bluuebunny/crossref_metadata_2025_split huggingface dataset:
A High Dose of Erythropoietin Induces the Expansion of CD34+CD133+ Cells in Human Umbilical Cord Blood.
Authors: Noguchi Mitsuho , Tashiro Haruko , Shirasaki Ryosuke , Gotoh Moritaka , Kawasugi Kazuo , Shirafuji Naoki 
Published: April 2020

Abstract
Objectives: We compared the responsiveness to the high dose stimulation of human EPO between the transplant grafts in an in vitro culture system to clarify the characteristics of cord blood and to make clear the repopulation characteristics on human hematopoietic stem/progenitor cells.
Methods: Cells (cord blood, bone marrow and peripheral blood stem/progenitor cells) were cultured for six days with G-CSF (100 ng/ml), EPO (10 U/ml), or without additives, and were sorted with antihuman CD133-1 monoclonal antibody. The obtained fractions were further labeled with antihuman CD34 monoclonal antibody and CD133-2 antibody as well as the lineage markers, and their expressions were analyzed. Cells were also assayed with ordinal colony-formation. For the analysis of long-term culture-initiating cells (LTC-ICs), Dexter’s long-term culture method was carried out. The expression of the specific proteins was also analyzed with RT-PCR method.
Results. The ratio of the CD34+ cell count to the total cultured mononuclear cell count was not changed between CB, BM and PBSC. However, the ratio of the CD133+ cell count to the total cultured mononuclear cell count was 1.4-fold that of when CB cells were cultured with a high dose of EPO. No significance was observed for BM and PBSC. The count of CD34+CD133+ cells was 1.9-fold that of when CB was cultured with EPO. Dexter’s long-term culture system demonstrated that CB cultured with EPO contained more LTC-ICs and colony-forming cells with a high proliferation potential than that cultured without additives.
Discussion: These results indicate that the CB has unique characteristics to respond to a high dose of EPO.
The Role of Telomerase Activity and Telomere Length in Cellular Sensitivity to DNA Damaging Agents.
Authors: Rice Greg T. , Beasley Michael A. , Akabogu Ike I. , Westin Erik R. , Winnike Dale A. , Klingelhutz Aloysius J. , Goldman Frederick D. 
Published: April 2020

Abstract
Dyskeratosis congenita (DC) is a premature aging syndrome characterized by progressive bone marrow failure, abnormal skin pigmentation and nail dystrophy. We have described an autosomal dominant form of DC (AD DC) in a large three-generation kindred that is due to a mutation in the gene encoding human telomerase RNA (hTR). While telomere shortening is a normal consequence of the aging process, DC patients display extremely short telomeres in many somatic cell types, including hematopoietic cells, and they often suffer from bone marrow failure. Allogeneic hematopoietic stem cell transplant (HSCT) remains the only curative therapy for marrow failure in DC. However, HSCT in DC is generally poorly tolerated and associated with significant morbidity, perhaps as a consequence of increased sensitivity of dividing cells to cytotoxic agents during myeloablative therapy. To test this hypothesis, we characterized lymphocytes from various AD DC patients and age matched controls that had been placed in long term culture following in vitro exposure to irradiation (137Cs) and varying doses of Taxol, Adriamycin, and Etoposide. Cell proliferation and viability were quantified by direct visual counting on a hemocytometer, and flow cytometry was employed to assess apoptosis and cell surface expression of senescent markers. In addition to DC lymphocytes having a decreased proliferative capacity and higher basal apoptotic levels, an increased sensitivity to irradiation, Taxol, Adriamycin, and Etoposide was noted. These results suggest that telomere shortening may be an important factor in determining cellular tolerance to cytotoxic therapy and support the concept of reduced intensity HSCT regimens in both aged individuals and DC patients. Further studies have been initiated to determine whether reconstitution of telomere length in DC cells alters response to cytotoxic agents.
Cell-Cycle-Dependent Oscillation of GATA2 Expression in Hematopoietic Cells.
Authors: Koga Shinichiro , Yamaguchi Nobuhiro , Abe Tomoko , Minegishi Masayoshi , Tsuchiya Shigeru , Yamamoto Masayuki , Minegishi Naoko 
Published: April 2020

