Decreased MHC-II Expression and Immune Dysfunction by Mesenchymal Stem Cells in the Bone Marrow of Patients with Myeloproliferative Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4647-4647
Author(s):  
Marianne D. Castillo ◽  
Pranela Rameshwar
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Hematological Malignancy ◽  
Flow Cytometric Analysis ◽  
Myeloproliferative Disorders ◽  
Bone Marrow Biopsies ◽  
Hematological Disorders ◽  
Mhc Ii ◽  
Immune Mediated

Abstract The etiology of hematological disorders has been studied at the cellular and molecular levels. These studies have led to an understanding of the effects by the bone marrow microenvironment on the pathophysiology of myeloproliferative disorders. The overarching hypothesis states that resident bone marrow Mesenchymal Stem Cells (MSCs) are important in the development of myeloproliferative disorders and are also involved in the development of fibrosis. The specific hypothesis is that MHC-II expression is decreased in patients MSCs. This makes them unable to act as antigen presenting cells and to suppress immune mediated mechanisms that lead to the development of some myeloproliferative disorders. MSCs were expanded from bone marrow aspirates of patients with AML (n=10), CML (n=10), and MDS (n=10). Flow cytometric analysis showed decreased MHC-II expression in MSCs from all patients as compared to MSCs from patients without hematological malignancy. The flow cytometry results were verified in functional studies using the MSCs as stimulators in a one way mixed lymphocyte reaction. Compared to MSCs from non-hematological malignancy patients, MSCs from study subjects showed reduced ability to elicit allogeneic responses. Retrospective analyses of bone marrow biopsies using immunohistochemistry showed an increase in the amount of MSCs in myelofibrosis patients, compared to patients without evidence of fibrosis, alluding to their role in the development of this condition. These results suggest that MSCs may be dysfunctional in patients with hematological disorders. Variations in the immune properties and the increased amount of MSCs in these patients open an avenue for a lingering question on the etiology of the development of some hematological disorders. Do dysfunctions of MSCs precede myeloproliferative disorders and leukemia or does the opposite occur? In summary, we provide insight into the immune-mediated mechanisms related to the pathophysiology of these disorders, which may have clinical implications for future therapies of bone marrow related disease.

Biological Characteristics of ZUC3 Antigen Expressed on Rat Bone Marrow Mesenchymal Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4254-4254
Author(s):  
He Huang ◽  
Jing Zheng ◽  
Xiaoyu Lai ◽  
Junli Cao ◽  
Jianling Fan
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Flow Cytometric Analysis ◽  
Osteoblastic Differentiation ◽  
Significant Decline ◽  
Human Mscs ◽  
Down Regulation ◽  
Flow Cytometric ◽  
Marrow Mesenchymal Stem Cells

