Sequential Treatment with Rigosertib Followed By Azacitidine Maximizes the Effects on the Interferon Signaling Pathway in Hematopoietic Cells in Myelodysplastic Syndrome (MDS)

Abstract Background: MDS is a clonal stem cell disorder characterized by abnormal maturation and differentiation of hematopoietic cells. Azacitidine (AZA), a hypomethylating agent (HMA), was approved by the FDA for patients (pts) with MDS and is the standard of care as 1st line therapy for higher- risk disease. About 50% of MDS pts respond to AZA, with a median duration of response of 14 - 24 month. For those pts responding to an HMA, most either relapse or progress with worsening bone marrow failure (BMF) and have a median survival of 4 to 6 months after treatment failure. Both primary and secondary resistance remains significant clinical problems and result in poor survival. A recent study reveals that AZA activates several immunomodulatory pathways leading to activation of the antiviral defense pathway and upregulation of interferon signaling (IFN) (Chiappinelli et al, 2017). INF has multiple effects on hematopoiesis and appears highly regulated. Interferon can stimulate hematopoietic stem cells (HSC) which may be countered through activation of p38 MAPK; inhibition of the latter may improve hematopoiesis. Short-term effects of IFN stimulate myeloid and erythroid hematopoiesis. Rigosertib (RIG) is a Ras-mimetic that blocks the activation of Ras effector proteins. It has been identified as a novel anti-cancer drug which inhibits cell cycle progression and induces apoptosis of cancer cells (Athuluri-Divakar et al, 2016). It activates apoptosis related pathways by ameliorating several signaling pathways like Akt, p38, MAPK/ JNK, STAT3, β-catenin, GSK3α/β and PLK1 that can be dysregulated in MDS pts (Xu et al, 2014). In vitro, the combination of RIG with AZA (Skidan et al, AACR 2006) showed synergistic effect in inducing cell death in a sequence dependent manner requiring RIG priming. We have reported that it acts as a histone deacetylase inhibitor with chromatin modifying activity (Chaurasia et al, ASCO 2016). In a Phase I/II study the combination of RIG and AZA (NCT 01926587) produced an ORR of 85% in pts who were HMA naïve and importantly 62% in HMA failures (Navada et al, EHA 2017). The ability to reverse the clinical resistance phenotype is a novel observation with important clinical implications and understanding the mechanisms is critical to develop ways of reversing resistance. Methods: We investigated the in vitro effects of RIG combined with AZA on two cell lines one resistant and one sensitive to AZA: BW90, an AZA resistant line and MDS-L, AZA sensitive and in specimens from 2 pts treated on a clinical trial of RIG and AZA in combination, to determine effects on hematopoietic cells and IN signaling. Results: QT-PCR studies demonstrated that individual treatment of MDS-L and BW90 with RIG or combined with AZA in sequential treatment (AZA/RIG or RIG/AZA) altered chromatin remodeler (KDM2a, SET1, JMJD3 and LRWD1) transcript levels in a cell line specific context. Exposure of cells from a bone marrow from a patient prior to treatment with RIG alone or RIG/AZA failed to induce expansion of CD34+ cells and yielded maximum aldehyde dehydrogenase (ALDH) activity, a marker of primitive hematopoietic stem and progenitor cells (HSCs) (ANOVA, p=0.006). AZA alone yielded a 3.8 fold expansion of CD34+, with marked decrease in ALDH activity that was inversely proportional to the expansion of CD34+ cells. Expansion of CD34+ cells led to ≥2 fold increase in pluripotent genes (SOX2, OCT4, NANOG and ZIC3) expression levels. QT PCR studies of the effects of AZA or RIG alone or in combination on INF yielded differential effects on INFa and INFb expression as follows compared to control reported as percent fold increase for INFa - AZA 2x; RIG 0.75x, AZA/RIG 1.5x; RIG/AZA 2.5x; INFb AZA 1.3x; RIG 0.6x, AZA/RIG 1.2x; RIG/AZA 0.7x. There was a significant increase in INFa (p=<0.0001) but not INFb, with RIG priming compared to AZA alone or AZA/RIG. Conclusion: Treatments with RIG either alone or combined with AZA results in epigenetic reprogramming of pluripotency genes, and expansion of primitive HSPC. This may serve to regulate HSPC pluripotency and expression of a maturation program. A significant increase in expression of IFNa seen after exposure to RIG/AZA may lead to enhanced hematopoietic function and may explain the differences in the alternative sequence of AZA/RIG. Further studies are underway to define more specific effects on these pathways to explain the impact on the resistance phenotype. Disclosures Navada: Onconova: Research Funding. Silverman:Mount Sinai School of Medicine: Employment; Onconova Therapeutics Inc.: Patents & Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding; Johnson and Johnson: Research Funding; Bayer: Research Funding.

