TRPC1 channel modulates mechanical stretch-induced bone marrow mesenchymal stem cell proliferation through Ca2+-dependent ERK1/2 activation.

SAT0169 Impaired migration and proliferation of bone marrow-derived mesenchymal stem cells from patients with systemic lupus erythematosus were mediated by IKK-β

Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for use in regenerative medicine. Several studies have shown that low-level laser irradiation (LLLI) could affect the differentiation and proliferation of MSCs. The aim of this study was to examine the influence of LLLI at different energy densities on BMSCs differentiation into neuron and osteoblast. Human BMSCs were cultured and induced to differentiate to either neuron or osteoblast in the absence or presence of LLLI. Gallium aluminum arsenide (GaAlAs) laser irradiation (810 nm) was applied at days 1, 3, and 5 of differentiation process at energy densities of 3 or 6 J/cm(2) for BMSCs being induced to neurons, and 2 or 4 J/cm(2) for BMSCs being induced to osteoblasts. BMSCs proliferation was evaluated by MTT assay on the seventh day of differentiation. BMSCs differentiation to neurons was assessed by immunocytochemical analysis of neuron-specific enolase on the seventh day of differentiation. BMSCs differentiation to osteoblast was tested on the second, fifth, seventh, and tenth day of differentiation via analysis of alkaline phosphatase (ALP) activity. LLLI promoted BMSCs proliferation significantly at all energy densities except for 6 J/cm(2) in comparison to control groups on the seventh day of differentiation. LLLI at energy densities of 3 and 6 J/cm(2) dramatically facilitated the differentiation of BMSCs into neurons (p < 0.001). Also, ALP activity was significantly enhanced in irradiated BMSCs differentiated to osteoblast on the second, fifth, seventh, and tenth day of differentiation (p < 0.001 except for the second day). Using LLLI at 810 nm wavelength enhances BMSCs differentiation into neuron and osteoblast in the range of 2-6 J/cm(2), and at the same time increases BMSCs proliferation (except for 6 J/cm(2)). The effect of LLLI on differentiation and proliferation of BMSCs is dose-dependent. Considering these findings, LLLI could improve current in vitro methods of differentiating BMSCs prior to transplantation.

Growth and proliferation of caprine bone marrow mesenchymal stem cells on different culture media

Hypoxia Preconditioned Mesenchymal Stem Cells Improve Vascular and Skeletal Muscle Fiber Regeneration After Ischemia Through a Wnt4-dependent Pathway

Transient receptor potential canonical 5 (TRPC5) forms cationic channels that are polymodal sensors of factors including oxidized phospholipids, hydrogen peroxide, and reduced thioredoxin. The aim of this study was to expand knowledge of the chemical-sensing capabilities of TRPC5 by investigating dietary antioxidants. Human TRPC5 channels were expressed in HEK 293 cells and studied by patch clamp and intracellular Ca2+ recording. GFP- and HA-tagged channels were used to quantify plasma membrane localization. Gallic acid and vitamin C suppressed TRPC5 activity if it was evoked by exogenous hydrogen peroxide or lanthanide ions but not by lysophosphatidylcholine or carbachol. Catalase mimicked the effects, suggesting that lanthanide-evoked activity depended on endogenous hydrogen peroxide. Trans-resveratrol, by contrast, inhibited all modes of TRPC5, and its effect was additive with that of vitamin C, suggesting antioxidant-independent action. The IC50 was ∼10 μm. Diethylstilbestrol, a related hydroxylated stilbene, inhibited TRPC5 with a similar IC50, but its action contrasted sharply with that of resveratrol in outside-out membrane patches where diethylstilbestrol caused strong and reversible inhibition and resveratrol had no effect, suggesting indirect modulation by resveratrol. Resveratrol did not affect channel surface density, but its effect was calcium-sensitive, indicating an action via a calcium-dependent intermediate. The data suggest previously unrecognized chemical-sensing properties of TRPC5 through multiple mechanisms: (i) inhibition by scavengers of reactive oxygen species because a mode of TRPC5 activity depends on endogenous hydrogen peroxide; (ii) direct channel blockade by diethylstilbestrol; and (iii) indirect, antioxidant-independent inhibition by resveratrol.

ObjectivesIncreasing evidences shown the important role of bone marrow-derived mesenchymal stem cells (BM-MSCs) in wound healing and vascular remodelling in vivo. However, their mechanism in the development of atherosclerosis and...

Acute lymphoblastic and myeloblastic leukemias activate lymphotoxin beta receptor in mesenchymal stem cells in the bone marrow to turn off interleukin-7 production and lymphopoiesis and gain competitive advantage.