Abstract
Manipulation of hematopoietic stem cells (HSC) is a key issue in transplantation therapy and regenerative medicine, and methods enhancing proliferation of HSC with keeping pluripotency (self-renewal) are awaited. GATA2 is a transcription factor controlling proliferation with keeping pluripotency of HSC (self-renewal), although simple overexpression experiments of GATA2 do not induce proliferation of HSC. In this study, we showed the cell-cycle dependent oscillation of GATA2 expression, high in S phase but low in G1/S and M phase, in leukemic cells, and also in cord-blood CD34+ cells cultured under stimulation of Notch signals. Using green fluorescent protin-GATA2 fusion protein, we demonstrated the cell-cycle specificity of proteasome- dependent degradation of GATA2, which was inhibited by the treatment with Roscovitine, a selective inhibitor of cyclin- dependent kinase (CDK). Immunoprecipitation- immunoblotting analysis demonstrated phosphorylation of GATA2 at CDK-consensus motifs, S0P+1 and T0P+1, and the interaction of cyclin A, Cdk2, and Cdk4 with GATA2. Furthermore, mutants in CDK-consensus phosphorylation motifs exhibited altered expression profiles of GFP fusion proteins. These results indicate cell-cycle dependent control of GATA2 expression through the phosphorylation and interactions with cyclin-CDK systems, suggesting the possible effect of cell-cycle-specific induction of GATA2 expression on the self-renewal of HSC.
Contribution of STAT3 to Activation of Survivin by Granulocyte-Macrophage Colony-Stimulating Factor in CD34+ Cells.
Authors: Gu Lubing , Chiang Kuang-Yueh , Zhu Ningxi , Findley Harry W. , Zhou Muxiang 
Published: April 2020

Abstract
Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to specifically stimulate proliferation and differentiation of CD34+ hematopoietic progenitor cells. Although STAT3 was thought to be essential for the transduction of GM-CSF-induced cell proliferation, the downstream signaling mediated by STAT3 to support cell proliferation and growth has not been completely understood. Because the inhibitor of apoptosis protein (IAP) survivin is believed to regulate cell proliferation and survival via its anti-apoptotic function, we chose to study the link between STAT3 signaling and survivin expression in CD34+ cells. We constructed plasmids containing the survivin promoter sequence and performed luciferase reporter assay in CD34+ KG-1 cells stimulated with GM-CSF. These experiments showed that GM-CSF stimulated survivin promoter activity. Chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assay (EMSA) revealed that STAT3 binds to the core survivin promoter containing a STAT response element (SRE) TT(N)5AA at sites −264 to −256. Mutation or deletion of this SRE completely abolished the effect of GM-CSF on survivin promoter activity. Furthermore, specific JAK inhibitor and STAT3 siRNA inhibited GM-CSF-induced survivin promoter activity and survivin expression. Inhibition of survivin by STAT3 siRNA or by withdrawal of GM-CSF in a GM-CSF-dependent CD34+ line TF-1 resulted in decreased cell growth and induction of apoptosis. These results suggest that the anti-apoptotic protein survivin is a transcriptional target of STAT3, and that GM-CSF stimulated-CD34+ cell proliferation is regulated by the JAK/STAT3/survivin signaling pathway.
All-Trans Retinoic Acid Accelerates Migration of MOLT3 to SDF-1 α/CXCL12 through the Phosphatidylinositol 3 Kinase (PI3K)/Akt Pathway.
Authors: Jimi Shiro , Matsumoto Taichi , Suzumiya Junji , Hara Shuji , Takamatsu Yasushi , Tamura Kazuo 
Published: April 2020

