Thomas H. Müller

A novel embryonic stem cell line derived from the common marmoset monkey (Callithrix jacchus) exhibiting germ cell-like characteristics

BACKGROUNDnEmbryonic stem cells (ESC) hold great promise for the treatment of degenerative diseases. However, before clinical application of ESC in cell replacement therapy can be achieved, the safety and feasibility must be extensively tested in animal models. The common marmoset monkey (Callithrix jacchus) is a useful preclinical non-human primate model due to its physiological similarities to human. Yet, few marmoset ESC lines exist and differences in their developmental potential remain unclear.nnnMETHODSnBlastocysts were collected and immunosurgery was performed. cjes001 cells were tested for euploidy by karyotyping. The presence of markers for pluripotency was confirmed by immunofluorescence staining and RT-PCR. Histology of teratoma, in vitro differentiation and embryoid body formation revealed the differentiation potential.nnnRESULTSncjes001 cells displayed a normal 46,XX karyotype. Alkaline phosphatase activity, expression of telomerase and the transcription factors OCT4, NANOG and SOX2 as well as the presence of stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor rejection antigens (TRA)-1-60, and TRA-1-81 indicated pluripotency. Teratoma formation assay displayed derivatives of all three embryonic germ layers. Upon non-directed differentiation, the cells expressed the germ cell markers VASA, BOULE, germ cell nuclear factor and synaptonemal complex protein 3 and showed co-localization of VASA protein within individual cells with the germ line stem cell markers CD9, CD49f, SSEA-4 and protein gene product 9.5, respectively.nnnCONCLUSIONSnThe cjes001 cells represent a new pluripotent ESC line with evidence for enhanced spontaneous differentiation potential into germ cells. This cjes001 line will be very valuable for comparative studies on primate ESC biology.

Two pathogen reduction technologies—methylene blue plus light and shortwave ultraviolet light—effectively inactivate hepatitis C virus in blood products

BACKGROUND: Contamination of blood products with hepatitisu2003C virus (HCV) can cause infections resulting in acute and chronic liver diseases. Pathogen reduction methods such as photodynamic treatment with methylene blue (MB) plus visible light as well as irradiation with shortwave ultraviolet (UVC) light were developed to inactivate viruses and other pathogens in plasma and platelet concentrates (PCs), respectively. So far, their inactivation capacities for HCV have only been tested in inactivation studies using model viruses for HCV. Recently, a HCV infection system for the propagation of infectious HCV in cell culture was developed.

Glycan stem-cell markers are specifically expressed by spermatogonia in the adult non-human primate testis

BACKGROUNDnThe glycan cell surface molecules, stage-specific embryonic antigen (SSEA)-1, -3 and -4 and tumor-rejection antigen (TRA)-1-60 and -1-81, are expressed in specific combinations by undifferentiated pluripotent cells, i.e. embryonic stem cells, induced pluripotent stem cells, embryonal carcinoma cells, primordial germ cells and embryonic germ cells. Upon differentiation of the cells, these markers vanish. Recently, it has been shown that also neonatal and adult mouse testes contain pluripotent cells. Here, we aimed at identifying in situ possibly pluripotent cells in the adult primate testis.nnnMETHODSnMonoclonal antibodies raised against the glyco-epitopes SSEA-1, -3 and -4 and TRA-1-60 and -1-81, respectively, were tested to detect cells expressing the antigens, by immunohistochemistry on Bouins-fixed and paraffin-embedded adult primate testes. Man, the new-world monkey, Callithrix jacchus (common marmoset), and the old-world monkey species, Macaca mulatta (Rhesus macaque) and Macaca silenus (Lion-tailed macaque), were included. The percentage of SSEA-4-positive cells in three adult marmoset testes was determined using flow cytometry.nnnRESULTSnSpermatogonia in the testes of C. jacchus were labeled by SSEA-4, TRA-1-60 and -1-81-antibodies. In the macaques, spermatogonia were detected by SSEA-4 and TRA-1-81-antibodies. TRA-1-61 did not bind to macaque spermatogonia. Also, SSEA-1 and -3 did not bind to spermatogonia in any species. In human testes, we never obtained any clear staining. The total percentage of SSEA-4-positive cells in marmoset testes was 8.6 +/- 1.61%.nnnCONCLUSIONSnSSEA-4 and TRA-1-81-antibodies may be very well suited for the identification and isolation of spermatogonia, and possibly also germline stem cells, in the non-human primate testis.

Evaluation of single-nucleotide polymorphisms as internal controls in prenatal diagnosis of fetal blood groups.

BACKGROUND: Determination of fetal blood groups in maternal plasma samples critically depends on adequate amplification of fetal DNA. We evaluated the routine inclusion of 52 single‐nucleotide polymorphisms (SNPs) as internal reference in our polymerase chain reaction (PCR) settings to obtain a positive internal control for fetal DNA.

