Transcriptional and Histochemical Signatures of Bone Marrow Mononuclear Cell-Mediated Resolution of Synovitis

Osteoarthritis (OA) may result from impaired ability of synovial macrophages to resolve joint inflammation. Increasing macrophage counts in inflamed joints through injection with bone marrow mononuclear cells (BMNC) induces lasting resolution of synovial inflammation. To uncover mechanisms by which BMNC may affect resolution, in this study, differential transcriptional signatures of BMNC in response to normal (SF) and inflamed synovial fluid (ISF) were analyzed. We demonstrate the temporal behavior of co-expressed gene networks associated with traits from related in vivo and in vitro studies. We also identified activated and inhibited signaling pathways and upstream regulators, further determining their protein expression in the synovium of inflamed joints treated with BMNC or DPBS controls. BMNC responded to ISF with an early pro-inflammatory response characterized by a short spike in the expression of a NF-ƙB- and mitogen-related gene network. This response was associated with sustained increased expression of two gene networks comprising known drivers of resolution (IL-10, IGF-1, PPARG, isoprenoid biosynthesis). These networks were common to SF and ISF, but more highly expressed in ISF. Most highly activated pathways in ISF included the mevalonate pathway and PPAR-γ signaling, with pro-resolving functional annotations that improve mitochondrial metabolism and deactivate NF-ƙB signaling. Lower expression of mevalonate kinase and phospho-PPARγ in synovium from inflamed joints treated with BMNC, and equivalent IL-1β staining between BMNC- and DPBS-treated joints, associates with accomplished resolution in BMNC-treated joints and emphasize the intricate balance of pro- and anti-inflammatory mechanisms required for resolution. Combined, our data suggest that BMNC-mediated resolution is characterized by constitutively expressed homeostatic mechanisms, whose expression are enhanced following inflammatory stimulus. These mechanisms translate into macrophage proliferation optimizing their capacity to counteract inflammatory damage and improving their general and mitochondrial metabolism to endure oxidative stress while driving tissue repair. Such effect is largely achieved through the synthesis of several lipids that mediate recovery of homeostasis. Our study reveals candidate mechanisms by which BMNC provide lasting improvement in patients with OA and suggests further investigation on the effects of PPAR-γ signaling enhancement for the treatment of arthritic conditions.

Download Full-text

Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo

Blood ◽

10.1182/blood.v88.11.4102.4102 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4102-4109 ◽

Cited By ~ 8

Author(s):

CI Civin ◽

G Almeida-Porada ◽

MJ Lee ◽

J Olweus ◽

LW Terstappen ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Population ◽

Mononuclear Cells ◽

Cell Subset ◽

Fetal Sheep ◽

Adult Human

Abstract Data from many laboratory and clinical investigations indicate that CD34+ cells comprise approximately 1% of human bone marrow (BM) mononuclear cells, including the progenitor cells of all the lymphohematopoietic lineages and lymphohematopoietic stem cells (stem cells). Because stem cells are an important but rare cell type in the CD34+ cell population, investigators have subdivided the CD34+ cell population to further enrich stem cells. The CD34+/CD38-cell subset comprises less than 10% of human CD34+ adult BM cells (equivalent to < 0.1% of marrow mononuclear cells), lacks lineage (lin) antigens, contains cells with in vitro replating capacity, and is predicted to be highly enriched for stem cells. The present investigation tested whether the CD34+/CD38-subset of adult human marrow generates human hematopoiesis after transfer to preimmune fetal sheep. CD34+/ CD38- cells purified from marrow using immunomagnetic microspheres or fluorescence-activated cell sorting generated easily detectable, long- term, multilineage human hematopoiesis in the human-fetal sheep in vivo model. In contrast, transfer of CD34+/CD38+ cells to preimmune fetal sheep generated only short-term human hematopoiesis, possibly suggesting that the CD34+/CD38+ cell population contains relatively early multipotent hematopoletic progenitor cells, but not stem cells. This work extends the prior in vitro evidence that the earliest cells in fetal and adult human marrow lack CD38 expression. In summary, the CD34+/ CD38-cell population has a high capacity for long-term multilineage hematopoietic engraftment, suggesting the presence of stem cells in this minor adult human marrow cell subset.

