Bone Marrow Mesenchymal Stroma Cells Research Articles

Arsenic Trioxide Reprograms the Bone-Marrow Microenvironment to Sensitize Minimal Residual Disease in Acute Myeloid Leukemia

Eliminating minimal residual disease (MRD) in order to prevent relapse is an unmet need in acute myeloid leukemia (AML). One of the success stories is acute promyelocytic leukemia (APL), a unique subtype of AML. The use of arsenic trioxide (ATO) in combination with all-trans retinoic acid (ATRA) has essentially eliminated disease relapse (<1% ATRA+ATO vs. ~18% ATRA+ chemotherapy - NEJM (2013), 369(2), 111-121). This is even more impressive as neither agent alone results in significant cure of patients with APL. In fact, single agent ATRA induces remission without cure due to bone marrow stroma expression of CYP26 enzymes (PloS one (2015), 10(6), e0127790), which are induced by ATRA, inactivate retinoids and protect APL MRD (Leukemia (2020), 34(11), 3077-3081). While ATO may synergize with ATRA by restoring PML nuclear bodies (Nature medicine (2014), 20(2), 167-174) and rescuing RAR-α dependent transcription (Nature reviews. Cancer (2018), 18(2), 117-127) in the bulk of the APL tumor cells, it is unclear how these mechanisms contribute to elimination of MRD, particularly given the hypothesized lack of ATRA in the bone marrow niche. It was previously reported that combination of ATRA with ATO may be associated with high incidence of transaminitis and headaches (NEJM (2013), 369(2), 111-121). Since these may be clinical signs of ATRA toxicity, we investigated if addition of ATO changes the pharmacokinetics (PKs) of ATRA in patients with APL. We determined the area under the plasma concentration versus time curve (AUC) for ATRA in patients suspected of APL and treated with single agent ATRA (“preATO”) and in the same patients after the diagnoses of APL was confirmed and the patients received ATO (“postATO”). Even though ATRA should have an expected drop in AUC during the first 3-4 days of therapy (JPET (2017), 361(2), 246-258), addition of ATO resulted in improved ATRA PKs (Figure A). Since ATRA levels are mainly controlled by clearance via hepatic CYP26s, we tested if treatment with ATO changes the levels of CYP26 or its induction by ATRA. We found that exposure to ATO decreases both baseline and ATRA-induced upregulation of CYP26B1 in HepG2 cells (Figure B). Similarly, ATO decreases CYP26B1 levels in human bone marrow derived mesenchymal stroma cells (MSCs) at baseline (21%±7% of NO ATO control, n=3, p<0.01) and after ATRA-induced upregulation (66%±14% of ATRA only control, n=3, p<0.01). These changes resulted in improved ATRA PKs in vitro in the presence of bone marrow MSCs (Figure C). Next, we used NB4-EvAsR cells (NB4R), ATO-resistant APL cells to test if ATO-induced improvement of MSC-dependent ATRA PKs may contribute to elimination of APL MRD. We found that ATO sensitizes NB4R to ATRA in the presence of BM MSCs but not in stroma free conditions (Figure D). ATO had no effect on ATRA-induced differentiation of NB4R in the presence of CYP26B1 knockout stroma (this stroma offers no protection from ATRA) or if stroma expresses CYP26B1 under the control of a viral promoter and thus, resistant to ATO downregulation. This data suggest that ATO-induced changes of BM MSC expression of CYP26 may contribute to elimination of MRD in APL. Stromal CYP26 protects not only APL but also non-APL AML cells from retinoids (Leukemia (2020), 34(11), 3077-3081). Retinoids are synergistic with FLT3 inhibitors to eliminate mutant FLT3 AML cells (Blood (2016), 127(23), 2867-2878) and stromal CYP26 breaks this synergism (Blood (2015) 126 (23): 790). We tested if ATO sensitizes mutant FLT3 AML cells, MV4;11 to gilteritinib in the presence of BM MSCs. For this, we derived MV4.11 cells resistant to ATO (MV4.11R) via long term exposure to increasing concentration of ATO. As seen with NB4R, ATO treatment synergizes with gilteritinib to decrease the IC50 of gilteritinib against the clonogenic activity of MV4.11R in the presence of stroma but not in stroma free conditions (Figure E). In an effort to fully explore the translational potential of these findings, we are currently investigating the mechanism by which ATO changes CYP26 levels.Taken together these findings support a model in which ATO changes the bone marrow niche and it's ability to metabolize retinoids and thus, sensitizes MRD in AML to targeted therapy. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor. Ghiaur: Syros Pharmaceuticals: Consultancy; Menarini Richerche: Research Funding.