Abstract
The chemokine stromal cells derived factor-1 (SDF-1)/CXCL12 stimulates lymphocytes, hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC) through the corresponding chemokine receptor CXCR4, which modulates homing, retention, and engraftment into the bone marrow. However, regulatory factors for CXCR4 excluding cytokines are totally unknown. Besides, all-trans retinoic acid (ATRA), a drug inducing cell differentiation and apoptosis, increases motility with unknown mechanisms. In the present study, we investigated the intracellular regulatory mechanisms of SDF-1α-induced migration of ATRA-treated cells. We initially surveyed cells exhibiting strongly membrane-associated CXCR4 (M-CXCR4), and found 3 out of 10 different cell lines (MOLT3, U266B1, and Raji). Of those, MOLT3 was identified as the only one enabled to respond to SDF-1α chemoattraction, accompanied by an increase in actin polymerization. When M-CXCR4 in MOLT3 was neutralized or antagonized by an antibody or the antagonist AMD3100, the migration activity to SDF-1α, analyzed using the Boyden chamber method, was totally abolished. ATRA pretreatment for 3 days induced growth inhibition of MOLT3, but apoptosis and differentiation did not occur. ATRA treatment alone had no effects on either CXCR4 gene expression or its total protein expression, and cell motility also did not increase; however, ATRA treatment dose dependently increased M-CXCR4 expression. When SDF-1α was added to the lower chamber, migration activity of cells pretreated by different doses of ATRA increased dose dependently and reached up to 5 times greater compared with cells lacking ATRA pretreatment. The increased migration activity was blocked by wortmannin, which inhibits phosphatidylinositol 3 kinase (PI3K), AKT inhibitor, and cytochalasin D, which inhibits actin polymerization; U0126, an inhibitor of MEK1/2, did not block the effects of ATRA and SDF-1α. The results indicate that M-CXCR4 is quite important for cell migration, and the intracellular signaling for the M-CXCR4/SDF-1 axis primarily involves the PI3K/Akt pathway, which ATRA positively affected. This is the fist time to show that migration activity to SDF-1 was accelerated by ATRA. Clinical application of this phenomenon may help to augment HSC/HPC homing after transplantation.
PBI-1402: A New Candidate for the Treatment of Anemia.
Authors: Gagnon Lyne , Barabé Jean , Lagraoui Mouna , Grouix Brigitte , Julien Nathalie , Asselin Michel , Gaudreau Dannyck , Sarra-Bournet François , Zacharie Boulos , Penney Christopher , Laurin Pierre , Morin Marie-Josée , Krosl Gorazd , Roy Denis-Claude 
Published: April 2020

Abstract
PBI-1402 is an orally active low molecular weight synthetic compound which is currently in a phase Ib/II clinical trial in patients with anemia associated with cancer and/or chemotherapy. PBI-1402 stimulates the in vitro/ex vivo proliferation and maturation of hematopoietic progenitors (erythroid and myeloid populations) with an activity comparable to EPO. Furthermore, an additive effect is observed in combination with EPO. PBI-1402 enhances the differentiation of pluripotent stem cells: CFU-GEMM, CFU-GM with a predominant effect on BFU-E. Clinical phase I study showed that PBI-1402 is devoid of significant side effects. In addition, the clinical phase I results showed statistical increase (100%, p&lt;0.0001) of relative and absolute reticulocyte count in healthy volunteers after 21 days of oral treatment compared to placebo. Preclinical results demonstrated that PBI-1402 yields an increase in erythrocytes in the systemic circulation. In myeloblated mice that received a syngeneic bone marrow transplant, oral treatment with PBI-1402 resulted in a significant increase in peripheral erythrocyte count, hemoglobin, platelet and bone marrow erythroid progenitor cells. PBI-1402 is targeted as an adjunct to cytotoxic drugs, radiotherapy and bone marrow transplantation for the treatment of anemia.
Mitotic Arrest Deficiency 2 (Mad2) Is Required for Proliferative Synergy of GM-CSF Plus SCF in Hematopoietic Progenitors Via Effects on Apoptosis and Is Mediated by Ligand-Dependent Association of Mad2 with SCF and GM-CSF Receptors.
Authors: Ito Shigeki , Mantel Charlie , Ishida Yoji , Broxmeyer Hal E. 
Published: April 2020

Abstract
Mitotic arrest deficiency 2 (Mad2) is a component of the mitotic spindle checkpoint complex and is essential for accurate chromosome segregation. We previously reported that Mad2 is involved in synergistic proliferation of hematopoietic progenitor cells in response to SCF combined with GM-CSF, but the underlying mechanism remains unclear. Recently, it was reported that Mad2 physically associates with common β chain (βc) of GM-CSF receptor. Because c-Kit constitutively binds to βc, we hypothesized that Mad2 protein physically associates with c-Kit receptor. We examined if Mad2 interacts with c-Kit using human growth factor-dependent cell line, MO7e, which expresses both c-Kit and GM-CSF receptors. Immunoprecipitation assays demonstrated interaction of c-Kit with Mad2. We then examined whether the interaction of Mad2 with c-Kit is cytokine dependent. Mad2 dissociates from c-Kit after stimulation with SCF plus GM-CSF, but not with SCF or GM-CSF alone. Additionally, because bone marrow hematopoietic progenitor cells from Mad2+/− mice lack synergy in response to SCF plus GM-CSF, it is possible that interaction of Mad2 with c-Kit mediates their synergistic proliferative effects. To address this, we examined intracellular cytokine signaling and apoptosis in MO7e cells depleted of Mad2 by RNA interference. We observed no difference between control and Mad2-depleted cells in phosphorylation of Erk1/2 at Thr202/Tyr204 and Akt at Ser473 after synergistic stimulation with SCF plus GM-CSF. In contrast, the percent of apoptosis of Mad2-depleted cells was significantly higher than that of control (26.2±0.7% vs. 20.3±0.4%, P&lt;0.01). This was compatible with the results of apoptosis assays using c-Kit+Lineage− bone marrow cells from Mad2+/− and wild type mice. These results suggest that Mad2 may be involved in synergistic proliferation of hematopoietic progenitor cells via regulation of apoptosis rather than early cytokine signaling. These effects are likely mediated through Mad2 interaction with c-Kit and the beta chain of the GM-CSF receptor.
G2E3 Is Required for Early Embryonic Development.
Authors: Brooks William S. , Kesterson Robert A. , Schoeb Trenton R. , Crawford David F. 
Published: April 2020