Acute lymphoblastic and myeloblastic leukemias activate lymphotoxin beta receptor in mesenchymal stem cells in the bone marrow to turn off interleukin-7 production and lymphopoiesis and gain competitive advantage.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract Acute lymphoblastic and myeloblastic leukemias (ALL and AML) have been known to modify the bone marrow microenvironment and disrupt non-malignant hematopoiesis. However, the molecular mechanisms driving these alterations remain poorly defined. Using mouse models of ALL and AML, here we show that leukemic cells turn off lymphopoiesis and erythropoiesis shortly after colonizing the bone marrow. ALL and AML cells express lymphotoxin α1β2 and activate lymphotoxin beta receptor (LTβR) signaling in mesenchymal stem cells (MSCs), which turns off IL7 production and prevents non-malignant lymphopoiesis. We show that the DNA damage response pathway and CXCR4 signaling promote lymphotoxin α1β2 expression in leukemic cells. Genetic or pharmacological disruption of LTβR signaling in MSCs restores lymphopoiesis but not erythropoiesis, reduces leukemic cell growth, and significantly extends the survival of transplant recipients. Similarly, CXCR4 blocking also prevents leukemia-induced IL7 downregulation and inhibits leukemia growth. These studies demonstrate that acute leukemias exploit physiological mechanisms governing hematopoietic output as a strategy for gaining competitive advantage. Editor's evaluation This study investigates a novel pathway by which leukemic cells remodel the bone marrow niche to promote their expansion at the expense of normal hematopoiesis. Feng X, Pereira JP et al. convincingly demonstrate a positive feedback loop between leukemic cells and stromal cells mediated by lymphotoxin produced by cancer cells and its receptor expressed by bone marrow stromal cells. The authors provide compelling evidence suggesting that this pathway disrupts normal blood production and provides a competitive advantage to leukemic cells. https://doi.org/10.7554/eLife.83533.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Blood cell production is a tightly regulated process important for organismal homeostasis. All blood cells develop from a dedicated hematopoietic stem cell (HSC) that colonizes specialized niches in the bone marrow (BM) formed predominantly by mesenchymal stem cells (MSCs) and endothelial cells (ECs) (Morrison and Scadden, 2014; Pinho and Frenette, 2019; Sugiyama et al., 2019). Within these niches, HSCs and hematopoietic progenitors receive critical signals for long-term HSC maintenance and for differentiation into lymphoid, myeloid, and erythroid lineages (Sugiyama et al., 2019; Miao et al., 2020). However, most hematopoietic cytokines act in a short-range manner, and thus hematopoietic stem and progenitor cells rely on localization cues such as CXCL12 for accessing growth factors produced by MSCs and ECs (Noda et al., 2011; Tzeng et al., 2011; Ding and Morrison, 2013; Greenbaum et al., 2013; Cordeiro Gomes et al., 2016). While HSCs and uncommitted hematopoietic progenitors are critically dependent on stem cell factor (SCF, encoded by Kitl), committed progenitors require lineage-specific signals, such as IL7 for lymphocytes, IL15 for NK cells, or M-CSF for monocytes and macrophages. Importantly, most hematopoietic cytokines are produced by MSCs and by a subset of ECs in the BM (Miao et al., 2020; Noda et al., 2011; Cordeiro Gomes et al., 2016; Ding et al., 2012; Baryawno et al., 2019; Comazzetto et al., 2019; Tikhonova et al., 2019). The production of hematopoietic cytokines and chemokines by MSCs and ECs is relatively stable during homeostasis but can change significantly under certain perturbations. For example, systemic inflammation caused by infections enforces the downregulation of multiple hematopoietic cytokines and CXCL12 in the BM (Ueda et al., 2004; Ueda et al., 2005; Manz and Boettcher, 2014). Likewise, acute lymphoblastic and myeloblastic leukemias (ALL and AML) also promote the downregulation of multiple cytokines and CXCL12 produced by MSCs and ECs (Baryawno et al., 2019; Hanoun et al., 2014; Fistonich et al., 2018; Zehentmeier and Pereira, 2019). During systemic infection, the coordinated downregulation of certain cytokines (e.g. IL7) and CXCL12 causes a temporary pause in lymphopoiesis that is necessary for an emergent production of short-lived neutrophils and monocytes (Manz and Boettcher, 2014). In leukemic states, however, the mechanism(s) promoting cytokine and chemokine downregulation are not well defined, and neither is known if these changes are protective or harmful for the host. In humans and in mouse models of B-ALL, leukemic cells use CXCR4 to home to the BM (Juarez et al., 2007; Colmone et al., 2008; van den Berk et al., 2014). However, B-ALL cells do not distribute randomly and seem to reside and proliferate in certain perivascular niches (Colmone et al., 2008; Sipkins et al., 2005). Importantly, CXCL12 production is measurably reduced exclusively in BM niches colonized by B-ALL cells (Colmone et al., 2008; van den Berk et al., 2014). Furthermore, intact CXCR4 signaling presumably in B-ALL cells is required for downregulation of CXCL12 expression in BM niche cells (van den Berk et al., 2014). The fact that CXCR4 expression levels in B-ALL cells inversely correlate with patient outcome suggests that B-ALL-induced changes in the BM microenvironment may favor leukemia progression (van den Berk et al., 2014; Cancilla et al., 2020). The BM microenvironment