Joana Frobel

Epigenetic Rejuvenation of Mesenchymal Stromal Cells Derived from Induced Pluripotent Stem Cells

Summary Standardization of mesenchymal stromal cells (MSCs) remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between studies difficult. In contrast, induced pluripotent stem cells (iPSCs) assimilate toward a ground state and may therefore give rise to more standardized cell preparations. We reprogrammed MSCs into iPSCs, which were subsequently redifferentiated toward MSCs. These iPS-MSCs revealed similar morphology, immunophenotype, in vitro differentiation potential, and gene expression profiles as primary MSCs. However, iPS-MSCs were impaired in suppressing T cell proliferation. DNA methylation (DNAm) profiles of iPSCs maintained donor-specific characteristics, whereas tissue-specific, senescence-associated, and age-related DNAm patterns were erased during reprogramming. iPS-MSCs reacquired senescence-associated DNAm during culture expansion, but they remained rejuvenated with regard to age-related DNAm. Overall, iPS-MSCs are similar to MSCs, but they reveal incomplete reacquisition of immunomodulatory function and MSC-specific DNAm patterns—particularly of DNAm patterns associated with tissue type and aging.

Epigenetic Classification of Human Mesenchymal Stromal Cells

Summary Standardization of mesenchymal stromal cells (MSCs) is hampered by the lack of a precise definition for these cell preparations; for example, there are no molecular markers to discern MSCs and fibroblasts. In this study, we followed the hypothesis that specific DNA methylation (DNAm) patterns can assist classification of MSCs. We utilized 190 DNAm profiles to address the impact of tissue of origin, donor age, replicative senescence, and serum supplements on the epigenetic makeup. Based on this, we elaborated a simple epigenetic signature based on two CpG sites to classify MSCs and fibroblasts, referred to as the Epi-MSC-Score. Another two-CpG signature can distinguish between MSCs from bone marrow and adipose tissue, referred to as the Epi-Tissue-Score. These assays were validated by site-specific pyrosequencing analysis in 34 primary cell preparations. Furthermore, even individual subclones of MSCs were correctly classified by our epigenetic signatures. In summary, we propose an alternative concept to use DNAm patterns for molecular definition of cell preparations, and our epigenetic scores facilitate robust and cost-effective quality control of MSC cultures.

DNA-methylation changes in replicative senescence and aging: two sides of the same coin?

Replicative senescence is often considered to be a hallmark of aging [1] – but this assumption needs to be challenged by new insights into associated epigenetic modifications. Primary cells can undergo only a limited number of cell divisions in vitro before they enter the state of replicative senescence, which is reflected by unequivocal cell cycle arrest. It was first described half a century ago by Leonard Hayflick [2] – therefore often referred to as ‘Hayflick limit’ – and since then it has been speculated that replicative senescence is tightly associated with aging of the organism [3]. In fact, cellular aging during in vitro culture reflects various molecular features that seem to be indicative for aging, such as telomere attrition, activation of the p53/p21CIP1 and p16INK4A/pRb signaling pathways, alteration of cell morphology and metabolism, increased senescence-associated β-galactosidase activity, loss of differentiation potential, formation of senescence-associated heterochromatin foci and the senescenceassociated secretory phenotype [1]. Cell samples from elderly donors recapitulate many of these parameters, including a higher number of positive staining for senescence-associated β-galactosidase [4]; slower proliferation rate and senescence-like morphological changes already at the initial cell passage [5]; reduced colony-forming unit frequency [6] and concordant gene expression changes [7]. These findings fueled the perception, that replicative senescence in vitro and aging in vivo are governed by the same conserved mechanism – although carried out at a different pace. Cellular changes in the course of culture expansion are therefore often considered as a good in vitro model to unravel the molecular mechanisms that drive the process of aging. Replicative senescence and aging are both reflected by highly reproducible epigenetic changes – particularly in the DNA methylation (DNAm) pattern of developmental genes [8]. DNAm is nowadays the best-characterized epigenetic modification: it represents a covalent addition of methyl groups to cytosine residues in the context of CG dinucleotides, referred to as ‘CpG site.’ Senescence-associated DNAm changes are significantly enriched in genomic regions with repressive histone marks (H3K9me3 and H3K27me3) and at target sites of Polycomb group proteins [9,10]. Similar findings have also been reported for age-associated DNAm changes [11,12]. In fact, direct correlation of age-associated and senescenceassociated DNAm changes in mesenchymal stromal cells (MSCs) revealed a moderate but significant association of the two epigenetic processes [8]. On the other hand, replicative senescence and aging can both be tracked by very specific epigenetic modifications: for example, an ‘epigenetic-senescence-signature’ DNA-methylation changes in replicative senescence and aging: two sides of the same coin?