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Anti-CD123 Chimeric Antigen Receptor T Cells (CART-123) Provide A Novel Myeloablative Conditioning Regimen That Eradicates Human Acute Myeloid Leukemia In Preclinical Models

Blood ◽

10.1182/blood.v122.21.143.143 ◽

2013 ◽

Vol 122 (21) ◽

pp. 143-143 ◽

Cited By ~ 4

Author(s):

Saar Gill ◽

Sarah K Tasian ◽

Marco Ruella ◽

Olga Shestova ◽

Yong Li ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Research Funding ◽

Conditioning Regimen ◽

Hematopoietic Stem ◽

Gm Csf ◽

Nsg Mice ◽

Cd34 Cells ◽

Society Research

Abstract Engineering of T cells with chimeric antigen receptors (CARs) can impart novel T cell specificity for an antigen of choice, and anti-CD19 CAR T cells have been shown to effectively eradicate CD19+ malignancies. Most patients with acute myeloid leukemia (AML) are incurable with standard therapies and may benefit from a CAR-based approach, but the optimal antigen to target remains unknown. CD123, the IL3Rα chain, is expressed on the majority of primary AML specimens, but is also expressed on normal bone marrow (BM) myeloid progenitors at lower levels. We describe here in vitro and in vivostudies to evaluate the feasibility and safety of CAR-based targeting of CD123 using engineered T cells (CART123 cells) as a therapeutic approach for AML. Our CAR consisted of a ScFv derived from hybridoma clone 32716 and signaling domains from 4-1-BB (CD137) and TCR-ζ. Among 47 primary AML specimens we found high expression of CD123 (median 85%, range 6-100%). Quantitative PCR analysis of FACS-sorted CD123dim populations showed measurable IL3RA transcripts in this population, demonstrating that blasts that are apparently CD123dim/neg by flow cytometry may in fact express CD123. Furthermore, FACS-sorted CD123dimblasts cultured in methylcellulose up-regulated CD123, suggesting that anti-CD123 immunotherapy may be a relevant strategy for all AML regardless of baseline myeloblast CD123 expression. CART123 cells incubated in vitro with primary AML cells showed specific proliferation, killing, and robust production of inflammatory cytokines (IFN-α, IFN-γ, RANTES, GM-CSF, MIP-1β, and IL-2 (all p<0.05). In NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with the human AML cell line MOLM14, CART123 treatment eradicated leukemia and resulted in prolonged survival in comparison to negative controls of saline or CART19-treated mice (see figure). Upon MOLM14 re-challenge of CART123-treated animals, we further demonstrated robust expansion of previously infused CART123 cells, consistent with establishment of a memory response in animals. A crucial deficiency of tumor cell line models is their inability to represent the true clonal heterogeneity of primary disease. We therefore engrafted NSG mice that are transgenic for human stem cell factor, IL3, and GM-CSF (NSGS mice) with primary AML blasts and treated them with CART123 or control T cells. Circulating myeloblasts were significantly reduced in CART123 animals, resulting in improved survival (p = 0.02, n=34 CART123 and n=18 control animals). This observation was made regardless of the initial level of CD123 expression in the primary AML sample, again confirming that apparently CD123dimAML may be successfully targeted with CART123 cells. Given the potential for hematologic toxicity of CART123 immunotherapy, we treated mice that had been reconstituted with human CD34+ cells with CART123 cells over a 28 day period. We observed near-complete eradication of human bone marrow cells. This finding confirmed our finding of a significant reduction in methylcellulose colonies derived from normal cord blood CD34+ cells after only a 4 hour in vitro incubation with CART123 cells (p = 0.01), and was explained by: (i) low level but definite expression of CD123 in hematopoietic stem and progenitor cells, and (ii) up-regulation of CD123 upon myeloid differentiation. In summary, we show for the first time that human CD123-redirected T cells eradicate both primary human AML and normal bone marrow in xenograft models. As human AML is likely preceded by clonal evolution in normal or “pre-leukemic” hematopoietic stem cells (Hong et al. Science 2008, Welch et al. Cell 2012), we postulate that the likelihood of successful eradication of AML will be enhanced by myeloablation. Hence, our observations support CART-123 as a viable therapeutic strategy for AML and as a novel cellular conditioning regimen prior to hematopoietic cell transplantation. Figure 1. Figure 1. Disclosures: Gill: Novartis: Research Funding; American Society of Hematology: Research Funding. Carroll:Leukemia and Lymphoma Society: Research Funding. Grupp:Novartis: Research Funding. June:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding. Kalos:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding.