Abstract Objective: Bone marrow mesenchymal stem cells (MSCs) are widely studied in recent years. As an important part of cell identification, specific surface markers of MSCs have been paid a lot of attention to for long, but no breakthrough as yet. Monoclonal antibodies (McAb) against surface of certain cells have been used to characterize cell lineages. ZUC3, a novel murine McAb was produced by hybridoma technology previously, which was specifically reactive with both human MSCs and rat MSCs. Studying the expression of ZUC3 antigen on rat MSCs after passage and differentiation, it was to define whether ZUC3 antigen would be available for the identification of rat MSCs or their differentiation lineages. Methods: Rat MSCs isolated by a single step of adhesion to cell culture plastic, and purified via replacement of medium and a serial of passage, then the cells were identified by surface molecules CD90, CD44 and CD45 by flow cytometry. Enzyme immunocytochemistry and indirect immunofluorescence were used to evaluate the availability of ZUC3 expression by rat MSCs as a surface marker. Then further exploratory researches were carried out concerning ZUC3 expression by rat MSCs during passages (P1 to P5) and multiple differentiation (neuron, osteoblasts and adipocytes) in the certain condition. Results: Homogeneous rat MSCs could be obtained in vitro, which were uniformly positive for adhesion molecules CD90, CD44, and negative for CD45. The McAb was specifically reactive with rat MSCs as the positive cells were more than 99% by immunohistochemistry and immunofluorescence staining, and ZUC3 antigen located on the membrane of rat MSCs. The flow cytometric analysis show ZUC3 antigen expression by rat MSCs from P1 to P5 were all more than 85%. Analysis by multiple comparison, it was found some differences between P2 and P1 (93.95±2.44% v.s. 86.90±1.80%, P<0.01). The maximal expression was reached at P3 (97.10±1.25%), and the flow cytometric analysis showed a single symmetrical peak. Data of P4 (94.50±2.23%) population were slightly lower than P3 (P>0.05). By contrast, P5 (88.35±2.99%) showed a significant decline comparing with the former passages (P<0.01). Furthermore, rat MSCs could be successfully induced to differentiate into neuron-like cells, osteoblasts, and adipocytes and there was to some extent a downward trend of ZUC3 expression after differentiation (P<0.01). More than 90% rat MSCs could transform to an neuron-like appearance which were positive for NeuN, NF-M after treated with alpha-thioglycerol, and there was some downward degree of ZUC3 expression (97.77±1.03% to 80.07±2.70%, P<0.01). During the osteoblastic differentiation, it was observed an obvious down-regulation of ZUC3 expression from the 10th day (96.63±1.03% to 90.07±2.40%, P<0.01 ) and percentage on the 10th (90.07±2.40%), 15th (84.43±2.80%), 20th (64.53±7.63%) and 25th (53.40±10.02%) day were significantly lower than their anterior time respectively (P<0.05). The results of adipogenic differentiation after MSCs incubated with proper medium were similar to what observed during osteoblastic differentiation and ZUC3 expression were down-regulation on the 7th (84.33±2.70%), 14th (75.90±2.00%) and 21st (70.57±0.47%) day compared with their anterior dots respectively (P<0.01). Conclusion: ZUC3 antigen could be used for identification of rat MSCs. Significant decline of ZUC3 expression had be observed after rat MSCs were induced to differentiate along neuronal, osteoblastic and adipogenic pathways, which indicated that ZUC3 antigen would be a marker of progenitor.

Mesenchymal Stem Cells from Bone Marrow of Children with Malignant and Non Malignant Hematological Disorders Are Not Quantitatively and Qualitatively Affected.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4312-4312
Author(s):  
Helen Dimitriou ◽  
Emmanouela Linardakis ◽  
Georgia Martimianaki ◽  
Eftichia Stiakaki ◽  
Anthi Fillipidi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Bone Marrow Involvement ◽  
Hematological Disorders ◽  
Lineage Differentiation ◽  
Hematopoietic Reconstitution ◽  
Multipotent Progenitor Cells

Abstract Mesenchymal Stem Cells (MSCs) are multipotent progenitor cells within the bone marrow (BM) capable of differentiating into various tissue specific cells including adipocytes (A), osteoblasts (O), chondrocytes (C), myoblasts, hepatocytes and possibly neuronal tissue. MSCs form an integral part of the BM stroma, have immunomodulatory functions and play an important role in the support of hematopoeisis. Their multipotentiality and ease of ex vivo expansion has raised great interest in the clinical use of MSCs for tissue repair and gene therapy. In order to evaluate if malignant and non malignant hematological diseases quantitatively and qualitatively affect BM derived MSCs, bone marrow from children with Acute Lymphoblastic Leukemia (ALL diagnosis n=9, different stages of treatment n=14, end of therapy n=9), Idiopathic Thrombocytopenic Purpura (ITP, n=12), autoimmune neutropenia (n= 9) and control patients (solid tumors without bone marrow involvement, n=19) was harvested and the mononuclear cell (MNC) fraction isolated. MSCs were expanded in aMEM supplemented with 10% selected FCS, characterized and compared in terms of their phenotypic characteristics, clonogenicity and ability to differentiate into A, O and C lineages. MNCs at day 0 of culture expressed high levels of CD34, CD45, CD29 and CD44, and very low levels of CD14, CD105 and CD90. Expression of hematopoietic markers (CD34, CD45 and CD14) on cells at passage 1 (P1) and thereafter progressively diminished while expression of CD29, CD44, CD90 and CD105 increased approaching 100%. High clonogenicity was observed in all samples at all passages as shown by the presence of CFU-F colonies (>50 cells) with the exception of ALL samples at diagnosis which showed impaired proliferation and clonogenicity that returned to normal at the following stages of treatment till the end of therapy. At P2 or P3, MSCs were differentiated towards the A, O, and C lineages by using specific induction media. Differentiation was assessed by histochemistry (Oil red O for A, von Kossa and Alkaline phosphatase (ALP) for O and Alcian Blue for C) and RT-PCR (LPL and aP2 for A, osteoprotegerin, osteocalcin and ALP for O, aggrecan and Col II for C). P2 or P3 MSCs from all groups exhibited bi- or tri-lineage differentiation. These results indicate that blood diseases of childhood do not affect the characteristics of MSCs and therefore could have clinical uses particularly in hematopoietic reconstitution following allotransplantation.

