Reverse Transcriptase-PCR Analysis of Gene Expression in Hematopoietic Stem Cells

2003 ◽  
pp. 287-299 ◽  
Author(s):  
Donald Orlic
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Reverse Transcriptase ◽  
Hematopoietic Stem Cells ◽  
Pcr Analysis ◽  
Hematopoietic Stem ◽  
Reverse Transcriptase Pcr

scholarly journals A highly efficient reporter system for identifying and characterizing in vitro expanded hematopoietic stem cells

2021 ◽  
Author(s):  
James Lok Chi Che ◽  
Daniel Bode ◽  
Iwo Kucinski ◽  
Alyssa H Cull ◽  
Fiona Bain ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gene Expression Signature ◽  
The Body ◽  
Molecular Profile ◽  
Functional Screening ◽  
Hematopoietic Stem ◽  
Wide Range ◽  
Single Clone

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved more than 200-fold expansion of functional HSCs, but their molecular characterization has not been possible due to the substantial majority of cells being non-HSCs and single cell-initiated cultures displaying substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. Linking single clone functional transplantation data with single clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to actively cycling fetal liver HSCs and shares a gene expression signature with functional HSCs from all sources, including Prdm16, Fstl1 and Palld. This new tool can now be applied to a wide-range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

scholarly journals Hematopoietic stem cells exhibit a specific ABC transporter gene expression profile clearly distinct from other stem cells

2010 ◽  
Vol 10 (1) ◽  
pp. 12 ◽  
Author(s):  
Leilei Tang ◽  
Saskia M Bergevoet ◽  
Christian Gilissen ◽  
Theo de Witte ◽  
Joop H Jansen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gene Expression Profile ◽  
Abc Transporter ◽  
Expression Profile ◽  
Transporter Gene ◽  
Hematopoietic Stem

Comparative gene expression analysis of zebrafish and mammals identifies common regulators in hematopoietic stem cells

Blood ◽  
2010 ◽  
Vol 115 (2) ◽  
pp. e1-e9 ◽  
Author(s):  
Isao Kobayashi ◽  
Hiromasa Ono ◽  
Tadaaki Moritomo ◽  
Koichiro Kano ◽  
Teruyuki Nakanishi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Side Population ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Junction ◽  
Hematopoietic Stem

Abstract Hematopoiesis in teleost fish is maintained in the kidney. We previously reported that Hoechst dye efflux activity of hematopoietic stem cells (HSCs) is highly conserved in vertebrates, and that Hoechst can be used to purify HSCs from teleost kidneys. Regulatory molecules that are strongly associated with HSC activity may also be conserved in vertebrates. In this study, we identified evolutionarily conserved molecular components in HSCs by comparing the gene expression profiles of zebrafish, murine, and human HSCs. Microarray data of zebrafish kidney side population cells (zSPs) showed that genes involved in cell junction and signal transduction tended to be up-regulated in zSPs, whereas genes involved in DNA replication tended to be down-regulated. These properties of zSPs were similar to those of mammalian HSCs. Overlapping gene expression analysis showed that 40 genes were commonly up-regulated in these 3 HSCs. Some of these genes, such as egr1, gata2, and id1, have been previously implicated in the regulation of HSCs. In situ hybridization in zebrafish kidney revealed that expression domains of egr1, gata2, and id1 overlapped with that of abcg2a, a marker for zSPs. These results suggest that the overlapping genes identified in this study are regulated in HSCs and play important roles in their functions.

scholarly journals Hyperglycemia induces abnormal gene expression in hematopoietic stem cells and their progeny in diabetic neuropathy

FEBS Letters ◽  
2014 ◽  
Vol 588 (6) ◽  
pp. 1080-1086 ◽  
Author(s):  
Miwako Katagi ◽  
Tomoya Terashima ◽  
Junko Okano ◽  
Hiroshi Urabe ◽  
Yuki Nakae ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Diabetic Neuropathy ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem

Relationship between Radiosensitivity and Nrf2 Target Gene Expression in Human Hematopoietic Stem Cells

2010 ◽  
Vol 174 (2) ◽  
pp. 177-184 ◽  
Author(s):  
Kengo Kato ◽  
Kenji Takahashi ◽  
Satoru Monzen ◽  
Hiroyuki Yamamoto ◽  
Atsushi Maruyama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Target Gene ◽  
Hematopoietic Stem ◽  
Target Gene Expression ◽  
Nrf2 Target Gene

