Patient Serum Stimulates Hematopoietic Regeneration
Final Report Abstract
Hematopoietic stress - e.g. after chemotherapy - evokes enormous regenerative potential of the hematopoietic system. The signals that recruit hematopoietic stem and progenitor cells (HSPCs) into cell cycle and that balance self-renewal and differentiation are so far poorly understood. We identified 23 small RNA molecules (miRNAs) that are differentially expressed in serum after chemotherapy. Particularly, miRNA-320c and miRNA-1275 were downregulated, whereas miRNA-3663-3p was upregulated. Inhibition of miRNA-320c seemed to increase HSPC-proliferation in vitro. Metabolomic profiling demonstrated that 44 metabolites were less abundant, whereas three (including 2-hydroxybutyrate and taurocholenate sulphate) increased in serum upon chemotherapy, but no clear effect on HSPCs could be observed in vitro. We therefore anticipated that these factors may act in concert to regulate hematopoiesis, rather than individually. In analogy, we addressed hematopoiesis-stimulating signals in serum of patients with myelodysplastic syndrome (MDS). We developed a mathematical model to simulate different modifications in MDS-initiating cells and systemic feedback signals during disease development. This model indicated that the disease initiating cells must have higher self-renewal rates than normal HSPCs to eventually outgrow healthy hematopoiesis. Experimental validation supported the predictions of our model. We also analyzed epigenetic modification upon in vitro culture of HSPCs. The culture conditions of CD34+ cells, with or without co-cultivation with mesenchymal stromal cells, had relatively little impact on DNA-methylation (DNAm), although proliferation is greatly increased by stromal support. Our results demonstrate that HSPCs acquire high DNAm at specific sites in the genome, and this is relevant for the rapid loss of primitive properties (‘stemness’) during in vitro manipulation. Aberrant hypermethylation in culture expansion was particularly observed in DNMT3A. Subsequently, we focused on DNAm at this region in AML patients and identified aberrant hypermethylation in DNMT3A in about 40% of AML patients. This “epimutation” in DNMT3A is rather mutually exclusive with somatic mutations in DNMT3A. Both modifications have similar sequel on specific splice variants of DNMT3A and seem to evoke similar changes in gene expression profiles (including HOX clusters). We could demonstrate that age-associated DNAm changes in blood, which can be used to track the chronological age of the donor, are cell intrinsically regulated and hardly affected by the hematopoietic environment. Furthermore, we demonstrated that this aging signature is also applicable to cell-free DNA in patient serum. Taken together, we have pursued all lines of research proposed in the initial grant application. However, our research did not give rise to a unique parameter that can be used to reliably expand HSPCs in vitro or in vivo. Our results indicate that epigenetic modifications, reflected in the DNAm pattern, play a central role for regulation of hematopoiesis. Splice variants of DNMT3A seem be involved in this regulatory process and we want to continue this research in the future.
Publications
- Hematopoietic stem and progenitor cells acquire distinct DNA-hypermethylation during in vitro culture. Sci Rep. 2013 Nov 28;3:3372
Weidner CI, Walenda T, Lin Q, Wölfler MM, Denecke B, Costa IG, Zenke M, Wagner W
(See online at https://doi.org/10.1038/srep03372) - Epimutations mimic genomic mutations of DNMT3A in acute myeloid leukemia. Leukemia. 2014 Jun;28(6):1227-34
Jost E, Lin Q, Weidner CI, Wilop S, Hoffmann M, Walenda T, Schemionek M, Herrmann O, Zenke M, Brümmendorf TH, Koschmieder S, Wagner W
(See online at https://doi.org/10.1038/leu.2013.362) - Feedback signals in myelodysplastic syndromes: increased selfrenewal of the malignant clone suppresses normal hematopoiesis. PLoS Comput Biol. 2014 Apr 24;10(4):e1003599
Walenda T, Stiehl T, Braun H, Fröbel J, Ho AD, Schroeder T, Goecke TW, Rath B, Germing U, Marciniak-Czochra A, Wagner W
(See online at https://doi.org/10.1371/journal.pcbi.1003599) - Epigenetic Aging Signatures are Coherently Modified in Cancer. PLoS Genetics 2015; 11: e1005334
Lin Q, Wagner W
(See online at https://doi.org/10.1371/journal.pgen.1005334) - Epigenetic aging upon allogeneic transplantation: the hematopoietic niche does not affect age-associated DNA methylation. Leukemia. 2015 Apr;29(4):985-8
Weidner CI, Ziegler P, Hahn M, Brümmendorf TH, Ho AD, Dreger P, Wagner W
(See online at https://doi.org/10.1038/leu.2014.323) - MicroRNA expression profiles of serum from patients before and after chemotherapy. Genomics Data. 2015; 6: 125- 127
Diener Y, Walenda T, Jost E, Brümmendorf TH, Bosio A, Wagner W, Bissels U
(See online at https://doi.org/10.1016/j.gdata.2015.08.018) - MicroRNAs and Metabolites in Serum Change after Chemotherapy: Impact on Hematopoietic Stem and Progenitor Cells. PLoS One. 2015 May 29;10(5):e0128231
Walenda T, Diener Y, Jost E, Morin-Kensicki E, Goecke TW, Bosio A, Rath B, Brümmendorf TH, Bissels U, Wagner W
(See online at https://doi.org/10.1371/journal.pone.0128231)
