Culture expansion of CAR T cells results in aberrant DNA methylation that is associated with adverse clinical outcome.

Chimeric antigen receptor (CAR) T cells provide new perspectives for treatment of hematological malignancies. Manufacturing of these cellular products includes culture expansion procedures, which may affect cellular integrity and therapeutic outcome. In this study, we investigated culture-associated epigenetic changes in CAR T cells and found continuous gain of DNAm, particularly within genes that are relevant for T cell function. Hypermethylation in many genes, such as TCF7, RUNX1, and TOX, was reflected by transcriptional downregulation. 332 CG dinucleotides (CpGs) showed an almost linear gain in methylation with cell culture time, albeit neighboring CpGs were not coherently regulated on the same DNA strands. An epigenetic signature based on 14 of these culture-associated CpGs predicted cell culture time across various culture conditions. Notably, even in CAR T cell products of similar culture time higher DNAm levels at these CpGs were associated with significantly reduced long-term survival post transfusion. Our data demonstrate that cell culture expansion of CAR T cells evokes DNA hypermethylation at specific sites in the genome and the signature may also reflect loss of potential in CAR T cell products. Hence, reduced cultivation periods are beneficial to avoid dysfunctional methylation programs that seem to be associated with worse therapeutic outcome.

Hematopoietic differentiation persists in human iPSCs defective in de novo DNA methylation.

BACKGROUND: DNA methylation is involved in the epigenetic regulation of gene expression during developmental processes and is primarily established by the DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B). DNMT3A is one of the most frequently mutated genes in clonal hematopoiesis and leukemia, indicating that it plays a crucial role for hematopoietic differentiation. However, the functional relevance of Dnmt3a for hematopoietic differentiation and hematological malignancies has mostly been analyzed in mice, with the specific role for human hematopoiesis remaining elusive. In this study, we therefore investigated if DNMT3A is essential for hematopoietic differentiation of human induced pluripotent stem cells (iPSCs). RESULTS: We generated iPSC lines with knockout of either exon 2, 19, or 23 and analyzed the impact of different DNMT3A exon knockouts on directed differentiation toward mesenchymal and hematopoietic lineages. Exon 19-/- and 23-/- lines displayed an almost entire absence of de novo DNA methylation during mesenchymal and hematopoietic differentiation. Yet, differentiation efficiency was only slightly reduced in exon 19-/- and rather increased in exon 23-/- lines, while there was no significant impact on gene expression in hematopoietic progenitors (iHPCs). Notably, DNMT3A-/- iHPCs recapitulate some DNA methylation patterns of acute myeloid leukemia (AML) with DNMT3A mutations. Furthermore, multicolor genetic barcoding revealed growth advantage of exon 23-/- iHPCs in a syngeneic competitive differentiation assay. CONCLUSIONS: Our results demonstrate that iPSCs with homozygous knockout of different exons of DNMT3A remain capable of mesenchymal and hematopoietic differentiation-and exon 23-/- iHPCs even gained growth advantage-despite loss of almost the entire de novo DNA methylation. Partial recapitulation of DNA methylation patterns of AML with DNMT3A mutations by our DNMT3A knockout iHPCs indicates that our model system can help to elucidate mechanisms of clonal hematopoiesis.

Investigation of measurable residual disease in acute myeloid leukemia by DNA methylation patterns.

Assessment of measurable residual disease (MRD) upon treatment of acute myeloid leukemia (AML) remains challenging. It is usually addressed by highly sensitive PCR- or sequencing-based screening of specific mutations, or by multiparametric flow cytometry. However, not all patients have suitable mutations and heterogeneity of surface markers hampers standardization in clinical routine. In this study, we propose an alternative approach to estimate MRD based on AML-associated DNA methylation (DNAm) patterns. We identified four CG dinucleotides (CpGs) that commonly reveal aberrant DNAm in AML and their combination could reliably discern healthy and AML samples. Interestingly, bisulfite amplicon sequencing demonstrated that aberrant DNAm patterns were symmetric on both alleles, indicating that there is epigenetic crosstalk between homologous chromosomes. We trained shallow-learning and deep-learning algorithms to identify anomalous DNAm patterns. The method was then tested on follow-up samples with and without MRD. Notably, even samples that were classified as MRD negative often revealed higher anomaly ratios than healthy controls, which may reflect clonal hematopoiesis. Our results demonstrate that targeted DNAm analysis facilitates reliable discrimination of malignant and healthy samples. However, since healthy samples also comprise few abnormal-classified DNAm reads the approach does not yet reliably discriminate MRD positive and negative samples.

