Expression Of Hematopoietic Markers Research Articles

Autologous Micro-Fragmented Adipose Tissue as Stem Cell-Based Natural Scaffold for Cartilage Defect Repair

Osteoarthritis (OA) poses a tough challenge worldwide. Adipose-derived stem cells (ASCs) have been proved to play a promising role in cartilage repair. However, enzymatic digestion, ex vivo culture and expansion, with significant senescence and decline in multipotency, limit their application. The present study was designed to obtain micro-fragmented adipose tissue (MFAT) through gentle mechanical force and determine the effect of this stem cell-based natural scaffold on repair of full-thickness cartilage defects. In this study, ASCs sprouted from MFAT were characterized by multi-differentiation induction and flow cytometry. Scratch and transwell migration assays were operated to determine whether MFAT could promote migration of chondrocytes in vitro. In a rat model, cartilage defects were created on the femoral groove and treated with intra-articular injection of MFAT or PBS for 6 weeks and 12 weeks (n = 12). At the time points, the degree of cartilage repair was evaluated by histological staining, immunohistochemistry and scoring, respectively. Two unoperated age-matched animals served as native controls. ASCs derived from MFAT possessed properties to differentiate into adipocytes, osteocytes and chondrocytes, with expression of mesenchymal stem cell markers (CD29, 44, 90) and no expression of hematopoietic markers (CD31, 34, 45). In addition, MFAT could significantly promote migration of chondrocytes. MFAT-treated defects showed improved macroscopic appearance and histological evaluation compared with PBS-treated defects at both time points. After 12 weeks of treatment, MFAT-treated defects displayed regular surface, high amount of hyaline cartilage, intact subchondral bone reconstruction and corresponding formation of type I, II, and VI collagen, which resembled the normal cartilage. This study demonstrates the efficacy of MFAT on cartilage repair in an animal model for the first time, and the utility of MFAT as a ready-to-use therapeutic alternative to traditional stem cell therapy.

A novel hiPSC-derived system for hematoendothelial and myeloid blood toxicity screens identifies compounds promoting and inhibiting endothelial-to-hematopoietic transition

The exposure to toxic environmental and pharmaceutical substances can pose a long-term risk to human's health. In this study, we sought to investigate the potential of our recently developed method for induction of myeloid hematoendothelial and blood cells by overexpression of two transcription factors, GATA2 and ETV2, in human induced pluripotent stem cells (hiPSCs) for toxicity screening. For the primary screen in a high-throughput format, we selected twenty-two chemicals with various degrees of cytotoxicity available from the NIEHS National Toxicology Program (Tox21). The compounds were applied during the endothelial-to-hematopoietic transition and to differentiated myeloid progenitors growing in suspension. The system was capable of identifying compounds with both inhibitory and favorable effects on hematopoietic network, changes in expression of hematopoietic markers, and mitochondrial and cytotoxicity. The findings were confirmed and further investigated by secondary screens, colony forming cell assay, and gene expression profiling. The hematoendothelial toxicity of 5-fluorouracil, berberine chloride, and benzo(a)pyrene is characterized by the inhibition of cell division and a shift of hematopoietic programming to non-hemogenic endothelial and mesenchymal fates. This study demonstrates the feasibility of transcription factor (TF)-based differentiation systems to monitor endothelial and hematotoxicity and serves as an informative platform for screening myelosuppressive or stimulatory drugs and mechanistic studies of their action.

