scholarly journals Genotoxic Bystander Signals from Irradiated Human Mesenchymal Stromal Cells Mainly Localize in the 10–100 kDa Fraction of Conditioned Medium

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 827
Author(s):  
Vanessa Kohl ◽  
Alice Fabarius ◽  
Oliver Drews ◽  
Miriam Bierbaum ◽  
Ahmed Jawhar ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Heat Inactivation ◽  
Control Medium ◽  
Sample Mean ◽  
Hematopoietic Stem ◽  
Human Mesenchymal Stromal Cells ◽  
Γh2ax Foci ◽  
Cd34 Cells

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) < 10 kDa, (II) 10–100 kDa and (III) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10–100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ∨ 3.8 ± 0.3 aberrant metaphases per CD34+ cell sample; mean ± SEM) when compared to (I) < 10 kDa (0.19 ± 0.01 ∨ 0.3 ± 0.2) or (III) > 100 kDa fractions (0.23 ± 0.04 ∨ 0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ∨ 0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10–100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.

scholarly journals Irradiated mesenchymal stromal cells induce genetic instability in human CD34+ cells

2020 ◽  
Author(s):  
Vanessa Kohl ◽  
Oliver Drews ◽  
Victor Costina ◽  
Miriam Bierbaum ◽  
Ahmed Jawhar ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Genetic Instability ◽  
Extracellular Medium ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Radiation Induced

AbstractRadiation-induced bystander effects (RIBE) in human hematopoietic stem and progenitor cells may initiate myeloid neoplasms (MN). Here, the occurrence of RIBE caused by genotoxic signaling from irradiated human mesenchymal stromal cells (MSC) on human bone marrow CD34+ cells was investigated. For this purpose, healthy MSC were irradiated in order to generate conditioned medium containing potential genotoxic signaling factors. Afterwards, healthy CD34+ cells from the same donors were grown in conditioned medium and RIBE were analyzed. Increased DNA damage and chromosomal instability were detected in CD34+ cells grown in MSC conditioned medium when compared to CD34+ cells grown in control medium. Furthermore, reactive oxygen species and distinct proteome alterations, e.g., heat-shock protein GRP78, that might be secreted into the extracellular medium, were identified as potential RIBE mediators. In summary, our data provide evidence that irradiated MSC induce genetic instability in human CD34+ cells potentially resulting in the initiation of MN. Furthermore, the identification of key bystander signals, such as GRP78, may lay the framework for the development of next-generation anti-leukemic drugs.

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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4358-4358
Author(s):  
Manal Alsheikh ◽  
Roya Pasha ◽  
Nicolas Pineault
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Soluble Factors ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
The Impact ◽  
Myeloid Progenitors

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.

scholarly journals Increased IL-6 secretion by aged human mesenchymal stromal cells disrupts hematopoietic stem and progenitor cells' homeostasis

Oncotarget ◽  
2016 ◽  
Vol 7 (12) ◽  
pp. 13285-13296 ◽  
Author(s):  
Kelsey O’Hagan-Wong ◽  
Stéphanie Nadeau ◽  
Audrey Carrier-Leclerc ◽  
Felipe Apablaza ◽  
Reggie Hamdy ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Hematopoietic Stem ◽  
Human Mesenchymal Stromal Cells ◽  
Stem And Progenitor Cells

Human Placental Derived Mesenchymal Stromal Cells (MSC) Grown in 3D-Culture (PLX-I), Promotes Engraftment of Human Umbilical Cord Blood (hUCB) Derived CD34+ Cells in NOD/SCID Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1416-1416 ◽  
Author(s):  
Ora Burger ◽  
Galit F. Ashtamker ◽  
Noam Bercovich ◽  
Marina Rusanovsky ◽  
Lena Pinzur ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
3D Culture ◽  
Scid Mice ◽  
Mice Model ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
I Cells

