Network-driven discovery yields new insight into Shox2-dependent cardiac rhythm control.

The homeodomain transcription factor SHOX2 is involved in the development and function of the heart's primary pacemaker, the sinoatrial node (SAN), and has been associated with cardiac conduction-related diseases such as atrial fibrillation and sinus node dysfunction. To shed light on Shox2-dependent genetic processes involved in these diseases, we established a murine embryonic stem cell (ESC) cardiac differentiation model to investigate Shox2 pathways in SAN-like cardiomyocytes. Differential RNA-seq-based expression profiling of Shox2+/+ and Shox2-/- ESCs revealed 94 dysregulated transcripts in Shox2-/- ESC-derived SAN-like cells. Of these, 15 putative Shox2 target genes were selected for further validation based on comparative expression analysis with SAN- and right atria-enriched genes. Network-based analyses, integrating data from the Mouse Organogenesis Cell Atlas and the Ingenuity pathways, as well as validation in mouse and zebrafish models confirmed a regulatory role for the novel identified Shox2 target genes including Cav1, Fkbp10, Igfbp5, Mcf2l and Nr2f2. Our results indicate that genetic networks involving SHOX2 may contribute to conduction traits through the regulation of these genes.

A novel approach to genetic engineering of T-cell subsets by hematopoietic stem cell infection with a bicistronic lentivirus.

Lentiviral modification of hematopoietic stem cells (HSCs) paved the way for in vivo experimentation and therapeutic approaches in patients with genetic disease. A disadvantage of this method is the use of a ubiquitous promoter leads not only to genetic modification of the leukocyte subset of interest e.g. T-cells, but also all other subsequent leukocyte progeny of the parent HSCs. To overcome this limitation we tested a bicistronic lentivirus, enabling subset specific modifications. Designed novel lentiviral constructs harbor a global promoter (mPGK) regulating mCherry for HSCs selection and a T-cell specific promoter upstream of eGFP. Two T-cell specific promoters were assessed: the distal Lck-(dLck) and the CD3δ-promoter. Transduced HSCs were FACS sorted by mCherry expression and transferred into sublethally irradiated C57/BL6 mice. Successful transplantation and T-cell specific expression of eGFP was monitored by peripheral blood assessment. Furthermore, recruitment response of lentiviral engineered leukocytes to the site of inflammation was tested in a peritonitis model without functional impairment. Our constructed lentivirus enables fast generation of subset specific leukocyte transgenesis as shown in T-cells in vivo and opens new opportunities to modify other HSCs derived subsets in the future.

Isolation of human mesenchymal stromal cells is more efficient by red blood cell lysis.

BACKGROUND: Human mesenchymal stromal cells (MSC) have raised high hopes for tissue engineering and clinical therapy. Their isolation usually involves density fractionation of mononuclear cells (MNC) but this is difficult to standardize, especially under good manufacturing practice (GMP) conditions. MSC represent a heterogeneous mixture of cell types and the composition of subpopulations is affected by the initial steps of cell preparation. METHODS: This study describes a straightforward method for isolation of human MSC based on red blood cell (RBC) lysis with ammonium chloride. Colony formation was compared directly with Ficoll density fractionation and culture of an untreated whole bone marrow (BM) aspirate. RESULTS: After 7 days the number of fibroblastic colony-forming units (CFU-F) per milliliter of BM aspirate was slightly higher upon RBC lysis and the colonies were significantly larger compared with density fractionation, possibly because of maintenance of platelets. In contrast, colony formation was much lower in untreated BM. The heterogeneous composition of subpopulations was reflected by differences between the initial colonies with regard to growth pattern (tight or disperse) and cell morphology (round or elongated). This heterogeneous composition was not affected by the three different isolation methods. Furthermore, enrichment of CD271(+) cells resulted in the same morphologic heterogeneity. All cell preparations demonstrated the same immunophenotype using a panel of surface markers and displayed adipogenic and osteogenic differentiation potential. DISCUSSION: This study demonstrates that human MSC can be efficiently isolated by RBC lysis. This technique is faster and can be standardized more easily for clinical application of MSC.

Improved lentiviral transduction of human mesenchymal stem cells for therapeutic intervention in pancreatic cancer.

Genetic modification of human bone marrow mesenchymal stem cells (MSC) is highly valuable for their exploitation in basic science and therapeutic applications, for example in cancer. We present here a new, fast and easy-to-use method to enrich a functional population of lentiviral (LV)-transduced MSC expressing enhanced green fluorescent protein (eGFP). We replaced the eGFP gene by a fusion gene of puromycin acetyltransferase and eGFP. Upon LV gene transfer and puromycin selection, we quickly obtained a pure transduced MSC population, in which growth, differentiation capacity and migration preferences were not compromised. Furthermore, we are the first to report the migration velocity of MSC among which 30% were moving and velocity of about 15 mum h(-1) was not altered by LV transduction. Manipulated MSC underwent senescence one passage earlier than non-transduced cells, suggesting the use for therapeutic intervention in early passage numbers. Upon tail vein application in nude mice, the majority of LV-transduced MSC could be detected in human orthotopic pancreatic tumor xenografts and to a minor extent in mouse liver, kidney and lung. Together, LV transduction of genes to MSC followed by puromycin selection is a powerful tool for basic research and improves the therapeutic prospects of MSC as vehicles in gene therapy.

