Identification of New Megakaryocytic Subpopulations in Mouse Bone Marrow.

Abstract Platelets (PLT) are produced from megakaryocytes (Mks) via proplatelet formation (PPF). However, the molecular mechanisms from Mks to PPF are not clearly elucidated, because the maturational steps of the Mks in bone marrow (BM) are not analyzed in detail. Until now, mouse Mks have been only isolated as acetylcholinesterase (AchE) positive cells and they are understood as well maturated population. In this study, we found the presence of different megakaryocytic subpopulations in BM by flowcytometry. To isolate the Mks, first we depleted lineage marker (CD4, CD8a, CD11b, B220, CD71, CD90, TER119, Gr-1, F4/80, 7/4) positive cells from BM cells of BALB/c mice. The analysis of the expression-pattern of CD41, CD45 and CD61 in the lineage negative (Lin−) cells showed the presence of two types of megakaryocytic subpopulations. By sorting, they were identified as Lin−CD41+/45+/61+ cells (AchE negative) and Lin−CD41++/45+/61++ cells (partially AchE positive), respectively. To assess the maturational stages of the subpopulations, each population was cultured with 10ng/mL of TPO followed by counting of PPF and PLT production. Both PPF and PLT production were observed in Lin−CD41+/45+/61+ cells later than those in Lin−CD41++/45+/61++ cells. On the other hand, CFU-Mk was scarcely detected in each subpopulation. The results indicate that both populations are the committed megakaryocytes and Lin−CD41+/45+/61+ cells are more immature population than Lin−CD41++/45+/61++ cells. Then to characterize these subpopulations in detail, gene expression profiling was performed against four-megakaryocytic lineage-populations, Lin−CD41−Thy1lowc-kit+ cells as stem/progenitor, Lin−CD41+/45+/61+ cells, Lin−CD41++/45+/61++ cells and PLT using GeneChipU74 or RT-PCR. These analyses revealed that many PLT-specific genes including gpIb/IX, P-selectin, thrombin-R and ADP-R were already expressed on Lin−CD41+/45+/61+ cells but less than Lin−CD41++/45+/61++ cells. Especially, beta-1 tubulin that is necessary for PPF was only expressed on Lin−CD41++/45+/61++ cells. On the contrary, the expression of c-kit gene was gradually decreasing from stem/progenitor fraction to PLT. In conclusion, we succeeded in the isolation of new subpopulations distinguishable between immature Mks and more matured Mks beginning to prepare PLT. The present finding can contribute to elucidate the molecular mechanisms during terminal maturation.

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Gene expression profiling related to the enhanced erythropoiesis in mouse bone marrow cells

Journal of Cellular Biochemistry ◽

10.1002/jcb.21620 ◽

2008 ◽

Vol 104 (1) ◽

pp. 295-303 ◽

Cited By ~ 9

Author(s):

Hee-Young Yang ◽

Dong Kee Jeong ◽

Seok-Ho Kim ◽

Kyoung-Jin Chung ◽

Eun-Jin Cho ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Bone Marrow Cells ◽

Mouse Bone Marrow ◽

Mouse Bone Marrow Cells ◽

Marrow Cells

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Gene Expression Profiling of the Bone Marrow Mononuclear Cells from Patients with Melodysplastic Syndrome.

Blood ◽

10.1182/blood.v104.11.3429.3429 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3429-3429

Author(s):