Early Growth Response (EGR)-1 Is a Key Regulator of Human Primary Bone Marrow Stroma Cells with a Dual Role in Proliferation and Hematopoietic Stroma Support Function

Hematopoietic stem cell (HSC) niches are specialized local tissue microenvironments that maintain HSCs and regulate HSC functions. Bone marrow mesenchymal stroma cells (BMSCs) are essential constituents of the HSC niche and they give rise to skeletal lineage cells as well as the hematopoietic stroma. Despite recent progress in the identification of the primary bone fide BMSC in human bone marrow, knowledge regarding the regulation of BMSC proliferation, differentiation and hematopoietic support function is incomplete.We have previously shown that the expression of Early Growth Response 1 (EGR1) in primary BMSCs is substantially higher compared to the non-colony-forming cells and cultured stromal cells, respectively, indicating a possible role of EGR1 in BMSC regulation (Li et al., Stem Cell Reports, 3(6):965-74, 2014). EGR1 encodes a zinc finger transcription factor that exemplifies a group of immediate early response genes, which have diverse functions in cell growth, development and stress response. EGR1 regulates murine HSC proliferation and localization (Min et al., Cell Stem Cell, 2(4):380-91, 2008), but its role in stroma cell regulation has thus far not been investigated. This study therefore aimed to investigate the functional role of EGR1 in human primary BMSCs.Quantitative gene expression analysis demonstrated that EGR1 expression was 126.9 ± 9.1-fold and 2.8 ± 0.2-fold higher in highly CFU-F (colony-forming unit, fibroblast) enriched human primary linneg/CD45neg/CD271pos/CD140aneg bone marrow cells compared to the non-colony forming CD45neg/CD271neg cell population and CD45neg/CD271pos/CD140apos cells with minimum CFU-F activity. Furthermore, we confirmed that EGR1 expression in cultured cells was down-regulated in comparison with primary BMSCs.Multi-lineage differentiation capacity and surface marker expression profiles of BMSCs were not affected by shRNA-induced EGR1 down-regulation. However, colony-forming capacity was enhanced (1.9 ± 0.1-fold) and population doubling times were reduced by EGR1 knockdown compared to scramble controls. Furthermore, the HSC stroma supporting function as measured by production of transplantable HSC in co-culture experiments (4-day serum-free culture supplemented with SCF 25 ng/ml, TPO 25 ng/ml, and FLT3L 25 ng/ml, 1×104 CD34pos seeded cord blood cells) was reduced when EGR1 knockdown cells were used as stroma layer.On the other hand, whereas CFU-F formation was virtually absent in EGR1 overexpressing cells and population doubling times increased by 2.4 ± 0.3-fold, the hematopoietic stroma support function was considerably improved by EGR1 overexpression as indicated by increased percentages and absolute numbers of transplantable CD34posCD90pos cells produced in co-cultures with EGR1 overexpressing BMSCs as stromal cells (12,496 ± 887 for EGR1 overexpression vs 6,867 ± 1242 for GFP control). Accordingly, CD34pos cells expanded on EGR1 overexpressing stroma exhibited improved in-vivo long-term reconstitution levels in NSG mice compared with cells grown on control stroma.To investigate how EGR1 regulates proliferation and hematopoiesis support in BMSCs, transwell experiments were performed showing that both soluble factors and cell-to-cell contact were important for EGR1-mediated hematopoiesis support. Furthermore, proteomic analysis revealed that a group of proteins related to oxidative-reduction processes was downregulated whereas reactive oxygen species (ROS) levels were elevated in EGR1 knockdown cells. In addition, gene expression profiling analysis identified a number of hematopoietic support genes that were upregulated in EGR1 overexpressing cells compared with control cells.In summary, our data indicate that EGR1 is an important BMSC regulator with a dual role on proliferation and hematopoietic stroma support function. EGR1 expression negatively regulated BMSC proliferation while positively regulating hematopoietic support function, which points to its possible role in coordinating the specific functions of BMSC in their different biological contexts. DisclosuresNo relevant conflicts of interest to declare.

