Characterization of the Hematopoietic Supporting Activity of Osteoblasts Derived from Bone Marrow Mesenchymal Stromal Cells

Abstract Osteoblasts (OST) found within the endosteal niche are important regulators of Hematopoietic Stem and Progenitor Cells (HSPC) under steady state and during hematopoietic reconstitution. OST are derived from mesenchymal stromal cells (MSC) following osteogenic differentiation. MSC and OST secrete a wide array of soluble factors that sustain hematopoiesis. Recently, we showed that media conditioned with OST derived from MSC (referred as M-OST) after 6 days of osteogenic differentiation were superior to MSC conditioned media (CM) for the expansion of cord blood (CB) progenitors, and CB cells expanded with M-OST CM supported a more robust engraftment of platelets in NSG mice after transplantation. These findings raised the possibility that M-OST could be superior to MSC for the ex vivoexpansion HSPC. In this study, we set out to test the hypothesis that the growth modulatory activity of M-OST would vary as a function of their maturation status. The objectives were to first monitor the impact of M-OST differentiation and maturation status on the expression of soluble factors that promote HSPC expansion and in second, to investigate the capacity of M-OST CMs prepared from M-OST at distinct stages of differentiation to support the expansion and differentiation of HSPCs in culture. M-OST at distinct stages of differentiation were derived by culturing bone marrow MSC in osteogenic medium for various length of time (3 to 21 days). All CB CD34+ enriched (92±7% purity) cell cultures were done with serum free media conditioned or not with MSC or M-OST and supplemented with cytokines SCF, TPO and FL. We first confirmed the progressive differentiation and maturation of M-OST as a function of osteogenic culture length, which was evident by the induction of the osteogenic transcription factors Osterix, Msx2 and Runx2 mRNAs, the gradual increase in osteopontin and alkaline phosphatase positive cells and quantitative increases in calcium deposit. Next, we investigated the expression in MSC and M-OSTs of genes known to collaborate for the expansion of HSPCs by Q-PCR. Transcript copy numbers for IGFBP-2 increased swiftly during osteogenic differentiation, peaking at day-3 (˃100-fold vs MSC, n=2) and returning below MSC level by day-21. In contrast, ANGPTL members (ANGPTL-1, -2, -3 and -5) remained superior in M-OSTs throughout osteogenic differentiation with expression levels peaking around day 6 (n=2). Next, we tested the capacity of media conditioned with primitive (day-3, -6), semi-mature (day-10, -14) and mature M-OST (day-21) to support the growth of CB cells. All M-OST CMs increased (p˂0.03) the growth of total nucleated cells (TNC) after 6 days of culture compared to non-conditioned medium used as control (mean 2.0-fold, n=4). Moreover, there was a positive correlation between cell growth and M-OST maturation status though differences between the different M-OST CMs tested were not significant. The capacity of M-OST CMs to increase (mean 2-fold, n=4) the expansion of CD34+ cells was also shared by all M-OST CMs (p˂0.05), as supported by significant increases with immature day-3 (mean ± SD of 18 ± 6, p˂0.02) and mature day-21 M-OST CMs (14 ± 5, p˂0.05) vs. control (8 ± 3, n=4). Conversely, expansions of TNC and CD34+ cells in MSC CM cultures were in-between that of control and M-OST CMs cultures. Interestingly, M-OST CMs also modulated the expansion of the HSPC compartment. Indeed, while the expansion of multipotent progenitors defined as CD34+CD45RA+ was promoted in control culture (ratio of 4.5 for CD34+CD45RA+/CD34+CD45RA- cells), M-OST CMs supported greater expansion of the more primitive CD34+CD45RA- HSPC subpopulation reducing the ratio to 3.3±0.4 for M-OST cultures (cumulative mean of 10 cultures, n=2). Moreover, the expansions of CD34+CD38- cells and of the long term HSC-enriched subpopulation (CD34+CD38-CD45RA-Thy1+) in M-OST CM cultures were respectively 2.7- and 2.8-fold greater than those measured in control cultures (n=2-4). Finally, the impact of M-OST CMs on the expansion of myeloid progenitors was investigated using a colony forming assay; expansion of myeloid progenitors were superior in all M-OST CM cultures (1.6±0.2 fold, n=2). In conclusion, our results demonstrate that M-OST rapidly acquire the expression of growth factors known to promote HSPC expansion. Moreover, the capacity of M-OST CMs to support the expansion of HSPCs appears to be a property shared by M-OST at various stages of maturation. Disclosures No relevant conflicts of interest to declare.