DNA methylation changes during long-term in vitro cell culture are caused by epigenetic drift.

Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated during long-term culture of mesenchymal stem cells (MSCs) and other cell types. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpopulations. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no enriched interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results support the notion that DNAm changes during culture-expansion are not directly regulated by a targeted mechanism but rather resemble epigenetic drift.

PRDM8 reveals aberrant DNA methylation in aging syndromes and is relevant for hematopoietic and neuronal differentiation.

BACKGROUND: Dyskeratosis congenita (DKC) and idiopathic aplastic anemia (AA) are bone marrow failure syndromes that share characteristics of premature aging with severe telomere attrition. Aging is also reflected by DNA methylation changes, which can be utilized to predict donor age. There is evidence that such epigenetic age predictions are accelerated in premature aging syndromes, but it is yet unclear how this is related to telomere length. DNA methylation analysis may support diagnosis of DKC and AA, which still remains a challenge for these rare diseases. RESULTS: In this study, we analyzed blood samples of 70 AA and 18 DKC patients to demonstrate that their epigenetic age predictions are overall increased, albeit not directly correlated with telomere length. Aberrant DNA methylation was observed in the gene PRDM8 in DKC and AA as well as in other diseases with premature aging phenotype, such as Down syndrome and Hutchinson-Gilford-Progeria syndrome. Aberrant DNA methylation patterns were particularly found within subsets of cell populations in DKC and AA samples as measured with barcoded bisulfite amplicon sequencing (BBA-seq). To gain insight into the functional relevance of PRDM8, we used CRISPR/Cas9 technology to generate induced pluripotent stem cells (iPSCs) with heterozygous and homozygous knockout. Loss of PRDM8 impaired hematopoietic and neuronal differentiation of iPSCs, even in the heterozygous knockout clone, but it did not impact on epigenetic age. CONCLUSION: Taken together, our results demonstrate that epigenetic aging is accelerated in DKC and AA, independent from telomere attrition. Furthermore, aberrant DNA methylation in PRDM8 provides another biomarker for bone marrow failure syndromes and modulation of this gene in cellular subsets may be related to the hematopoietic and neuronal phenotypes observed in premature aging syndromes.

Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells.

BACKGROUND: The use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. Generation of MSC-like cells from induced pluripotent stem cells (iPSCs) may circumvent these limitations, and such iPSC-derived MSCs (iMSCs) are already tested in clinical trials. So far, a comparison of MSCs and iMSCs was particularly addressed in bulk culture. Despite the high hopes in cellular therapy, only little is known how the composition of different subclones changes in these cell preparations during culture expansion. METHODS: In this study, we used multicolor lentiviral genetic barcoding for the marking of individual cells within cell preparations. Based on this, we could track the clonal composition of syngenic MSCs, iPSCs, and iMSCs during culture expansion. Furthermore, we analyzed DNA methylation patterns at senescence-associated genomic regions by barcoded bisulfite amplicon sequencing. The proliferation and differentiation capacities of individual subclones within MSCs and iMSCs were investigated with limiting dilution assays. RESULTS: Overall, the clonal composition of primary MSCs and iPSCs gradually declined during expansion. In contrast, iMSCs became oligoclonal early during differentiation, indicating that they were derived from few individual iPSCs. This dominant clonal outgrowth of iMSCs was not associated with changes in chromosomal copy number variation. Furthermore, clonal dynamics were not clearly reflected by stochastically acquired DNA methylation patterns. Limiting dilution assays revealed that iMSCs are heterogeneous in colony formation and in vitro differentiation potential, while this was even more pronounced in primary MSCs. CONCLUSIONS: Our results indicate that the subclonal diversity of MSCs and iPSCs declines gradually during in vitro culture, whereas derivation of iMSCs may stem from few individual iPSCs. Differentiation regimen needs to be further optimized to achieve homogeneous differentiation of iPSCs towards iMSCs.

Senescence-Associated Metabolomic Phenotype in Primary and iPSC-Derived Mesenchymal Stromal Cells.

Long-term culture of primary cells is characterized by functional and secretory changes, which ultimately result in replicative senescence. It is largely unclear how the metabolome of cells changes during replicative senescence and if such changes are consistent across different cell types. We have directly compared culture expansion of primary mesenchymal stromal cells (MSCs) and induced pluripotent stem cell-derived MSCs (iMSCs) until they reached growth arrest. Both cell types acquired similar changes in morphology, in vitro differentiation potential, senescence-associated ß-galactosidase, and DNA methylation. Furthermore, MSCs and iMSCs revealed overlapping gene expression changes, particularly in functional categories related to metabolic processes. We subsequently compared the metabolomes of MSCs and iMSCs and observed overlapping senescence-associated changes in both cell types, including downregulation of nicotinamide ribonucleotide and upregulation of orotic acid. Taken together, replicative senescence is associated with a highly reproducible senescence-associated metabolomics phenotype, which may be used to monitor the state of cellular aging.

