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Mutations in the genetic sequence of the DNA de novo methyltransferase DNMT3A (DNA methyltransferase 3A) are found in many patients with acute myeloid leukemia (AML). They lead to dysfunction of DNMT3A protein and represent a marker for poor prognosis. Effects of genetic mutations can be mimicked by epigenetic modifications in the DNA methylation (DNAm) pattern. Using DNAm profiles of the Cancer Genome Atlas Research Network (TCGA), we identified aberrant hypermethylation at an internal promoter region of DNMT3A, which occurred in about 40% of AML patients. Bisulfite pyrosequencing assays designed for this genomic region validated hypermethylation specifically in a subset of our AML samples. High DNAm levels at this site are particularly observed in samples without genetic mutations in DNMT3A. Epimutations and mutations of DNMT3A were associated with related gene expression changes such as upregulation of the homeobox genes in HOXA and HOXB clusters. Furthermore, epimutations in DNMT3A were enriched in patients with poor or intermediate cytogenetic risk, and in patients with shorter event-free survival and overall survival (OS). Taken together, aberrant DNA hypermethylation within the DNMT3A gene, in analogy to DNMT3A mutations, is frequently observed in AML and both modifications seem to be useful for risk stratification or choice of therapeutic regimen.

The stromal cell-derived factor 1 (SDF-1) is essential for circulation, homing, and retention of hematopoietic stem cells in the bone marrow. Present evidence indicates that this factor might play an important role in leukemia cells as well. The aim of this study is to present a model of SDF-1-induced mobilization using leukemia cell lines. CXCR4 expression was compared in Kasumi-1, Jurkat, HL-60, KG-1a, and K562 cells by flow cytometry and Western blot. Migration was analyzed with Transwell assays, and adhesive cell-cell interaction was quantified with a standardized adhesion assay and flow cytometry. CXCR4 was expressed by all leukemic cell lines analyzed, although surface expression of this receptor was found in Kasumi-1 and Jurkat cells only. Correspondingly, SDF-1α effects on migration and cell-cell adhesion were observed in Kasumi-1 and Jurkat cells only, and this could be blocked by AMD3100 in a reversible manner. We have provided evidence that SDF-1α acts as a chemotactic and chemokinetic agent. In addition, surface expression of integrin-β2, activated leukocyte cell adhesion molecule and N-cadherin decreased after stimulation with SDF-1α. SDF-1α affects cell-cell adhesion and migration only in leukemia cells on which the CXCR4 receptor is present on the surface. An SDF-1 gradient is not necessarily required to induce migration, as chemokinesis can also occur. Upon stimulation with SDF-1, CXCR4 promotes modifications on the surface pattern of adhesion molecules, which have an influence on adhesion and migration.

Objective The number of hematopoietic stem and progenitor cells (HPCs) per cord blood unit is limited, and this can result in delayed engraftment or graft failure. In vitro expansion of HPCs provides a perspective to overcome these limitations. Cytokines as well as mesenchymal stromal cells (MSCs) have been shown to support HPCs ex vivo expansion, but a systematic analysis of their interplay remains elusive. Materials and Methods Twenty different combinations of growth factors (stem cell factor [SCF], thrombopoietin [TPO], fibroblast growth factor−1 [FGF-1], angiopoietin-like 5, and insulin-like growth factor-binding protein 2), either with or without MSC coculture were systematically compared for their ability to support HPC expansion. CD34 + cells were stained with carboxyfluorescein diacetate N-succinimidyl ester to monitor cell division history in conjunction with immunophenotype. Colony-forming unit frequencies and hematopoietic reconstitution of nonobese diabetic severe combined immunodeficient mice were also assessed. Results Proliferation of HPCs was stimulated by coculture with MSCs. This was further enhanced in combination with SCF, TPO, and FGF-1. Moreover, these conditions maintained expression of primitive surface markers for more than four cell divisions. Colony-forming unit−initiating cells were not expanded without stromal support, whereas an eightfold increase was reached by simultaneous cytokine-treatment and MSC coculture. Importantly, in comparison to expansion without stromal support, coculture with MSCs significantly enhanced hematopoietic chimerism in a murine transplantation model. Conclusions The supportive effect of MSCs on hematopoiesis can be significantly increased by addition of specific recombinant growth factors; especially in combination with SCF, TPO, and FGF-1.

Specific cell–cell junctions between hematopoietic stem cells (HSC) and their niche have been shown to regulate stem cell function. N-cadherin was suggested to play a central role in this process, whereas other studies indicated that it did not play an essential role in the murine model. We have analyzed the role of N-cadherin for interaction between hematopoietic progenitor cells (HPC) and supportive mesenchymal stromal cells (MSC) in a human–human setting. Expression of N-cadherin and of cadherin-11 (osteoblast cadherin) was analyzed in HPC by quantitative RT-PCR, Western blot, and flow cytometry. N-cadherin and cadherin-11 were expressed in HPC at a moderate level, whereas they were not detectable in differentiated cells. Confocal laser scanning microscopy revealed that N-cadherin and β-catenin are colocalized at the junction of HPC and MSC. siRNA knockdown of N-cadherin or cadherin-11 as well as treatment with the blocking function antibody decreased adhesive interaction of HPC to MSC. Furthermore, knockdown of N-cadherin or blocking function antibody impaired maintenance of long-term culture-initiating cells (LTC-IC) on coculture of HPC and MSC. These results indicate that N-cadherin is involved in the bidirectional interaction of human HPC with their cellular determinants in the niche.