MHC universal cells survive in an allogeneic environment after incompatible transplantation.

Cell, tissue, and organ transplants are commonly performed for the treatment of different diseases. However, major histocompatibility complex (MHC) diversity often prevents complete donor-recipient matching, resulting in graft rejection. This study evaluates in a preclinical model the capacity of MHC class I-silenced cells to engraft and grow upon allogeneic transplantation. Short hairpin RNA targeting β2-microglobulin (RN_shβ2m) was delivered into fibroblasts derived from LEW/Ztm (RT1l) (RT1-Al) rats using a lentiviral-based vector. MHC class I (RT1-A-) expressing and -silenced cells were injected subcutaneously in LEW rats (RT1l) and MHC-congenic LEW.1W rats (RT1u), respectively. Cell engraftment and the status of the immune response were monitored for eight weeks after transplantation. In contrast to RT1-A-expressing cells, RT1-A-silenced fibroblasts became engrafted and were still detectable eight weeks after allogeneic transplantation. Plasma levels of proinflammatory cytokines IL-1α, IL-1β, IL-6, TNF-α, and IFN-γ were significantly higher in animals transplanted with RT1-A-expressing cells than in those receiving RT1-A-silenced cells. Furthermore, alloantigen-specific T-cell proliferation rates derived from rats receiving RT1-A-expressing cells were higher than those in rats transplanted with RT1-A-silenced cells. These data suggest that silencing MHC class I expression might overcome the histocompatibility barrier, potentially opening up new avenues in the field of cell transplantation and regenerative medicine.

Growth Characteristics of the Nonhuman Primate Embryonic Stem Cell Line Cjes001 Depending on Feeder Cell Treatment

Embryonic stem cells (ESC) hold tremendous potential for therapeutic applications, including regenerative medicine, as well as for understanding basic mechanisms in stem cell biology. Since numerous experiments cannot be conducted in human ESC because of ethical or practical limitations, nonhuman primate ESC serve as invaluable clinically relevant models. The novel marmoset (Callithrix jacchus) ESC line cjes001 was characterized using different stem cell markers. The cells were stained positively with Oct4, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, and Sox-2 underscoring their status as undifferentiated ESC. ESC are typically grown on mouse embryonic fibroblasts (MEF) as feeder cells whose proliferation is arrested either by treatment with Mitomycin C or by gamma-irradiation. To assess the impact of these treatments on the ability of MEF to support the growth of undifferentiated ESC, we used an MTT assay to evaluate the cellular metabolic activity of growth arrested feeder cells. There was a significant (p < 0.02) difference in gamma-irradiated cells displaying a higher metabolic activity compared to Mitomycin C inactivation. Also we quantified 69 soluble factors in the supernatant of both Mitomycin-treated and gamma-irradiated MEF by bead-based multiplex analysis, and thus established a profile of MEF-secreted factors. The time course of secretion was analyzed by monitoring the supernatant at 0, 6, 12, and 24 h after changing the medium. Comparing gamma-irradiated and Mitomycin-treated MEF suggested higher amounts of some cytokines including FGF or SCF by the former. We also assessed whether the method of inactivation had an effect on growth kinetics and differentiation of primate ESC. There appeared to be a trend to a lower number of differentiated ESC colonies on the gamma-irradiated feeder cells, suggesting that this may be the preferable method of growth arrest.

Laboratory Evaluation of the Effectiveness of Pathogen Reduction Procedures for Bacteria

Bacterial contamination remains a leading factor for transfusion-associated serious morbidity and mortality. Pathogen reduction procedures offer a pro-active approach to prevent bacterial contamination of cellular blood components and especially of platelet concentrates. In the past, the laboratory evaluation of the effectiveness of the pathogen reduction procedures to minimise the bacterial load of blood components has been primarily based on log reduction assays similar to the assessment of antiviral activities. Bacteria strains with the ability to multiply in the blood components are seeded in highest possible cell numbers, the pathogen reduction procedure is applied, and the post-treatment number of bacteria is measured. The effectiveness of the procedure is characterised by calculating the log reduction of the post- to pre-treatment bacteria titres. More recently, protocols have been developed for experiments starting with a low bacteria load and monitoring the sterility of the blood component during the entire storage period of the blood component. Results for 3 different pathogen reduction technologies in these experimental models are compared and critical determinants for the results are addressed. The heterogeneity of results observed for different strains suggests that the introduction of international transfusion-relevant bacterial reference strains may facilitate the validity of findings in pathogen reduction experiments.

Platelet recovery and survival measured in patients by quantitative polymerase chain reaction of mitochondrial DNA.

Mitochondrial (mt) DNA markers have been identified as potential targets for the quantification of endogenous and allogeneic platelets (PLTs) in the blood of individuals who received transfusions. Our goal was to develop a routine polymerase chain reaction (PCR) assay for ex vivo monitoring of PLT survival in patients after transfusion.