Differentiation of Induced Pluripotent Stem Cells towards Mesenchymal Stromal Cells is Hampered by Culture in 3D Hydrogels.

Directed differentiation of induced pluripotent stem cells (iPSCs) towards specific lineages remains a major challenge in regenerative medicine, while there is a growing perception that this process can be influenced by the three-dimensional environment. In this study, we investigated whether iPSCs can differentiate towards mesenchymal stromal cells (MSCs) when embedded into fibrin hydrogels to enable a one-step differentiation procedure within a scaffold. Differentiation of iPSCs on tissue culture plastic or on top of fibrin hydrogels resulted in a typical MSC-like phenotype. In contrast, iPSCs embedded into fibrin gel gave rise to much smaller cells with heterogeneous growth patterns, absence of fibronectin, faint expression of CD73 and CD105, and reduced differentiation potential towards osteogenic and adipogenic lineage. Transcriptomic analysis demonstrated that characteristic genes for MSCs and extracellular matrix were upregulated on flat substrates, whereas genes of neural development were upregulated in 3D culture. Furthermore, the 3D culture had major effects on DNA methylation profiles, particularly within genes for neuronal and cardiovascular development, while there was no evidence for epigenetic maturation towards MSCs. Taken together, iPSCs could be differentiated towards MSCs on tissue culture plastic or on a flat fibrin hydrogel. In contrast, the differentiation process was heterogeneous and not directed towards MSCs when iPSCs were embedded into the hydrogel.

Tracking of epigenetic changes during hematopoietic differentiation of induced pluripotent stem cells.

BACKGROUND: Differentiation of induced pluripotent stem cells (iPSCs) toward hematopoietic progenitor cells (HPCs) raises high hopes for disease modeling, drug screening, and cellular therapy. Various differentiation protocols have been established to generate iPSC-derived HPCs (iHPCs) that resemble their primary counterparts in morphology and immunophenotype, whereas a systematic epigenetic comparison was yet elusive. RESULTS: In this study, we compared genome-wide DNA methylation (DNAm) patterns of iHPCs with various different hematopoietic subsets. After 20 days of in vitro differentiation, cells revealed typical hematopoietic morphology, CD45 expression, and colony-forming unit (CFU) potential. DNAm changes were particularly observed in genes that are associated with hematopoietic differentiation. On the other hand, the epigenetic profiles of iHPCs remained overall distinct from natural HPCs. Furthermore, we analyzed if additional co-culture for 2 weeks with syngenic primary mesenchymal stromal cells (MSCs) or iPSC-derived MSCs (iMSCs) further supports epigenetic maturation toward the hematopoietic lineage. Proliferation of iHPCs and maintenance of CFU potential was enhanced upon co-culture. However, DNAm profiles support the notion that additional culture expansion with stromal support did not increase epigenetic maturation of iHPCs toward natural HPCs. CONCLUSION: Differentiation of iPSCs toward the hematopoietic lineage remains epigenetically incomplete. These results substantiate the need to elaborate advanced differentiation regimen while DNAm profiles provide a suitable measure to track this process.

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

Background: Transplantation 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. Results: We 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. Conclusions: Epigenetic 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.

Effects of senolytic drugs on human mesenchymal stromal cells.

BACKGROUND: Senolytic drugs are thought to target senescent cells and might thereby rejuvenate tissues. In fact, such compounds were suggested to increase health and lifespan in various murine aging models. So far, effects of senolytic drugs have not been analysed during replicative senescence of human mesenchymal stromal cells (MSCs). METHODS: In this study, we tested four potentially senolytic drugs: ABT-263 (navitoclax), quercetin, nicotinamide riboside, and danazol. The effects of these compounds were analysed during long-term expansion of MSCs, until replicative senescence. Furthermore, we determined the effect on molecular markers for replicative senescence, such as senescence-associated beta-galactosidase staining (SA-ß-gal), telomere attrition, and senescence-associated DNA methylation changes. RESULTS: Co-culture experiments of fluorescently labelled early and late passages revealed that particularly ABT-263 had a significant but moderate senolytic effect. This was in line with reduced SA-ß-gal staining in senescent MSCs upon treatment with ABT-263. However, none of the drugs had significant effects on the maximum number of population doublings, telomere length, or epigenetic senescence predictions. CONCLUSIONS: Of the four tested drugs, only ABT-263 revealed a senolytic effect in human MSCs-and even treatment with this compound did not rejuvenate MSCs with regard to telomere length or epigenetic senescence signature. It will be important to identify more potent senolytic drugs to meet the high hopes for regenerative medicine.