Regulation of human stem and progenitor cells by acute myeloid leukaemia in the human niche

In acute myeloid leukemia (AML) the suppression of normal blood production is a major factor contributing to treatment complications and mortality rates. Recently it was demonstrated that cell cycle inhibition of haematopoietic stem and progenitor cells (HSPCs) may be the underlying cause of suppression in both AML xenograft and transgenic mouse models. However, the mechanism in human HSPCs is still poorly understood. Using a fully humanized ex vivo system comprising AML cell lines, umbilical cord blood derived CD34+ HSPCs and bone marrow mesenchymal stroma cells (MSCs), we studied the interactions between AML, normal HSPCs and their stromal niche. In our ex vivo system, AML but not normal white blood cells rapidly suppress the proliferation and differentiation of normal HSPCs via cell cycle inhibition of both (CD34+CD38+) progenitors and (CD34+CD38-CD45RA-CD90+) stem cells. In line with the finding of the in vivo AML models, AML did not induce apoptosis in HSPCs. Transcriptomic interrogation of a stroma cell line after AML exposure identified a hypoxia-response signature and an upregulation of Hypoxia inducible factor -1 alpha (HIF-1α) target genes. Hypoxia-induced stabilization of HIF-1α in MSCs recapitulated AML induced HSPC suppression, which was reversible by HIF-1α knockdown. Furthermore, we identified Stanniocalcin 1 (STC1), a small secreted glycoprotein, to be upregulated via HIF-1α dependent mechanisms in MSCs after AML exposure. Recombinant STC1 alone reduced HSPC proliferation and differentiation, using a STC1 neutralizing antibody, we were able to significantly reduce the AML-induced cell cycle inhibition of stem cells and increase the number of progenitors. In summary, we developed a fully humanized ex vivo system to study the interaction between AML, normal HSPCs and the stroma niche. We identified HIF-1α stabilization and its downstream target STC1 in MSCs as key contributors to the suppression of HSPC differentiation and proliferation, providing a potential route to release normal blood production from AML-induced suppression.

Improved adipogenic in vitro differentiation: comparison of different adipogenic cell culture media on human fat and bone stroma cells for fat tissue engineering.

To date there is no sufficient in vitro fat tissue engineering and a protocol has not been well established for this purpose. Therefore, we evaluated the in vitro influence of two different adipogenic growth media for their stimulation potential on different cell lineages to clearly define the most potent adipogenic growth media for future in vitro tissue engineering approaches. The samples for differentiation were composed of human adipogenic-derived stroma cells (hADSCs) and human bone marrow mesenchymal stroma cells (hMSCs). A normal adipogenic medium (NAM) and a specific adipogenic medium (SAM) were tested for their adipogenic stimulation potential. After 10 days and 21 days the relative gene expression was measured for the adipogenic marker genes PPARγ2, C/EBPα, FABP4, LPL, and GLUT4 detected through real time reverse transcriptase polymease chain reaction (RT-PCR). Other study variables were the comparison between NAM and SAM and between the used cells hADSCs and hMSCs. Additionally an Oil-Red staining was performed after 21 days. Our results revealed that only SAM was significantly (P<0.05) superior in the differentiation process in contrast to NAM for 10 days and 21 days. As well was SAM superior to differentiate the used cell lineages. This was evaluated by the detected marker genes PPARγ2, C/EBPα, FABP4, LPL, and GLUT4 through real time RT-PCR and by Oil-Red staining. In addition, the hMSCs proofed to be equal donor cells for adipogenic differentiation especially when stimulated by SAM. The results suggest that the SAM should be established as a new standard medium for a more promising in vitro adipogenic differentiation.