Download Full-text

Donor Origin of Multipotent Adult Progenitor Cells in Radiation Chimeras.

Blood ◽

10.1182/blood.v106.11.1395.1395 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1395-1395

Author(s):

Morayma Reyes ◽

Jeffrey S. Chamberlain

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Y Chromosome ◽

Stromal Cells ◽

Mononuclear Cells ◽

Multipotent Adult Progenitor Cells

Abstract Multipotent Adult Progenitor Cells (MAPC) are bone marrow derived stem cells that can be extensively expanded in vitro and can differentiate in vivo and in vitro into cells of all three germinal layers: ectoderm, mesoderm, endoderm. The origin of MAPC within bone marrow (BM) is unknown. MAPC are believed to be derived from the BM stroma compartment as they are isolated within the adherent cell component. Numerous studies of bone marrow chimeras in human and mouse point to a host origin of bone marrow stromal cells, including mesenchymal stem cells. We report here that following syngeneic bone marrow transplants into lethally irradiated C57Bl/6 mice, MAPC are of donor origin. When MAPC were isolated from BM chimeras (n=12, 4–12 weeks post-syngeneic BM transplant from a transgenic mouse ubiquitously expressing GFP), a mixture of large and small GFP-positive and GFP-negative cells were seen early in culture. While the large cells stained positive for stroma cell markers (smooth muscle actin), mesenchymal stem cell makers (CD73, CD105, CD44) or macrophages (CD45, CD14), the small cells were negative for all these markers and after 30 cell doublings, these cells displayed the classical phenotype of MAPC (CD45−,CD105−, CD44−, CD73−, FLK-1+(vascular endothelial growth factor receptor 2, VEGFR2), Sca-1+,CD13+). In a second experiment, BM obtained one month post BM transplant (n=3) was harvested and mononuclear cells were sorted as GFP-positive and GFP-negative cells and were cultured in MAPC expansion medium. MAPC grew from the GFP-positive fraction. These GFP positive cells displayed the typical MAPC-like immunophenotypes, displayed a normal diploid karyotype and were expanded for more than 50 cell doublings and differentiated into endothelial cells, hepatocytes and neurons. To rule out the possibility that MAPC are the product of cell fusion between a host and a donor cell either in vivo or in our in vitro culture conditions, we performed sex mismatched transplants of female GFP donor BM cells into a male host. BM from 5 chimeras were harvested 4 weeks after transplant and MAPC cultures were established. MAPC colonies were then sorted as GFP-positive and GFP- negative and analyzed for the presence of Y-chromosome by FISH analysis. As expected all GFP-negative (host cells) contained the Y-chromosome whereas all GFP-positive cells (donor cells) were negative for the Y-chromosome by FISH. This proves that MAPC are not derived from an in vitro or in vivo fusion event. In a third study, BM mononuclear cells from mice that had been previously BM-transplanted with syngeneic GFP-positive donors (n=3) were transplanted into a second set of syngeneic recipients (n=9). Two months after the second transplant, BM was harvested and mononuclear cells were cultured in MAPC medium. The secondary recipients also contained GFP-positive MAPC. This is the first demonstration that BM transplantation leads to the transfer of cells that upon isolation in vitro generate MAPCs and, whatever the identity of this cell may be, is eliminated by irradiation. We believe this is an important observation as MAPC hold great clinical potential for stem cell and/or gene therapy and, thus, BM transplant may serve as a way to deliver and reconstitute the MAPC population. In addition, this study provides insight into the nature of MAPC. The capacity to be transplantable within unfractionated BM transplant renders a functional and physiological distinction between MAPC and BM stromal cells. This study validates the use of unfractionated BM transplants to study the nature and possible in vivo role of MAPC in the BM.

Download Full-text

p38MAPK Inhibitor LSN2322600 Modulates the Bone Marrow Microenvironment and Inhibits Osteoclastogenesis in Multiple Myeloma.