scholarly journals Mesenchymal Stem Cells in Immune-Mediated Bone Marrow Failure Syndromes

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Maria-Christina Kastrinaki ◽  
Konstantia Pavlaki ◽  
Aristea K. Batsali ◽  
Elisavet Kouvidi ◽  
Irene Mavroudi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Bone Marrow Failure ◽  
Bone Marrow Failure Syndromes ◽  
Immune Mediated

scholarly journals NFĸB Targeting in Bone Marrow Mesenchymal Stem Cell-Mediated Support of Age-Linked Hematological Malignancies.

2021 ◽  
Author(s):  
Pranela Rameshwar ◽  
Lauren S. Sherman ◽  
Shyam A. Patel ◽  
Marianne Castillo ◽  
Rachel Unkovic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Hematological Malignancies ◽  
High Risk Patient ◽  
Hematological Disorders ◽  
Mhc Ii ◽  
Potential Target ◽  
Allogeneic Stimulation ◽  
Acute Myeloid

Abstract Mesenchymal stem cells (MSCs) can become dysfunctional in patients with hematological disorders. An unanswered question is whether age-linked disruption of the bone marrow (BM) microenvironment is secondary to hematological dysfunction or vice versa. We therefore studied MSC function in patients with different hematological disorders and found decreased MHC-II except from one sample with acute myeloid leukemia (AML). The patients’ MSCs were able to exert veto properties except for AML MSCs. While the expression of MHC-II appeared to be irrelevant to the immune licensing of MSCs, AML MSCs lost their ability to differentiate upon contact and rather, continued to proliferate, forming foci-like structures. We performed a retrospective study that indicated a significant increase in MSCs, based on phenotype, for patients with BM fibrosis. This suggests a role for MSCs in patients transitioning to leukemia. NFĸB was important to MSC function and was shown to be a potential target to sensitize leukemic CD34+/CD38- cells to azacitidine. This correlated with their lack of allogeneic stimulation. This study identified NFĸB as a potential target for combination therapy to treat leukemia stem cells and showed that understanding MSC biology and immune response could be key in determining how the aging BM might support leukemia. More importantly, we show how MSCs might be involved in transitioning the high risk patient with hematological disorder to AML.