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Targeting the Inflammatory Niche in MDS By Tasquinimod Restores Hematopoietic Support and Suppresses Immune-Checkpoint Expression in Vitro

Blood ◽

10.1182/blood-2021-150455 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2596-2596

Author(s):

Manja Wobus ◽

Ekaterina Balaian ◽

Uta Oelschlaegel ◽

Russell Towers ◽

Kristin Möbus ◽

...

Keyword(s):

Bone Marrow ◽

Mrna Expression ◽

Immunohistochemical Staining ◽

Research Funding ◽

Fold Increase ◽

Low Risk ◽

Hematopoietic Stem ◽

Healthy Donors ◽

The Impact

Abstract Introduction Myelodysplastic syndromes (MDS) belong to the most common hematological neoplasms in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. There is increasing evidence that an aberrant innate immune response and a proinflammatory bone marrow (BM) microenvironment play a critical role in the pathogenesis of MDS. The alarmin S100A9, a key player for regulation of inflammatory responses, has been shown to be elevated in MDS patients. It directs an inflammatory cell death (pyroptosis) by increased NF-kB mediated transcription and secretion of proinflammatory, hematopoiesis-inhibitory cytokines and production of reactive oxygen species. Tasquinimod (TASQ, Active Biotech) is a novel, oral small molecular drug with S100A9 inhibitory activity and it is currently investigated in a phase Ib/IIa trial in relapsed/refractory multiple myeloma (NCT04405167). TASQ has demonstrated anti-angiogenic, antitumor and immunomodulatory properties in a broad range of preclinical solid tumor models; however, little is known about its effects in myeloid malignancies. Aim We investigated the role of S100A9 in cellular models of MDS and the potential of TASQ to target S100A9 within the MDS stroma in vitro. Methods Immunohistochemical staining of S100A9, CD271+ mesenchymal stromal cells (MSCs), CD68+ macrophages and CD66b+ neutrophils in BM tissues from MDS patients and healthy donors was performed with multiplex immunohistochemistry and analyzed with the VECTRA imaging system. MSCs from patients with either low-risk MDS, CMML or age-adjusted healthy donors were exposed to S100A9 (1.5µg/ml) in the presence or absence of TASQ (10µM). Subsequently, TLR4 downstreaming molecules such as IRAK1, gasdermin and NF-kB-p65 were analyzed by Western blot. Moreover, the mRNA expression of further proinflammatory molecules (IL-1b, IL-18, caspase1) and PD-L1 was quantified by real-time PCR. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with S100A9 ± TASQ before CD34+ hematopoietic stem and progenitor cells (HSPCs) were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly followed by a CFU-GEMM assay in methylcellulose medium. Additionally, PD-1 mRNA expression was quantified in cocultured HSPCs. Results Immunohistochemical staining of BM tissue demonstrated S100A9 expression mainly by CD66b+ neutrophils and with less extent by CD68+ macrophages. In line with this, we could not detect relevant S100A9 mRNA expression in cultured MDS or healthy MSCs in vitro. Exposure of MDS and healthy MSCs with S100A9 induced TLR4 downstream signalling as demonstrated by increased expression of IRAK1 and NF-kB-p65. We further detected a higher expression of gasdermin, an inductor of pyroptosis, in S100A9 exposed MSCs. Addition of TASQ abolished these effects and inhibited the expression of the mentioned proteins, indicating an alleviation of inflammation. Furthermore, we detected a 2-fold increase of mRNA expression of the proinflammatory cytokines IL-1b and IL-18 as well as a 5-fold increase of their activator caspase 1 in MSCs after treatment with S100A9, which could be prevented by TASQ. Interestingly, PD-L1 as a potential downstream target was induced by S100A9 by 2.5-fold and could be suppressed by TASQ to about 50%. To evaluate the impact on the hematopoietic support of MSCs, we analysed MSC/HSPC cocultures after treatment with S100A9. We observed a decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay, indicating a disturbed hematopoietic support by S100A9 treated MSCs. Interestingly, both the number of CAF-C and CFU could be increased by TASQ pre-treatment. Finally, the PD-1 expression in co-cultured HSPCs was regulated in the same way as its ligand in treated MSCs, nominating this interaction as a potential target of S100A9/TASQ in the MDS BM. Conclusion In summary, we provide evidence that the pathological inflammasome activation in the myelodysplastic bone marrow can be rescued by TASQ at least in part by inhibition of the S100A9 mediated TLR4 downstream signalling including NF-kB-p65 transcription and PD-L1 expression. These effects result in an improved hematopoietic support by MSCs, suggesting a potential efficacy to improve cytopenia in low-risk MDS patients. Disclosures Balaian: Novartis: Honoraria. Törngren: Active Biotech: Current Employment. Eriksson: Active Biotech: Current Employment. Platzbecker: AbbVie: Honoraria; Takeda: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Geron: Honoraria. Röllig: Novartis: Honoraria, Research Funding; Jazz: Honoraria; Janssen: Honoraria; Bristol-Meyer-Squibb: Honoraria, Research Funding; Amgen: Honoraria; AbbVie: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Research Funding.