Prostaglandin E2 (PGE2) Regulates Osteoblastic Jagged1 and Expands Primitive Hematopoietic Cells In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 89-89 ◽  
Author(s):  
Laura M. Calvi ◽  
Benjamin J. Frisch ◽  
Benjamin J. Gigliotti ◽  
Christina A. Christianson ◽  
Jonathan M. Weber ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cortical Bone ◽  
Osteoblastic Cells ◽  
Pcr Analysis ◽  
Inhibitory Peptide ◽  
Hematopoietic Stem ◽  
Hsc Niche

Abstract Parathyroid Hormone (PTH) targets osteoblastic cells (OBs) in the bone marrow microenvironment and expands hematopoietic stem cells (HSC) through Notch activation. Since PTH stimulates the Notch ligand Jagged1 (J1) in OBs, we have focused on the signaling pathways involved in this PTH effect in order to identify novel activators of the HSC niche. Osteoblastic Protein Kinase A (PKA) activation is required for the PTH-dependent J1 increase in OBs. Therefore, we hypothesized that alternative PKA activators could also regulate osteoblastic J1, alter the HSC niche, and provide additional pharmacologic tools to expand HSC in vivo. Consistent with this hypothesis, direct PKA agonists 8-bromo-cAMP and dibutyryl-cAMP stimulated J1 in osteoblastic UMR106 cells. In addition, PGE2, a member of the prostaglandin family known to stimulate PKA in OBs, was studied in vivo and in vitro. By real-time RT-PCR analysis, J1 mRNA was increased up to 5 fold at 2 hours in UMR106 cells when treated with PGE2 (10−7 M) compared to vehicle. J1 protein was also increased after treatment with PGE2. The PGE2-dependent J1 increase was blocked in the presence of the specific PKA inhibitors H89 and myristoylated PKA Inhibitory Peptide (14–22)(PKI) (200ug/ml), demonstrating that PKA is necessary for osteoblastic J1 stimulation by PGE2. Since systemic PGE2 is known to have bone anabolic effects in both humans and animal models, adult wild-type FVB/N male mice were treated with PGE2 (6mg/kg/day i.p.) for 12 days. This regimen has previously been shown to have bone anabolic effects in rats. At day 12, histologic analysis demonstrated an anabolic effect mainly on cortical bone, as was evident in the femurs and tibiae of PGE2-treated mice compared to control. This histologic finding was confirmed by histomorphometry (trabecular bone area means 41% vs 12%,p=0.0916, n=3 in both groups; cortical thickness means 138 vs 85 μm, p=0.0071, n=3 in both groups). Frequency of hematopoietic stem cells (c-Kit+, Sca1+, lin−) was increased in bone marrow from PGE2-treated vs control mice by over 20% (p=0.0018, n=8 in both groups). In summary, PGE2 stimulates J1 in osteoblastic cells through PKA activation and increases mainly cortical bone in vivo. Ongoing studies will confirm whether in vivo PGE2 treatment expands HSC, and whether osteoblastic J1 regulates this process. This study identifies PGE2 as a novel regulator of osteoblastic J1, and as a potential new microenvironmental modulator of HSC, which could be used for in vivo therapeutic HSC niche manipulation.

Identification and Characterization of Osteoblastic Niche Cells That Regulate Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1412-1412
Author(s):  
Yuka Nakamura ◽  
Fumio Arai ◽  
Yumiko Gomei ◽  
Kentarou Hosokawa ◽  
Hiroki Yoshihara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Expression Profiles ◽  
Osteoblastic Cells ◽  
Calcium Deposition ◽  
Pcr Analysis ◽  
Isolated Cells ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Osteoblastic Lineage