Blood ◽

10.1182/blood.v108.11.5042.5042 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5042-5042

Author(s):

Kenji Ishitsuka ◽

Teru Hideshima ◽

Paola Neri ◽

Sonia Vallet ◽

Norihiko Shiraishi ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Mononuclear Cells ◽

Severe Combined Immunodeficiency ◽

Marrow Stromal Cells ◽

Skeletal Complications

Abstract The interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment plays a crucial role not only in proliferation and survival of MM cells, but also in osteoclastogenesis. In this study, we examined diverse potential of novel p38MAPK inhibitor LSN2322600 (LSN) for MM therapy in vitro and in vivo. The cytotoxic activity of LSN against MM cell lines was modest; however, LSN significantly enhances the cytotoxicity of Bortezomib by down-regulating Bortezomib-induced heat shock protein (HSP) 27 phosphorylation. We next examined the effects of LSN on cytokine secretion in MM cells, bone marrow stromal cells and osteoclast precursor cells. LSN inhibited IL-6 secretion from long-term cultured-bone marrow stromal cells (LT-BMSCs) and bone marrow mononuclear cells (BMMNCs) from MM patients in remission. LSN also inhibited MIP-1 α secretion by fresh tumor cells, BMMNCs and CD14 positive cells. Since these cytokines mediate osteoclastogenesis, we further examined whether LSN could inhibit osteoclastogenesis. Importantly, LSN inhibited in vitro osteoclastogenesis induced by macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of nuclear factor- κ B ligand (sRANKL), as well as osteoclastogenesis in the severe combined immunodeficiency (SCID)-Hu mouse model of human MM. These results suggest that LSN represents a promising novel targeted strategy to reduce skeletal complications as well as to sensitize or overcome resistance to Bortezomib.

Download Full-text

SGX70393 Inhibits Bcr-AblT315I In Vitro and In Vivo and Completely Suppresses Resistance When Combined with Nilotinib or Dasatinib.

Blood ◽

10.1182/blood.v110.11.535.535 ◽

2007 ◽

Vol 110 (11) ◽

pp. 535-535 ◽

Cited By ~ 3

Author(s):

Thomas O’Hare ◽

Christopher A. Eide ◽

Jeffrey W. Tyner ◽

Amie S. Corbin ◽

Matthew J. Wong ◽

...

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Kinase Inhibitor ◽

Single Agent ◽

Resistance Profile ◽

Murine Bone Marrow ◽

Abl Kinase ◽

A Cell

Abstract Overview: Bcr-AblT315I is detected in the majority of CML patients who relapse after dasatinib- or nilotinib-based second-line Bcr-Abl kinase inhibitor therapy. SGX70393, an azapyridine-based Abl kinase inhibitor, is effective against Bcr-Abl and Bcr-AblT315I at low nanomolar concentrations in vitro and in cell lines. Here, we comprehensively profiled SGX70393 against native and mutant Bcr-Abl in vitro and in vivo. We also used a cell-based mutagenesis screen to evaluate the resistance profile of SGX70393 alone and in combination with imatinib, nilotinib, or dasatinib. Methods: We assessed colony formation in the presence of SGX70393 by murine bone marrow infected with retroviruses for expression of Bcr-Abl, Bcr-AblT315I, or a variety of other mutants. Toxicity was tested in clonogenic assays of normal bone marrow. SGX70393 effects on cellular tyrosine phosphorylation were measured by immunoblot and FACS in primary Bcr-AblT315I cells isolated from patients with CML or Ph+ B-ALL. In vivo activity was evaluated in a xenograft model using Ba/F3 cells expressing Bcr-AblT315I. Lastly, the resistance profile of SGX70393 was evaluated alone and in dual combinations with imatinib, nilotinib, or dasatinib in a cell-based mutagenesis assay. Results: Colony formation by murine bone marrow cells expressing Bcr-AblT315I (IC50: 180 nM) was reduced by SGX70393 in a dose dependent manner, while no toxicity was observed in colony forming assays of normal human or murine mononuclear cells at concentrations up to 2 μM. Ex vivo exposure of human Bcr-AblT315I mononuclear cells to SGX70393 decreased CrkL phosphorylation, while imatinib, nilotinib, or dasatinib had no effect. SGX70393 inhibited Bcr-AblT315I-driven tumor growth in mice and this was correlated with reduced levels of pCrkL in tumor tissue, while imatinib was ineffective. A cell-based mutagenesis screen revealed a profile of resistant clones confined to four p-loop residues and position 317. SGX70393 in combination with imatinib contracted the spectrum of resistant mutations relative to either single agent, though outgrowth could not be completely suppressed. Combining SGX70393 with low concentrations of nilotinib or dasatinib narrowed the resistance profile still further (residues 248 and 255 for nilotinib; 317 for dasatinib) and, with clinically achievable doses of either second drug, completely abrogated emergence of resistant subclones. Conclusions: SGX70393, a potent inhibitor of Bcr-AblT315I, exhibits a resistance profile centered around the p-loop and residue 317 of Bcr-Abl. Remarkably, in combination with nilotinib or dasatinib, outgrowth of resistant clones is completely suppressed. Single-agent therapy with an effective T315I inhibitor may provide a viable option for patients who relapse with Bcr-AblT315I. However, as a broader spectrum of mutations accounts for imatinib resistance, patients with acquired dasatinib or nilotinib resistance may continue to harbor residual mutant clones other than T315I. Thus, the full clinical potential of SGX70393 may be realized in combinations with a second Abl kinase inhibitor. Our findings provide the first demonstration that Abl kinase inhibitor combinations that include a T315I-targeted component such as SGX70393 have the potential to pre-empt Bcr-Abl-dependent resistance.