has also been reported to be severely affected in AML patients and in mouse models of AML. Of note, hematopoietic cytokines and chemokines are significantly downregulated along with the re-programing of MSC and EC transcriptomes (Baryawno et al., 2019; Hanoun et al., 2014; Chandran et al., 2015; Geyh et al., 2016). Although no specific mechanisms have been identified for explaining how AML cells dysregulate MSCs and ECs, some evidence suggests that this may be mediated by direct AML-niche cell interactions (Hérault et al., 2017). Thus, a model emerges where leukemia cells attracted to CXCL12-producing BM niches physically interact and re-program MSCs and ECs to reduce CXCL12 levels, possibly reduce hematopoietic output, and in this way favor leukemic cell expansion. However, the molecular mechanisms utilized by leukemia for MSC and EC re-programing and for reducing non-malignant hematopoiesis remain poorly defined. In this study, we show that ALL and AML cells preferentially turn off lymphopoiesis and erythropoiesis shortly after seeding the BM. We demonstrate that both B-ALL and AML cells express LTα1β2, the membrane-bound ligand of lymphotoxin beta receptor (LTβR), which enforces IL7 downregulation in LTβR-expressing MSCs. Genetic or pharmacological blockade of LTβR signaling in MSCs restores lymphopoiesis but not erythropoiesis at the onset of leukemia, which in turn reduces leukemic cell growth and extends survival of transplant recipients. These studies demonstrate that leukemic cells exploit molecular mechanisms that confer flexibility in blood cell production to suppress normal hematopoiesis. Results ALL inhibits non-leukemic hematopoiesis Although leukemia alters BM niches, whether these changes directly affect hematopoietic cell production has not been carefully studied. To determine if and which hematopoietic cell lineages are affected by leukemia, we transplanted 3 million pre-B-cell precursor ALL cells expressing the BCR-ABL1 oncogene (from here on referred as ALL; BCR-ABL1 reported by YFP expression) into non-irradiated C57Bl6/J mice and analyzed its impact on lymphoid, myeloid, and erythroid cell production over time. As expected, ALL cells expanded rapidly in BM (Figure 1A). Conversely, non-leukemic developing B cells and mature recirculating B cells declined sharply 2 weeks after ALL transplantation (Figure 1B). Monocyte numbers reduced between the first and second weeks by four- to fivefold, but cell numbers recovered to normal levels at 3 weeks (Figure 1C). This contrasted with a moderate twofold decline in neutrophil numbers at 2 weeks that remained stable until 3 weeks (Figure 1D). Changes in immature erythrocytes (Ter119+CD71+ cells) were similar to the reductions seen in B cell progenitors: erythroid cells progressively reduced at 6, 14, and 21 days after ALL transplantation, reaching >10-fold reductions at 3 weeks (Figure 1E). In summary, ALL expansion induces a strong decline in lymphopoiesis and erythropoiesis while their impact on myeloid cell production is modest. Figure 1 Download asset Open asset Kinetics of B-ALL growth and impact on hematopoiesis. (A) B-ALL number. (B) Number of non-malignant developing B cell subsets. (C) Inflammatory monocytes. (D) Neutrophils. (E) Immature (Ter119+CD71+) and mature (Ter119+CD71-) red blood cells. Data in all panels show bone marrow cell numbers obtained from wild-type (WT) mice transplanted with 3×106 BCR-ABL-expressing B-ALL cells. In all panels, X-axis indicates time (days) after B-ALL transplantation. Bars indicate mean, circles depict individual mice. Data are representative of two independent experiments. **p<0.005; ***p<0.0005 unpaired, two-sided, Student’s t test. ###p<0.0005 Mann–Whitney test. ALL, acute lymphoblastic leukemia. ALL induces LTβR signaling in MSCs, downregulates Il7 expression, and modulates lymphopoiesis In previous studies we noted that transplanted ALL cells and Artemis-deficient (pre-leukemic) pre-B cells led to IL7 and CXCL12 downregulation in MSCs (Fistonich et al., 2018), which could explain the negative impact of ALL in non-malignant lymphopoiesis. While the mechanism(s) responsible for IL7 and CXCL12 downregulation remained undefined, earlier studies suggested a role for LTβR signaling in BM stromal cells in development of some lymphoid lineages (Wu et al., 2001; Kim et al., 2014). In recent studies, we found that MSCs express LTβR and that LTβR signaling controls IL7 expression in vivo (Zehentmeier et al., 2022). Furthermore, when mRNA levels of LTB were analyzed in pediatric samples of B-ALL (Children’s Oncology Group Study 9906 for High-Risk Pediatric ALL) and associated with clinical outcome at the time of diagnosis, we noted an inverse correlation between LTB transcript abundance and relapse-free survival that reached statistical significance (Figure 2—figure supplement 1A). These observations led us to hypothesize that leukemic cells express LTβR ligands and induce LTβR signaling in MSCs in vivo. In mice, BCR-ABL1 expressing pre-B ALL cells also express higher LTα/LTβ amounts than non-leukemic pre-B, immature, and mature B cells (Figure 2A and B). The presence of ALL cells in the BM environment did not change LTα and LTβ expression on non-leukemic host pre-B cells (Figure 2A). Importantly, when mouse ALL cells were engineered to over-express LTα/LTβ, these ALLs induced stronger IL7 downregulation in BM MSCs and were lethal more quickly than empty vector transduced ALL cells (Figure 2—figure supplement 1B–E). Combined, these studies suggest a