Molecular Alterations in Bone Marrow Mesenchymal Stroma Cells of AML Patients

It is increasingly recognized that the tumor microenvironment plays a pivotal role in cancer initiation and progression. In mouse models it was shown that a genetically altered bone marrow (BM) micro milieu was sufficient to induce leukemia (Raaijmakers, Nature 2010); however, the pathogenic role and contribution of the BM stroma in leukemia initiation and during disease progression warrants further investigation. To address this, we have performed gene expression, methylation, RNAseq, whole exome sequencing (WES) in BM mesenchymal stroma cells (BM-MSC) and leukemic cells from AML patients (pts) to unravel underlying molecular alterations.We collected BM hematopoietic cells (BM-HC) as well as plastic-adherent BM-MSC from aspirates from AML pts and healthy donors (HD). BM-MSC were expanded to passage 4 and defined as CD73+/CD105+/CD271+/low/CD45-/CD33-. We investigated gene expression profiles (Affymetrix) of BM-MSC from newly diagnosed AML pts (n=20) and compared these to BM-MSC from HD (n=4). BM-MSC from AML pts displayed an altered expression signature with 191and 175genesbeingsignificantly 2-fold over- and under-expressed. KEGG analysis of differentially expressed genes in BM-MSC from AML pts exhibited enrichment for TGF-ß signalling, whereas downregulated genes were enriched for cytokine receptor interactions. Several of these candidates were validated in a larger set of BM-MSC samples by RT-PCR. One putative stroma-leukemia interaction molecule, lumican (LUM) was highly overexpressed in BM-MSC (n=60) from AML pts compared to HD (n=5; p value =0.019) indicating that LUM may affect the BM niche in AML.To explore the altered expression pattern in AML BM-MSC compared to HD BM-MSC, global methylation analyses (Illumina Infinium HumanMethylation 450 bead chip arrays) were performed in 5 AML pts where we had collected BM-HC and BM-MSC at 3 sequential time points [initial diagnosis (ID), remission (CR), relapse (REL); n=30] as well as in BM-HC and BM-MSC from HDs (n=6). A significantly different methylation profile was evident comparing AML BM-HC to the corresponding AML BM-MSC samples, the latter showing a homogenous pattern during the course of disease. When AML BM-MSC were compared to a set of HD BM-MSC, we identified 2416 differentially methylated CpG sites (p value <0.01) indicating that an epigenetic deregulation contributes to the altered gene expression profile observed in AML BM-MSC.These 30 AML BM-MSC/BM-HC samples were subsequently analyzed by WES to unravel genetic alterations in the compartments of the mesenchymal and hematopoietic cell fractions. In WES (HiSeq2000, 100bp paired-end), we obtained an average of 100 reads for the target region; more than 90% of the exome target region was covered at least 30-fold. When the AML BM-HC CR sample was used as germline control, a median of 3 SNVs were detected in AML BM-MSC samples. The only BM-MSC-specific alteration present in one AML patient at all time points (ID, CR, REL) was a mutation in the plectin gene (PLEC). This mutation in the ROD domain of this cytoskeletal linker protein is located in the hot spot for mutations described in epidermolysis bullosa. This mutation was validated by Sanger sequencing, however in a larger cohort of 50 AML ID BM-MSC, no additional PLEC mutation at the same position was found. The set of AML BM-MSC (n=15) samples further allowed us to identify lesions (SNVs, Indels) in the corresponding BM-HC (n=15). When we used the AML BM-MSC as germline control we identified in total 43 lesions in the AML BM-HC fractions, which were not found when the corresponding BM-HC CR sample was used as germline control. This unraveled pre-leukemic lesions present in the AML BM-HC at remission: importantly using this approach, lesions in ASXL1 (Y591*) and DNMT3A (R882H), and in another patient a DNMT3A (M880V) mutation were revealed.In conclusion, the altered gene expression profile and methylation signature of AML BM-MSC provide novel insights into the pathogenic role of the leukemic BM microenvironment. Genetic alterations explored by WES revealed only very few genetic hits that will require further functional exploration. However, the low number of genetic alterations suggests that the transcriptional and epigenetic alterations are directed by extrinsic factors. At the same time, AML BM-MSC provides a non-hematopoietic derived germline control that allows to unravel pre-leukemic lesions in BM-HC. DisclosuresNo relevant conflicts of interest to declare.

Reconstitution with Bone Marrow Cells is Consistently Defective in the Tibia of Aged Monkeys

Biological aging is associated with a progressive loss of regulation of cellular, tissue and organ interaction, ultimately resulting in senescence. Fat gradually occupies bone cavity with age. Studies of the hematopoietic system using nonhuman primates have provided important information for understanding the mechanism of human hematopoiesis. We previously reported that excellent reconstitution of donor hematopoietic cells in a collagen gel group was observed in the long term in mice. We therefore investigated whether the method would be helpful for reconstituting hematopoiesis in the fatty tibias of monkeys. Nine- to 11-year-old monkeys were used for this study to examine if hematopoiesis could be restored in the fatty marrow of old monkeys. Bone marrow cells were collected from the humerus using the perfusion method, mixed with cultured bone marrow mesenchymalstroma cells in collagen gel, and then injected into the tibia of the same monkey. Clot sections were made from the tibias 1, 2 and 6 weeks after the bone marrow transplantation. However, no bone marrow cells were observed at any of the time points. These results suggested that bone marrow reconstitution is consistently defective in aged monkeys.