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Direct Comparison of Wharton Jelly and Bone Marrow Derived Mesenchymal Stromal Cells to Enhance Engraftment of Cord Blood CD34+ Transplants

Blood ◽

10.1182/blood.v126.23.5410.5410 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5410-5410

Author(s):

Mark van der Garde ◽

Melissa Van Pel ◽

Jose Millan Rivero ◽

Alice de Graaf-Dijkstra ◽

Manon Slot ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Waste Product ◽

Human Platelets ◽

Human Umbilical Cord ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Co-transplantation of CD34+ hematopoietic stem and progenitor cells (HSPC) and mesenchymal stromal cells (MSC) enhances HSPC engraftment. For these applications, MSC are mostly obtained from bone marrow. However, MSC can also be sourced from Wharton's jelly (WJ) of the human umbilical cord, which as a 'waste product', is cheaper to acquire and without significant burden to the donor. Here, we evaluated the ability of WJ MSC to enhance HSPC engraftment. First, we compared cultured human WJ MSC with human bone marrow-derived MSC (BM MSC) for in vitro marker expression, immunomodulatory capacity and differentiation into three mesenchymal lineages. Although we confirmed that WJ MSC have a more restricted differentiation capacity, both WJ MSC and BM MSC expressed similar levels of surface markers and exhibited similar immune inhibitory capacities. Co-transplantation of either WJ MSC or BM MSC with CB CD34+ cells into NOD-SCID mice showed faster recovery of human platelets and CD45+ cells in the peripheral blood and a 3-fold higher engraftment in the BM, blood and spleen six weeks after transplantation when compared to transplantation of CD34+ cells alone. Upon co-incubation, both MSC sources increased the expression of adhesion molecules on CD34+ cells, although SDF-1-induced migration of CD34+ cells remained unaltered. Interestingly, there was an increase in CFU-GEMM when CB CD34+ cells were cultured on monolayers of WJ MSC in the presence of exogenous thrombopoietin, and an increase in BFU-E when BM MSC replaced WJ MSC in such cultures. Our results suggest that WJ MSC is likely to be a practical alternative for BM MSC to enhance CB CD34+ cell engraftment. Disclosures No relevant conflicts of interest to declare.

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Bone Marrow Mesenchymal Stromal Cells and Inflammation Contribute to ETV6-RUNX1+ Preleukemic Cells Persistence and DNA Damaging

Blood ◽

10.1182/blood-2018-99-114791 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3918-3918

Author(s):

Linda Beneforti ◽

Erica Dander ◽

Silvia Bresolin ◽

Clara Bueno ◽

Geertruy te Kronnie ◽

...

Keyword(s):

Bone Marrow ◽

B Cell ◽

Inflammatory Cytokines ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Fold Increase ◽