Abstract
G2E3 is a putative ubiquitin ligase (E3) identified in a microarray screen for genes encoding mitotic regulatory proteins and DNA damage responsive genes. This protein was predicted to function as an ubiquitin ligase based on the presence of a c-terminal HECT domain, a common E3 catalytic domain. Three n-terminal PHD domains likely function as protein-protein interaction domains. We have demonstrated that G2E3 localizes to the nucleus and nucleolus of HeLa cells with dynamic localization responsive to external stimuli. For example, G2E3 rapidly delocalized from the nucleolus after treatment with DNA damaging agents, a property shared with several other proteins involved in DNA damage responses. The protein is extremely labile as a result of proteasome-mediated degradation, accumulating in nucleoli and aggresomes after proteasome inhibition. In this study, we determined the effect of G2E3 gene disruption. Mice were generated with a germline mutation inactivating the G2E3 gene. Animals with a heterozygous inactivation of the G2E3 gene were phenotypically normal with no overt change in development, growth, longevity, or fertility. In contrast, mice with disruption of both alleles did not survive to birth. We genotyped mice from G2E3+/− intercrosses at several embryonic stages and found that no homozygous mutant animals were identified as early as E8.5. To determine whether G2E3−/− animals survive through the earliest stages of development, blastocysts were isolated and genotyped by nested PCR. At E3.5, G2E3 gene disruption does not appear to affect viability since the numbers of wild-type, heterozygous knockout, and homozygous knockout blastocysts are similar to the expected 1:2:1 ratio. Therefore, G2E3 gene disruption does not appear to impair viability in the earliest stages of development but causes embryonic lethality prior to or at about the time of implantation. We further show by β-galactosidase staining that G2E3 is predominantly expressed within the central nervous system of the developing embryo and in the early stages of limb bud formation. These studies show that G2E3 plays an essential role in early mammalian development. The development of conditional knockout animals may be very valuable in determining the precise function of G2E3 in development and the DNA damage response.
G-CSF Mobilized Human Mesenchymal Stem Cells Are Found in the Peripheral Blood and Have Telomere Limited Growth Potential.
Authors: Lund Troy C. , Baso Lisa , Orchard Paul J. 
Published: April 2020

Abstract
Mesenchymal stem cells (MSC) are multipotent cells found lining the bone marrow cavity. Their primary function is to support the growth and differentiation of hematologic progenitors. MSC have been shown to differentiate into a variety of cell types including: bone, adipocytes, cartilage, neuron-like, and muscle-like cells. There is mounting evidence that these cells can, under the right circumstances, enter the peripheral circulation. However, MSC have not been routinely isolated from peripheral blood. Granulocyte colony stimulating factor (G-CSF) is commonly used to mobilize hematopoietic stem cells from the bone marrow into the peripheral circulation. We show that G-CSF mobilized peripheral blood also contains a small percentage of MSC although lower than that of bone marrow derived MSC (BMMSC): 0.012% vs 0.04%. Isolates were morphologically similar to BMMSC and were successfully expanded and shown to differentiate into osteogenic and adipogenic lineages in the appropriate differentiation conditions. FACS analysis showed that the cells reliably expressed cell surfaces markers commonly found on MSC including CD105, CD29, CD166, and CD13. They were negative for CD14, CD34, CD133, and CD45. Mobilized peripheral blood derived MSC (MPB-MSC) had limited expansion potential when compared with bone marrow isolated MSC. Most cells appeared to cease cell division 20–25 days after isolation. MPB-MSC did not have any detectable telomerase activity (as determined by TRAP assay) and consequently were found to have undergone significant telomere shortening (shown by Southern analysis.) The rarity of this cell in G-CSF-mobilized peripheral blood and the subsequent tremendous pressure to divide in cell culture are likely contributing factors leading to the telomere loss seen in MPB-MSC. This phenomenon probably also accounts for the observed senescence observed in vitro. While we have conclusively shown that MSC can be found in G-CSF mobilized peripheral blood, the use of such cells for transplant or gene therapy may be of limited potential due to the telomere-restricted capability of expansion.
Characteristics of Adipose Tissue-Derived Mesenchymal Stem Cells.
Authors: Jang Jun Ho , Lee Hyun Woo , Eom Young-Woo , Kang Seok Yun , Park Joon Seong , Choi Jin Hyuk , Kim Hugh C. 
Published: April 2020

