Intact human amniotic membrane differentiated towards the chondrogenic lineage.

Human amniotic membrane (hAM) represents a tissue that is well established as biomaterial in the clinics with potential for new applications in regenerative medicine. For tissue engineering (TE) strategies, cells are usually combined with inductive factors and a carrier substrate. We have previously recognized that hAM represents a natural, preformed sheet including highly potent stem cells. In the present approach for cartilage regeneration we have induced chondrogenesis in hAM in vitro. For this, hAM biopsies were cultured for up to 56 days under chondrogenic conditions. The induced hAM was characterized for remaining viability, glycosaminoglycan (GAG) accumulation using histochemical analysis, and a quantitative assay. Collagen I, II and X was immunohistochemically determined and cartilage-specific mRNA expression of (sex determining region Y-) box 9, cartilage oligomeric matrix protein (COMP), aggrecan (AGC1), versican (CSPG2), COL1A1, COL9A2, melanoma inhibitory activity (MIA), and cartilage-linking protein 1 (CRTL1) analyzed by quantitative real-time polymerase chain reaction. Human AM was successfully induced to accumulate GAG, as demonstrated by Alcianblue staining and a significant (p < 0.001) increase of GAG/viability under chondrogenic conditions peaking in a 29.9 ± 0.9-fold induction on day 56. Further, upon chondrogenic induction collagen II positive areas were identified within histological sections and cartilage-specific markers including COMP, AGC1, CSPG2, COL1A1, COL9A2, MIA, and CRTL1 were found upregulated at mRNA level. This is the first study, demonstrating that upon in vitro induction viable human amnion expresses cartilage-specific markers and accumulates GAGs within the biomatrix. This is a promising first step towards a potential use of living hAM for cartilage TE.

Human immunodeficiency virus type 1 drug resistance testing: Evaluation of a new ultra-deep sequencing-based protocol and comparison with the TRUGENE HIV-1 Genotyping Kit.

Genotypic HIV-1 drug resistance testing with standard Sanger sequencing is limited to the detection of mutations with >20% prevalence. A new protocol for variant detection of protease and reverse transcriptase genes of HIV-1 genotype B samples with ultra-deep sequencing on the GS-FLX sequencer (Roche 454 Life Sciences, Branford, CT) was evaluated. The new technology was compared with the standard Sanger sequencing method. For accuracy testing, genotype B samples obtained from proficiency panels were examined with ultra-deep sequencing. Reproducibility was determined by repeat GS-FLX sequencing of 21 clinical samples. Clinical performance was evaluated with 44 samples and the results were compared to the TRUGENE HIV-1 Genotyping Kit (Siemens Healthcare Diagnostics, Tarrytown, NY). Sequences generated with both protocols were analyzed using the Stanford University HIV drug resistance database. When accuracy was tested, 316 of 317 mutation codons included in the analysis of proficiency panels could be identified correctly with ultra-deep sequencing. Reproducibility testing resulted in a correlation value of R(2)=0.969. Analysis of 44 routine clinical samples with the Stanford University HIV drug resistance database revealed a total number of 269 and 171 mutations by the ultra-deep and standard Sanger sequencing, respectively. Drug resistance interpretations showed differences for 11 samples. With ultra-deep sequencing, total time to result was four times longer in comparison to standard Sanger sequencing. Manual work was increased significantly using the new protocol. The ultra-deep sequencing protocol showed good accuracy and reproducibility. However, automation and shorter time to obtain results are essential for use in the routine diagnostic laboratory.

Sequence capture and next generation resequencing of the MHC region highlights potential transplantation determinants in HLA identical haematopoietic stem cell transplantation.

How cells coordinate the immune system activities is important for potentially life-saving organ or stem cell transplantations. Polymorphic immunoregulatory genes, many of them located in the human major histocompatibility complex, impact the process and assure the proper execution of tolerance-versus-activity mechanisms. In haematopoietic stem cell transplantation, on the basis of fully human leukocyte antigen (HLA)-matched donor-recipient pairs, adverse effects like graft versus leukaemia and graft versus host are observed and difficult to handle. So far, high-resolution HLA typing was performed with Sanger sequencing, but for methodological reasons information on additional immunocompetent major histocompatibility complex loci has not been revealed. Now, we have used microarray sequence capture and targeted enrichment combined with next generation pyrosequencing for 3.5 million base pair human major histocompatibility complex resequencing in a clinical transplant setting and describe 3025 variant single nucleotide polymorphisms, insertions and deletions among recipient and donor in a single sequencing experiment. Taken together, the presented data show that sequence capture and massively parallel pyrosequencing can be used as a new tool for risk assessment in the setting of allogeneic stem cell transplantation.