Following Autologous Stem Cell Transplantation (1 Yr) Mesenchymal Stem Cells Are Functionally Impaired with Reduced Hematopoietic Support

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3888-3888
Author(s):  
Catharina Hazenberg ◽  
Fiona A.J. van den Heuvel ◽  
Edo Vellenga ◽  
Annet Z. Brouwers-Vos ◽  
Gerbrig Berger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Autologous Stem Cell Transplantation ◽  
Population Doubling ◽  
Bone Marrow Biopsies

Abstract Autologous stem cell transplantation (ASCT) is frequently applied in patients with multiple myeloma and malignant lymphoma. Although adequate steady state hematopoiesis with normal peripheral blood counts is attained after ASCT, marked cytopenias may occur in times of stress such as sepsis or re-exposure to chemotherapy. Our group has previously shown impairment of the hematopoietic stem cell (HSC) compartment 1 year post ASCT (pASCT), reflected by reduced HSC frequency and quiescence, and increased ROS production (Haematologica 2013;98:1264). Considering the essential role for mesenchymal stem cells (MSCs) in supporting hematopoiesis, we studied the MSC compartment 1 year post ASCT. Bone marrow biopsies from pASCT patients (n=17) were studied and compared to normal bone marrow from healthy donors (NBM, n=20) by performing immunohistochemistry staining of endothelial cells by CD34 (indicating microvessel density, MVD) and MSCs by nestin, CD146 (Melanoma Cell Adhesion Molecule, MCAM) and CD271 (Nerve Growth Factor Receptor, NGFR). A significant increase in CD271+ MSCs was observed in pASCT bone marrow biopsies compared to NBM (p<0.0001), while the expression of additional markers did not differ between pASCT vs. NBM. MSCs were cultured from the CD34- fraction of bone marrow mononuclear cells, obtained from pASCT patients (n=17) and MSCs derived from NBM (n=20). MSCs were selected by their plastic-adherency and replated to generate MSCs. Although pASCT MSCs and NBM MSCs had similar population doubling times (1.92±0.22 and 3.52±1.02 in passage 4 (P4), pASCT MSCs cultured in vitro demonstrated a change in morphology from the onset of P4. We also observed premature exhaustion of growth in 45% of the studied patients at P5 (vs. 18% in NBM) and increased senescence shown by B-galactosidase staining in P5/P6 (p=0.04). Differentiation assays did not show impairment in differentiation towards osteoblasts or adipocytes of pASCT MSCs. Gene expression analysis on early passage MSCs showed upregulation of pro-inflammatory and cell cycle genes, such as IL6 and p21, in pASCT MSCs compared to NBM MSCs. Co-culture studies with cord blood-derived CD34+ cells on pASCT MSCs showed a significant reduction in output in CFC assays and significant reduction in number of cobblestone-area forming cells in pASCT co-cultures versus NBM (p < 0.05). Given the higher incidence of MDS and AML after ASCT, we questioned whether the observed phenotype of pASCT MSCs resembles MSCs from patients with MDS and AML. Therefore the endothelial and mesenchymal compartments of MDS (n=20) and AML (n=23) patients were studied. An increase in MVD was detected in MDS/AML bone marrow biopsies in contrast to NBM and pASCT (p < 0.05), while the expression of CD146, CD271 and nestin in MDS/AML patients was not significantly increased. 25% of AML MSC cultures showed no growth in the first passage. When MSC growth did occur, the remaining cultures did not show a difference in population doubling time or expansion. However, a change in morphology of MDS/AML MSCs similar to pASCT MSCs was observed. Studies of early passages of MDS/AML MSCs demonstrated a significantly increased gene expression of IL-6 and p21 comparable to pASCT MSCs. In addition PITX2 and Foxc1 expression was increased but no difference was observed in pASCT vs. MDS/AML MSCs. PITX2 has been linked to increased senescence of MDS MSCs while Foxc1 is linked to adipo-osteoprogenitor cell differentiation thereby affecting the HSC compartment. Since none of the pASCT patients did develop MDS, immunohistochemical stainings were also performed on bone marrow biopsies of patients that developed therapy related (t-)MDS/AML following ASCT for lymphoma and myeloma (n=7), after a mean of 117 (MDS) and 50 months (AML). An increase in MVD was observed shortly before or during MDS/AML development, which is probably related to the emergence of malignant cells. No major changes in the phenotype of the MSC compartment were observed before or during the emergence of t-MDS/AML, indicating that t-MDS/AML is preceded by an increase in MVD without distinct changes in the MSC compartment. In summary our results demonstrate that MSCs are affected after ASCT, as shown by expression pattern and functionality. These changes result in a pro-inflammatory phenotype with premature senescence and impaired support of hematopoietic cells, which may account for the reduced bone marrow reserve observed in pASCT patients. Disclosures No relevant conflicts of interest to declare.