Download Full-text

Therapeutic angiogenesis by autologous adipose-derived regenerative cells: comparison with bone marrow mononuclear cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00310.2014 ◽

2014 ◽

Vol 307 (6) ◽

pp. H869-H879 ◽

Cited By ~ 25

Author(s):

Changning Hao ◽

Satoshi Shintani ◽

Yuuki Shimizu ◽

Kazuhisa Kondo ◽

Masakazu Ishii ◽

...

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Umbilical Vein ◽

Rabbit Model ◽

Bone Marrow Mononuclear Cell ◽

M2 Macrophages ◽

Hindlimb Ischemia ◽

Macrophages Polarization ◽

Ischemic Muscle

Transplantation of adipose-derived regenerative cell (ADRC) enhances ischemia-induced angiogenesis, but the underlying mechanism remains unknown. Here, we compared the efficacy between ADRC and bone marrow mononuclear cell (BM-MNC) transplantation in rabbits model of hindlimb ischemia and examined the possible roles of alternative phenotypic macrophages polarization in ADRC-mediated angiogenesis using mice model of hindlimb ischemia. ADRCs and BM-MNCs were isolated from New Zealand White rabbits and C57BL/6J mice. In rabbit studies, our data showed that ADRCs could incorporate into the endothelial vasculature in vitro and in vivo. Both ADRC-conditioned media (CM) and BM-MNC-CM enhanced the migratory ability and interrupted the process of apoptosis in human umbilical vein endothelial cells. Four weeks after cell transplantation, augmentation of postnatal neovascularization was observed in the ischemic muscle injected with either ADRCs or BM-MNCs. In mice studies, we presented that ADRCs polarized into the IL-10-releasing M2 macrophages through PGE2-EP2/4 axis and suppressed the expressions of TNF-α and IL-6 in the ischemic muscle. Gene expressions of several angiogenic cytokines were amplified in the macrophages cultured in ADRC-CM rather than BM-MNC-CM. Blockade of IL-10 using neutralizing MAb attenuated the ADRC-mediated angiogenesis and caused muscle apoptosis in vivo. In conclusion, ADRC transplantation harvested similar effect of neovascularization augmentation compared with BM-MNC in experimental rabbit model of hindlimb ischemia; ADRC displayed a unique immunoregulatory manner of accelerating IL-10-releasing M2 macrophages polarization through the PGE2-EP2/4 axis.

Download Full-text

In Vitro Toxicity of Fludarabine on Normal Bone Marrow (BM) Progenitors Suggests Its Involvement in the Impairment of Hematopoietic Progenitor (HP) Mobilization.

Blood ◽

10.1182/blood.v110.11.1192.1192 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1192-1192

Author(s):

Céline Richard ◽

Véronique Maguer-Satta ◽

Juliette Berger ◽

Sandrine Jeanpierre ◽

Franck E. Nicolini ◽

...