pathogenic role for the LTβR pathway in leukemia progression. Figure 2 with 1 supplement see all Download asset Open asset Lymphotoxin α1β2 expression in B-ALL cells and therapeutic effect of lymphotoxin beta receptor (LTβR) blocking. (A) Histograms of LTα and LTβ expression in B-ALL cells and in pre-B cells. Purple, B-ALL; green, non-malignant pre-B cells (CD19+CD93+IgM-cKit-) in bone marrow (BM) of wild-type (WT) mice transplanted with B-ALL cells; blue, non-malignant pre-B cells in BM of WT mice (no B-ALL); filled gray, non-malignant Ltb-deficient pre-B cells in BM of Ltb-/- mice. (B) LTα and LTβ mean fluorescence intensity (MFI) in developing B cells and ALLs isolated from BM of ALL transplanted mice. (C) Experimental design of data described in panels D–H. (D) Number of non-malignant developing B cell subsets in BM. (E) Immature and mature erythrocyte number. (F) Neutrophils. (G) Monocytes. (H) B-ALL number. Data in panels D–H show BM numbers from WT mice transplanted with 3×106 BCR-ABL-expressing B-ALL cells and treated with HEL-Ig or LTβR-Ig (150 µg/mouse) on day 0 and day 5; mice were analyzed on day 8 post ALL transplantation. (I) Frequency of mouse survival after B-ALL transplantation following pre-treatment with either HEL-Ig or LTβR-Ig (n=5 per group). Mice were treated with HEL-Ig or LTβR-Ig (150 µg/mouse) every 5 days until endpoint. Bars indicate mean, circles depict individual mice. Data are representative of two independent experiments. *p<0.05; **p<0.005; ***p<0.0005 unpaired, two-sided, Student’s t test. ###p<0.0005 Mann–Whitney test. To test if LTβR signaling impacts ALL growth and non-malignant hematopoiesis, we transplanted 3 million ALL cells into WT syngeneic recipient mice (C57BL6/J) treated weekly with a soluble LTβR-Ig decoy (a fusion between LTβR ectodomain and the Fc domain of a mouse IgG1 recognizing Hen Egg Lysozyme) or with control Hel-Ig. Transplanted ALL cells reduced lymphopoiesis significantly, which was reverted with LTβR-Ig treatment (Figure 2C and D). In contrast, LTβR signaling blockade did not restore erythropoiesis or myelopoiesis (Figure 2E–G). Importantly, ALL growth was significantly reduced at 2 weeks (Figure 2H), which reflected in a small but significant extension of mouse survival (Figure 2I). To gain further insight into the mechanisms used by ALL cells for reducing non-malignant hematopoiesis, we analyzed the MSC transcriptome in homeostasis, during ALL expansion, and in mice with ALL but treated with LTβR-Ig. To identify gene expression differences between the three groups, we performed principal component analyses (PCA) on the transcriptome datasets from three to four independent replicates. The first two principal components (PC1 and PC2) represent the main axes of variation within these datasets and explained 46% and 17% of variation, respectively. Samples from control and ALL groups separated by PC1, and within ALL cohorts, samples from LTβR-Ig versus Hel-Ig treated ALL also segregated from each other, thus indicating major transcriptional changes induced by ALL growth in vivo, of which a significant fraction was sensitive to LTβR blocking (Figure 3A). Unsupervised clustering of the top 1000 most variable genes also independently segregated the three groups (Figure 3B). Comparisons between control and ALL treated with Hel-Ig samples revealed 322 differentially expressed genes (DEGs; Padj <0.05, |log2FC|>1), of which 74 were downregulated and 248 were upregulated in MSCs of control mice (Supplementary file 1). Comparisons between the ALL groups (Hel-Ig versus LTβR-Ig) revealed 226 DEGs of which 149 were upregulated and 77 were downregulated in MSCs of mice with ALL and treated with Hel-Ig (Supplementary file 2). Gene set enrichment analyses revealed a strong inflammatory gene signature induced by ALL with a strong statistical significance in interferon α- and γ-induced genes, complement, and cytokines IL2, IL6, and TNFα signaling (Figure 3C). Of note, LTβR blocking further increased the interferon stimulated gene signature, while it reduced the expression of genes associated with TNFα signaling (Figure 3C), consistent with the fact that LTβR is a TNF superfamily member that activates canonical and non-canonical nuclear factor kappa-binding transcription factors (NFκB) (Norris and Ware, 2007). Importantly, of the several hematopoietic cytokines expressed by MSCs, KITL, IL7, IGF1, and CSF1 were significantly downregulated by ALL cells (Figure 3D). These transcriptional changes in MSCs were similar to those described in mice with acute myeloid leukemia (Baryawno et al., 2019). However, of these hematopoietic cytokines, only IL7 downregulation was blocked by LTβR-Ig treatment (Figure 3D). Furthermore, blocking other NFκB-inducing cytokines, such as TNFα and IL1β, did not prevent IL7 downregulation nor did it rescue non-malignant lymphopoiesis or myelopoiesis (Figure 3—figure supplement 1A–C) and did not impact ALL expansion in vivo (Figure 3—figure supplement 1D). Even though ALL cells promoted an interferon-induced gene expression signature in MSCs (Figure 3C), blocking IFNα or IFNγ signaling did not rescue IL7 downregulation and non-malignant hematopoiesis, nor did it reduce ALL growth in vivo (Figure 3—figure supplement 1E–I). Combined, these results show a major impact of ALL expansion in the MSC transcriptome, with a large fraction of DEGs being sensitive to LTβR blocking. Figure 3 with 1 supplement see all Download asset Open asset Lymphotoxin beta receptor (LTβR)-dependent and -independent