Mrna Levels ◽

Soluble Factors ◽

Cd34 Cells

Abstract INTRODUCTION ETV6-RUNX1 (ER), generated from translocation t(12;21), is the most frequent fusion gene in pediatric cancers, exclusively leading to B-Cell Precursor Acute Lymphoblastic Leukemia. Translocation occurs in fetal hematopoietic stem-progenitor cells (HSPC) but it is insufficient for disease. ER, in fact, is an aberrant transcription factor that expands a silent preleukemic clone with enhanced self-renewal and partial B cell differentiation. Secondary hits are thus required to complete transformation. Epidemiological and experimental data indicate that infections/inflammation play an important role in the preleukemia to leukemia transition. We previously demonstrate that TGFβ1, a pleiotropic cytokine produced after inflammation, favored the persistence of ER+Ba/F3 cells and selected putative preleukemic stem cells in ER+umbilical cord blood (UCB) CD34+cells. We also demonstrated that ER+Ba/F3 showed altered expression of adhesion molecules and impaired migration towards CXCL12. Migration, physical interactions and response to soluble factors determine HSPC fate in the Bone Marrow (BM) niche. BM Mesenchymal Stromal Cells (MSC) are non-redundant regulators of HSPC in the niche; in addition, they possess pro- and anti-inflammatory properties, representing a bridge between hemopoiesis and inflammation. Finally, dysfunctions in MSC can induce myelodisplasia and secondary myeloid leukemia, while MSC inflammation cause genotoxicity in HSPC predicting myeloid leukemia evolution in predisposing syndromes. On that basis, we questioned if interaction between ER+cells, MSC and inflammation could favor preleukemic clone persistence and progression. METHODS The murine proB cell line Ba/F3 was transfected to generate an inducible ER-V5tag expressing model (Ford A, Palmi C, 2009). BM-MSC were characterized and cultured for controlled passages. UCB-CD34+cells were immunomagnetically isolated and lentivirally transduced with pRRL-eGVP or pRRL-ER-eGFP constructs. Cells were treated with IL6/IL1β/TNFα inflammatory cytokines. RESULTS Gene Expression Profile shows that ER affects pathways involved in inflammatory response, cell cycle, apoptosis and migration in Ba/F3. In particular, ER+ cells overexpress CXCR2, a chemokine receptor also implicated in cancer, (MFI: ER=1378±807 vs ctr=284±167, p<0.05) and highly migrate toward its ligand CXCL1 (% migrated cell/input: ER=21.5±6.7 vs ctr=2.2±1.8, p<0.01). Interestingly, MSC increases CXCL1 secretion after inflammatory stimulation (murine MSC, pg/mL: basal=78±28 vs +infl.ck=30162±4760, p<0.01). In accordance, ER+ Ba/F3 are highly attracted by inflamed MSC supernatants (% migrated cell/input: ER=30.2±9.1 vs ctr=14.3±9.6, p<0.01) in a CXCR2-dependent manner. Coculturing control and ER+ Ba/F3 with MSC and inflammatory cytokines favored the persistence of preleukemic cells in the coculture (% ER+ fold increase: +MSC vs +MSC+infl.ck = 2.62±0.94, p<0.01). The effect is mediated by soluble factors and results from decreased survival in control (% ann-V negative cells: +MSC=68.4±5.7 vs +MSC+infl.ck=48.2±1.3, p<0.05) but not ER+ Ba/F3; cell proliferation was reduced in both, but the effect was stronger on control Ba/F3 (CSFE MFI fold increase +MSC vs +MSC+infl.ck: ER=2.2±0.6, p<0.001; ctr=4.4±1.8, p<0.05). However, CXCL1 is not implicated. Phosphorilation of histone H2AX and AID mRNA levels, which are basally higher in ER+ Ba/F3, further increase in both normal and ER+ Ba/F3 cocultured with MSC and inflammatory cytokines, confirming the genotoxicity of MSC inflammation (γH2AX MFI fold increase +MSC vs +MSC+infl.ck: ER=2.5±1, p<0.05; ctr=2.8±1.2, p<0.01) (AID mRNA fold increase basal vs +MSC+infl.ck: ER=6.3±1.6, p<0.05; ctr=14.6±11). Finally, preliminary data show a higher migration towards inflamed MSC also in ER+ UCB-CD34+cells (% migrated cell/input: ER=21.2±2.4 vs ctr=5.2±0.6, p<0.01). CONCLUSIONS ER expression increases migration towards inflamed BM-MSC supernatants in murine proB cells. Interestingly, MSC and inflammation create favoring microenvironmental conditions for preleukemic cells persistence and DNA damage accumulation. Preliminary results show that inflamed MSC highly attract human ER-expressing UCB-CD34+as well. Collectively, our data support the importance of ER-driven alterations in hematopoietic/BM stromal cells interactions in the leukemogenic process. Disclosures No relevant conflicts of interest to declare.

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Chd1 Regulates Primitive State of Bone Marrow Mesenchymal Stromal Cells without Affecting Hematopoietic Support; An Insight on the Dissociation Between Primitive State and Niche Function

Blood ◽

10.1182/blood.v124.21.1587.1587 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1587-1587

Author(s):

Il-Hoan Oh ◽

Hyun-Kyung Choi

Keyword(s):

Bone Marrow ◽

Chromatin Remodeling ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Expression Level ◽