Human mesenchymal stromal cells (MSC) have raised high hopes for tissue engineering and clinical therapy. Their isolation usually involves density fractionation of mononuclear cells (MNC) but this is difficult to standardize, especially under good manufacturing practice (GMP) conditions. MSC represent a heterogeneous mixture of cell types and the composition of subpopulations is affected by the initial steps of cell preparation.This study describes a straightforward method for isolation of human MSC based on red blood cell (RBC) lysis with ammonium chloride. Colony formation was compared directly with Ficoll density fractionation and culture of an untreated whole bone marrow (BM) aspirate.After 7 days the number of fibroblastic colony-forming units (CFU-F) per milliliter of BM aspirate was slightly higher upon RBC lysis and the colonies were significantly larger compared with density fractionation, possibly because of maintenance of platelets. In contrast, colony formation was much lower in untreated BM. The heterogeneous composition of subpopulations was reflected by differences between the initial colonies with regard to growth pattern (tight or disperse) and cell morphology (round or elongated). This heterogeneous composition was not affected by the three different isolation methods. Furthermore, enrichment of CD271(+) cells resulted in the same morphologic heterogeneity. All cell preparations demonstrated the same immunophenotype using a panel of surface markers and displayed adipogenic and osteogenic differentiation potential.This study demonstrates that human MSC can be efficiently isolated by RBC lysis. This technique is faster and can be standardized more easily for clinical application of MSC.

Scientific reports 3, 3372 (2013). doi:10.1038/srep03372, Published by Nature Publ. Group, London

The composition of mesenchymal stromal cells (MSCs) changes in the course of in vitro culture expansion. Little is known how these cell preparations are influenced by culture media, plating density, or passaging. In this study, we have isolated MSCs from human adipose tissue in culture medium supplemented with either fetal calf serum (FCS) or human platelet lysate (HPL). In addition, culture expansion was simultaneously performed at plating densities of 10 or 10,000 cells/cm2. The use of FCS resulted in larger cells, whereas HPL significantly enhanced proliferation. Notably, HPL also facilitated expansion for more population doublings than FCS (43 ± 3 vs. 22 ± 4 population doubling; p < 0.001), while plating density did not have a significant effect on long-term growth curves. To gain further insight into population dynamics, we conceived a cellular automaton model to simulate expansion of MSCS. It is based on the assumptions that the number of cell divisions is limited and that due to contact inhibition proliferation occurs only at the rim of colonies. The model predicts that low plating densities result in more heterogeneity with regard to cell division history, and favor subpopulations of higher migratory activity. In summary, HPL is a suitable serum supplement for isolation of MSC from adipose tissue and facilitates more population doublings than FCS. Cellular automaton computer simulations provided additional insights into how complex population dynamics during long-term expansion are affected by plating density and migration.

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34(+)CD38(-) fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34(+), CD133(+) and CD38(-)) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC-MSC interaction might further enhance cord blood expansion on MSC.

The regenerative potential diminishes with age and this has been ascribed to functional impairments of adult stem cells. Cells in culture undergo senescence after a certain number of cell divisions whereby the cells enlarge and finally stop proliferation. This observation of replicative senescence has been extrapolated to somatic stem cells in vivo and might reflect the aging process of the whole organism. In this study we have analyzed the effect of aging on gene expression profiles of human mesenchymal stromal cells (MSC) and human hematopoietic progenitor cells (HPC). MSC were isolated from bone marrow of donors between 21 and 92 years old. 67 genes were age-induced and 60 were age-repressed. HPC were isolated from cord blood or from mobilized peripheral blood of donors between 27 and 73 years and 432 genes were age-induced and 495 were age-repressed. The overlap of age-associated differential gene expression in HPC and MSC was moderate. However, it was striking that several age-related gene expression changes in both MSC and HPC were also differentially expressed upon replicative senescence of MSC in vitro. Especially genes involved in genomic integrity and regulation of transcription were age-repressed. Although telomerase activity and telomere length varied in HPC particularly from older donors, an age-dependent decline was not significant arguing against telomere exhaustion as being causal for the aging phenotype. These studies have demonstrated that aging causes gene expression changes in human MSC and HPC that vary between the two different cell types. Changes upon aging of MSC and HPC are related to those of replicative senescence of MSC in vitro and this indicates that our stem and progenitor cells undergo a similar process also in vivo.

Culture medium for mesenchymal stromal cells (MSC) is frequently supplemented with fetal calf serum (FCS). FCS can induce xenogeneic immune reactions, transmit bovine pathogens and has a high lot-to-lot variability that hampers reproducibility of results. Several studies have demonstrated that pooled human platelet lysate (HPL) provides an attractive alternative for FCS. However, little is known about the variation between different platelet lysates.We compared activities of individual HPL on initial fibroblastoid colony-forming units (CFU-F), proliferation, in vitro differentiation and long-term culture. These data were correlated with chemokine profiles of HPL.Isolation of MSC with either HPL or FCS resulted in similar CFU-F frequency, colony morphology, immunophenotype and adipogenic differentiation potential. Osteogenic differentiation was even more pronounced in HPL than FCS. There were significant differences in MSC proliferation with different HPL, but it was always higher in comparison with FCS. Cell growth correlated with the concentration of platelet-derived growth factor (PDGF) and there was a moderate association with platelet counts. All HPL facilitated expansion for more than 20 population doublings.Taken together, reliable long-term expansion was possible with all HPL, although there was some variation in platelet lysates of individual units. Therefore the use of donor recipient-matched or autologous HPL is feasible for therapeutic MSC products.