Analysis of Expansion Mesenchymal Stromal Cells in patients with low risk myelodysplastic syndrome

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis, cytopenias and dysplasia and one or more lineages. The stratification of MDS is made based on the percentage of bone marrow blasts, number of cytopenias and karyotype at diagnosis. Somatic mutations in the p53 tumor suppressor gene are found in approximately 50% of all human tumors, making it the most commonly mutated gene. The expression of p53 protein and the study of mutations is especially needed in the prognosis of MDS. In this context, the study aims to evaluate the expansion of mesenchymal stromal cells (MSCs) and the expression of p53 protein in patients with SMD, low risk, according to the International Prognostic System (IPSS), in order to demonstrate the importance of these evaluations also diagnostics. This is a cross-sectional analytical study with review 3 adult patients of both sexes, the diagnosis of low-risk MDS receiving outpatient treatment at the University Hospital Walter Cantídio (HUWC). MSCs were characterized by immunophenotyping and screening of mutation of the p53 gene by Real Time PCR System (Applied Biosystems). For data analysis, the statistical software was used GraphPadPrism 5.0. Statistical differences between groups were checked by Student t or Mann-Whitney’s test significance level was p < 0.05 for all analyzes. The results showed a smaller expansion of MSCs in the bone marrow of patients with MDS compared with a control group. A survey of mutation of the p53 gene was negative in all patients. The results demonstrate an impairment in the growth of MSCs in patients with MDS, collaborating with the hypothesis that medullary microenvironment in MDS may be compromised contributing greater understanding of disease mechanisms. However studies with larger sample should be conducted in order to establish the best results.

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Molecular Imaging for Comparison of Different Growth Factors on Bone Marrow-Derived Mesenchymal Stromal Cells’ Survival and ProliferationIn Vivo

BioMed Research International ◽

10.1155/2016/1363902 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10 ◽

Cited By ~ 5

Author(s):

Hongyu Qiao ◽

Ran Zhang ◽

Lina Gao ◽

Yanjie Guo ◽

Jinda Wang ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factors ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Bioluminescence Imaging ◽

Firefly Luciferase ◽

Control Group ◽

Induced Apoptosis

Introduction. Bone marrow-derived mesenchymal stromal cells (BMSCs) have emerged as promising cell candidates but with poor survival after transplantation. This study was designed to investigate the efficacy of VEGF, bFGF, and IGF-1 on BMSCs’ viability and proliferation bothin vivoandin vitrousing bioluminescence imaging (BLI).Methods. BMSCs were isolated fromβ-actin-Fluc+transgenic FVB mice, which constitutively express firefly luciferase. Apoptosis was induced by hypoxia preconditioning for up to 24 h followed by flow cytometry and TUNEL assay. 106BMSCs with/without growth factors were injected subcutaneously into wild type FVB mice’s backs. Survival of BMSCs was longitudinally monitored using bioluminescence imaging (BLI) for 5 weeks. Protein expression of Akt, p-Akt, PARP, and caspase-3 was detected by Western blot.Results. Hypoxia-induced apoptosis was significantly attenuated by bFGF and IGF-1 compared with VEGF and control groupin vitro(P<0.05). When combined with matrigel, IGF-1 showed the most beneficial effects in protecting BMSCs from apoptosisin vivo.The phosphorylation of Akt had a higher ratio in the cells from IGF-1 group.Conclusion. IGF-1 could protect BMSCs from hypoxia-induced apoptosis through activation of p-Akt/Akt pathway.

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Significant Engraftment of Immature Hematopoietic Cells From Patients with Low Risk Myelodysplastic Syndromes (MDS) in Immunodeficient Mice

Blood ◽

10.1182/blood.v120.21.1694.1694 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1694-1694

Author(s):

Hind Medyouf ◽

Florian Nolte ◽

Maximilian Mossner ◽

Verena Nowak ◽

Bettina Zens ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mesenchymal Stromal Cells ◽