Abstract
Background: The aim of this study was to examine the differentiation potentials and characteristics of adipose tissue-derived stem cells (ASCs) in other to evaluate for the ASCs to be used as alternative cell sources of mesenchymal stem cells.
Methods: ASCs were isolated from lipo-aspirated adipose tissues by treatment of collagenase A and cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM). To know the characteristics of ASCs, the expression of cell surface antigens was analyzed by flow cytometry, and the proliferation potentials were estimated by colony forming abilities or capacities of population doubling. The differentiation potentials into adipocytes or osteoblasts were confirmed by accumulation of neutral lipid vacuoles stained with Oil-red O and expression of alkaline phosphatases.
Results: When the nucleated cells were isolated by collagenase treatment after lipo-aspiration, the mean cell yield was about 3.1 X 106 or 1.2 X 106 cells per gram of lipo-aspirate (n=8) processed at 3 or 21 hours, respectively. But cells processed at 21 hours were able to form more colonies than those at 3 hours. Flowcytometric analysis showed that Adipose tissue-derived stem cells (ASCs) have a marker expression that is similar to that of bone marrow stromal cells (BMSCs). ASCs expressed CD44, CD73, CD90, and CD105 and were absent for CD14, CD31 and CD45 expression. When primary cells were plated at 50 or 1000 cells/cm2 on 6-well plates, cumulative population doublings were about 50 times until passage 7 or 13 (approximately 130 days), respectively, and ASCs expanded to 1018 cells. ASCs were multipotent, differentiating to the adipocyte and osteoblast lineages. ASCs did not provoke in vitro alloreactivity of incompatable lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogen.
Conclusion: Our results suggested that ASCs have highly proliferative potentials, multiple differentiation potentials, and immunosuppressive properties like BMSCs. Therefore, ASCs-based reconstructive therapy could employ allogeneic cells and because of their immunosuppressive properties, ASCs could be an alternative source of BMSCs to treat allogeneic conflicts.
Human Platelet-Derived Factors Regulate Mesenchymal Stem Cell Gene Expression.
Authors: Malischnik Christina , Schallmoser Katharina , Rohde Eva , Reinisch Andreas , Guelly Christian , Lanzer Gerhard , Linkesch Werner , Strunk Dirk 
Published: April 2020

Abstract
The use of animal-derived products during human stem cell processing bears the evident risk of xenogeneic prion, virus, or zoonose contamination. Human platelet lysate (HPL) has recently been recognized as a rich source of cytokines and growth factors with the potential to replace fetal bovine serum (FBS) during ex vivo stem cell manipulation. In this study we compared the gene expression profile of human multipotent mesenchymal stromal/stem cells (MSC) during ex vivo expansion for clinical applications under the aegis of either FBS or HPL.
The Applied Biosystems 1700 Expression Array System was used for full genome expression profiling of MSC after a 12–14 day expansion period in a previously optimized low density expansion system. Data have been obtained from biological as well as technical replicates. A starting amount of 40μg total RNA was directly labeled and DIG-labeled cDNA was hybridized to Human Genome Survey Microarray V2.0. Attribution of regulated genes to biological processes and pathways was done using the PANTHER® db analysis software.
We identified more than 300 genes that are differentially regulated upon culture of MSC in HPL compared to FBS. Biological processes specifically activated in HPL culture include mesoderm development, cell cycle control, hematopoiesis and angiogenesis which interestingly correspond to a considerable proportion of the regenerative function of MSC. In contrast, processes related to cell adhesion and adhesion-mediated signaling, cell structure, cell motility and cell communication are significantly upregulated in MSC after FBS in comparison to HPL culture.
Replacing FBS with HPL not only avoids bovine prion, viral and zoonose contamination of MSC for clinical use. The tightly regulated gene expression profiles under the aegis of human growth factors and cytokines provided by HPL may even help to develop new stem cell therapy strategies.