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Blood Platelet ◽

Biological Properties ◽

Lymphocytic Leukemia ◽

In Vitro Toxicity ◽

Bone Marrow Niche ◽

Dose Dependent

Abstract Fludarabine phosphate (FDR) is considered the most effective drug for treating aggressive B-cell Chronic Lymphocytic Leukemia (B-CLL). Nevertheless, several groups have reported negative effects on BM HP mobilization by G-CSF alone after several months and sometimes correlated with the blood platelet count. As it was previously reported that in vivo FDR-induced cytopenia suggesting toxicity towards HP, we postulate that FDR could impair two major cell components of the bone marrow niche - mesenchymal (MP) and hematopoietic cells - and durably alter the HP egress process. We assessed the effects of increasing doses of FDR (for 5 days) on normal BM MP and HP biological properties, on HP adherence to fibronectin (Fn) or stromal cells and on SDF-1-induced in vitro migration. The expression of molecules involved in HP egress, i.e. CXCR4, CD49d and CD106, was evaluated by flow cytometry. As we demonstrated previously that all MP express CD73, an ecto-nucleotidase probably involved in FDR metabolism, we then tested the effect of a specific CD73 inhibitor (α, b methylene adenosine 5′-diphosphate (MADP)) on MP response to FDR treatment. In two independent series, we found a dose-dependent toxic effect of FDR on BM mononuclear cells, particularly on clonogenic mesenchymal progenitors (MP) (n=8) and hematopoietic progenitors (HP) (n=9). The most sensitive progenitors were MP, BFU-E and CFU-Mk (from 1mM dose) but other progenitors (CFU-GM, CFU-Mix), including the most primitive (LTC-IC) (n=3), were also dose-dependently sensitive. We found that toxicity of FDR on MP was CD73-independent since no improvement in cell survival was observed in presence of MADP (n=4). Interestingly, after expanding the surviving cells, we observed that FDR-induced impairment of the proliferative capacity of input MP was transmitted to cell progeny during the following passages. This means that progeny-derived cells, that have not been directly in contact with FDR, are still affected by the initial dose of FDR in a dose-dependent fashion. In the hematopoietic compartment, FDR had no effect on mononuclear cell adhesion, but there was an increase in the adhesion of HP colony-forming cells (CFC) which correlated with an inhibition of SDF-1 induced migration. However, FDR did not modify the expression of CXCR4, CD49d or CD106 on mesenchymal (CD45CD14) − /CD73+ cells or hematopoietic CD34+ cells. In conclusion, FDR appeared toxic towards clonogenic MP and HP, and profoundly impaired cell metabolism, since the effect persisted in cell progeny. The high sensitivity of the mesenchymal component suggests a possible impairment of BM stem cell niches. Although there was no modification of expression of molecules involved in egress, increased CFC adhesion and inhibition of HP migration suggest a FDR-induced retention of HSC in bone marrow. We are currently evaluating these parameters in MP and HP cells isolated from the bone marrow of CLL patients.

Download Full-text

Chronic Myelogenous Leukemia Cells Contribute to a Bone Marrow-Stroma in In Vivo NOD/SCID Mouse System.

Blood ◽

10.1182/blood.v114.22.2191.2191 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2191-2191

Author(s):

Ryosuke Shirasaki ◽

Haruko Tashiro ◽

Yoko Oka ◽

Toshihiko Sugao ◽

Nobu Akiyama ◽

...

Keyword(s):