transcriptomic changes in mesenchymal stem cells (MSCs) induced by B-ALL. (A) Principal component analysis (PCA) distribution plot. (B) Unsupervised hierarchical clustering and heatmap representation of top 1000 differentially expressed genes. (C) GSEA-KEGG pathway alterations in MSCs. (D) Hematopoietic cytokines and chemokine mRNA expression. Data in all panels were generated from analyses of MSC bulk RNA sequencing. *p<0.05; **p<0.005; unpaired, two-sided, Student’s t test. ALL, acute lymphoblastic leukemia. To test if LTβR signaling in MSCs impacts ALL growth, non-malignant hematopoiesis, and mouse survival, we transplanted ALL cells into mice conditionally deficient in Ltbr in MSCs (Ltbrfl/fl; LeprCre/+ mice, from here on referred as LTβR∆) that also report Il7 transcription via GFP expression (Il7GFP/+). We ruled out a role for LTβR signaling in MSCs in promoting ALL homing to the BM by transplanting 3×106 ALL cells into control or LTβR∆ mice (Figure 4A), in agreement with prior studies showing that LTβR signaling in MSCs does not control CXCL12 expression under homeostatic conditions (Zehentmeier et al., 2022). Transplanted ALL cells induced IL7 downregulation in control mice (WT, Lepr+/+; Ltbrfl/fl) but not in LTβR∆ mice (Figure 4B), as expected (Figure 3D). These changes in IL7 production corresponded with reduced lymphopoiesis in WT mice whereas lymphopoiesis was largely unaffected in LTβR∆ mice (Figure 4C–E). In contrast, ALL-induced reductions in myeloid and erythroid lineages were largely independent of LTβR signaling in MSCs (Figure 4G–J). The inability to induce LTβR signaling in MSCs also impacted ALL growth in vivo (Figure 4F and K) such that it extended mouse survival by approximately 1 week (Figure 4L). To further test if ALL cells directly induce LTβR signaling in MSCs, we generated ALL cells genetically deficient in Ltb (Figure 4M). Indeed, Ltb-deficient ALL cells were unable to induce IL7 downregulation in MSCs and to block non-malignant lymphopoiesis (Figure 4N and O). Furthermore, Ltb-deficient ALL cells proliferated significantly less than Ltb-sufficient ALL cells (Figure 4P), which extended mouse survival significantly (Figure 4Q). Finally, to account for reduced Ltb-deficient ALL growth in vivo, we measured changes in Il7 expression in mice transplanted with 3×106 Ltb+/+ ALLs, 3×106 Ltb-/- ALLs, and 9×106 Ltb-/- ALLs (3×Ltb-/-). Importantly, IL7 expression was unchanged even in mice transplanted with threefold higher number of Ltb-deficient ALLs (Figure 4R and S). Combined, these studies show that the ALL-induced IL7 downregulation that we reported in previous studies (Fistonich et al., 2018; Zehentmeier and Pereira, 2019) is mediated by direct delivery of lymphotoxin ligands to LTβR expressed on BM MSCs. Figure 4 with 1 supplement see all Download asset Open asset Effects of mesenchymal stem cell (MSC)-intrinsic lymphotoxin beta receptor (LTβR) signaling in lymphopoiesis and B-ALL growth. (A) Measurements of ALL homing to the bone marrow (BM): 3×106 BCR-ABL ALLs were transferred into control (red) or LTβR∆ (green) mice and allowed to home into the BM for 24 hr. (B) Il7-GFP expression in MSCs. (C–E) Number of non-malignant developing B cell subsets. (C) ProB cells. (D) Pre-B cells. (E) Immature B cells. (F) B-ALL frequency in BM. (G–K) Myeloid and erythroid cell numbers in BM. (G) Neutrophils. (H) Monocytes. (I) Immature RBCs. (J) Mature RBCs. (K) Total ALL number. (L) Probability of wild-type (WT) or LTβR∆ mouse survival after B-ALL transplantation (n=8 mice/group). Mice were transplanted with 3×106 BCR-ABL-expressing B-ALL cells and analyzed on day 8 after transplantation. (M) Histogram of LTβR ligand expression in ALL cells. Green, Ltb-sufficient; brown, Ltb-deficient. (N) Il7-GFP expression in MSCs. (O) Number of non-malignant developing B cell subsets. (P) ALL number. (Q) Frequency of WT mouse survival after Ltb-deficient or Ltb-sufficient ALL transplantation (n=7/group). (R and S) Effects of Ltb-expressing ALLs in MSCs. (R) Ltb+/+ and Ltb-/- B-ALL numbers in BM 3 weeks after transplantation into Il7GFP/+ mice. (S) Il7-GFP expression in MSCs. Gray bar indicates control Il7GFP/+ mice (no ALL); green bar represents Il7GFP/+ mice recipient of 3×106 Ltb+/+ ALLs; brown bar indicates Il7GFP/+ mice recipient of 3×106 Ltb-/- ALLs; red bars depict Il7GFP/+ mice recipient of 9×106 Ltb-/- ALLs (3×Ltb-/-). Bars indicate mean, circles depict individual mice. Data in all panels are representative of two independent experiments. *p<0.05; **p<0.005; ***p<0.0005 unpaired, two-sided, Student’s t test. ALL, acute lymphoblastic leukemia. To test if increased lymphopoiesis due to excess IL7 is directly responsible for reduced ALL growth in vivo, we treated mice transplanted with B-ALL cells with recombinant IL7 complexed with a neutralizing anti-IL7 (aIL7, clone M25) monoclonal antibody (Figure 4—figure supplement 1A), which increases the half-life of recombinant IL-7 in vivo (Boyman et al., 2008). Indeed, mice treated with IL7/aIL7 had significantly higher numbers of developing B cell subsets in the BM (Figure 4—figure supplement of and pre-B cells. Conversely, ALL numbers in BM were significantly which reflected in significant reductions in blood and (Figure 4—figure supplement 1C). Combined, these data demonstrate that the of IL7 production reduces which results in ALL growth. and used an mouse model of Artemis-deficient mice with mice and with mice to study the impact of DNA pathway in B cell In these studies, reported in pre-B cells that could