Control Group ◽

Open Chromatin ◽

Hematopoietic Stem ◽

Primitive State

Abstract Mesenchymal stromal cells (MSCs) are characterized by heterogeneity in the proliferation/self-renewal potentials and hematopoietic supporting activity among subpopulations. Numerous studies have suggested that a primitive state of MSC subpopulation are correlated to its niche function to support hematopoietic stem cells (HSCs), but the mechanisms regulating primitive state of MSCs remains poorly understood. In the present study, we examined the role of a chromatin remodeling enzyme, chd1 in the maintenance of open chromatin and undifferentiated state of MSCs. We analyzed for expression in MSCs, the expression level of chd1 progressively decreased during in-vitro subculture (from 7 to 18 passages) in a manner proportional to the passage numbers. Moreover, chd1 expression was down regulated in the MSCs during their differentiation into adipogenic or osteogenic lineages, compared to proliferative state, indicating the correlations between MSC proliferation potentials and expression level of chd1. Next, we transduced human bone marrow-derived MSCs with shRNAs against chd1 and found that chd1 knock down MSCs (chd1-KD) exhibit significant loss of colony forming activity (CFU-F), decrease of cell proliferation and loss of multi-lineage differentiation towards osteogenic or adipogenic lineages. Moreover, chd1-KD MSCs exhibited lower level expression of pluripotency-related genes, oct-4, sox-2 and nanog, with concomitant increase of H3K9me3 on the promoters and decreased chromatin accessibility in the oct-4 promoter, suggesting that chd1 regulate open chromatin and multi-lineage potential of MSCs. However, KD of chd1 in MSCs did not affect the HSC-supporting activity of MSCs; human cord blood-derived CD34+ cells co-cultured on chd1-KD MSCs exhibited rather higher maintenance of primitive phenotype (CD34+90+) and higher repopulating activity in NOD/SCID-ɤC KO mice compared to those co-cultured on control group MSCs. Together, these results show that, while primitive state of MSCs are regulated by chromatin remodeling complex,chd1, the hematopoietic niche activity of MSCs is not directly influenced by the primitive state of MSCs, raising a questions on the prevailing notion that undifferentiated MSCs can better support hematopoietic function. Disclosures No relevant conflicts of interest to declare.

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Oxygen Tension Regulates Hematopoietic Stem Cell Migration Into An in-Vitro Niche Beneath Mesenchymal Stromal Cells.

Blood ◽

10.1182/blood.v114.22.1443.1443 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1443-1443

Author(s):

Duohui Jing ◽

Nael Alakel ◽

Martin Bornhauser ◽

Gerhard Ehninger ◽

Rainer Ordemann

Keyword(s):

Stem Cell ◽

Oxygen Tension ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Hypoxic Conditions ◽

Migratory Capacity ◽

The Impact

Abstract Abstract 1443 Poster Board I-466 Background Hematopoietic stem cells (HSCs) are located mainly in the bone marrow interacting with a specific microenvironment called “stem cell niche”. The niche has been proven to be critical for stem cell regulation. Coculture with mesenchymal stromal cells (MSCs) has been used as an in vitro model to investigate the interaction between HSCs and MSCs. In our study we investigated the impact of normoxia and hypoxia on the distribution of HSC subsets with regard to their spatial localization in cocultures during ex-vivo expansion. Design and Methods Three HSC subsets are defined: (i) cells in supernatant (non-adherent cells); (ii) cells adhering on the MSC layer surface (phase-bright cells); (iii) cells beneath the MSC layer (phase-dim cells). Using pimonidazole binding we investigated the spatial distribution of hypoxic cells in various cell subsets. Cell cycle, cell division, immunophenotype and migratory capacity of the three HSC subsets under distinct oxygen tension were studied. In addition the impact of oxygen tension on HSCs via VEGF-A and SDF-1 were analyzed by ELISA and gene knockdown with siRNA. Results First we could show that phase-bright cells contained the highest proportion of cycling progenitors. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype. Next pimonidazole binding revealed that the most hypoxic area in the coculture is the compartment beneath MSC layer. Then we investigated the impact of hypoxia conditions on HSCs in cocultures. We could demonstrate that under hypoxic conditions phase-bright cells were significantly diminished and phase-dim cells were increased. Interestingly, the migratory capacity of phase-bright cells from cocultures performed under hypoxic conditions was consistently enhanced in comparison to normoxia (32.7 ±2.2.0% vs 17.6 ±2.6%, p<0.01). Surprisingly, the SDF-1 concentration was lower after hypoxic coculture (189 ±33μg/ml vs 352 ±40μg/ml, p<0.05). In contrast, the VEGF-A concentration was significantly increased compared to normoxic conditions (7.7 ±1.2ng/ml vs 4.5 ±1.0ng/ml, p<0.01). In addition we could demonstrate that the lower adhesion and higher migratory capacity of HSCs under hypoxia can be partially inversed by silencing VEGF-A with siRNA in MSCs. Conclusions Our data indicate that under our experimental conditions, the MSC surface is the dominant location where HSCs proliferate, whereas the compartment beneath the MSC layer seems to be a hypoxic niche dedicated to the maintenance of HSC stemness. The lower levels of SDF-1 in the supernatant may be explained by the increased internalization of SDF-1 by MSCs when cultured together with HSC. This hypothesis will require the concomitant analysis of protein and SDF-1 mRNA in MSC. In addition our data suggest that low oxygen tension facilitates HSC migration into the in-vitro niche provided by MSCs which preserves immaturity of HSCs and modifies the cytokine profile of MSCs. Disclosures No relevant conflicts of interest to declare.