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Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽

10.1182/blood.v89.11.3919 ◽

1997 ◽

Vol 89 (11) ◽

pp. 3919-3924 ◽

Cited By ~ 328

Author(s):

Jean C.Y. Wang ◽

Monica Doedens ◽

John E. Dick

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Allogeneic Transplantation ◽

Functional Assay ◽

Hematopoietic Stem ◽

Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

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Primitive hematopoietic cells in murine bone marrow express the CD34 antigen

Blood ◽

10.1182/blood.v88.10.3774.bloodjournal88103774 ◽

1996 ◽

Vol 88 (10) ◽

pp. 3774-3784 ◽

Cited By ~ 65

Author(s):

F Morel ◽

SJ Szilvassy ◽

M Travis ◽

B Chen ◽

A Galy

Keyword(s):

Stem Cells ◽

Monoclonal Antibody ◽

Bone Marrow ◽

Progenitor Cells ◽

Significant Proportion ◽

Hematopoietic Cells ◽

Full Detail ◽

Hematopoietic Stem ◽

Cd34 Antigen

The CD34 antigen is expressed on most, if not all, human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and its use for the enrichment of HSCs with repopulating potential is well established. However, despite homology between human and murine CD34, its expression on subsets of primitive murine hematopoietic cells has not been examined in full detail. To address this issue, we used a novel monoclonal antibody against murine CD34 (RAM34) to fractionate bone marrow (BM) cells that were then assayed in vitro and in vivo with respect to differing functional properties. A total of 4% to 17% of murine BM cells expressed CD34 at intermediate to high levels, representing a marked improvement over the resolution obtained with previously described polyclonal anti-CD34 antibodies. Sixty percent of CD34+ BM cells lacked lineage (Lin) markers expressed on mature lymphoid or myeloid cells. Eighty-five percent of Sca-1+Thy-1(10)Lin- /10 cells that are highly enriched in HSCs expressed intermediate, but not high, levels of CD34 antigen. The remainder of these phenotypically defined stem cells were CD34-. In vitro colony-forming cells, day-8 and -12 spleen colony-forming units (CFU-S), primitive progenitors able to differentiate into B lymphocytes in vitro or into T lymphocytes in SCID mice, and stem cells with radioprotective and competitive long-term repopulating activity were all markedly enriched in the CD34+ fraction after single-parameter cell sorting. In contrast, CD34-BM cells were depleted of such activities at the cell doses tested and were capable of only short-term B-cell production in vitro. The results indicate that a significant proportion of murine HSCs and multilineage progenitor cells express detectable levels of CD34, and that the RAM34 monoclonal antibody is a useful tool to subset primitive murine hematopoietic cells. These findings should facilitate more direct comparisons of the biology of CD34+ murine and human stem and progenitor cells.

