAECs compared to those cultured on MEF or without any feeder cells. Furthermore, we demonstrated that the CpG islands of the Nanog and Oct-4 promoters were hypomethylated in cells cultured on hAECs. In addition, mouse SSCs cultured on hAECs exhibited higher levels of H3AC and H3K4Me3 in the Nanog and Oct-4 promoters than those cultured on MEF or without feeder cells. Taken together, these results suggest that the hAEC-induced epigenetic modifications at the Nanog and Oct-4 locus could be a key mechanism for maintaining mouse SSCs in an undifferentiated state capable of self-renewal.     

A Novel Feeder-Free Culture System for Expansion of Mouse Spermatogonial Stem Cells

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.     

Effective and specific control of aml1/eto gene expression in acute myeloid leukemia cells by lentivecior-based RNA-interference

In presented work, new approach for the control of aml1/eto gene expression in t(8;21)(q22;q22)-positive acute myeloid leukemia cells has been developed. The technique is based on using the RNA-interference and lentiviral transduction methodology. Two new lentiviral vector sets for induction of constitutive anti-aml1/eto RNA-interference in acute myeloid leukemia cells have been developed and tested. The first set was based on use of artificial microRNAs (miRNAs) and second one was intended for production of short hairpin RNAs (shRNAs). It was shown that Kasumi-1 and SKNO-1 leukemia cells can be efficiency transduced by each new lentiviral vector. Moreover, the percent of modified leukemia cells that may be easily evaluated in multiplicity of infection (MOI) test achieved more than 90% for Kasumi-1 and SKNO-1 cells at MOI 40 and 20, respectively. Comparative study elucidated that the anti-aml1/eto shRNA-based approach induced a stronger knock-down of aml1/eto gene in Kasumi-1 and SKNO-1 cells than the miRNA-based method did. We hope that the proposed approach may become useful instrument for controlling the aml1/eto gene expression in vitro as well as in vivo investigations of function and biological role of the gene.     

Mechanical pressure-induced phosphorylation of p38 mitogen-activated protein kinase in epithelial cells via Src and protein kinase C

The aim of this study was to determine the impact of lentiviral transduction on primary murine B cells. Studying B cell activities in vivo or using them for tolerance induction requires that the cells remain unaltered in their biological behavior except for expression of the transgene. As we show here, murine B cells can efficiently be transduced by lentiviral, VSV-G-pseudotyped vectors without the necessity of prior activation. Culture with LPS gave enhanced transduction efficiencies but led to the upregulation of CD86 and proliferation of the cells. Transduction of naive B cells by lentiviral vectors was dependent on multiplicity of infection and did not lead to a concomitant activation. Furthermore, the transduced cells could be used for studies in the NOD mouse system without altering the onset of diabetes. We conclude that lentiviral gene transfer into naive B cells is a powerful tool for manipulation of B cells for therapeutic applications.     

Establishment of the Embryo-derived Stem (ES) Cell Lines from Mouse Blastocysts: Effects of the Feeder Cell Layer.

The ES ceii lines are embryo-derived stem cell lines directly isolated from the inner cell mass of mouse blastocysts using feeder cell layer. We have established a number of ES cell lines from 129 or C57BL/6 strain mice by using the feeder layer of the STO cells (from ATCC) or the primary embryonic fibroblasts, which was obtained by trypsinizing the 16-day-old BALB/c mouse fetus. The ES cell lines established on the STO feeder layer showed differentiation into various tissues in solid tumors when injected into syngenic mice. Karyotype was, however, nearly tetraploid. The ES cell lines established on the primary fibroblasts exhibited differentiation into larger variety of tissues in solid tumors. Karyotype was almost diploid and majority of the cells kept normal set of chromosomes in G-banding. We conclude that the primary fibroblasts are better feeder layer than the STO cells for establishment and maintenance of the ES cell lines.     

Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro

The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro.     

DNA-Dependent protein kinase is not required for efficient lentivirus integration

How DNA is repaired after retrovirus integration is not well understood. DNA-dependent protein kinase (DNA-PK) is known to play a central role in the repair of double-stranded DNA breaks. Recently, a role for DNA-PK in retroviral DNA integration has been proposed (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644-647, 1999). Reduced transduction efficiency and increased cell death by apoptosis were observed upon retrovirus infection of cultured scid cells. We have used a human immunodeficiency virus (HIV) type 1 (HIV-1)-derived lentivirus vector system to further investigate the role of DNA-PK during integration. We measured lentivirus transduction of scid mouse embryonic fibroblasts (MEF) and xrs-5 or xrs-6 cells. These cells are deficient in the catalytic subunit of DNA-PK and in Ku, the DNA-binding subunit of DNA-PK, respectively. At low vector titers, efficient and stable lentivirus transduction was obtained, excluding an essential role for DNA-PK in lentivirus integration. Likewise, the efficiency of transduction of HIV-derived vectors in scid mouse brain was as efficient as that in control mice, without evidence of apoptosis. We observed increased cell death in scid MEF and xrs-5 or xrs-6 cells, but only after transduction with high vector titers (multiplicity of infection [MOI], >1 transducing unit [TU]/cell) and subsequent passage of the transduced cells. At an MOI of <1 TU/cell, however, transduction efficiency was even higher in DNA-PK-deficient cells than in control cells. Taken together, the data suggest a protective role of DNA-PK against cellular toxicity induced by high levels of retrovirus integrase or integration. Another candidate cellular enzyme that has been claimed to play an important role during retrovirus integration is poly(ADP-ribose) polymerase (PARP). However, no inhibition of lentivirus vector-mediated transduction or HIV-1 replication by 3-methoxybenzamide, a known PARP inhibitor, was observed. In conclusion, DNA-PK and PARP are not essential for lentivirus integration.