Myelodysplastic Syndromes ◽

Stromal Cells ◽

Hematopoietic Cells ◽

Low Risk ◽

Cell Phenotype

Abstract Abstract 1694 Introduction: Myelodysplastic syndromes are a heterogeneous group of malignant clonal hematologic disorders characterized by ineffective hematopoiesis, peripheral cytopenias and dysplastic bone marrow cells, with frequent progression to acute myeloid leukemia. Because of its heterogeneous nature, modeling of this disease has proven to be very difficult in cell culture systems as well as mice. In addition, attempts to generate a xenotransplant model in immuno-compromised mice have only achieved very low levels of engraftment that are often transient, making it very difficult to study the biology of this disease in vivo. Recent studies in mice have shown that conditional impairment of the small RNA processing enzyme Dicer in mouse osteolineages induced a stromal niche that promoted myelodysplasia, leading to the hypothesis that abnormal bone marrow stromal cells might provide a “fertile soil“ for the expansion of the malignant clone. Patients and Methods: To the date of writing, a total of 12 primary hematopoietic stem cell- and mesenchymal stroma cell (MSCs) samples selected from patients with MDS have been isolated and xenotransplanted into NOD.Cg-Prkdscid Il2rgtm1Wjl/Szj (NSG) mice: MDS 5q- (n=7), MDS RCMD (n=3), MDS RAEB I (n=1), MDS-U (n=1). Engraftment was monitored by FACS using human specific antibodies to CD45, CD34 and CD38. In addition cell cycle behavior was analyzed by Ki67/Hoechst staining. Mesenchymal stromal cells were characterized using previously described stromal markers: CD105, CD271, CD73, CD166, CD90, CD146 and CD44. To isolate genomic DNA and RNA for molecular analyses, MDS xenografts were flow sorted based on human CD45 expression. Molecular characterization of primary MDS samples and xenotransplants was carried out by serial copy number analysis using Affymetrix SNP 6.0 Arrays, metaphase cytogenetics and direct sequencing of known mutations in the transplanted MDS samples. Results: We show, that the concomitant transplantation of MDS-derived mesenchymal stromal cells with the corresponding hematopoietic patient stem/progenitor cells leads to significant and long-term engraftment (0.1 – 15% for up to 23 weeks) of cells isolated from IPSS low and intermediate risk MDS patients. In addition to the bone marrow, MDS hematopoietic cells also infiltrate other hematopoietic compartments of the mouse including the spleen. Significant engraftment of cells with progenitor (CD34+CD38+) as well as stem cell phenotype (CD34+CD38-) was observed, which is consistent with engraftment of an MDS stem cell that sustains long-term hematopoiesis. SNP array analysis confirmed the clonal origin of the engrafted cells as MDS xenografts harboring the identical genomic lesions as present in the patient disease. Conclusion: We present a robust MDS xenograft model of low risk MDS entities based on the concomitant transplantation of primary MDS hematopoietic cells with MSCs from the same patients. This model does not only allow to study the biology of this disease in vivo but also the molecular and cellular interactions between MSCs and hematopoietic MDS cells. In addition it provides a useful platform to study the effects of new experimental therapeutic agents for the treatment of MDS in molecularly defined MDS cells. Disclosures: No relevant conflicts of interest to declare.

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Chd1 Regulates Primitive State of Bone Marrow Mesenchymal Stromal Cells without Affecting Hematopoietic Support; An Insight on the Dissociation Between Primitive State and Niche Function

Blood ◽

10.1182/blood.v124.21.1587.1587 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1587-1587

Author(s):

Il-Hoan Oh ◽

Hyun-Kyung Choi

Keyword(s):

Bone Marrow ◽

Chromatin Remodeling ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Expression Level ◽

Control Group ◽

Open Chromatin ◽

Hematopoietic Stem ◽

Primitive State

Abstract Mesenchymal stromal cells (MSCs) are characterized by heterogeneity in the proliferation/self-renewal potentials and hematopoietic supporting activity among subpopulations. Numerous studies have suggested that a primitive state of MSC subpopulation are correlated to its niche function to support hematopoietic stem cells (HSCs), but the mechanisms regulating primitive state of MSCs remains poorly understood. In the present study, we examined the role of a chromatin remodeling enzyme, chd1 in the maintenance of open chromatin and undifferentiated state of MSCs. We analyzed for expression in MSCs, the expression level of chd1 progressively decreased during in-vitro subculture (from 7 to 18 passages) in a manner proportional to the passage numbers. Moreover, chd1 expression was down regulated in the MSCs during their differentiation into adipogenic or osteogenic lineages, compared to proliferative state, indicating the correlations between MSC proliferation potentials and expression level of chd1. Next, we transduced human bone marrow-derived MSCs with shRNAs against chd1 and found that chd1 knock down MSCs (chd1-KD) exhibit significant loss of colony forming activity (CFU-F), decrease of cell proliferation and loss of multi-lineage differentiation towards osteogenic or adipogenic lineages. Moreover, chd1-KD MSCs exhibited lower level expression of pluripotency-related genes, oct-4, sox-2 and nanog, with concomitant increase of H3K9me3 on the promoters and decreased chromatin accessibility in the oct-4 promoter, suggesting that chd1 regulate open chromatin and multi-lineage potential of MSCs. However, KD of chd1 in MSCs did not affect the HSC-supporting activity of MSCs; human cord blood-derived CD34+ cells co-cultured on chd1-KD MSCs exhibited rather higher maintenance of primitive phenotype (CD34+90+) and higher repopulating activity in NOD/SCID-ɤC KO mice compared to those co-cultured on control group MSCs. Together, these results show that, while primitive state of MSCs are regulated by chromatin remodeling complex,chd1, the hematopoietic niche activity of MSCs is not directly influenced by the primitive state of MSCs, raising a questions on the prevailing notion that undifferentiated MSCs can better support hematopoietic function. Disclosures No relevant conflicts of interest to declare.