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Bone Marrow Cell Aggregates: A Way of Collecting the Adult Human Hematopoietic Stem Cell Niche

Blood ◽

10.1182/blood.v124.21.4363.4363 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4363-4363

Author(s):

Alexandre Janel ◽

Nathalie Boiret-Dupré ◽

Juliette Berger ◽

Céline Bourgne ◽

Richard Lemal ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Confocal Microscopy ◽

Hematopoietic Stem Cell ◽

Biological Samples ◽

Mesenchymal Cells ◽

Hematopoietic Stem ◽

Bm Niche ◽

Cd34 Cells

Abstract Hematopoietic stem cell (HSC) function is critical in maintaining hematopoiesis continuously throughout the lifespan of an organism and any change in their ability to self-renew and/or to differentiate into blood cell lineages induces severe diseases. Postnatally, HSC are mainly located in bone marrow where their stem cell fate is regulated through a complex network of local influences, thought to be concentrated in the bone marrow (BM) niche. Despite more than 30 years of research, the precise location of the HSC niche in human BM remains unclear because most observations were obtained from mice models. BM harvesting collects macroscopic coherent tissue aggregates in a cell suspension variably diluted with blood. The qualitative interest of these tissue aggregates, termed hematons, was already reported (first by I. Blaszek's group (Blaszek et al., 1988, 1990) and by our group (Boiret et al., 2003)) yet they remain largely unknown. Should hematons really be seen as elementary BM units, they must accommodate hematopoietic niches and must be a complete ex vivo surrogate of BM tissue. In this study, we analyzed hematons as single tissue structures. Biological samples were collected from i) healthy donor bone marrow (n= 8); ii) either biological samples collected for routine analysis by selecting bone marrow with normal analysis results (n=5); or iii) from spongy bone collected from the femoral head during hip arthroplasty (n=4). After isolation of hematons, we worked at single level, we used immunohistochemistry techniques, scanning electronic microscopy, confocal microscopy, flow cytometry and cell culture. Each hematon constitutes a miniature BM structure organized in lobular form around the vascular tree. Hematons are organized structures, supported by a network of cells with numerous cytoplasmic expansions associated with an amorphous structure corresponding to the extracellular matrix. Most of the adipocytes are located on the periphery, and hematopoietic cells can be observed as retained within the mesenchymal network. Although there is a degree of inter-donor variability in the cellular contents of hematons (on average 73 +/- 10 x103 cells per hematon), we observed precursors of all cell lines in each structure. We detected a higher frequency of CD34+ cells than in filtered bone marrow, representing on average 3% and 1% respectively (p<0.01). Also, each hematon contains CFU-GM, BFU-E, CFU-Mk and CFU-F cells. Mesenchymal cells are located mainly on the periphery and seem to participate in supporting the structure. The majority of mesenchymal cells isolated from hematons (21/24) sustain in vitro hematopoiesis. Interestingly, more than 90% of the hematons studied contained LTC-ICs. Furthermore, when studied using confocal microscopy, a co-localization of CD34+ cells with STRO1+ mesenchymal cells was frequently observed (75% under 10 µm of the nearest STRO-1+ cell, association statistically highly significant; p <1.10-16). These results indicate the presence of one or several stem cell niches housing highly primitive progenitor cells. We are confirming these in vitro data with an in vivo xenotransplantation model. These structures represent the elementary functional units of adult hematopoietic tissue and are a particularly attractive model for studying homeostasis of the BM niche and the pathological changes occurring during disease. Disclosures No relevant conflicts of interest to declare.

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In Vitro Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line

Stem Cells International ◽

10.1155/2018/9896142 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Hong Kiat Lim ◽

Pravin Periasamy ◽

Helen C. O’Neill

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Stromal Cell ◽

Hematopoietic Cells ◽

Model Systems ◽

Hematopoietic Stem ◽

Cell Production ◽

Stromal Cell Line ◽

Reticular Cells

There are very few model systems which demonstrate hematopoiesis in vitro. Previously, we described unique splenic stromal cell lines which support the in vitro development of hematopoietic cells and particularly myeloid cells. Here, the 5G3 spleen stromal cell line has been investigated for capacity to support the differentiation of hematopoietic cells from progenitors in vitro. Initially, 5G3 was shown to express markers of mesenchymal but not endothelial or hematopoietic cells and to resemble perivascular reticular cells in the bone marrow through gene expression. In particular, 5G3 resembles CXCL12-abundant reticular cells or perivascular reticular cells, which are important niche elements for hematopoiesis in the bone marrow. To analyse the hematopoietic support function of 5G3, specific signaling pathway inhibitors were tested for the ability to regulate cell production in vitro in cocultures of stroma overlaid with bone marrow-derived hematopoietic stem/progenitor cells. These studies identified an important role for Wnt and Notch pathways as well as tyrosine kinase receptors like c-KIT and PDGFR. Cell production in stromal cocultures constitutes hematopoiesis, since signaling pathways provided by splenic stroma reflect those which support hematopoiesis in the bone marrow.