Zi-xing Chen ◽

Jun Qian ◽

Wei Wang ◽

Jian-nong Cen ◽

Yong-quang Xue

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Gene Expression Profiling ◽

Expression Pattern ◽

Expression Profiling ◽

Cdna Clones ◽

Rt Pcr ◽

Transcript Levels ◽

Significant Difference ◽

Normal Controls

Abstract Myelodysplastic syndrome (MDS), a clonal disorder of hematopoietic stem cells, is characterized by cytopenia in at least one lineage of peripheral blood and dysplastic features in BM cells. It has been considered as a preleukemic condition for a high risk of transformation to AML. The FAB classification has divided MDS into five clinical categories: RA, RAS, RAEB, RAEB-t and CMML. To date, the key genes involved in the pathogenesis and its progression to acute leukemia has not been clearly identified. This was at least partially caused by a narrow scale of genes which could only been studied once a time by using traditional methods. The present study was to investigate the molecular mechanism of MDS by using cDNA microarray techniques. We have first applied total RNA of bone marrow monoculear cells (BMNCs) from 2 MDS patients (one RA and one RAS) to a BioStar H141 microarray ( United Gene Holdings Co. LTD, Shanghai, China) containing 13484 gene cDNA clones and ets. Based on the preliminary results of these assay, 500 genes which were shown most remarkably differentially expressed in MDS compare to normal control, and with known functions and potential involvement in hematopoiesis regulation, were selected to design and compose 10 cDNA chips. These arrays were then used to analyze the gene expression pattern of BMNCs from 10 patients with MDS, including 4 RA, 1 RTC, 4 RAEB, and 1 RAEBt. To confirm the microarray results and to evaluate the disease relevance of some selected genes from array results, a semiquantitative RT-PCR was performed to analyze gene expression in fifty addi-tional patients with MDS (28 RA, 15 RAEB, 7 RAEBt), 38 acute myeloid leukemia (7 M1, 12 M2, 4 M3, 4 M4, 5 M5, 6 M6), 15 atypical anaplastic anemia (AA), and 12 hypercellular anemia. Our results revealed that 95 genes were abnormally expressed in at least five MDS patients compared to normal controls, involving cell growth and differentiation regulation, cell cycle control, signaling, redox, such as thrombospondin 1 (THBS1), phosphatase and tensin homolog (PTEN), MAD, DNA-damage-inducible transcript 3 (DDIT3), ets variant gene 1 (ETV1), and G1 to S phase transition 1 (GSPT1). These MDS patients in different risk groups could be clustered into two groups overall by hierarchical clustering, wherein a case with isolated thrombocytopenia and other RA patients were clustered into two subgroups 5 genes (RNAHP, DDIT3, FOXO3A, GSPT1, and ETV1) which displayed a most marked differential expression pattern in most MDS patients, were selected as "candidate genes". Consistent expression patterns of 3 (60%) in 5 genes were confirmed by semiquantitative RT-PCR. Further analysis showed different transcript levels of RNAHP, DDIT3 among patients with MDS in different risk group, AML, and normal controls. Meanwhile, the transcript levels of five genes were also compared in the patients with RA, AA and other hypercelluler anemia patients. There was significant difference in RNAHP levels between RA and CAA, or other hypercelluler anemia (P<0.05), similar diversity also seen in ETV1 levels between RA and AA (P<0.01). However, significant differences in DDIT3, FOXO3A, and GSPT1 levels were not observed. Our study suggested that gene expression profiling of MDS patients may reveal a specific transcription patterns for BMNCs in MDS. The abnormal expression of RNAHP, DDIT3 and ETV1 may play roles in the patho-genesis of MDS and may provide useful biomarkers for the molecular diagnosis of MDS.

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Identification of molecular mechanisms underlying mood stabilization through genome-wide gene expression profiling

The International Journal of Neuropsychopharmacology ◽

10.1017/s1461145705006176 ◽

2005 ◽

Vol 9 (03) ◽

pp. 263 ◽

Cited By ~ 3

Author(s):

Rulun Zhou ◽

Carlos A. Zarate ◽

Husseini K. Manji

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Molecular Mechanisms ◽

Genome Wide ◽

Mood Stabilization ◽

Genome Wide Gene Expression

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256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab256 ◽

2004 ◽

Vol 16 (2) ◽

pp. 248

Author(s):

C. Wrenzycki ◽

T. Brambrink ◽

D. Herrmann ◽

J.W. Carnwath ◽

H. Niemann

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Expression Profiles ◽

Cdna Array ◽

Preimplantation Embryos ◽

Average Insert Size ◽

Rt Pcr ◽

Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

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Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation

European Journal of Medical Genetics ◽

10.1016/j.ejmg.2017.04.003 ◽

2017 ◽

Vol 60 (6) ◽

pp. 326-334 ◽

Cited By ~ 5

Author(s):

Carla Martins Kaneto ◽

Patrícia S. Pereira Lima ◽

Karen Lima Prata ◽

Jane Lima dos Santos ◽

João Monteiro de Pina Neto ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenesis Imperfecta ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Osteoblast Differentiation ◽

Marrow Mesenchymal Stem Cells

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Molecular mechanisms associated with breast cancer based on integrated gene expression profiling by bioinformatics analysis

Journal of Obstetrics and Gynaecology ◽

10.3109/01443615.2015.1127902 ◽

2016 ◽

Vol 36 (5) ◽

pp. 615-621 ◽

Cited By ~ 4

Author(s):

Di Wu ◽

Bing Han ◽

Liang Guo ◽

Zhimin Fan

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Molecular Mechanisms ◽

Bioinformatics Analysis

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Gene Expression Profiling Utilizing Microarray Technology and RT-PCR

BioMEMS and Biomedical Nanotechnology ◽

10.1007/978-0-387-25843-0_2 ◽

2007 ◽

pp. 23-46 ◽

Cited By ~ 2

Author(s):

Dominick Sinicropi ◽

Maureen Cronin ◽

Mei-Lan Liu

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Microarray Technology ◽

Rt Pcr

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Global gene expression profiling in early-stage polycystic kidney disease in the Han:SPRD Cy rat identifies a role for RXR signaling

AJP Renal Physiology ◽

10.1152/ajprenal.00470.2010 ◽

2011 ◽

Vol 300 (1) ◽

pp. F177-F188 ◽

Cited By ~ 7

Author(s):

Masanori Kugita ◽

Kazuhiro Nishii ◽

Miwa Morita ◽

Daisuke Yoshihara ◽

Hiroe Kowa-Sugiyama ◽

...

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Kidney Disease ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Early Stage ◽

Global Gene Expression ◽

Rt Pcr ◽

Polycystic Kidney ◽

Global Gene Expression Profiling

Han:SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated “Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation,” “LPS/IL-1-Mediated Inhibition of RXR Function,” and “Liver X Receptor (LXR)/RXR Activation.” These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.

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