224 CHARACTERIZATION OF EQUINE ENDOMETRIAL-DERIVED MESENCHYMAL STROMAL CELLS

Equine mesenchymal stromal cells (MSC) are mainly harvested from bone marrow and adipose tissue, requiring surgical procedures. Although human endometrium is known to harbor mesenchymal precursor cells, the presence of MSC in equine endometrium, a dynamic tissue, has not been investigated. This study reports for the first time the culture and characterisation of MSC from equine endometrium compared with equine bone marrow (BM)-derived MSC. Samples of equine endometrium (n = 6) and BM (n = 3) were collected postmortem. Endometrial tissue was digested using a dissociation medium containing collagenase I and DNase type I, and CD227 (mucin-1)-bound magnetic beads were utilised to separate epithelial (CD227+) from stromal (CD227–) cell fractions. Red blood cells from BM samples were excluded using a density gradient. All cell fractions were cultured in DMEM/F-12 containing 10% fetal bovine serum. After expansion, colony-forming unit (CFU) assay at passage 2, trilineage differentiation (adipogenic, chondrogenic, osteogenic), and flow cytometry analysis at passage 3/4 were performed for CD227– fractions and BM-MSC. Descriptive statistical analysis and 2-tailed t-test was performed with IBM SPSS Statistics 22 (SPSS Inc./IBM, Chicago, IL, USA). Both isolated cell fractions were plastic adherent and grew well under standard MSC culture conditions, although endometrial CD227– cells attached quicker to culture plasticware than did BM-MSC. The CFU assay at passage 2 showed no significant difference in cloning efficiency (CE) between BM-MSC (20.78 ± 2.86%) and CD227– (24.89 ± 3.04%) cell lines (P = 0.36). Flow cytometry showed the expression of MSC markers (CD29, CD44, CD90, CD105) and perivascular markers (CD146, NG2) but almost no expression of haematopoietic markers (CD34, CD45) in both cell lines (Table 1). No statistically relevant difference was seen except for the higher expression of NG2 in BM-MSC (P = 0.054). Trilineage differentiation was successfully induced in both cell lines. In conclusion, we showed the presence of putative MSC in equine endometrium. We successfully isolated and cultured these cells, which display comparable characteristics in MSC criteria as well-established BM-derived MSC. These endometrial-derived MSC may provide a convenient source for veterinary regenerative therapies in equine reproduction. Table 1.Flow cytometry marker expression (mean ± standard error) at passage 4

Rhabdomyosarcoma of breast resembles acute leukemia: a case report and review of the literature

Objective To investigate the clinical pathological characteristics, diagnosis and treatment of breast rhabdomyosarcoma, and to enhance the awareness of malignancy infiltration to bone marrow (BM). Methods The data of one case of Rhabdomyosarcoma of breast were analyzed retrospectively. BM aspirate and biopsy, morphology, immunology, cytogenetics, molecular biology (MICM) in different parts of BM, peripheral blood smear, fine puncture of breast mass, final biopsy of breast mass by Mammotome System and whole body PET-CT were performed. The immunochemistry stain of specimen of breast mass was used. Results The peripheral blood smear of this patient showed immature erythrocytes, leucocytes and classification of unknown cells which were consistent with BM morphology. The results of BM aspirate and biopsy depicted a hypercellular specimen with disseminated unknown cells infiltration. Unknown cells were positive for CD56 and negative for any hematopoietic markers by flow cytometry. The whole body PET-CT showed that uptake of 18F-FDG of bilateral breast and whole BM was increased, whereas the mass of breast was not presented by CT. PET-CT suggested a probable malignant hematologic disease. The enough specimen of breast mass got from Mammotome System showed embryonal rhabdomyosarcoma, and the tumor cells were positive for MyoD1, Vimentin and Desmin. Conclusions It is a challenge for early diagnosis of solid sarcoma with unknown origin which diffusely infiltrating into BM. Negative expression of hematopoietic markers by flow cytometry plays a role on differential diagnosis in this setting, whereas PET-CT only provides a valuable reference. Enough specimen and immunohistochemical staining could provide solid evidences of diagnosis. Key words: Rhabdomyosarcoma, embryonal; Adult; Neoplasma metastasis; Pathohistology; Diagnosis

DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish.