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Characteristics of bone marrow fibroblast colony-forming cells (CFU-F) and their progeny in patients with myeloproliferative disorders

Blood ◽

10.1182/blood.v59.5.1046.1046 ◽

1982 ◽

Vol 59 (5) ◽

pp. 1046-1054 ◽

Cited By ~ 87

Author(s):

H Castro-Malaspina ◽

RE Gay ◽

SC Jhanwar ◽

JA Hamilton ◽

DR Chiarieri ◽

...

Keyword(s):

Bone Marrow ◽

Philadelphia Chromosome ◽

Hematopoietic Cells ◽

Indirect Evidence ◽

Myeloproliferative Disorders ◽

Immunofluorescent Staining ◽

Type I ◽

Hematopoietic Stem ◽

Similar Cell

Abstract Chronic myeloproliferative disorders (MPD) are clonal diseases of the pluripotent hematopoietic stem cell frequently associated with myelofibrosis (MF). There is only indirect evidence indicating that the increased deposition of collagen in bone marrow matrix is a secondary phenomenon. A liquid culture system for cloning and growing bone marrow fibroblasts has permitted us to approach more directly the understanding of the pathogenesis of myelofibrosis by comparing the biophysical, growth, and functional characteristics of fibroblasts from normals, MPD patients without MF, and those with MF. In patients with MF, marrow fibroblast colony (CFU-F) formation could not be studied; fibroblasts were grown from marrow explants. CFU-E from normals and MPD patients exhibited similar cell density distribution and similar cell sedimentation rates. These similarities contrasted sharply with the differences seen when the erythroid and granulocyte-macrophage progenitors were studied by the same methods. There was a marked light density shift and a rapidly sedimenting shift of MPD hematopoietic colony-forming cells. Marrow fibroblasts from MPD patients with and without MF displayed the same in vitro growth characteristics as fibroblasts from normals. Both types of fibroblasts exhibited anchorage and serum dependence, and contact inhibition of growth. Marrow fibroblasts were also characterized for the presence and distribution of fibronectin and collagen types by immunofluorescent staining using monospecific antibodies. Extracellular matrix, membrane-, and cytoplasm- associated fibronectin, type I, type III, and type V collagen showed a similar staining pattern in both normal and myelofibrotic marrow fibroblasts. Plasminogen-dependent fibrinolytic activity elicited from normal and myelofibrotic marrow fibroblasts were equivalent. Chromosomal analysis of hematopoietic cells and marrow fibroblasts from Philadelphia chromosome positive chronic myelocytic leukemia patients with and without MF showed that the Philadelphia chromosome was present only in hematopoietic cells. The results of these studies taken together demonstrate that bone marrow collagen-producing cells from MPD patients with and without MF behave in vitro as do those from normals. These findings support the hypothesis that that the marrow fibrosis observed in patients with MPD results from a reactive process rather than from a primary disorder affecting the marrow collagen-producing cells.

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Engraftment of Human Mesenchymal Stem Cells upon Transplantation into Newborn Rag2−/−gc−/− Mice.

Blood ◽

10.1182/blood.v108.11.1652.1652 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1652-1652

Author(s):

Patrick Ziegler ◽

Steffen Boettcher ◽

Hildegard Keppeler ◽

Bettina Kirchner ◽

Markus G. Manz

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cord Blood ◽

Human Mesenchymal Stem Cells ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Gm Csf ◽