Tissue distribution and persistence in mice of plasmid DNA encapsulated in a PLGA-based microsphere delivery vehicle.

Information regarding the distribution and persistence of DNA encapsulated in poly-(lactide co-glycolide) microspheres was collected to provide additional information regarding the safety of DNA vaccines and to support the clinical testing of this new delivery system for DNA. Plasmid DNA was encapsulated in poly(lactide co-glycolide) microspheres and the distribution and persistence of plasmid in murine tissues resulting from parenteral administration were examined by a sensitive PCR assay. Encapsulated DNA delivered by intramuscular or subcutaneous injection can be detected for 100 days post-injection and is distributed primarily at the site of injection and the lymphoid organs. Intravenous administration results in more widespread dissemination with long term persistence limited to the lymphoid organs and those of the reticuloendothelial system. Specific cellular uptake of DNA by professional antigen presenting cells (APCs) following injection suggests the utility of microspheres as DNA delivery agents. Distribution and persistence studies support the safety of encapsulated DNA and the specific cellular uptake of DNA by professional APCs following injection suggests the utility of microspheres as DNA delivery agents.

[Cellular immune reactivity in xenogenic "human-anti-pig" transplant combination]. / Zellvermittelte Immunreaktivität in der xenogenen Transplantationskombination "Mensch-anti-Schwein".

The worldwide lack of human organ donors puts the pig as potential xenogeneic donor species into the prime of interest. Aim of the present in vitro study is the analysis of T-cell activation in the clinically attractive combination "pig-to-human". Peripheral human blood leukocytes (hPBL) and peripheral porcine blood leukocytes (pPBL) were co-cultured for 4-8 days in the xenogeneic mixed lymphocyte reaction (xMLR) and cell proliferation was measured by 3H-thymidine uptake. Both cell populations were separated into T-cells and antigen presenting cells (APC) to analyze direct and indirect antigen recognition. The results show that (a) activation of human T-cells occurs, (b) the strength of activation depends e.g. on the human responder ("high" and "low" responders), (c) the strength of activation is independent of the responder's HLA-DR status, and (d) direct T-cell activation dominates over indirect activation. Thus, T-cell activation is another immunological barrier that has to be overcome before xenotransplantation can be clinically approached.

Cross-matches on donor cadaver retinal pigment epithelial cells in corneal risk patients.

BACKGROUND: Allografts can be rejected either through the antibody-mediated or cellular pathways. The objective of this study was to look at the extent of antibody formation in patients awaiting re-keratoplasty using cross-matches on cadaver retinal pigment epithelial (RPE) cells. METHODS: Cadaver RPE cells were derived by trypsin digestion from donor eyes (n = 1200). After 3 days of cell cultivation, the cells were adherent and began to lose their pigment. By day 7 most cells were clear and grew as a polygonal monolayer. MHC class I expression by RPE cells was studied by the W6/32 (anti-HLA-A, B, C) monoclonal antibody (MoAb) and that of class II (HLA-DR) by the 136 MoAb. Normal RPE cells express few class I and no detectable class II antigens. For the induction of MHC expression, cells were subsequently stimulated with 250 U/ml of recombinant gamma-interferon for 5 days. Cells were used for tissue typing and also for cross-matches with recipient serum. Cross-matches were subsequently performed and measured by flow cytometry. RESULTS: Both class I and class II antigens were strongly enhanced, as could be shown by immunohistochemical staining. Some 20% of those patients awaiting rekeratoplasty (n = 60) were positive for anti-HLA antibodies. In one case anti-DR3 antibodies were detected in a recipient who had had several rejection episodes after keratoplasty. CONCLUSIONS: RPE cells are not only useful for cadaver post-mortem HLA typing but also for donor-specific cross-matches. The degree of antibody formation after keratoplasty in rejecting patients was, however, low. This may imply that anti-HLA antibodies are not the major cause of corneal graft loss after keratoplasty.

Analysis of tolerance inducing mechanism after application of oncogen-transformed macrophages.

The purpose of this study was to examine the expression of T cell receptors (TCR) and their V beta subclasses under the influence of the parental cell line P388D1 and its clones mos2 and mos3, using a mouse model. It was shown, that v-mos oncogene-transformed cells of this line (mos2) induced selective immunological unresponsiveness in vitro. Because the induction of tolerance is of a central importance for the organ transplantation, this phenomenon, found in vitro, was also studied in vivo. We found that the in vivo injection of mos2 cells into mice induced a state of selective noncreativity. To further analyse these effects, we studied whether specific tolerance is the consequence of a decreased number of essential receptors or receptor families. For this purpose C57BL/6 mice were immunized with cells of the parental line P388D1 or mos2 and mos3 clones. Their spleen and thymus cells were examined phenotypically. The most impressive result of this study was a clearly changed amount of T cells receptors in mos2 immunized mice, in which a state of tolerance was induced. In these mice only the expression of CD3 T receptors as well as that of the V beta 11 chains was reduced. In spleen of these mice the CD3 expression was decreased, compared to D1 or nonimmunized control animals by 54-58% and compared to mos3 mice by 38-40%. Even though the differences in the thymus were not very pronounced, we still saw a decrease in CD3 stained cells selective in mos2 immunized C57B1/6. The expression of V beta 11 chains on the surface of spleen cells of mos2 animals was reduced by 33.3%, on the thymocytes even by 50% comparing to that in nonimmunized mice. Whether the reduced expression of T receptor V beta families is due to changes in the genetic material (cDNA), has to be studied.