not due to in et al., 2008; et al., 2016). In previous studies this we that Artemis-deficient pre-B cells could also induce in MSCs (Fistonich et al., 2018; Zehentmeier and Pereira, suggesting that the LTβR pathway may also be in with this when the transcriptome of Artemis-deficient and pre-B cells, we noted that Artemis-deficient pre-B cells also expressed significantly higher amounts of LTα and LTβ et al., 2008; et al., 2016). In agreement with these we higher amounts of LTα and LTβ on the cell of pre-B cells than on pre-B cells (Figure supplement 1A). of BM cells into Il7GFP/+ LTβR∆ mice or control revealed Il7 downregulation (Figure supplement which in a increased numbers of Artemis-deficient B cells (Figure supplement 1C). In contrast, did not impact production (Figure supplement and cells unable to which causes a of the DNA damage response pathway and To test if the DNA damage response controls LTα and LTβ expression, we treated ALL cells with a that prevents ALLs upregulated LTα on the cell in an (Figure supplement and The DNA damage response pathway signals of treatment with (a small of significantly reduced lymphotoxin α1β2 expression in ALLs (Figure supplement Combined, these studies demonstrate that LTα and LTβ expression can be by DNA damage response CXCR4 signaling ALL studies have that receptor signaling in cells lymphotoxin α1β2 expression et al., of LTβR expressed on lymphoid stromal cells increases the production of B cell which further increases lymphotoxin α1β2 expression in B cells, thus a loop et al., et al., To test if CXCR4 signaling in ALLs lymphotoxin α1β2 expression, we treated ALLs in with a of CXCL12 and measured Indeed, ALLs upregulated LTα after to CXCL12 (Figure Furthermore, LTα expression was further increased in ALLs treated with CXCL12 and (Figure Although prior studies have also that signaling can promote lymphotoxin α1β2 expression in lymphoid cells et al., in ALL cells signaling was not for LTα even at IL7 even though it promoted (Figure supplement 2A and B). To test if CXCR4 signaling is required for lymphotoxin α1β2 expression in ALLs in vivo, we transplanted 3×106 ALL cells into WT mice for 1 week and treated with or prior to Indeed, lymphotoxin α1β2 expression was significantly reduced in ALLs and in non-leukemic pre-B cells of mice (Figure Conversely, increased CXCR4 expression in ALL and non-leukemic pre-B cells (Figure as expected et al., 2005; et al., 2014). has a half-life in vivo thus it for long-term treatment in vivo et al., 2014; et al., To test if CXCR4 also prevents ALL-induced IL7 downregulation in BM MSCs, we transferred 3×106 ALL cells into Il7GFP/+ mice and treated with an CXCR4 et al., or with by (Figure While mice ALL-induced Il7-GFP downregulation in MSCs, mice treated with CXCR4 IL7 expression within the normal of mice ALL (Figure Similarly, developing B cells were significantly reduced in mice, but their numbers were normal in CXCR4 treated mice (Figure In contrast, ALL numbers were significantly in the BM and of mice treated with CXCR4 (Figure which with extended mouse survival (Figure Figure 5 with 2 see all Download asset Open asset CXCR4 signaling and its impact on acute lymphoblastic leukemia growth in vivo. (A) Histograms of LTα expression in B-ALL cells treated for with CXCL12 at the in (B) Histograms of LTα expression and mean intensity as of cells) in B-ALL cells treated with 1 or in with CXCL12 in and LTα (C) and CXCR4 expression (D) in ALLs and non-leukemic pre-B cells in the bone marrow (BM) of mice transplanted with 3×106 Ltb+/+ ALLs were allowed to in vivo for Mice were treated with or prior to (E) Experimental design of data described in panels Mice were transplanted with 3×106 BCR-ABL-expressing B-ALL cells and treated with CXCR4 on day 2 and until day mice were analyzed on day (F) Il7-GFP expression in mesenchymal stem cells (G) Number of non-malignant developing B cell subsets. (H) Total ALL number in BM and B-ALL in blood (I) Frequency of mouse survival after B-ALL transplantation into mice treated with or CXCR4 (J) CXCR4 expression on developing B cells and ALLs in BM. (K) In of developing B cells and Data in panels and are from BM of mice 1 week after ALL transplantation. Data are representative of two independent experiments. *p<0.05; **p<0.005; ***p<0.0005 unpaired, two-sided, Student’s t test. Mann–Whitney test. studies have that CXCR4 ALL homing and in the BM (Juarez et al., 2007; Sipkins et al., 2005). studies revealed that ALLs induce a but downregulation of CXCL12 expression in vivo (Figure 3D). However, when CXCR4 levels between ALLs and non-leukemic B cell progenitors developing in the we noted that ALLs express significantly higher amounts of CXCR4 in vivo (Figure and a CXCL12 in significantly more than non-leukemic developing B cell subsets (Figure These observations suggest that ALLs are more in the BM than non-leukemic B cell LTβR signaling AML growth and As AML also induces the downregulation of multiple hematopoietic cytokines expressed by MSCs, IL7 (Baryawno et al.,