Does soft really matter? Differentiation of induced pluripotent stem cells into mesenchymal stromal cells is not influenced by soft hydrogels.

Induced pluripotent stem cells (iPSCs) can be differentiated toward mesenchymal stromal cells (MSCs), but this transition remains incomplete. It has been suggested that matrix elasticity directs cell-fate decisions. Therefore, we followed the hypothesis that differentiation of primary MSCs and generation of iPSC-derived MSCs (iMSCs) is supported by a soft matrix of human platelet lysate (hPL-gel). We demonstrate that this fibrin-based hydrogel supports growth of primary MSCs with pronounced deposition of extracellular matrix, albeit it hardly impacts on gene expression profiles or in vitro differentiation of MSCs. Furthermore, iPSCs can be effectively differentiated toward MSC-like cells on the hydrogel. Unexpectedly, this complex differentiation process is not affected by the substrate: iMSCs generated on tissue culture plastic (TCP) or hPL-gel have the same morphology, immunophenotype, differentiation potential, and gene expression profiles. Moreover, global DNA methylation patterns are essentially identical in iMSCs generated on TCP or hPL-gel, indicating that they are epigenetically alike. Taken together, hPL-gel provides a powerful matrix that supports growth and differentiation of primary MSCs and iMSCs - but this soft hydrogel does not impact on lineage-specific differentiation.

Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells.

Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements - it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

Senescence-associated DNA methylation is stochastically acquired in subpopulations of mesenchymal stem cells.

Replicative senescence has a major impact on function and integrity of cell preparations. This process is reflected by continuous DNA methylation (DNAm) changes at specific CpG dinucleotides in the course of in vitro culture, and such modifications can be used to estimate the state of cellular senescence for quality control of cell preparations. Still, it is unclear how senescence-associated DNAm changes are regulated and whether they occur simultaneously across a cell population. In this study, we analyzed global DNAm profiles of human mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) to demonstrate that senescence-associated DNAm changes are overall similar in these different cell types. Subsequently, an Epigenetic-Senescence-Signature, based on six CpGs, was either analyzed by pyrosequencing or by bar-coded bisulfite amplicon sequencing. There was a good correlation between predicted and real passage numbers in bulk populations of MSCs (R2  = 0.67) and HUVECs (R2  = 0.97). However, when we analyzed the Epigenetic-Senescence-Signature in subclones of MSCs, the predictions revealed high variation and they were not related to the adipogenic or osteogenic differentiation potential of the subclones. Notably, in clonally derived subpopulations, the DNAm levels of neighboring CpGs differed extensively, indicating that these genomic regions are not synchronously modified during senescence. Taken together, senescence-associated DNAm changes occur in a highly reproducible manner, but they are not synchronously co-regulated. They rather appear to be acquired stochastically-potentially evoked by other epigenetic modifications.

The lncRNA HOTAIR impacts on mesenchymal stem cells via triple helix formation.

There is a growing perception that long non-coding RNAs (lncRNAs) modulate cellular function. In this study, we analyzed the role of the lncRNA HOTAIR in mesenchymal stem cells (MSCs) with particular focus on senescence-associated changes in gene expression and DNA-methylation (DNAm). HOTAIR binding sites were enriched at genomic regions that become hypermethylated with increasing cell culture passage. Overexpression and knockdown of HOTAIR inhibited or stimulated adipogenic differentiation of MSCs, respectively. Modification of HOTAIR expression evoked only very moderate effects on gene expression, particularly of polycomb group target genes. Furthermore, overexpression and knockdown of HOTAIR resulted in DNAm changes at HOTAIR binding sites. Five potential triple helix forming domains were predicted within the HOTAIR sequence based on reverse Hoogsteen hydrogen bonds. Notably, the predicted triple helix target sites for these HOTAIR domains were also enriched in differentially expressed genes and close to DNAm changes upon modulation of HOTAIR Electrophoretic mobility shift assays provided further evidence that HOTAIR domains form RNA-DNA-DNA triplexes with predicted target sites. Our results demonstrate that HOTAIR impacts on differentiation of MSCs and that it is associated with senescence-associated DNAm. Targeting of epigenetic modifiers to relevant loci in the genome may involve triple helix formation with HOTAIR.

Epigenetic Classification of Human Mesenchymal Stromal Cells.

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.