Efficacy and Mechanisms of Apoptosis Induction by Simultaneous Inhibition of PI3K with GDC-0941 and Blockade of Bcl-2 (ABT-737) or FLT3 (Sorafenib) In AML Cells In the Hypoxic Bone Marrow Microenvironment

AbstractAbstract 777Hypoxia and the interactions with bone marrow (BM) stromal cells have emerged as important mechanisms of leukemia cell survival and chemoresistance. The PI3K-Akt-mTOR-HIF1a signaling pathway has been shown to be activated in response to hypoxia in several cancer models including AML. Therefore, effective targeting of PI3K/Akt signaling pathway could suppress AML cell survival in the hypoxic BM microenvironment. Furthermore, concomitant intra-pathway blockade, or inhibition of the key pro-survival pathways may be more effective. In this study, we investigated the anti-leukemia effects and molecular mechanisms of apoptosis induction in the context of hypoxic bone marrow microenvironment by simultaneous use of a selective Class I PI3K inhibitor GDC-0941 (Genentech) with BH3 mimetic ABT-737 (Abbott) or FLT3 inhibitor sorafenib in AML cells. For hypoxia experiments, AML cells with FLT3-ITD mutation and wild-type FLT3 (FLT3-ITD: MOLM13, MV4;11, wt-FLT3: HL60) were cultured under 1.0% O2 for at least 14 days to assure their continuous proliferation and survival. Under hypoxia, more cells accumulated in G0/G1 phase, indicating that hypoxic conditions promote cell cycle quiescence in leukemic cells.GDC-0941 treatment in normoxia resulted in a reduction of cell proliferation with G0/G1 cell cycle arrest and minimal apoptosis induction, in a time and concentration-dependent manner (IC50 at 48 hrs; 0.7 μM for HL-60, 0.9 μM for MOLM13, 0.7 μM for MV4;11). In hypoxia, GDC-0941 further enhanced cell growth inhibition and G0/G1 cell cycle arrest. Importantly, co-culture with BM mesenchymal stroma cells (MSC), which protected AML cells from cytarabine induced apoptosis, did not affect cell growth inhibition by GDC-0941 both in normoxia and hypoxia. Combined treatment of GDC-0941 synergistically enhanced the ABT-737- or sorafenib induced apoptosis under both normoxic- and hypoxic conditions (p<0.05). Combination Index of GDC-0941/ABT-737 was 0.25 for HL-60 and 0.86 for MOLM13 cells; GDC-0941/sorafenib 0.58 for MV4;11 and 0.65 for MOLM13. In FLT3/ITD harboring MOLM13 and MV4;11 cells, ABT-737 and GDC-0941/ABT-737, or sorafenib and GDC-0941/sorafenib-induced apoptosis was partially reversed by MSC co-culture in normoxia, but not in hypoxia.We next examined the molecular mechanisms of apoptosis induction by simultaneous blockade of PI3K and mutant FLT3 in MOLM13 cells. GDC-0941 inhibited phospho-AktSer473, irrespective of oxygen tension. Hypoxia increased/induced phosphorylation of AktSer473, and of the pro-survival serine/threonine-protein kinase Pim-1, known to promote hypoxia-induced chemoresistance. Pim-1 is a downstream target of Akt and FLT-3 signaling that promotes cell survival and inhibits apoptosis through Bcl-2-dependent mechanisms. While GDC-0941 or ABT-737 alone did not affect hypoxia-induced Pim-1 levels, the GDC-0941/ABT-737 combination down-regulated Pim-1 by 86% (24 hrs). Treatment with sorafenib induced Pim-1 down-regulation by itself by 70%. Under hypoxia, expression levels of Bcl-2 family proteins Bcl-2 and BIM were not significantly changed by any treatment and cell culture conditions. In turn, Mcl-1 expression was upregulated by MSC co-cultures. Further, sorafenib alone but not GDC-0941 or ABT-737 decreased Mcl-1 protein levels, which was reversed by MSC co-culture in hypoxia; in turn, the GDC-0941/sorafenib or GDC-0941/ABT-737 combination potently suppressed Mcl-1 expression under MSC/hypoxia conditions. These changes were associated with the observed apoptotic responses.In summary, our findings indicate that hypoxic conditions of BM microenvironment result in stimulation of the pro-survival Akt and PIM kinase pathways and increase anti-apoptotic Mcl-1 protein in AML cells with mutated FLT3. In turn, simultaneous blockade of PI3K and FLT3 pathways, or of PI3K and Bcl-2 results in inhibition of these signaling modules and synergistic induction of apoptosis in AML. This data suggest that combined inhibition of Bcl-2 with PI3K or FLT3-ITD may constitute a targeted approach to eradicate chemoresistant AML cells sequestered in the hypoxic BM niches. Disclosures:No relevant conflicts of interest to declare.