Del(5q) Myelodysplastic Stem Cells Exhibit Their Clonal Advantage Via Increased Adhesion to the Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 790-790
Author(s):  
Christian Scharenberg ◽  
Valentina Giai ◽  
Andrea Pellagatti ◽  
Leonie Saft ◽  
Goran Karlsson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Cell Cycle Distribution ◽  
Matricellular Protein ◽  
Hematopoietic Stem ◽  
Bone Marrow Biopsies ◽  
Induced Changes

Abstract Abstract 790 Lenalidomide has emerged as a very effective therapy for del(5q) myelodysplasia. Its mechanism of action, however, has hitherto remained elusive. Interestingly, the more primitive hematopoietic compartment seems to possess a clonal advantage where del(5q) HSC are able to outcompete remaining normal HSC. We have previously demonstrated that lenalidomide is able to abrogate this clonal advantage and found that lenalidomide restored expression of the matricellular protein SPARC, a gene located within the commonly deleted region on chromosome 5q. We hypothesized that the decreased expression of SPARC in del(5q) HSC leads to increased adhesion of HSC to their respective niche cells, translating to increased rates of proliferation, partly explaining the competitive advantage against non-del(5q) HSC. We conducted a prospective study analyzing the del(5q) HSC/progenitor compartment from 23 patients before, during and after (refractory phase) lenalidomide, in order to test whether a hematopoietic stem cell (HSC)-intrinsic decrease of SPARC explains the why and how a clone of cells inherently defective at spawning functioning cellular descendants is not selected against, but rather exhibits a clonal advantage. In addition, we studied whether treatment with lenalidomide induced changes in the microenvironment these cells reside in. We analyzed cell cycle distribution, frequency of apoptosis, and expression of adhesion markers on normal and del(5q) HSPC by multi-parameter flow cytometry. These experiments revealed a slight increase in proliferation of del(5q) versus normal HSC, as well as complex changes in the expression of adhesion markers in HSC of patients treated with lenalidomide. We studied the functional adhesion of normal and del(5q) HSPC to defined matrix components of the microenvironment and observed that HSPC from del(5q) patients exhibited stronger adhesion than normal bone marrow cells to fibronectin and VCAM-1. Recombinant SPARC protein abrogated adhesion to VCAM-1 specifically in a subset of patients, while having no significant effect on normal HSPC. To study whether SPARC plays a role in the clonal dominance, we used lentiviral transduction to overexpress SPARC in HSPC and found that increased expression of SPARC led to severely reduced engraftment in NSG-mice. We also analyzed how lenalidomide impacts the microenvironmental niche. To this end, we compared the gene expression profile of mesenchymal stem cells (MSC) obtained from del(5q) patients by DNA microarray and found only 36 genes to be differentially expressed by more than 2-fold, with 15 and 21 genes up- or downregulated in del(5q) MSC, respectively. Using longitudinal bone marrow biopsies from 5 patients on and off treatment, we analyzed whether lenalidomide induced changes in the frequency of candidate niche cells such as mesenchymal stem cells (MSC), macrophages, endothelium and megakaryocytes, by using immunohistochemical markers for the aforementioned cells. Lenalidomide induced no significant changes in the number of nestin+ MSC but seemed to decrease the number of Factor-VIII+ megakaryocytes. Taken together, these studies suggest that decreased expression of SPARC leads to increased adhesion of del(5q) HSC/progenitor cells to defined components of the microenvironment and may explain why del(5q) HSC are able to outcompete the remaining healthy HSC. Our studies implicate that lenalidomide is able to abrogate this clonal advantage partly via its increase in SPARC expression with a consecutive decrease in adhesion. Disclosures: No relevant conflicts of interest to declare.

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