Bone Marrow ◽

Scid Mouse ◽

Mononuclear Cells ◽

Bone Marrow Cells ◽

Myelogenous Leukemia ◽

Bone Marrow Stroma ◽

Marrow Stroma ◽

Mouse System

Abstract Abstract 2191 Poster Board II-168 Aims: The stroma-forming cells in a bone marrow are derived from hematopoietic stem cells. We reported previously that non-adherent leukemia blast cells converted into myofibroblasts to create a microenvironment for proliferation of leukemia blasts in vitro. In this report we demonstrate that with severe combined immunodeficiency (SCID) mouse system chronic myelogenous leukemia (CML) cells are also differentiated into myofibroblasts to contribute to a bone marrow-stroma in vivo. Materials and Methods: Bone marrow cells were collected from informed CML patients, from which mononuclear cells were separated with density-gradient sedimentation method. After discarded an adherent cell-fraction, non-adherent mononuclear cells were injected to the priory 2.5 Gray-irradiated non-obese diabetes (NOD)/SCID mice intravenously. For the inactivation of NK cells, anti-Asialo GM1 antibody was injected intra-peritoneally prior to the transplantation, and on each 11th day thereafter. Blood was collected to monitor Bcr-Abl transcript, and mice were sacrificed after chimeric mRNA was demonstrated. Bone marrow cells were obtained, and sorted with anti-human CD133 antibody and -CD106 to select CML-derived human stromal myofibroblasts referred to the in vitro data. The isolated positive fraction was further cultured, and the biological and the molecular characteristics were analyzed. Results and Discussion: When non-adherent CML cells were transplanted to NOD/SCID mice, CML cells were engrafted after 2 months. In the murine bone marrow human stromal cells were identified, in which BCR and ABL gene was fused with FISH analysis. When the parental CML cells were cultured on the CML-derived myofibroblasts, CML cells grew extensively in a vascular endothelial growth factor-A-dependent fashion. These results indicate that CML cells can create their own microenvironment for proliferation in vivo. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Primary Bone Marrow-Derived MSC Subsets Have Distinct Wnt-Signatures Compared to Conventionally Cultured MSC and Differ in Their Capacity to Support Hematopoiesis in Vitro,

Blood ◽

10.1182/blood.v118.21.3414.3414 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3414-3414 ◽

Cited By ~ 1

Author(s):

Marijke W Maijenburg ◽

Marion Kleijer ◽

Kim Vermeul ◽

Erik P.J. Mul ◽

Floris P.J. van Alphen ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Wnt Signaling ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Cell Types ◽

Hematopoietic Stem ◽

Wnt Proteins

Abstract Abstract 3414 Mesenchymal stromal cells (MSC) are of promising therapeutic use to suppress immunogenic responses following transplantation, and to support expansion of hematopoietic stem- and progenitors cells (HSPC) from small transplants derived for instance from cord blood. Culture-expanded MSC produce a wide variety and quantity of Wnt-proteins and the crucial role of Wnt-signaling in the hematopoietic stem cell niche is well established. However, studies addressing Wnt-signaling in MSC have (i) only focused on culture-expanded MSC and (ii) did not discriminate between phenotypically distinct subpopulations which are present in bulk cultures of expanded MSC. Recently we identified three new subpopulations of MSC in human bone marrow (BM) based on expression of CD271 and CD146: CD271brightCD146−, CD271brightCD146+, CD271−CD146+. These fractions co-express the “classical” MSC markers CD90 and CD105 and lack expression of CD45 and CD34 (Maijenburg et al, Blood 2010, 116, 2590). We and others demonstrated that the adult BM-derived CD271brightCD146− and CD271brightCD146+ cells contain all colony forming units-fibroblasts (Maijenburg et al, Blood 2010, 116, 2590; Tormin et al, Blood 2010, 116, 2594). To investigate how these primary subsets functionally compare to conventional, culture-expanded MSC, we investigated their Wnt-signature and hematopoietic support capacity. To this end, we sorted CD271brightCD146− and CD271brightCD146+ cells from human adult BM (n=3) and compared their Wnt-signatures obtained by Wnt-PCR array to the profiles from cultured MSC from the same donors. Fifteen genes were consistently differentially expressed in the two sorted uncultured subsets compared to their conventionally cultured counterparts. Expression of CCND1, WISP1 and WNT5B was strongly increased, and WNT5A was only detected in the conventionally cultured MSC. In contrast, WNT3A was exclusively expressed by sorted primary CD271brightCD146− and CD271brightCD146+ cells, that also expressed higher levels of JUN, LEF1 and WIF1. The differences in Wnt (target)-gene expression between CD271brightCD146− and CD271brightCD146+ cells were more subtle. The Wnt-receptors LRP6 and FZD7 were significantly higher expressed in CD271brightCD146+ cells, and a trend towards increased expression in the same subset was observed for CTNNB1, WNT11 and MYC. When the sorted subsets were cultured for 14 days (one passage), the differences in Wnt-related gene expression between the subsets was lost and the expanded sorted cells acquired an almost similar Wnt-signature as the MSC cultured from BM mononuclear cells from the same donors. The cultured subsets lost the expression of Wnt3a and gained the expression of Wnt5a, similar to the unsorted MSC cultured from the same donors in parallel. Despite the loss of a distinct Wnt-signature, co-culture experiments combining the sorted MSC subsets with human HSPC revealed that CD271brightCD146+ cells have a significantly increased capacity to support HSPC in short-term co-cultures (2 weeks) compared to CD271brightCD146− cells (p<0.021, n=3), which was analyzed in hematopoietic colony assays following co-culture. In contrast, a trend towards better long-term hematopoietic support (co-culture for 6 weeks) was observed on CD271brightCD146− cells. In conclusion, we demonstrate for the first time that primary sorted uncultured MSC subsets have a distinct Wnt-signature compared to cultured unsorted MSC and display differences in hematopoietic support. As it was recently shown that CD271brightCD146− and CD271brightCD146+ MSC localize to separate niches in vivo (Tormin et al, Blood 2011), our data indicate that the two MSC subsets are not necessarily distinct cell types and that the different Wnt-signature may be a reflection of these distinct microenvironments. Cell culturing for only one passage dramatically changed the Wnt-signature of the sorted MSC subsets, indicating that Wnt-signaling in in vitro expanded MSC does not resemble the Wnt-signature in their tissue resident counterparts in vivo. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Targeting STAT5 Expression Resulted in Molecular Response Improvement in Patients with Chronic Phase CML Treated with Imatinib