Analysis of Chromosomal Aberrations and DNA Mutations in Bone Marrow Mesenchymal Stroma Cells from Patients with Myelodysplastic Syndrome Amd Acute Myeloid Leukemia

Introduction: The biology of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is not well understood. In MDS, ineffective hematopoiesis may result from disturbed interactions between hematopoietic cells (HC) and the hematopoietic microenvironment. Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. BMSC from patients with hematological disorders display functional and quantitative alterations. However, the question whether BMSC in MDS/AML have cytogenetic abnormalities is discussed controversially.Methods: In the present study, we have collected BMSC from 51 MDS and 42 AML patients at the time of initial diagnosis. Chromosome preparation was performed after cell culture for 30 days and analyzed by conventional cytogenetic (G-banding) and by different types of FISH-techniques. Furthermore, FLT3 and NPM1 mutation analysis was performed in HC and BMSC. As a control we have studied BMSC from 25 healthy individuals.Results: Cytogenetic analysis of BMSC was successfully performed in 90 of the 93 cases. Clonal structural chromosomal aberrations, including t(1;7), t(1;10), t(1;2), t(7;9), i(1q), inv(X), del(7q), del(13q), del(17p), and others, were detectable in BMSC of 15% of patients. All cytogenetic markers were confirmed by FISH with specific probes and M-FISH. Interestingly, cytogenetic markers in BMSC differed from the aberrations in HC from the same individual. We have found cytogenetic abnormalities in BMSC from patients presenting with cytogenetic alterations in their HC as well as from those with normal karyotype. In BMSC we could not detect specific mutations of NPM1 and FLT3 (ITD and TKD), independent from the mutation status of HC. For control analysis, BMSC cultures from 25 healthy donors were prepared under the same conditions (time of culture, number and frequency of passages). BMSC from healthy donors did show normal diploid karyotypes and absence of DNA-mutations of NPM1 and FLT3.Conclusions: Our data indicate that BMSC from MDS and AML patients are characterized by genetic instability. Lack of aberrations as detected in HC and appearance of novel clonal rearrangements in BMSC may suggest enhanced genetic susceptibility and potential involvement of BMSC in the pathogenesis of MDS and AML.

Cytogenetic and Molecular Genetic Aberrations in Bone Marrow Mesenchymal Stroma Cells from Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia.

Introduction : The biology of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is heterogeneous. Ineffective hematopoiesis results from complex interactions between hematopoietic cells (HC) and the hematopoietic microenvironment. Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. The question of whether BMSC from patients with hematological disorders have cytogenetic abnormalities is discussed controversially.Methods: We performed standard cytogenetic analyses (G-banding), FISH, M-FISH, and FLT3 mutation examinations of both HC and BMSC from 56 patients (30 MDS and 26 AML) and 9 healthy individuals. For BMSC selection, mononuclear cells were isolated from fresh bone marrow aspirates at the time of initial diagnosis and were further expanded in cell culture.Results: Clonal cytogenetic aberrations were observed in HC from 14 (46%) MDS and 14 (53%) AML patients. Cytogenetic analyses of BMSC were successfully performed in 50 of the 56 cases. Structural chromosomal aberrations, including t(1;7); t(1;3); t(1;10); t(4;7); t(7;9); t(7;19); i(1q); inv(X); del(1q); del(2q); del(3p); del(4p); del(11q); del(13q); del(17p), and others were detectable in BMSC from 42% of patients. The breakpoints of chromosomes in BMSC were typical for leukemia aberrations. Six patients showed clonal chromosomal markers: t(1;7), t(1;10), t(7;9), inv(X), del(17p), and monosomy 4. Interestingly, cytogenetic markers in BMSC differed to the aberrations in HC from the same individual. No deletions or monosomy of chromosomes 5 or 7 in BMSC (FISH, 500 cells) were found in BMSC, even in those patients, who showed these aberrations in HC. M-FISH confirmed chromosomal aberrations in BMSC. We have found cytogenetic abnormalities in BMSC from patients presenting with cytogenetic alterations in their HC as well as from those with normal karyotype. We did not find structural chromosomal alterations in BMSC cultures of healthy bone marrow donors. Fourteen percent of AML patients showed FLT3 mutations in HC, but no FLT3 mutations were found in BMSC.Conclusion: We showed that BMSC from AML and especially from MDS patients are characterized by genetic instability. The breakpoints of chromosomes in BMSC were typical for leukemia aberrations. The fact that BMSC showed typical chromosomal changes may suggest enhanced genetic susceptibility and potential involvement in the pathophysiology of MDS. Characterization of BMSC may help us to better understand the biology of this disease.