Abstract Currently, the niche for long-term hematopoietic stem cells (HSCs) is thought to consist conceptually of two parts: the endosteal surface (the osteoblastic niche) and a sinusoidal endothelium (the vascular niche), and a subset of osteoblasts functions as a key component of the hematopoietic stem cell niche. However, it is still unclear that the precise cellular and molecular contribution of osteoblastic cells on the HSC supportive microenvironment. In this study, we try to characterize the osteoblastic cells and investigate the property of osteoblastic niche cells. For isolation of osteoblastic cells, we treated the bone fragments of femur and tibiae with collagenase following flush-out of the bone marrow (BM). Non-hematopoietic and non-endothelial cells were then enriched by magnetic cell sorting of the CD45-CD31-Ter119- population, and expression of Sca-1 and platelet derived growth factor receptor α (PDGFRα) was analyzed. FACS analysis showed that CD45-CD31-TER119- cells were subdivided into three fractions: Sca-1+PDGFRα+, Sca-1-PDGFRα-, Sca-1-PDGFRα+. First we examined the multilineage differentiation potential of three populations. Although Sca-1- fractions efficiently differentiated into the osteoblastic lineage and showed calcium deposition, these cells hardly differentiated into adipocytes. In contrast to the Sca-1- cells, we found that Sca-1+PDGFRα+ cells can differentiate into osteoblastic and adipocytic lineages, suggesting that Sca-1+ cells have multi-potency. Next we examined the expression of osteoblastic marker expression by quantitative RT-PCR analysis, and found that Sca-1- populations expressed Runx2 and OB-cadherin. Alkaline phosphatase (ALP) staining of freshly isolated cells showed that Sca-1- fractions expressed ALP, while Sca-1+ cells did not express ALP. These data suggest that Sca-1- populations were the cell fractions, which were already committed to osteoblastic lineage. In addition, osteocalcin was expressed in PDGFRα+ fraction in Sca-1- cells, indicating that Sca-1-PDGFRα+ cells are more mature osteoblastic cells than Sca-1-PDGFRα-cells. Furthermore, N-cadherin was specifically expressed in Sca-1-PDGFRα+ cells, suggesting that N-cadherin was up-regulated with the maturation of osteoblastic cells. In addition, N-cadherin expression was up-regulated in Sca-1-PDGFRα+ cells with the postnatal development of BM. Interestingly, in the freshly isolated cells, we found that Sca-1+PDGFRα+ cells showed higher expression of Angiopoietin-1 (Ang-1), compared to Sca-1- fractions. Ang-1 expression was up-regulated in Sca-1-PDGFRα+ cells after over night incubation. Next we investigated the ability of these fractionated cells to support hematopoiesis. We examined the capacity of these fractionated cells on maintenance of colony formation ability of BM linage-Sca-1+c-Kit+ cells after 5 days of co-culture. Although CFU-C formation was supported Sca-1+PDGFRα+ cells, Sca-1-PDGFRα+ cells maintained CFU-Mix formation compared to the Sca-1+PDGFRα+ and Sca-1-PDGFRα-cells. From these data above, we hypothesize that multiple osteoblastic populations form a “niche complex” and collaborate with other supporting cells, such as CXCL12-abundant reticular (CAR) cells, to support HSCs, and that N-cadherin+ osteoblastic cells provide a foothold for anchoring of quiescent HSCs. Now we are investigating the gene expression profiles of these three populations and are tying to clarify the changes of characteristics of osteoblastic cells during postnatal BM development.

MicroRNA-29a Is An Oncogene in Acute Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 683-683
Author(s):  
Christopher Y. Park ◽  
Yoon-Chi Han ◽  
Govind Bhagat ◽  
Jian-Bing Fan ◽  
Irving L Weissman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Mirna Expression ◽  
Myeloid Leukemia ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract microRNAs (miRNAs) are short, non-protein encoding RNAs that bind to the 3′UTR’s of target mRNAs and negatively regulate gene expression by facilitating mRNA degradation or translational inhibition. Aberrant miRNA expression is well-documented in both solid and hematopoietic malignancies, and a number of recent miRNA profiling studies have identified miRNAs associated with specific human acute myeloid leukemia (AML) cytogenetic groups as well as miRNAs that may prognosticate clinical outcomes in AML patients. Unfortunately, these studies do not directly address the functional role of miRNAs in AML. In fact, there is no direct functional evidence that miRNAs are required for AML development or maintenance. Herein, we report on our recent efforts to elucidate the role of miRNAs in AML stem cells. miRNA expression profiling of AML stem cells and their normal counterparts, hematopoietic stem cells (HSC) and committed progenitors, reveals that miR-29a is highly expressed in human hematopoietic stem cells (HSC) and human AML relative to normal committed progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors is sufficient to induce a myeloproliferative disorder (MPD) that progresses to AML. During the MPD phase of the disease, miR-29a alters the composition of committed myeloid progenitors, significantly expedites cell cycle progression, and promotes proliferation of hematopoietic progenitors at the level of the multipotent progenitor (MPP). These changes are manifested pathologically by marked granulocytic and megakaryocytic hyperplasia with hepatosplenomegaly. Mice with miR-29a-induced MPD uniformly progress to an AML that contains a leukemia stem cell (LSC) population that can serially transplant disease with as few as 20 purified LSC. Gene expression analysis reveals multiple tumor suppressors and cell cycle regulators downregulated in miR-29a expressing cells compared to wild type. We have demonstrated that one of these genes, Hbp1, is a bona fide miR-29a target, but knockdown of Hbp1 in vivo does not recapitulate the miR-29a phenotype. These data indicate that additional genes are required for miR-29a’s leukemogenic activity. In summary, our data demonstrate that miR-29a regulates early events in normal hematopoiesis and promotes myeloid differentiation and expansion. Moreover, they establish that misexpression of a single miRNA is sufficient to drive leukemogenesis, suggesting that therapeutic targeting of miRNAs may be an effective means of treating myeloid leukemias.

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