Blood ◽

10.1182/blood.v120.21.696.696 ◽

2012 ◽

Vol 120 (21) ◽

pp. 696-696 ◽

Cited By ~ 1

Author(s):

Philippe Rousselot ◽

Lydia Roy ◽

Gabriel Etienne ◽

Laurence Legros ◽

Aude Charbonnier ◽

...

Keyword(s):

Bone Marrow ◽

Cumulative Incidence ◽

Research Funding ◽

Molecular Response ◽

Undetectable Level ◽

Ppar Γ ◽

Cd34 Cells ◽

Daily Dose

Abstract Abstract 696 Background. We have previously demonstrated that PPAR-γ agonists (such as pioglitazone) negatively regulate the level of Stat5A and Stat5Bgene expression in normal CD34+ bone marrow progenitors (Prost, Le Dantec et al. 2008). Aim. We investigated whether targeting Stat5 expression with pioglitazone may impact clonogenic activity of Chronic Myelogenous Leukemia (CML) cells in vitro and result in molecular response improvement in vivo. Patients and methods. Preliminary in vitro studies tested the ability of pioglitazone to impact viability and clonogenicity of CD34+ primary cells from CML patients (pts). We conducted clonogenic and LTC-IC studies. Cultivated Ph+ -CD34+cells were characterized by facs and analyzed by CSFE assay. BCR-ABL and Stat5 expression were quantified by real time PCR. Control experiments were conducted using lentiviral vectors and siRNA assay for Stat5 and PPAR-g. CML pts were eligible in the ACTIM trial (EudraCT 2009–011675-79) if they were treated by imatinib for at least 2 years with a stable daily dose for at least 3 months and in major molecular response without having achieved CMR4.5 (defined by a BCR-ABL/ABL IS ratio ≤ 0.0032%). After inclusion, pts received imatinib (no dose modification) and started pioglitazone (Actos®) 30 mg/d during 2 months and 45 mg/d thereafter for 12 months. BCR-ABL transcript level was monitored every 3 months during the study period. Primary objective was the proportion of patients achieving a confirmed undetectable level of BCR-ABL transcript. Secondary objectives included cumulative incidence of CMR4.5 and safety. A companion biologic study evaluated imatinib through levels, Stat5 expression in bone marrow at baseline, months 6 and 12 and clonogenic activity of bone marrow mononuclear cells at baseline, months 6 and 12. Results. From the in vitro studies we first demonstrated that pioglitazone at pharmacological doses inhibited cell growth of the Bcr-Abl positive cell line K562 through the activation of the PPAR-g/STAT5 pathway. We next showed that PPAR-g / STAT5 pathway induced a clonogenic defect in CD34+ cells from CML patients. Moreover, the activation of the PPAR-g / STAT5 pathway also induced a clonogenic and a proliferative defect in CML LTC-IC. We then confirmed that imatinib induced a selection of insensitive quiescent CML cells and showed that this effect was abrogated by the activation of the PPAR-g / STAT5 pathway. Twenty seven pts were enrolled in the clinical trial and 24 were evaluable (1 was excluded in CMR4.5, 1 pt was not in MMR and 1 pt had consent withdrawal). Median age was 61.6 years (24.1–79) and median follow-up after inclusion was 13 months (9.8–21). All evaluable pts started pioglitazone as planned. Seven pts (29.2%) discontinued pioglitazone before 12 months, 6 following investigator decision after the warning of the French ministry of health regarding the risk of bladder cancer and 1 after its own decision. No pt discontinued due to