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Targeting Bone Marrow Mesenchymal Stromal Cells Using Cre-Recombinase Transgenes

Blood ◽

10.1182/blood.v126.23.2401.2401 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2401-2401

Author(s):

Jingzhu Zhang ◽

Daniel C. Link

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Stromal Cell ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Sympathetic Nerves ◽

Cell Populations ◽

Hematopoietic Stem ◽

Lineage Mapping

The bone marrow microenvironment contains hematopoietic niches that regulate the proliferation, differentiation, and trafficking of hematopoietic stem/progenitors cells (HSPCs). These hematopoietic niches are comprised of a heterogeneous population of stromal cells that include, endothelial cells, osteoblasts, CXCL12-abundant reticular (CAR) cells, mesenchymal stem cells (MSCs), arteriolar pericytes, and sympathetic nerves. Emerging data suggest that specific stromal populations may regulate distinct types of HPSCs. Thus, it is important to have validated approaches to interrogate and target specific stromal cell populations. Prior studies have shown that Prx1-Cre, Osx-Cre, Lepr-Cre, and Nes-Cre broadly target mesenchymal stromal cells in the bone marrow. Here, we rigorously define the stromal cell populations targeted by two Cre-transgenes that are commonly used to target osteolineage cells (Ocn-Cre, and Dmp1-Cre) and introduce a new Cre-transgene (Tagln-Cre) that efficiently targets bone marrow pericytes. For each Cre-transgene, we performed lineage mapping using ROSA26Ai9/Ai9 mice, in which cells that have undergone Cre-mediated recombination express tdTomato. In some cases, we further crossed these mice to introduce the Cxcl12gfp transgene, which can be used to define GFP-bright CAR cells. Immunostaining of bone sections and flow cytometry were used to define the target stromal cell population(s) in these mice. Osteocalcin (Bglap, Ocn) is primarily expressed in mature osteoblasts. Accordingly, Ocn-Cre is widely used to specifically target osteoblasts. However, our lineage mapping studies show that Ocn-Cre targets not only all osteoblasts, but also 72 ± 4.0% of CAR cells. Ocn-Cre also targets a subset of NG2+ arteriolar pericytes. Dentin matrix acidic phosphoprotein 1 (Dmp1) is expressed primarily in osteocytes, and Dmp1-Cre has been widely used to specifically target osteocytes. However, we show that Dmp1-Cre also efficiently targets endosteal osteoblasts and approximately 40% of CAR cells. To target bone marrow pericytes, we tested several Cre-transgenes, ultimately focusing on Tagln-Cre. Transgelin (Tagln, SM22a) is broadly expressed in pericytes, smooth muscle cells, and cardiomyocytes. Lineage-mapping studies show that Tagln-Cre targets all arteriolar and venous sinusoidal pericytes in the bone marrow. It also targets osteoblasts and 75 ± 5.2% of CAR cells. There are several recent studies that have ascribed specific functions to osteoblasts or osteocytes based on targeting using Ocn-Cre or Dmp1-Cre, respectively. In light of our data, these conclusions need to be re-evaluated. Ocn-Cre, Dmp1-Cre, and Tagln-Cre each target a subset of CAR cells. Studies are underway to determine whether these CAR subsets have unique expression profiles and functions. Finally, Talgn-Cre represents a new tool for investigators in the field to efficiently target bone marrow pericytes. Disclosures No relevant conflicts of interest to declare.