Human mesenchymal stem cells from adipose tissue and amnion influence T-cells depending on stimulation method and presence of other immune cells.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells exerting immunomodulatory effects on cells of the innate and adaptive immune system. It has been shown that an inflammatory milieu is required for the activation of MSC-mediated immunomodulation, and interferon-γ (IFN-γ) plays an important role in this process. We determined the influence of IFN-γ on human adipose-derived stem cells (ASCs) and human amniotic mesenchymal stromal cells (hAMSCs). We further evaluated the effect of MSCs on stimulated T-cells and peripheral blood mononuclear cells (PBMCs) in a cell-contact independent setting. On IFN-γ treatment, ASCs and hAMSCs possessed significantly higher antiproliferative properties and showed surface characteristics of nonprofessional antigen presenting cells (HLA-DR(+)CD40(med+)CD54(high)) with a possible regulatory phenotype (PD-L1(+)PD-L2(+)). The effect of ASCs and hAMSCs on cytokine secretion and T-cell activation was dependent on stimulation method and cellular context. Although ASCs and hAMSCs highly inhibited cytokine secretion of stimulated PBMCs, this was not observed in the case of purified T-cells. The presence of ASCs even favored the secretion of pro-inflammatory cytokines including IFN-γ by T-cells, although T-cell proliferation was efficiently inhibited. Further, ASCs enhanced the number of CD69(+) T-cells independent of the stimuli and cellular context. Interestingly, ASCs significantly suppressed CD25 expression on phytohemagglutinin stimulated PBMCs but had no effect on αCD3/αCD28 stimulated cells. Depending on the stimulation method and cellular context, immune cells create a specific cytokine milieu in vitro, thus differently influencing MSCs and, in turn, affecting their action on immune cells.

Rapid high-throughput human leukocyte antigen typing by massively parallel pyrosequencing for high-resolution allele identification.

Transplantation and, notably, hematopoietic stem cell transplantation require high-resolution human leukocyte antigen (HLA) typing and, because of the heterozygous genomic DNA samples, are dependent on clonal analytical methods. High-resolution HLA typing is a necessity for accomplishing the best possible histocompatibility match between donor and recipient, because mismatches strongly increase the risk of severe acute graft-versus-host disease. We describe the development and first application in a clinical setting of a novel, HLA sequence-based typing method by exploring the next-generation sequencing technology as provided by the Genome Sequencer FLX system (Roche/454 Life Sciences, Branford, CT). The developed system allows for ambiguity-free, high-throughput, high-resolution HLA-A and -B typing with the potential for automation. Primers and Genome Sequencer FLX specific adapters were lengthened with donor-identifying barcode sequences to identify each of eight Caucasian reference donors within one single multiplex sequencing run. Compared with normal SBT HLA typing, results indicate that every patient was identified correctly with an average of 1000 reads per amplicon. Furthermore, current investments for increased read lengths and fully automated molecular diagnostic software tools, using original GS-FLX data file formats, will enhance this novel HLA typing strategy in the near future.

Clinical significance of HLA-E*0103 homozygosity on survival after allogeneic hematopoietic stem-cell transplantation.

BACKGROUND: Hematopoietic stem-cell transplantation is a well-established treatment in various hematologic malignancies, but the outcome depends on disease relapse, infections, and the development and severity of acute and chronic graft-versus-host disease. Some evidence has revealed an important role for the nonclassical major histocompatibility complex class I molecules in transplantation, most notably human leukocyte antigen (HLA)-E. This study evaluates the impact of HLA-E alleles on transplantation outcome after HLA-matched allogeneic HSCT. METHODS: We genotyped DNA for HLA-E polymorphism from 83 recipients and their respective donors by real-time polymerase chain reaction after melting curve analysis and compared the results with clinical outcome. RESULTS: HLA-E*0103 homozygous patients showed a higher probability of overall survival (P=0.003) and disease-free survival (P=0.001) in a univariate model. Cox regression analysis confirmed HLA-E*0103, 0103 (P=0.006; relative risk 1.12; 95% confidence interval 0.31-1.94) and early stage of disease (P=0.005; relative risk 1.16; 95% confidence interval 0.45-1.86) as independent factors improving overall survival. Moreover, homozygosity for HLA-E*0103 was associated with a significant decreased incidence of transplant-related mortality (P=0.01). CONCLUSIONS: We found an association between HLA-E*0103 homozygosity and the significant reduction of transplant-related mortality in related and unrelated HSCT. The risk of posttransplant complications was significantly reduced when the donor possesses the HLA-E*0103, 0103 genotype, and this was translated in a better overall survival.