BackgroundDNA methyltransferase 1 (Dnmt1) regulates expression of many critical genes through maintaining parental DNA methylation patterns on daughter DNA strands during mitosis. It is essential for embryonic development and diverse biological processes, including maintenance of hematopoietic stem and progenitor cells (HSPCs). However, the precise molecular mechanism of how Dnmt1 is involved in HSPC maintenance remains unexplored.MethodsAn N-ethyl-N-nitrosourea (ENU)-based genetic screening was performed to identify putative mutants with defects in definitive HSPCs during hematopoiesis in zebrafish. The expression of hematopoietic markers was analyzed via whole mount in situ hybridization assay (WISH). Positional cloning approach was carried out to identify the gene responsible for the defective definitive hematopoiesis in the mutants. Analyses of the mechanism were conducted by morpholino-mediated gene knockdown, mRNA injection rescue assays, anti-phosphorylated histone H3 (pH3) immunostaining and TUNEL assay, quantitative real-time PCR, and bisulfite sequencing analysis.ResultsA heritable mutant line with impaired HSPCs of definitive hematopoiesis was identified. Positional cloning demonstrated that a stop codon mutation was introduced in dnmt1 which resulted in a predicted truncated Dnmt1 lacking the DNA methylation catalytic domain. Molecular analysis revealed that expression of CCAAT/enhancer-binding protein alpha (C/ebpa) was upregulated, which correlated with hypomethylation of CpG islands in the regulation regions of cebpa gene in Dnmt1 deficient HSPCs. Overexpression of a transcriptional repressive SUMO-C/ebpa fusion protein could rescue hematological defects in the dnmt1 mutants. Finally, dnmt1 and cebpa double null embryos exhibited no obvious abnormal hematopoiesis indicated that the HSPC defects triggered by dnmt1 mutation were C/ebpa dependent.ConclusionsDnmt1 is required for HSPC maintenance via cebpa regulation during definitive hematopoiesis in zebrafish.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0115-7) contains supplementary material, which is available to authorized users.

Abstract 242: Subcutaneous and Visceral Adipose-Derived Stem Cells Have Similar Biological Properties and Both Improve Cardiac Function of Infarcted Rat Hearts

Background: Adipose stem cells (ASC) from subcutaneous and visceral adipose tissues have been studied individually. However, it is unclear whether ASC from the two sources have different biological properties and, more importantly, whether one sub-type of ASC is more effective in treatment of CHF. This study was designed to address these concerns. Methods: Morphology, yield, proliferation, surface markers, and cytokine secretion of rat subcutaneous ASC (S-ASC) and visceral ASC (V-ASC) were analyzed. A rat model of myocardial infarction (MI) was established by occlusion of the LAD. 7 days after MI, S-ASC (n = 11), V-ASC (n = 11), and cell culture medium (Control, n = 7) were injected into the infarct rim, respectively. Cardiac function of the infarcted hearts was monitored with MRI for 6 months. Results: Both S-ASC and V-ASC exhibited a fibroblast-like morphology and expressed stromal cell markers (CD29, CD90 and CD105). No significant expression of hematopoietic markers (CD11b, CD34 and CD45) was found. Under appropriate conditions, both cells could differentiate to adipocyte- and osteocyte-like cells. Both of them expressed a significant level of HGF, IGF-1 and VEGF. As to their differences, V-ASC had approximately 3-times greater cell yield and a lower colony-formation rate (9.8±1.0% vs.13.5±2.6%) relative to S-ASC. In contrast, S-ASC showed a significantly greater growth rate (Doubling Time: 17.9 h vs. 26.0 h) relative to V-ASC. Both S-ASC and V-ASC-treated hearts showed a significantly greater left ventricular ejection fraction (LVEF, 58.3% and 56.7%) than the control group (LVEF, 47.2%) at end of 6 months. LVEF between the two ASC-treated groups was not significantly different. Finally, the implanted stem cells were readily detected in vivo with MRI for at least 6 months. Myocardial tissue sections showed existence of ASC and their locations matched with MRI signals. Conclusions: S-ASC and V-ASC share several biological characteristics. Both provide comparable significant improvement on cardiac function. Moreover, these implanted cells can be reliably tracked for at least 6 months using MRI. We conclude that the S-ASC and V-ASC are equally effective for treatment of heart failure.

Biomechanical forces promote embryonic haematopoiesis

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3-5), a master regulator of haematopoiesis, and give rise to haematopoietic cells. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41(+)c-Kit(+) haematopoietic progenitor cells, concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the para-aortic splanchnopleura/aorta-gonads-mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development.

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

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.