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Alteration of the Hematopoietic Components, Mesenchymal Stromal Cells and Vascular Elements in the Bone Marrow Niche in Myelodysplastic Syndrome: Refractory Cytopenia with Multilineage Dysplasia

Blood ◽

10.1182/blood.v128.22.5516.5516 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5516-5516

Author(s):

Salar Abbas ◽

Aparna Venkatraman ◽

Sanjay Kumar ◽

Archana Kini ◽

Marie Therese Manipadam ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Proliferation Index ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Monosomy 7 ◽

Multilineage Dysplasia ◽

Vascular Elements

Abstract Introduction:The myelodysplastic syndromes (MDS) are clonal disorders characterized by cytopenias and abnormal hematopoiesis. Though there are reports of perturbations in the hematopoietic stem cells (HSC) and the mesenchymal stromal cells (MSC) as well as other elements of the bone marrow (BM) niche in an instructive or permissive manner leading to the genesis of ineffective hematopoiesis in this condition, most studies have evaluated single elements. Here we demonstrate altered HSC, MSC and the vascular niche elements in patients with MDS - Refractory cytopenia with multilineage dysplasia (RCMD). Methods and results: Bone marrow aspirates from patients with RCMD (n=12, mean age 54.33±14.51 years) were compared with those from age-matched controls (n=18, mean age 47.78±18.60 years) who had a 'normal' marrow obtained for other diagnostic purposes. Cytogenetic analysis showed abnormal karyotypes in seven patients and normal karyotypes in five. The abnormalities seen were as follows: deletion 7q/monosomy 7 (four patients), deletion 5q (three patients), loss of Y (two patients), monosomy 5, deletion 20q (one each), complex karyotypes (four patients). Phenotypic enumeration of HSPCs revealed a marked decrease in the frequency of highly purified HSCs (Lin-CD34+CD38-CD90+CD45RA-) in RCMD (0.04135±0.01748%, n=11) when compared to controls (0.3168±0.05266%, n=13, p<0.0001) (Figure 1). Patients with RCMD also had increased common myeloid progenitors (CMP) (3.221 ± 0.7478%, n=8) compared to control (1.243±0.4463%, n=10, p<0.0302) and loss of granulocyte-macrophage progenitors (GMP) (0.4863±0.1638%, n=8) compared to controls (2.047±0.5422%, n=10, p<0.0242) (Figure 2). Assessment of the frequency of de novo MSCs (CD31-CD45/CD71- population) expressing CD271 and/or CD146, indicating the more primitive population, in total nucleated cells showed increased CD271+CD146-MSCs (0.632±0.2, n=5) compared to controls (0.2000±0.05158, n=6, p<0.0401) as also the CD271+CD146+ MSCs (0.2900±0.09803, n=5) in RCMD patients when compared to controls (0.02861±0.01354, n=6, p<0.0172) (Figure 3). We also evaluated in vitro cultured MSCs (P4) in these patients. CD271+CD146+ MSCs within total cultured MSCs were higher (0.1900±0.06429%, n=3) than in controls (0.0040±0.0040%, n=3, p<0.0447). RCMD MSCs had significantly lower proliferation index (32±3.7%, n=3) compared to controls (60±9.2%, n=3, p<0.0479). Cell cycle analysis of MSC showed significantly lower numbers in G0 in RCMD (0.1567 ± 0.07881 %, n=3) compared to control (0.6400 ± 0.1007 %, n=3; p<0.0194). Apoptosis was much higher in RCMD MSCs (2.700±0.8007%, n=3) compared to controls (0.03633±0.01802%, n=3, P<0.0292). No significant differences in expression profile of stem cell maintenance related cytokines and growth factors (CXCL12a, SCF, VEGF, ANGPT and LIF ) or components of Notch (Notch1, Notch3, Jagged-1, Delta like-1 and Hes1) and Wnt (Dkk1 and Dkk-2) pathways were found between RCMD and control MSCs within the limited numbers evaluated. Interestingly, unlike controls, immunofluorescence imaging of bone marrow trephine from MDS-RCMD revealed CD271+CD146+ MSCs co-localized with sinusoids and in direct contact with CD34+ (<5% blast) HSC/progenitor cells (HSPCs). Conclusion: Our data shows that in patients with MDS-RCMD, both qualitative and quantitative abnormalities exist in the HSC, HSPC and niche elements. We also show that the cytopenia could be related to decreased numbers of primitive HSCs and a differentiation arrest at the CMP stage. Primitive MSCs are reduced, and those that exist show poorer proliferative and survival features. Further studies are needed to understand the cause and effect relationship of these changes. Disclosures No relevant conflicts of interest to declare.