Extended Donor Typing by Pooled Capillary Electrophoresis: Impact in a Routine Setting

Background: PCR with sequence-specific priming using allele-specific fluorescent primers and analysis on a capillary sequencer is a standard technique for DNA typing. We aimed to adapt this method for donor typing in a medium-throughput setting. Methods: Using the Extract-N-Amp PCR system, we devised a set of eight multiplex allele-specific PCR with fluorescent primers for Fya/Fyb, Jka/Jkb, M/N, and S/s. The alleles of a gene were discriminated by the fluorescent color; donor and polymorphism investigated were encoded by product length. Time, cost, and routine performance were collated. Discrepant samples were investigated by sequencing. The association of new alleles with the phenotype was evaluated by a step-wise statistical analysis. Results: On validation using 312 samples, for 1.1% of antigens (in 5.4% of samples) no prediction was obtained. 99.96% of predictions were correct. Consumable cost per donor were below EUR 2.00. In routine use, 92.2% of samples were successfully predicted. Discrepancies were most frequently due to technical reasons. A total of 11 previously unknown alleles were detected in the Kell, Lutheran, and Colton blood group systems. In 2017, more than 20% of the RBC units prepared by our institution were from donors with predicted antigen status. A steady supply of Yt(a-), Co(a-) and Lu(b-) RBC units was ensured. Conclusions: Pooled capillary electrophoresis offers a suitable alternative to other methods for extended donor DNA typing. Establishing a large cohort of typed donors improved transfusion support for patients.

Digital PCR to assess platelet recovery after stem cell transplantation

Engraftment of platelets in patients after human hematopoietic stem cell (HSC) transplantation serves as an early and relevant sign of the success of the transplantation. The time to platelet engraftment appears to be a valid predictor of the probability of complications in transplanted patients and especially for allogeneic HSC transplantation [1]. At least two different criteria of platelet engraftment, i.e. definitions according to the CIBMTR and EBMT, have been widely applied. Both criteria utilize daily platelet counts to assess the time to a threshold count of more than 20,000/μL platelets in peripheral blood, provided that platelet transfusions have been terminated for either at least three [2] (EBMT) or at least seven [3] (CIBMTR) consecutive days. Here, we describe the development of a digital PCR assay to specifically quantify graft-derived platelets versus transfused single-donor platelets. The performance of this assay was then tested with samples from allogeneic HSC transplant patients. Samples of 2 mL EDTA anticoagulated blood from patients were mixed with an equal volume of 0.9% NaCl and centrifuged at 100×g for 10 min without brake (Hettich Rotina 46S, Hettich, Tuttlingen, Germany). A final centrifugation step at 1569×g for 5 min followed. The pellet was resuspended in 500 μL phosphate buffered saline (PBS, Invitrogen Karlsruhe, Germany). The number of platelets in the PRP was adjusted with PBS to 5 × 10–2.5 × 10 to extract the mt DNA from these platelet suspensions (500 μL). The MagnaPure Large Volume protocol (MagnaPure compact, Roche Diagnostics GmbH, Mannheim, Germany) was used to extract the mt DNA. Elution volume was 50 μL. The PCR strategy was based on generic amplification primers and allele-specific probes. The probes were designed to specifically detect alleles carrying SNPs at positions 73, 195, 295, 310, 16069, 16399 and 16,516 of the non-coding regions HVR 1 and HVR 2 of the mt genome. Droplet generation, amplification and detection of PCR products were performed according to the manufacturer’s instructions (QX200 ddPCR System, Bio-Rad Laboratories GmbH, München, Germany) using 5 μL extracted DNA. Specificity of the assay was evaluated in mt DNA samples pre-typed by Sanger sequencing. Sensitivity was tested by dilution series, described in detail elsewhere [4]. The reproducibility of the background signal (“false positive droplets”) was determined by calculation of mean and standard deviation of n= 50 non-template controls and n= 20 positive controls each for FAMor VIClabelled mt DNA samples. Residual white blood cells and their mt DNA content could interfere with the platelet quantification based on the mt DNA PCR. Samples prepared for mt DNA analysis were, therefore, tested for the presence of genomic DNA as an indicator for residual white blood cells as described before [4]. The absolute count of platelets was calculated from the readout of the droplet digital PCR system (copies/μL) as: concentration (copies/μL)/genome mass (pg) × dilution factor (PRP preparation; PCR assay). The cut-off value for quantification of platelets for all mt SNPs was set to 1000 platelets/μL based on dilution series. The third of three consecutive days with increasing platelet counts was defined as day of engraftment. A digital PCR for SNP detection was developed and validated to monitor the platelet engraftment in patients after allogeneic stem cell transplantation. DNA samples from 30 patients examined in our previous study for quantification of endogenous platelets with real-time PCR were used to validate the digital PCR assay [4]. The quantification of platelets in 150 samples was congruent in * Andrea Doescher doescher@bsd-nstob.de