Leukocyte counts based on site-specific DNA methylation analysis

The composition of white blood cells is usually assessed by histomorphological parameters or flow cytometric measurements. Alternatively, leukocyte differential counts (LDCs) can be estimated by deconvolution algorithms for genome-wide DNA methylation (DNAm) profiles. We identified cell-type specific CG dinucleotides (CpGs) that facilitate relative quantification of leukocyte subsets. Site-specific analysis of DNAm levels by pyrosequencing provides similar precision of LDCs as conventional methods, whereas it is also applicable to frozen samples and requires only very small volumes of blood. Furthermore, we describe a new approach for absolute quantification of cell numbers based on a non-methylated reference DNA. Our “Epi-Blood-Count” facilitates robust and cost effective analysis of blood counts for clinical application.

iPSC-derived mesenchymal stromal cells are less supportive than primary MSCs for co-culture of hematopoietic progenitor cells

In vitro culture of hematopoietic stem and progenitor cells (HPCs) is supported by a suitable cellular microenvironment, such as mesenchymal stromal cells (MSCs)—but MSCs are heterogeneous and poorly defined. In this study, we analyzed whether MSCs derived from induced pluripotent stem cells (iPS-MSCs) provide a suitable cellular feeder layer too. iPS-MSCs clearly supported proliferation of HPCs, maintenance of a primitive immunophenotype (CD34+, CD133+, CD38-), and colony-forming unit (CFU) potential of CD34+ HPCs. However, particularly long-term culture-initiating cell (LTC-IC) frequency was lower with iPS-MSCs as compared to primary MSCs. Relevant genes for cell-cell interaction were overall expressed at similar level in MSCs and iPS-MSCs, whereas VCAM1 was less expressed in the latter. In conclusion, our iPS-MSCs support in vitro culture of HPCs; however, under the current differentiation and culture conditions, they are less suitable than primary MSCs from bone marrow.

Epigenetic aging of human hematopoietic cells is not accelerated upon transplantation into mice

BackgroundTransplantation of human hematopoietic stem cells into immunodeficient mice provides a powerful in vivo model system to gain functional insights into hematopoietic differentiation. So far, it remains unclear if epigenetic changes of normal human hematopoiesis are recapitulated upon engraftment into such “humanized mice.” Mice have a much shorter life expectancy than men, and therefore, we hypothesized that the xenogeneic environment might greatly accelerate the epigenetic clock.ResultsWe demonstrate that genome-wide DNA methylation patterns of normal human hematopoietic development are indeed recapitulated upon engraftment in mice—particularly those of normal early B cell progenitor cells. Furthermore, we tested three epigenetic aging signatures, and none of them indicated that the murine environment accelerated age-associated DNA methylation changes.ConclusionsEpigenetic changes of human hematopoietic development are recapitulated in the murine transplantation model, whereas epigenetic aging is not accelerated by the faster aging environment and seems to occur in the cell intrinsically.

Variants of DNMT3A cause transcript-specific DNA methylation patterns and affect hematopoietic differentiation

The de novo DNA methyltransferase 3A (DNMT3A) plays pivotal roles in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing of DNMT3A has characteristic epigenetic and functional sequels. Specific DNMT3A transcripts were either downregulated or overexpressed in human hematopoietic stem and progenitor cells and this resulted in complementary and transcript-specific DNA methylation and gene expression changes. Functional analysis indicated that particularly transcript 2 (coding for DNMT3A2) activates proliferation and induces loss of a primitive immunophenotype, whereas transcript 4 interferes with colony formation of the erythroid lineage. Notably, in acute myeloid leukemia (AML) expression of transcript 2 correlates with its in vitro DNA methylation and gene expression signatures and is associated with overall survival, indicating that DNMT3A variants impact also on malignancies. Our results demonstrate that specific DNMT3A variants have distinct epigenetic and functional impact. Particularly DNMT3A2 triggers hematopoietic differentiation and the corresponding signatures are reflected in AML.