adverse events. Discontinuations occurred between month 3 and month 9. Median duration of pioglitazone therapy was 11.2 months (2.6–15.4) median daily dose was 39.9 mg. No interaction was observed between imatinib and pioglitazone in term of through level before (median 850 ng/ml) and after (median 927 ng/ml) pioglitazone initiation (p=ns). Main adverse events were weight gain and worsening fluid retention in 3 pts. Three pts (14%) obtained a confirmed undetectable level of BCR-ABL transcript. The one year cumulative incidence of CMR4.5 was 57%. Stat5 mRNA quantification was significantly diminished in pt samples at M6 and M12 compared to the baseline values and a reduction of the clonogenic potential was also observed in bone marrow cells at M6 and M12. We collected “control pts” with similar characteristic (n=20). None of these pts obtained a confirmed CMR and the cumulative incidence of CMR4.5 in this control group was 27% as compared to 57% in the pioglitazone group (p=0.02). Conclusion. We have extended our in vitro results showing that PPAR-γ agonists resulted in Stat5 down regulation in CML CD34+ cells and preferentially reduced their clonogenic and long term potency in CFCs and LTC-IC assays. We now demonstrated that these effects translate in vivo by the achievement of MMR in more than half of the pts treated with the combination of pioglitazone and imatinib suggesting that it may be possible to target quiescent CML cells in vivo and supporting the concept of stem cell pool erosion. Disclosures: Off Label Use: pioglitazone in CML. Roy:Novartis, BMS: Speakers Bureau. Legros:Novartis, BMS: Research Funding, Speakers Bureau. Coiteux:Novartis, BMS: Speakers Bureau. Mahon:Novartis, BMS: Consultancy, Research Funding.

Download Full-text

Chronic Myelogenous Leukemia Cells Contribute to the Stromal Myofibroblasts in Leukemic NOD/SCID MouseIn Vivo

Journal of Oncology ◽

10.1155/2012/901783 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Ryosuke Shirasaki ◽

Haruko Tashiro ◽

Yoko Oka ◽

Takuji Matsuo ◽

Tadashi Yamamoto ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Myelogenous Leukemia ◽

Mononuclear Cells ◽

Severe Combined Immunodeficiency ◽

Myelogenous Leukemia ◽

Mouse Bone Marrow ◽

Murine Bone Marrow ◽

Endothelial Growth Factor

We recently reported that chronic myelogenous leukemia (CML) cells converted into myofibroblasts to create a microenvironment for proliferation of CML cellsin vitro. To analyze a biological contribution of CML-derived myofibroblastsin vivo, we observed the characters of leukemic nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mouse. Bone marrow nonadherent mononuclear cells as well as human CD45-positive cells obtained from CML patients were injected to the irradiated NOD/SCID mice. When the chimericBCR-ABLtranscript was demonstrated in blood, human CML cells were detected in NOD/SCID murine bone marrow. And CML-derived myofibroblasts composed with the bone marrow-stroma, which produced significant amounts of human vascular endothelial growth factor A. When the parental CML cells were cultured with myofibroblasts separated from CML cell-engrafted NOD/SCID murine bone marrow, CML cells proliferated significantly. These observations indicate that CML cells make an adequate microenvironment for their own proliferationin vivo.

Download Full-text