Cd34 Cells ◽

Gene Transcripts

Abstract We recently demonstrated human T cell, B cell, dendritic cell, and natural interferon producing cell development and consecutive formation of primary and secondary lymphoid organs in Rag2−/−gc−/− mice, transplanted as newborns intra-hepatically (i.h.) with human CD34+ cord blood cells (Traggiai et al., Science 2004). Although these mice support high levels of human cell engraftment and continuous T and B cell formation as well as CD34+ cell maintenance in bone marrow over at least six month, the frequency of secondary recipient reconstituting human hematopoietic stem and progenitor cells within the CD34+ pool declines over time. Also, although some human immune responses are detectable upon vaccination with tetanus toxoid, or infection with human lymphotropic viruses such as EBV and HIV, these responses are somewhat weak compared to primary human responses, and are inconsistent in frequency. Thus, some factors sustaining human hematopoietic stem cells in bone marrow and immune responses in lymphoid tissues are either missing in the mouse environment, or are not cross-reactive on human cells. Human mesenchymal stem cells (MSCs) replicate as undifferentiated cells and are capable to differentiate to multiple mesenchymal tissues such as bone, cartilage, fat, muscle, tendon, as well as marrow and lymphoid organ stroma cells, at least in vitro (e.g. Pittenger et al., Science 1999). Moreover, it was shown that MSCs maintain CD34+ cells to some extend in vitro, and engraft at low frequency upon transplantation into adult immunodeficient mice or fetal sheep as detected by gene transcripts. We thus postulated that co-transplantation of cord blood CD34+ cells and MSCs into newborn mice might lead to engraftment of both cell types, and to provision of factors supporting CD34+ maintenance and immune system function. MSCs were isolated and expanded by plastic adherence in IMDM, supplemented with FCS and cortisone (first 3 weeks) from adult bone marrow, cord blood, and umbilical vein. To test their potential to support hemato-lymphopoiesis, MSCs were analyzed for human hemato-lymphotropic cytokine transcription and production by RT-PCR and ELISA, respectively. MSCs from all sources expressed gene-transcripts for IL-6, IL-7, IL-11, IL-15, SCF, TPO, FLT3L, M-CSF, GM-CSF, LIF, and SDF-1. Consistently, respective cytokines were detected in supernatants at the following, declining levels (pg/ml): IL-6 (10000-10E6) > SDF-1 > IL-11 > M-CSF > IL-7 > LIF > SCF > GM-CSF (0–450), while FLT3L and TPO were not detectable by ELISA. Upon i.h. transplantation of same passage MSCs (1X10E6) into sublethally irradiated (2x2 Gy) newborn Rag2−/−gc−/− mice, 2-week engraftment was demonstrated by species specific b2m-RT-PCR in thymus, spleen, lung, liver and heart in n=7 and additionally in thymus in n=3 out of 13 animals analyzed. Equally, GFP-RNA transcripts were detectable in the thymus for up to 6 weeks, the longest time followed, upon co-transplantation of same source CD34+ cells and retrovirally GFP-transduced MSCs in n=2 out of 4 animals. Further engraftment analysis of ongoing experiments will be presented. Overall, these results demonstrate that human MSC produce hemato-lymphoid cytokines and engraft in newborn transplanted Rag2−/−gc−/− mice, at least at early time-points analyzed. This model thus might allow studying hematopoietic cell and MSC-derived cell interaction, and might serve as a testing system for MSC delivered gene therapy in vivo.

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Muc1 Expression Identifies Human Self-Renewing Stem/Progenitor Populations and Is Elevated in AML CD34+ Cells.

Blood ◽

10.1182/blood.v110.11.4040.4040 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4040-4040

Author(s):

Szabolcs Fatrai ◽

Simon M.G.J. Daenen ◽

Edo Vellenga ◽

Jan J. Schuringa

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Fold Increase ◽

Marrow Stromal Cells ◽

Muc1 Expression ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Mucin1 (Muc1) is a membrane glycoprotein which is expressed on most of the normal secretory epithelial cells as well as on hematopoietic cells. It is involved in migration, adhesion and intracellular signalling. Muc1 can be cleaved close to the membrane-proximal region, resulting in an intracellular Muc1 that can associate with or activate various signalling pathway components such as b-catenin, p53 and HIF1a. Based on these properties, Muc1 expression was analysed in human hematopoietic stem/progenitor cells. Muc1 mRNA expression was highest in the immature CD34+/CD38− cells and was reduced upon maturation towards the progenitor stage. Cord blood (CB) CD34+ cells were sorted into Muc1+ and Muc1− populations followed by CFC and LTC-IC assays and these experiments revealed that the stem and progenitor cells reside predominantly in the CD34+/Muc1+ fraction. Importantly, we observed strongly increased Muc1 expression in the CD34+ subfraction of AML mononuclear cells. These results tempted us to further study the role of Muc1 overexpression in human CD34+ stem/progenitor cells. Full-length Muc1 (Muc1F) and a Muc1 isoform with a deleted extracellular domain (DTR) were stably expressed in CB CD34+ cells using a retroviral approach. Upon coculture with MS5 bone marrow stromal cells, a two-fold increase in expansion of suspension cells was observed in both Muc1F and DTR cultures. In line with these results, we observed an increase in progenitor counts in the Muc1F and DTR group as determined by CFC assays in methylcellulose. Upon replating of CFC cultures, Muc1F and DTR were giving rise to secondary colonies in contrast to empty vector control groups, indicating that self-renewal was imposed on progenitors by expression of Muc1. A 3-fold and 2-fold increase in stem cell frequencies was observed in the DTR and Muc1F groups, respectively, as determined by LTC-IC assays. To determine whether the above mentioned phenotypes in MS5 co-cultures were stroma-dependent, we expanded Muc1F and DTR-transduced cells in cytokine-driven liquid cultures. However, no proliferative advantage or increase in CFC frequencies was observed suggesting that Muc1 requires bone marrow stromal cells. In conclusion, our data indicate that HSCs as well as AML cells are enriched for Muc1 expression, and that overexpression of Muc1 in CB cells is sufficient to increase both progenitor and stem cell frequencies.