G-CSF-mobilized peripheral blood progenitor cells for allogeneic transplantation: comparison of T cell depletion strategies using different CD34+ selection systems or CAMPATH-1.

Allogeneic transplantation of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) appears to be an attractive alternative to allogeneic bone marrow transplantation (BMT). However, because vast amounts of potentially graft-vs.-host-reactive T cells are transfused with PBPC grafts, the use of PBPC in the allogeneic setting may be associated with an increased incidence or severity of graft-vs.-host disease (GVHD). To evaluate strategies for prevention of GVHD after PBPC allografting, we have studied T cell depletion (TCD) of G-CSF-mobilized PBPC samples harvested from six healthy donors and from five patients scheduled for autologous PBPC transplantation. Three approaches (CAMPATH-1 plus autologous complement [C], immunomagnetic CD34+ cell selection, and biotin-avidin-mediated CD34+ cell selection) were compared. TCD of PBPC samples with the monoclonal antibody (MAb) CAMPATH-1 plus autologous C resulted in a median elimination of 2.16 log CD3+ T cells, whereas 39% of CD56+ natural killer (NK) cells and 56% of CD34+ progenitor cells were recovered. TCD by CD34+ cell selection with the Isolex (Baxter, Munich, Germany) or Ceprate (CellPro, Bothell, WA) devices achieved median depletions (Isolex vs. Ceprate) of 4.04 vs. 3.12 log T cells and > 5 vs. 3.27 log NK cells while allowing the recovery of 36 vs. 27% CD34+ cells. The median purity of CD34+ cells in the final product was 1.7 (CAMPATH-1), 94 (Isolex), and 65% (Ceprate). We conclude that all methods tested effectively deplete T cells from PBPC preparations harvested from healthy donors. Whereas immunomagnetic CD34+ selection is most effective in terms of elimination of T cells, the less intensive T and NK cell depletions achieved with CAMPATH-1 might be advantageous with regard to retaining engraftment potential and graft-vs.-leukemia (GVL) activity of PBPC allografts.

G-CSF-mobilized peripheral blood progenitor cells for allogeneic transplantation: safety, kinetics of mobilization, and composition of the graft.

Allogeneic transplantation of peripheral blood progenitor cells (PBPC) makes the general anaesthesia of the donor unnecessary and may result in more rapid engraftment and faster recovery of the immune system. We have studied G-CSF-mediated PBPC mobilization in healthy donors and analysed the cellular composition of the resulting PBPC grafts. PBPC grafts were obtained from nine healthy donors (18-67 years old) for allogeneic or syngeneic transplantation. Six donors received 10 micrograms/kg G-CSF per day, the others 5-6 micrograms/kg. Mobilization and harvesting were well tolerated except for moderate bone pain which occurred in all donors primed with 10 micrograms/kg. With 10 micrograms/kg, a 31-fold (9-62) enrichment of circulating CD34+ cells was observed with peak values constantly occurring on day 5 after the start of G-CSF administration. Starting harvest on day 5, one to three collections on consecutive days yielded 5.5 x 10(6)/kg (0.9-10.7) CD34+ cells, 219 x 10(6)/kg (106-314) T cells, and 34 x 10(6)/kg (23-67) NK cells per 10 litres leukapheresis volume. Altogether, PBPC grafts contained 3 times more CD34+ cells, 7 times more T cells, and 20 times more NK cells than five allogeneic marrow grafts that were analysed for comparison. The yield of CD34+ cells per 10 litres apheresis volume as well as the height of the CD34+ peak in peripheral blood were inversely correlated to the age of the donor. In the donors primed with 5-6 micrograms/kg G-CSF the increase of circulating CD34+ cells (4-7-fold enrichment) and the CD34+ cell yield per 10 litres leukapheresis volume (1 x 10(6)/kg [0.8-2.2]) was much smaller compared with the 10 micrograms/kg group. In conclusion, sufficient amounts of PBPC capable of restoring haemopoiesis in allogeneic recipients can be mobilized safely by administration of G-CSF (10 micrograms/kg s.c. for 5 d) in healthy donors, and harvested with one or two leukapheresis procedures. Whether the large numbers of T-cells and NK cells that are contained in the collection products may influence graft-versus-host and graft-versus-leukaemia reactivities of PBPC grafts remains to be determined.