BackgroundHypoxia has been shown to be able to induce tenogenic differentiation and proliferation of mesenchymal stem cells (MSCs) which lead hypoxia-induced MSCs to be a potential treatment for tendon injury. However, little is known about the mechanism underlying the tenogenic differentiation and proliferation process of hypoxic MSCs, which limited the application of differentiation-inducing therapies in tendon repair. This study was designed to investigate the role of Mohawk homeobox (Mkx) in tenogenic differentiation and proliferation of hypoxic MSCs.MethodsqRT-PCR, western blot, and immunofluorescence staining were performed to evaluate the expression of Mkx and other tendon-associated markers in adipose-derived MSCs (AMSCs) and bone marrow-derived MSCs (BMSCs) under hypoxia condition. Small interfering RNA technique was applied to observe the effect of Mkx levels on the expression of tendon-associated markers in normoxic and hypoxic BMSCs. Hypoxic BMSCs infected with Mkx-specific short hair RNA (shRNA) or scramble were implanted into the wound gaps of injured patellar tendons to assess the effect of Mkx levels on tendon repair. In addition, cell counting kit-8 assay, colony formation unit assay, cell cycle analysis, and EdU assay were adopted to determine the proliferation capacity of normoxic or hypoxic BMSCs infected with or without Mkx-specific shRNA.ResultsOur data showed that the expression of Mkx significantly increased in hypoxic AMSCs and increased much higher in hypoxic BMSCs. Our results also detected that the expression of tenogenic differentiation markers after downregulation of Mkx were significantly decreased not only in normoxic BMSCs, but also in hypoxic BMSCs which paralleled the inferior histological evidences, worse biomechanical properties, and smaller diameters of collagen fibrils in vivo. In addition, our in vitro data demonstrated that the optical density values, the clone numbers, the percentage of cells in S phage, and cell proliferation potential of both normoxic and hypoxic BMSCs were all significantly increased after knockdown of Mkx and were also significantly enhanced in both AMSCs and BMSCs in hypoxia condition under which the expression of Mkx was upregulated.ConclusionsThese findings strongly suggested that Mkx mediated hypoxia-induced tenogenic differentiation of MSCs but could not completely repress the proliferation of hypoxic MSCs.

Emerging evidence suggests that microRNA plays a pivotal role in cell proliferation. Our previous research has certified that miR-146a attenuates osteoarthritis through the regulation of cartilage homeostasis. However, little information about the function of miR-146a in bone marrow-derived mesenchymal stem cells (BMSCs) proliferation and the underlying mechanism was available. Therefore, this study aims at investigating the role of miR-146a on the proliferation of BMSCs and the possible mechanisms involved. The function of miR-146a on BMSCs proliferation was studied through overexpression and knockdown of miR-146a or the indicated long noncoding RNAs (lncRNAs) in BMSCs and then the proliferation rate of the BMSCs were detected by Cell Counting Kit-8 assay, colony formation assay. Besides, flow cytometry was used to test the cell cycle state of BMSCs modified by overexpression or knockdown of miR-146a or lncRNA EPB41L4A-AS1 (EPB41L4A Antisense RNA 1) and small nucleolar RNA host gene 7 (SNHG7). The expression level of marker genes involved in modulating cell proliferation was evaluated by quantitative polymerase chain reaction and western blot analysis. We discovered that the knockdown of miR-146a significantly promoted BMSCs proliferation. Moreover, miR-146a could bind to and inhibit endogenous expression of EPB41L4A-AS1 and SNHG7. Further study demonstrated that overexpression of EPB41L4A-AS1 and SNHG7 significantly enhanced proliferation of BMSCs. For the first time, we certified that miR-146a suppressed BMSCs proliferation, but EPB41L4A-AS1 and SNHG7 promoted BMSCs proliferation in the present study. Mechanistically, miR-146a significantly inhibited BMSCs proliferation partly through miR-146a/EPB41L4A-AS1 SNHG7/cell proliferation signaling pathway axis.

Fetal bovine serum (FBS) is widely used to culture mesenchymal stem cells (MSCs) in the laboratory; however, FBS has been linked to adverse immune-mediated reactions prompting the search for alternative cell culture medium. Platelet lysate (PL) as an FBS substitute has been shown to promote MSCs growth without compromising their functionality. Fibrinogen contained in PL has been shown to negatively impact the immune modulating properties of MSCs; therefore, we sought to deplete fibrinogen from PL and compare proliferation, viability, and immunomodulatory capacities of MSCs in FBS or PL without fibrinogen. We depleted fibrinogen from equine platelet lysate (ePL) and measured platelet-derived growth factor-beta (PDGF-β), transforming growth factor-beta (TGF-β) and tumor necrosis factor-alpha (TNF-α) through ELISA. First, we determined the ability of 10% ePL or fibrinogen-depleted lysate (fdePL) compared with 10% FBS to suppress monocyte activation by measuring TNF-α from culture supernatants. We then evaluated proliferation, viability, and immunomodulatory characteristics of bone marrow-derived MSCs (BM-MSCs) cultured in FBS or ePL with or without fibrinogen. Growth factor concentrations decreased in ePL after fibrinogen depletion. Lipopolysaccharide (LPS)-stimulated monocytes exposed to ePL and fdePL produced less TNF-α than LPS-stimulated monocytes in 10% FBS. BM-MSCs cultured in fdePL exhibited lower proliferation rates, but similar viability compared with BM-MSCs in ePL. BM-MSCs in fdePL did not effectively suppress TNF-α expression from LPS-stimulated monocytes compared with BM-MSCs in FBS. Depleting fibrinogen results in a lysate that suppresses TNF-α expression from LPS-stimulated monocytes, but that does not support proliferation and immune-modulatory capacity of BM-MSCs as effectively as nondepleted lysate.