Abstract The use of UCB for transplantation in adults is limited due to the low cell number in each unit. Engraftment rates are lower and the time for neutrophil and platelet recovery is longer following CBT compared to bone marrow (BM) transplants. Co-transplantation of human hematopoietic stem/progenitor cells (HSC) with mesenchymal stromal cells in an irradiated NOD/SCID mice model has been demonstrated to promote HSC engraftment. Using PluriX technology, we expanded human placental derived MSC on 3D-carriers in a bioreactor (PLX-I) and evaluated its potential to promote UCB engraftment in the NOD/SCID mice model. Flow cytometer analysis detected high rates of expression of CD105, CD73, CD90 and CD29 and lacked expression of CD45, CD34 and CD19 surface markers in the PLX-I cells. PLX-I cells were capable of differentiation into bone, adipose tissue and cartilage under specific inductive conditions. Additionally, they possess both immune privileged and immune suppressive characteristics in a mixed lymphocyte reaction (MLR) assay. In this study 60 – 100x103 hUCB derived CD34+ cells were injected into the tail vein of 7–8 week old NOD/SCID mice along with 0.5x106 PLX-I cells. Human cell engraftment was tested in both sublethally irradiated (350 rad) and chemotherapy (50 mg/kg busulfan) treated NOD/SCID mice. Following 5–6 weeks, mice BM FACS analysis showed a significant increase in % hCD45+ rate in mice transplanted with PLX-I cells compared to mice transplanted with CD34+ cells alone: 13.6 vs. 31.7, p=0.01, (n=6) in the irradiation setting and 28.8 vs 6.3, p<0.05 (n=7) in the chemotherapy setting. These preclinical results demonstrate the potential of human placental-derived MSCs, grown as a 3D culture (PLX-I), to promote hUCB CD34+ cell engraftment in BM. Co-transplantation of PLX-I may be considered for improving the delayed engraftment using CB as the source of HSC.

scholarly journals Application of conditioned medium from mesenchymal stromal cells in the protocol for ex vivo production of megakaryocytes and platelets

2021 ◽  
Vol 66 (4) ◽  
pp. 526-538
Author(s):  
D. Yu. Klyuchnikov ◽  
M. Yu. Yazykova ◽  
A. A. Stepanov ◽  
S. E. Volchkov ◽  
O. V. Tyumina
Keyword(s):  
Cord Blood ◽  
Conditioned Medium ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Blood Platelets ◽  
Venous Blood ◽  
Hematopoietic Stem ◽  
Group 2 ◽  
Group 1

Introduction. Of interest is the use of a conditioned medium from mesenchymal stromal cells in order to increase the expansion of CD34+  hematopoietic stem cells (HSCs).Aim — to analyze the effi cacy of two methods of ex vivo production of human megakaryocytes and platelets from CD34+ cord blood HSC using conditioned media from mesenchymal stromal cells and IMDM. Methods. Two cultivation methods that differ from each other by medium composition were compared. As a control of antigen expression of the donor, venous blood platelets were used. CD34+ HSCs were isolated from mononuclear fraction of cord blood using the immunomagnetic selection technique. The resulting cells were introduced at a concentration of 1 × 104  cells/mL into 24-well plates and cultured at 39 °C and 10 % CO2  for the first 7 days, after which the conditions were changed to 37 °C and 5 % CO2  and cultured for 14 days. In Group 1, up to day 7, the culture was performed using conditioned medium from mesenchymal stromal cell containing TPO (30 ng/mL), SCF (2 ng/mL), IL-6 (7.5 ng/mL), IL-9 (13.5 ng/mL), and in Group 2 a IMDM medium with the same cytokine cocktail was used. The cells were calculated using haemocytometer. CD34, CD41a, CD42b expression was evaluated using fl ow cytometry. Statistic data was processed with using R-language. The differences were evaluated as statistically signifi cant at signifi cance level p < 0.05.Results. Megakaryocyte production was observed starting from day 7 of culture. The expression level using conditioned medium from mesenchymal stromal cells (Group 1) according to CD41a was 5.84 ± 0.33 % versus 10.43 ± 1.08 % using IMDM medium (Group 2). On day 13 the ratio increased up to 42.05 ± 1.71 % in Group 1 and 61.78 ± 1.71 % in Group 2. CD41a+ megakaryocytes of Group 1 expressed the CD42b marker at the level of 96.85 ± 1.06 % versus 88.7 ± 0.56 % in Group 2. With the application of MSC conditioned medium the average number of nucleated cells was signifi cantly higher on the day 11 and it was equal 326.016 ± 1.86 × 104  cells/mL vs 197.26 ± 10.55 × 104  cells/mL in IMDM medium. Proplatelet formation was observed with microscopy staring from the day 12. The ratio of CD41a+ /CD42b+  platelets was 59.5 ± 3.85 % in conditioned medium, 65.9 ± 8.72 % in IMDM, and 96.11 ± 0.89 % in control platelets derived from venous blood.Conclusion. It was demonstrated that the use of MSC conditioned medium leads to an increase in the expansion of nucleated cells, however it decreases the rate of differentiation in megakaryocytes. 

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