56. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Vorsitz

Prof. Dr.

Restoration of immune function in a model of familial hyperinflammation syndrome by editing of hematopoietic stem cells

eingeladene Redner

Vortrag

Toward clinical application of leukocyte counts based on targeted DNA methylation analysis

Prof. Dr. Dr.

Professor

HLA-silenced Megakaryocytes evade allogeneic T-cell immune responses

Stem Cells

Background Clinical demand on platelets (PLTs) to treat thrombocytopenic patients is rising. Transfusion of in vitro produced megakaryocytes (MKs) may be used as an alternative to donor PLTs. HLA class I-silenced MKs effectively produced PLTs in the circulation of a refractory mouse model. However, their capacity to upregulate HLA class II may lead to harmful allogeneic immune responses. Hence, we investigated the potential beneficial effect of producing HLA class I and II-silenced MKs.Methods HLA class I or HLA- class I and II-silenced iPSCs were generated using lentiviral vectors encoding short-hairpin RNAs (shRNA) targeting β2-microglobulin (shβ2m) and CIITA (shCIITA) respectively. Nontransduced or with a nonspecific shRNA (shNS) encoding vector transduced iPSCs were used as control. Expression levels of β2m, CIITA and HLA class II were evaluated by RT-qPCR. MKs and PLTs were differentiated and phenotyping was performed by flow cytometry. Allogeneic CD4 and CD8 T-cell responses were assessed in CPD-based proliferation assays followed by flow cytometric analysis. Granular T-cell mediated cytotoxicity was measured by quantifying Granzyme B levels by ELISA. MK production was upscaled in an Eppendorf DasBOX Mini bioreactor.Results Gene expression levels of β2m and CIITA in HLA class I or class I and II-silenced iPSCs were significantly reduced (p&lt;0.01). HLA-silencing had no effect on their differentiation capability. After incubation of primed T-cells with MKs, both HLA class I or HLA class I and II-silenced MKs led to significantly (p&lt;0.001) lower CD8- and CD4 T-cell proliferation compared to the controls. T-cell granzyme B secretion levels were significantly reduced by 25% (p&lt;0.01) after exposure to HLA class I-silenced MKs. Remarkably, release of Granzyme B-containing granules was even more pronouncedly reduced (52 %, p&lt;0.01) when HLA class I and II-silenced MKs were used. In addition, MKs were successfully produced in a bioreactor.Conclusion This study reports the effect of silencing HLA class I and II antigens to prevent allogeneic T-cell-mediated responses towards allogeneic iPSC-derived MKs. Furthermore, the feasibility of upscaling MK production in bioreactors, to meet clinical needs was shown. Use of in vitro manufactured HLA-universal MKs may open new frontiers in the management of highly sensitized thrombocytopenic patients.Offenlegung Interessenkonflikt: Es bestehen keinerlei Interessenskonflikte

Freier Vortrag

Deciphering enucleation: Analysis of RNA sequencing data for investigating differences in differentiation behavior between HSCs and erythroblast cell line "imBMEP"

Background An erythroid progenitor cell line capable of efficient in vitro production of mature red blood cells (RBCs) represents a promising alternative for transfusion medicine. Since an erythroblast line previously established in our lab (imBMEP) showed limitations in efficient maturation, knockout (K.O.) of enucleation inhibitor miR-30a-5p was performed. Although this modification led to a significant promotion of terminal erythropoiesis, insufficient enucleation remains a major challenge.Methods To systematically investigate possible causes of the inhibited final maturation a transcriptome analysis was performed. Samples for RNA Sequencing were obtained from imBMEP cells and hematopoietic stem cells (HSCs), acting as control. Extracted RNA was derived at different time points during erythroid differentiation in biological triplicates. RNA quality and concentration were determined. The following enrichment of mRNA was performed via ribosomal RNA depletion with RNAse H. After reverse transcription barcoded cDNA libraries were prepared. The following next generation sequencing was executed paired-ended. After read alignment to a human reference genome and quality control, differential gene expression (DGE) analysis was carried out.Results DGE analysis revealed high variance between imBMEP cells and HSC controls. Comparison of raw counts of the different RNA biotypes showed a significant elevation of lncRNAs during differentiation compared to corresponding HSC controls (8,4±0,5% vs. 14,4±1,2%). A notable number of cancer related lncRNAs are among the RNAs with the highest differences in expression between imBMEPs and HSCs. Furthermore, dysregulation of previously described factors involved in terminal erythropoiesis and enucleation, like HDAC5, FOXO3 or GATA2, was observed in imBMEP cells.Conclusion RNA Sequencing is a powerful tool for analysing differential gene expression. Comparison of sequencing data between imBMEP cells and HSCs gave first insights in dysregulated processes and possible candidates causing differences in differentiation behaviour. Planned ingenuity pathway analysis (IPA) and pathway enrichment analysis should now help to infer the underlying reasons of observed differences, predict downstream effects and find key candidates for further modifications of the cell line.Offenlegung Interessenkonflikt: There are no conflicts of interest to be disclosed.

Mesenchymal stromal cells-derived secretome rescues cisplatin-induced injury of proximal tubular epithelial cells while dampening macrophage activation in an indirect co-culture system

Background The interplay between renal proximal tubular epithelial cells (PTEC) and macrophages is important in the progression of kidney injury, caused by a chemotherapeutic drug, cisplatin. Upon injury, PTEC attract macrophages, where macrophages can either promote injury resolution or inflammation. Mesenchymal stromal cells (MSC), having pro-regenerative capacity, might rescue the injury by acting on PTEC and macrophages. We hypothesize MSC can modulate PTEC and macrophage crosstalk upon the injury.Methods MSC secretome was harnessed by producing MSC conditioned medium (CM). First, the effect of CM was tested on PTEC and macrophages upon cisplatin injury separately. To evaluate CM and cisplatin effect on PTEC, apoptosis, gene expression and reactive oxygen species of PTEC were assessed. On macrophages, the surface markers for M1 and M2 polarization, and their phagocytosis capacity were measured. Lastly, the interplay between PTEC and macrophages was investigated using an indirect co-culture system. The PTEC injury was evaluated by measuring their apoptosis, nuclei fragmentation, and TNF-α, while macrophage polarization was assessed by phagocytosis assay. The crosstalk of ciPTECs and macrophages was interrogated by LUMINEX assay.Results CM rescued cisplatin-induced PTEC death via gene expression modification and oxidative stress amelioration. On macrophages, CM promoted phagocytosis and the expression of M2-associated surface markers CD163 and CD206, while suppressing M1 markers CD86 and HLA-DR. In the co-culture system, CM suppressed PTEC death by inhibiting apoptosis and nuclei fragmentation. CM also downregulated pro-inflammatory response of PTEC, by lowering TNF-α release. While cisplatin inhibited macrophage phagocytosis, PTEC, and CM, to a greater extent, enhanced it. CM dampened macrophage cytokine secretion triggered by PTEC. Of note, despite CM presence, cisplatin caused macrophage death in long-term.Conclusion CM rescued cisplatin injury on PTEC and promoted M2 polarization of macrophages, individually. However, combining PTEC and macrophages did not boost CM amelioration of injury on PTEC. We conclude that MSC-CM overrules the fine-orchestrated crosstalk between PTEC and macrophages, at least in vitro.Offenlegung Interessenkonflikt: The authors declare no potential conflicts of interest.

MOA 01+02

Joint Session German Stem Cell Network

Abstractsitzungen

Joint Session German Stem Cell Network

Termin

Vorsitz

Programm