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Mesenchymal Stromal Cells Accelerate Hematopoietic Reconstitution and Mediate An IL6-Dependent Regeneration of the Intestinal Epithelium of Lethally Irradiated Mice.

Blood ◽

10.1182/blood.v116.21.3846.3846 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3846-3846

Author(s):

Moira Francois ◽

Elena Birman ◽

Daniel L. Coutu ◽

Kathy-Ann Forner ◽

Jacques Galipeau

Keyword(s):

Bone Marrow ◽

Radiation Exposure ◽

Mesenchymal Stromal Cells ◽

Intestinal Epithelium ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Control Group ◽

Hematopoietic Reconstitution ◽

Radiation Induced ◽

Epithelium Regeneration

Abstract Abstract 3846 Apoptosis of radiation-sensitive stem cells in the bone marrow and the gut are the main complications arising from radiation exposure, whether therapeutic or accidental. Bone marrow-derived mesenchymal stromal cells (MSC) were shown to exert regenerative properties through the secretion of factors which act in a paracrine manner. In this study, we demonstrated that a single intra-peritoneal injection of C57Bl/6 MSC rescued lethally irradiated Balb/c mice. Histological analysis and Ki67 immunostaining of jejunum sections collected 48 hours and 5 days post MSC injection indicated that MSC protected the gastro-intestinal epithelium from severe radiation-induced damages and prompted the regeneration of the gut by stimulating proliferation of the crypt stem cell pool. Using interleukin-6 null MSC, we demonstrated that IL-6 derived from the MSC played a role accelerating gastro-intestinal epithelium regeneration. We also observed that MSC injection prevented severe anemia and restored granulocyte and platelet counts to normal values faster than the control group. Accelerated regeneration of the bone marrow and extra-medullar hematopoiesis in the spleen of MSC transplanted mice corroborated this observation. Our results suggest that IL-6 derived from MSC or used on its own may be exploited to alleviate radiation induced injuries to patients undergoing radiotherapy or subject to accidental radiation exposure Disclosures: No relevant conflicts of interest to declare.

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Hepatoprotective effect of bone marrow-derived mesenchymal stromal cells in CCl4-induced liver cirrhosis

3 Biotech ◽

10.1007/s13205-021-02640-y ◽

2021 ◽

Vol 11 (2) ◽

Author(s):

Ashwini P. Aithal ◽

Laxminarayana K. Bairy ◽

Raviraja N. Seetharam ◽

Naveen Kumar

Keyword(s):

Bone Marrow ◽

Liver Cirrhosis ◽

Portal Vein ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Disease Model ◽

Hepatoprotective Effect ◽

Control Group ◽

Multipotent Stem Cells ◽

Tail Vein

AbstractBone marrow mesenchymal stromal cells (BM-MSCs) are multipotent stem cells which are ideal candidates for use in regenerative medicine. The objectives of this study were to evaluate the hepatoprotective effect of BM-MSC and its combination treatment with silymarin in carbon tetrachloride (CCl4)-induced liver cirrhosis animal model and to investigate whether tail vein or portal vein infusion was the ideal route for BM-MSC transplantation. 36 female Wistar rats were randomly divided into six groups (n = 6): Group 1 (normal control), Group 2 (received only CCl4, disease model), Group 3 (CCl4 + BM-MSCs through tail vein), Group 4 (CCl4 + BM-MSCs through portal vein), Group 5 (CCl4 + silymarin), Group 6 (CCl4 + BM-MSCs + silymarin). On the 21st day after treatment, blood samples were collected for biochemical estimations. After the experiment, the rats were sacrificed. Liver was dissected out and processed for histopathology and scanning electron microscopy studies. Liver enzyme and marker analysis, histopathological studies indicated that the combination of BM-MSCs and silymarin was effective in treating liver cirrhosis. Transplanted BM-MSCs in combination with silymarin ameliorated the liver tissue damage through their immunoregulatory activities. Among the two routes, the intravenous administration of cells through the tail vein was found to be more effective and safe.

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