Chromosomal aberrations in bone marrow mesenchymal stroma cells from patients with myelodysplastic syndrome and acute myeloblastic leukemia

Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. The question of whether BMSC from patients with hematological disorders have cytogenetic abnormalities is discussed controversially, some studies indicating that they are cytogenetically normal and others providing evidence of their aberrations. We performed standard and molecular cytogenetic analyses of both hematopoietic cells and BMSC from 31 patients with myelodysplastic syndrome (MDS, n = 18) and acute myeloid leukemia (AML, n = 13) and 7 healthy individuals. Mononuclear cells were isolated from fresh bone marrow aspirates at the time of initial diagnosis for cytogenetic analysis of hematopoietic cells (HC) and selection of BMSC. Clonal cytogenetic aberrations were observed in HC from 8 (44%) MDS and 8 (61%) AML patients. Cytogenetic analyses of BMSC were successfully performed in 27 of the 31 cases. Structural chromosomal aberrations, including t(1;7), t(4;7), t(7;9), t(7;10), t(7;19), t(15;17), and others, were detectable in BMSC from 7 of 16 (44%) MDS and 6 of 11 (54%) AML patients. The breakpoints of chromosomes in BMSC were typical for leukemia aberrations. Two patients showed clonal chromosomal markers. BMSC from MDS and AML patients show chromosomal abnormalities. Although the majority of cytogenetic aberrations in BMSC were not clonal and differed from chromosomal markers in HC from the same individual, detection of typical chromosomal changes in BMSC suggests enhanced genetic susceptibility of these cells in MDS/AML. This may indicate potential involvement of BMSC in the pathophysiology of MDS/AML.

Alteration on the Expression of IL-1, PDGF, TGF-β, EGF, and FGF Receptors and c-Fos and c-Myc Proteins in Bone Marrow Mesenchymal Stroma Cells from Advanced Untreated Lung and Breast Cancer Patients

Previously, we reported a deficient cloning capacity of the bone marrow (BM) mesenchymal stem cells to give colony-forming unit fibroblast (CFU-F) and an inefficient confluence capacity of BM stromal cells in advanced untreated lung cancer patients (LCP) and breast cancer patients (BCP). Moreover, a decreased level of bFGF at day 7 in the conditioned media from BM CFU-F cultures was found in both cancer groups when compared to the normal range. The current study was specially undertaken, to evaluate the percentage of subconfluent fibroblasts expressing receptors (R) of interleukin-1 (IL-1), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF-beta), epidermal growth factor (EGF), and the proteins c-Fos and c-Myc in BM primary cultures from untreated LCP and BCP. An immunocytochemical study on subconfluent BM fibroblast cultures from 13 healthy patients, 16 LCP, and 8 BCP was performed, using as primary antibodies, anti-type I of IL-1 R (IL-1R-1), anti-alpha, beta chains of PDGF R (PDGFR-alpha, PDGFR-beta), anti-type I of FGF R (FGFR-I), anti-type I, II, and III of TGF-beta R (TGF-betaR-I, TGF- betaR-II, and TGF-betaR-III), anti-EGF R, anti-c-Fos, and anti-c-Myc. A diminished percentage of subconfluent fibroblasts expressing PDGFR-alpha, TGFbetaR-I, II, III, EGFR, and FGFR-I was found in LCP and BCP compared to healthy patients. A diminished percentage of subconfluent fibroblasts expressing c-Fos and c-Myc was found in patients when compared to healthy patients. The alterations we describe could help to explain the deficiency regarding the proliferative and confluence capacity of BM stroma cells in cancer patients.