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Prospective Isolation and Functional Characterization of Human Bone Marrow-Derived Hematopoietic Stem Cell-Supportive Mesenchymal Stromal Cells.

Blood ◽

10.1182/blood.v116.21.3852.3852 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3852-3852

Author(s):

Yoshikazu Matsuoka ◽

Yutaka Sasaki ◽

Masaya Takahashi ◽

Ryusuke Nakatsuka ◽

Yasushi Uemura ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Scid Mice ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells

Abstract Abstract 3852 (Background) The identification of human CD34-negative (CD34−) SCID-repopulating cells (SRCs) provide a new concept for the hierarchy in the human HSC compartment (Blood 101:2924, 2003). Recently, we succeeded to highly purify these CD34-SRCs using 18 lineage specific antibodies (Blood 114:336, 2009). It has been suggested that human hematopoietic stem cell (HSC)-supportive microenvironment exist in the bone marrow (BM), which play a pivotal role in the maintenance of self-renewal capacity and dormancy of primitive HSCs. It was reported that osteoblasts and vascular endothelial cells played an important role to organize HSC niches. However, whether mesenchymal stromal cells (MSCs) contribute to organize HSC niches or not is not clearly understood, because MSCs are heterogeneous population. Therefore, it is important to clarify their origin and functional characteristics. (Objectives) The aim of this study was to prospectively isolate/identify human BM-derived MSCs and investigate their functional characteristics including HSC-supportive abilities. (Results) First, human BM-derived Lin−CD45− cells were subdivided into 4 fractions according to their expression levels of CD271 and SSEA-4 by FACS. We succeeded to isolate 3 MSC lines from these 4 fractions, including CD271+/−SSEA-4+/− cells. Approximately 1 out of 6 CD271+SSEA-4+ (DP) cells could form MSC-derived colony. These DP cells-derived MSCs could differentiate into osteoblasts and chondrocytes, but could not differentiate into adipocytes. In contrast, CD271+SSEA-4− cells and CD271−SSEA-4− cells-derived MSCs could differentiate into three lineages. Then, we assessed CD34− SRC-supportive activity of these 3 MSC lines. First, certain numbers of 18Lin−CD34− cells were cocultured with 3 MSC lines for 1 week, respectively. Next recovered cells were transplanted into NOD/SCID mice by intra-bone marrow injection (IBMI) to investigate SCID-repopulating cell (SRC) activity. After 8 weeks, the highest CD45+ human cell engraftments (0.1 % to 32.4 %, median 8.6 %) were observed in mice received 18Lin−CD34− cells cocultued with DP cells-derived MSCs. As recently reported (Cell Stem Cell 1:635,2007), Lin−CD34+CD38−CD45RA−CD90+ cells contained most primitive human CD34+CD38− SRCs. Very interestingly, these Lin−CD34+CD38−CD45RA−CD90+ cells were generated from the above mentioned cocultures. In order to evaluate SRC activity of these Lin−CD34+CD38−CD45RA−CD90+ cells generated from 18Lin−CD34− cells in vitro, Lin−CD34+CD38−CD45RA−CD90+/− cells were sorted by FACS and then transplanted into NOD/SCID mice by IBMI. Eight weeks after transplantation, 8 out of 16 mice received Lin−CD34+CD38−CD45RA−CD90+ cells (400 to 3000 cells/mouse) were repopulated with human cells. In contrast, only 2 out of 16 mice received Lin−CD34+CD38− CD45RA−CD90− cells (1500 to 7000 cells/mouse) were repopulated. These results demonstrated that human CB-derived 18Lin−CD34− cells could generate very primitive CD34+CD38− SRCs in vitro. (Conclusion) These findings elucidate that human BM-derived DP cell-derived MSCs can support very primitive human CB-derived CD34− SRCs in vitro and suggest that these CD34− SRCs seem to be more immature than CD34+CD38− SRCs. These results provide a new concept of hierarchy in the human primitive HSC compartment. Disclosures: No relevant conflicts of interest to declare.