Background and Aims: Bone marrow-derived mesenchymal stem cells (BM-MSCs) are a well-known source of multipotent adult stem cells. Despite using different methodologies of MSCs preparing for clinical applications, the top safest procedure to manipulate these cells, has not yet been determined. Recently, ex-vivo expansion of MSCs for their subsequent implantation, using some biological product, is suggested instead of fetal bovine serum (FBS). Previous studies have shown the effect of follicular fluid (FF) (a dynamic fluid in ovarian follicle) as an additive component in cell culture. Hence, this study aimed to decipher its role on the human BM-MSC proliferation.Materials and Methods: In this study, BM-MSCs at 3rd passage were cultivated in the presence of 20% FF (group I), 10% FF+ FBS 10% (group II) and FBS 20% as control group. The capacity of proliferation as calculating population doubling times and gene expression levels of stem cell factor, stromal cell-derived factor 1, and transforming growth factor beta were analyzed in osteogeneic media to examine the impacts of FF on osteogenesis of MSCs.Results: Our results corroborated an up-regulatory effect of FF on the proliferation of BM-MSCs by shorter population doubling times in the group II of treated cells and an increase in gene expression level of osteocalcin and transforming growth factor beta in the presence of higher concentrations of FF in cell culture FF 20% and 10%, respectively.Conclusions: FF is a potent mitogen for cell proliferation. FF may be an efficient substitution of FBS in ex-vivo cell culture, eliminating zoonotic infections and immunological reactions.

Long non coding RNAs (lncRNAs) show an encouraging trend in regulating the proliferation of bone marrow-derived mesenchymal stromal cells (BMSCs). The present study investigated the role of lncRNA low expression in tumor (LET) in BMSCs proliferation. Our result showed that LET was down-regulated in rapidly proliferated BMSCs (P < 0.05). Suppression of LET promoted BMSCs proliferation and over-expression of LET inhibited BMSCs proliferation (P < 0.05). LET negatively regulated the expression of transforming growth factor β1 (TGF-β1) in BMSCs (P < 0.05). Knockdown of TGF-β1 reversed the LET suppression-induced BMSCs proliferation (P < 0.05). Moreover, knockdown of TGF-β1 alleviated the LET suppression-induced activation of Wnt/β-catenin pathway in BMSCs. Therefore, we drew the conclusion that LET suppression promoted BMSCs proliferation by up-regulating the expression of TGF-β1 and activating Wnt/β-catenin pathway.

Objective To investigate the effect of heme oxygenase-1 (HO-1) gene modification on the proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) under the acute kidney injury (AKI) microenvironment in vitro and the possible mechanism. Methods Plasmids were constructed by the gateway technology that contained HO-1 target gene (eGFP as the tracing-gene) or only eGFP as the control, and were transfected into the 293FT cells through the liposome method to get the lenti-HO-1-eGFP/puro and the lenti-eGFP/puro, which then infected BMSCs to obtain HO-1-BMSCs and eGFP-BMSCs, testing the activity and differentiation potential of transfected cells. The ischemia/reperfusion-AKI kidney homogenate supernatant (KHS) and the control N-KHS were harvested and used to cultivate with BMSCs, eGFP-BMSCs, or HO-1-BMSCs, respectively, consisting of five groups: the blank group (BMSCs group), control group (BMSCs/N-KHS group), BMSCs/AKI-KHS group, eGFP-BMSCs/AKI-KHS group, and HO-1-BMSCs/AKI-KHS group, at 37 ℃ in 5% CO2 for 3 days. The MTT method was used to detect the growth inhibitory rate of BMSCs, flow cytometry to detect the cell apoptosis, the transmission electron microscope (TEM) to observe the cell ultrastructure changes, immunohistochemistry to detect the expression of cytokeratin 18 (CK18), and Western blot to detect the protein expressions of HO-1, phospho-AKT (pAKT), and phospho-ERK (pERK). Results The cell viability and differentiation potential of BMSCs were not changed by the gene modification. Compared with the BMSCs/N-KHS group, the growth inhibitory rate of the BMSCs/AKI-KHS group as well as the proportion of apoptotic cells increased significantly (t=12.581, t=16.283, P<0.05), which, after HO-1 gene modification, however, significantly decreased in the HO-1-BMSCs/AKI-KHS group (t=5.958, t=7.957, P<0.05). AKI-KHS induced ultrastructural change of the renal tubular epithelial differentiation in the cultured BMSCs with CK18 expression in the cytoplasm. The proportion of the CK18+ cells was the highest in the HO-1-BMSCs/AKI-KHS group (t=4.057, P<0.05). Compared with the BMSCs/AKI-KHS group, the HO-1-BMSCs/AKI-KHS had significantly increased cellular expression of HO-1 (t=4.163, P<0.05), pAKT (tpAKT=14.305, P<0.05), and pERK (tpERK=7.148; P<0.05). Conclusions In the AKI microenvironment, HO-1 gene modification could improve the proliferation of BMSCs, enhance the ability of BMSCs to differentiate into renal tubular epithelial cells, and decrease the apoptosis of BMSCs. HO-1 overexpression with the downstream AKT and ERK signaling pathway might be the possible mechanism. Key words: Heme oxygenase-1; Bone marrow-derived mesenchymal stem cells; Acute kidney injury