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Mesenchymal Stem and Progenitor Cells In the Mouse Bone Marrow Lack Expression of CD44.

Blood ◽

10.1182/blood.v116.21.2581.2581 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2581-2581

Author(s):

Hong Qian ◽

Mikael Sigvardsson

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Stromal Cells ◽

Stromal Cell ◽

Hematopoietic Cells ◽

Hematopoietic Stem ◽

Colony Forming Unit ◽

Stem And Progenitor Cells ◽

Cell Fractions

Abstract Abstract 2581 The bone marrow (BM) microenvironment consists of a heterogeneous population including mesenchymal stem cells and as well as more differentiated cells like osteoblast and adipocytes. These cells are believed to be crucial regulators of hematopoetic cell development, however, so far, their identity and specific functions has not been well defined. We have by using Ebf2 reporter transgenic Tg(Ebf2-Gfp) mice found that CD45−TER119−EBF2+ cells are selectively expressed in non-hematopoietic cells in mouse BM and highly enriched with MSCs whereas the EBF2− stromal cells are very heterogenous (Qian, et al., manuscript, 2010). In the present study, we have subfractionated the EBF2− stromal cells by fluorescent activated cell sorter (FACS) using CD44. On contrary to previous findings on cultured MSCs, we found that the freshly isolated CD45−TER119−EBF2+ MSCs were absent for CD44 whereas around 40% of the CD45−TER119−EBF2− cells express CD44. Colony forming unit-fibroblast (CFU-F) assay revealed that among the CD45−LIN−EBF2− cells, CD44− cells contained generated 20-fold more CFU-Fs (1/140) than the CD44+ cells. The EBF2−CD44− cells could be grown sustainably in vitro while the CD44+ cells could not, suggesting that Cd44− cells represents a more primitive cell population. In agreement with this, global gene expression analysis revealed that the CD44− cells, but not in the CD44+ cells expressed a set of genes including connective tissue growth factor (Ctgf), collagen type I (Col1a1), NOV and Runx2 and Necdin(Ndn) known to mark MSCs (Djouad et al., 2007) (Tanabe et al., 2008). Furthermore, microarray data and Q-PCR analysis from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca7,-8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the EBF2−CD44− cells, compared to the CD44+ cells indicating a relatively quiescent state of the CD44− cells ex vivo. This was confirmed by FACS analysis of KI67 staining. Furthermore, our microarray analysis suggested high expression of a set of hematopoietic growth factors and cytokines genes including Angiopoietin like 1, Kit ligand, Cxcl12 and Jag-1 in the EBF2−CD44− stromal cells in comparison with that in the EBF2+ or EBF2−CD44+ cell fractions, indicating a potential role of the EBF2− cells in hematopoiesis. The hematopoiesis supporting activity of the different stromal cell fractions were tested by in vitro hematopoietic stem and progenitor assays- cobblestone area forming cells (CAFC) and colony forming unit in culture (CFU-C). We found an increased numbers of CAFCs and CFU-Cs from hematopoietic stem and progenitor cells (Lineage−SCA1+KIT+) in culture with feeder layer of the EBF2−CD44− cells, compared to that in culture with previously defined EBF2+ MSCs (Qian, et al., manuscript, 2010), confirming a high capacity of the EBF2−CD44− cells to support hematopoietic stem and progenitor cell activities. Since the EBF2+ cells display a much higher CFU-F cloning frequency (1/6) than the CD44−EBF2− cells, this would also indicate that MSCs might not be the most critical regulators of HSC activity. Taken together, we have identified three functionally and molecularly distinct cell populations by using CD44 and transgenic EBF2 expression and provided clear evidence of that primary mesenchymal stem and progenitor cells reside in the CD44− cell fraction in mouse BM. The findings provide new evidence for biological and molecular features of primary stromal cell subsets and important basis for future identification of stage-specific cellular and molecular interaction pathways between hematopoietic cells and their cellular niche components. Disclosures: No relevant conflicts of interest to declare.

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