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Activation of Notch-Hes Signaling Pathway Impairs Osteogenic Differentiation and Hematopoietic Stem Cell-Supporting Capacitiy of Mesenchymal Stromal Cells Derived from Myelodysplastic Syndromes Patients

Blood ◽

10.1182/blood.v126.23.2857.2857 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2857-2857

Author(s):

Chunkang Chang ◽

Chengming Fei ◽

Juan Guo ◽

Youshan Zhao ◽

Shucheng Gu ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Osteogenic Differentiation ◽

Hematopoietic Stem Cell ◽

Mesenchymal Stromal Cells ◽

Myelodysplastic Syndromes ◽

Stromal Cells ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Healthy Donors

Abstract Ineffective hematopoiesis is a major characteristic of myelodysplastic syndromes (MDS). Bone marrow mesenchymal stromal cells(BMMSCs) and their progeny (i.e., osteoblasts, adipocytes, and reticular cells), which are considered as main cellular components of the bone marrow niche, have been shown to physiologically support hematopoiesis, but their contribution to the pathogenesis of MDS is controversially discussed. In this study, we examined the osteogenic differentiation and hematopoietic stem cell-supporting capacitiy of BMMSCs in patients with MDS (n=67) and healthy donors (n=22). After 21 days osteogenic induction differentiation, osteogenesis potential of BMMSCs was significantly reduced in cases with RARS(83.3%), RCMD(75.0%), RAEB I(44.4%), RAEB II (40%), indicated by cytochemical stainings and reduced expressions of Runx2. Moreover, we observed that in co-cultures with normal hematopoietic stem cells(HSCs) and MDS-BMMSCs, the colony number (CFU-GM、BFU-E and CFU-GEMM) was significantly lower in the presence of MDS-BMMSCs in comparison to the normal counterpart. Furthermore, in MDS-BMMSCs, we detected increased mRNA expression of several members of the Notch pathway, including Delta-like-1, Jagged-1, Notch1, Notch2, Hes1 and Hes5. Basically, the Notch-Hes pathway is the main regulator of the microenvironment dependent hematopoietic stem cell fate. Therefore we investigated if the activation of Notch-Hes pathway affected their osteogenesis and hematopoietic stem cell-supporting capacitiy of BMMSCs. By overexpression of Notch1 intracellular domain (NICD) in BMMSCs from healthy donors, we confirmed that Notch signaling negatively regulated BMMSCs osteogenesis through inhibition of Runx2 transcriptional activity. Importantly, treatment with the Notch1 inhibitor DAPT reversed the osteogenic differentiation and improved the hematopoiesis supporting capacitiy of MDS-BMMSCs. Taken together, our findings suggest that the ineffective hematopoiesis typical of MDS may be partly due to the impaired osteogenic differentiation of BMMSCs, and the activation of Notch-Hes signaling is involved in the impaired osteogenic differentiation and diminished hematopoietic stem cell-supporting capacitiy of MDS-BMMSCs, restoring the adequate Notch-Hes signaling could represent a potential therapeutic approach to MDS. Disclosures No relevant conflicts of interest to declare.

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Aging of Bone Marrow Mesenchymal Stromal Cells: Hematopoiesis Disturbances and Potential Role in the Development of Hematologic Cancers

Cancers ◽

10.3390/cancers13010068 ◽

2020 ◽

Vol 13 (1) ◽

pp. 68

Author(s):

Fulvio Massaro ◽

Florent Corrillon ◽

Basile Stamatopoulos ◽

Nathalie Meuleman ◽

Laurence Lagneaux ◽

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Keyword(s):

Bone Marrow ◽

Tumor Progression ◽

Cancer Progression ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Cell Communication ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Wide Range ◽

Major Player

Aging of bone marrow is a complex process that is involved in the development of many diseases, including hematologic cancers. The results obtained in this field of research, year after year, underline the important role of cross-talk between hematopoietic stem cells and their close environment. In bone marrow, mesenchymal stromal cells (MSCs) are a major player in cell-to-cell communication, presenting a wide range of functionalities, sometimes opposite, depending on the environmental conditions. Although these cells are actively studied for their therapeutic properties, their role in tumor progression remains unclear. One of the reasons for this is that the aging of MSCs has a direct impact on their behavior and on hematopoiesis. In addition, tumor progression is accompanied by dynamic remodeling of the bone marrow niche that may interfere with MSC functions. The present review presents the main features of MSC senescence in bone marrow and their implications in hematologic cancer progression.

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