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1
Koller, MR, MA Palsson, I. Manchel und BO Palsson. „Long-term culture-initiating cell expansion is dependent on frequent medium exchange combined with stromal and other accessory cell effects“. Blood 86, Nr. 5 (01.09.1995): 1784–93. http://dx.doi.org/10.1182/blood.v86.5.1784.bloodjournal8651784.
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Despite considerable effort, the expansion of long-term culture- initiating cells (LTC-ICs) in cultures of purified hematopoietic cells has not yet been achieved. In contrast, LTC-IC expansion has been attained in cultures of bone marrow mononuclear cells (MNC) using frequent medium exchange. The use of frequent medium exchange was, therefore, examined in cultures of CD34-enriched cells. In stromal- free, CD34-enriched cell cultures, medium exchange intervals ranging from 2 days to no feeding for 14 days gave similar results. Six different growth factor combinations, reported by other groups to give optimal expansion of CD34-enriched cells, were tested in comparison with the control combination of IL-3/GM-CSF/Epo/SCF. None of the combinations resulted in improved colony-forming unit-granulocyte macrophage (CFU-GM) expansion or LTC-IC maintenance, although two were equivalent. All stromal-free cultures resulted in loss of LTC-IC to half of input. Because of the limited effect of medium exchange and growth factor variations on CD34-enriched cell cultures, the effect of preformed stroma was next examined. Preformed stroma increased cell (3- fold), CFU-GM (5-fold), and LTC-IC (3-fold) output, but only when the medium was exchanged every other day. Under these conditions, the number of LTC-IC was maintained near input level. The lack of LTC-IC expansion in CD34-enriched cell cultures prompted experiments to examine the effect of cell purification. Parallel cultures were performed at CD34+lin- cell purities of 20%, 40%, 70%, and 95%, with each well containing exactly 4,000 CD34+lin- cells in addition to the CD34- accessory cells required to give the desired percentage. Also, MNC from the same source (approximately 2% CD34+lin-) were cultured at a concentration to give 4,000 CD34+lin- cells per well. As CD34+lin- cell purity was decreased from 95% to 2%, the output of cells, CFU-GM, and LTC-IC increased by threefold to fivefold. The loss of culture performance with purification was likely due to the removal of important accessory cells, because the levels of endogenously produced leukemia inhibitory factor and IL-6 were found to decline significantly with increasing CD34+lin- cell purity. In summary, preformed stroma abrogated the decrease in cell and CFU-GM output from cultured CD34- enriched cells and led to LTC-IC maintenance. In contrast, MNC inocula resulting in a growing stromal layer during the culture led to LTC-IC expansion (3.2-fold).(ABSTRACT TRUNCATED AT 400 WORDS)
2
Shibuya, A., K. Nagayoshi, K. Nakamura und H. Nakauchi. „Lymphokine requirement for the generation of natural killer cells from CD34+ hematopoietic progenitor cells“. Blood 85, Nr. 12 (15.06.1995): 3538–46. http://dx.doi.org/10.1182/blood.v85.12.3538.bloodjournal85123538.
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We have established a cell culture system without stromal cells that allows the CD34+ hematopoietic progenitor cells (HPC) to differentiate into natural killer (NK) cells. CD34+Lin (CD3, CD16, CD56)-cells were purified using fluorescence-activated cell sorting from normal adult bone marrow (BM) and cultured for 28 days in medium supplemented with interleukin-2 (IL-2) and stem cell factor (SCF). NK (CD3-CD16-CD56+) cells were generated in a dose-dependent manner in response to SCF. NK cells originated from CD34+CD33+Lin- cells, but they were barely detectable in cultures of CD34+CD33-Lin- cells. However, on addition of IL-3, an induced differentiation of NK cells from CD34+CD33-Lin- cells was observed, although at a lower frequency. Supplementing of the cell cultures with SCF alone or both SCF and IL-3 for the first 7 days followed by IL-2 for the next 21 days is essential for production of NK cells from CD34+CD33+Lin- cells and from CD34+CD33-Lin- cells, respectively. These data provide direct evidence that NK cells arise from CD34+HPC and show the minimum lymphokine requirement for their differentiation.
3
Gallacher, Lisa, Barbara Murdoch, Dongmei M. Wu, Francis N. Karanu, Mike Keeney und Mickie Bhatia. „Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7“. Blood 95, Nr. 9 (01.05.2000): 2813–20. http://dx.doi.org/10.1182/blood.v95.9.2813.009k20_2813_2820.
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Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.
4
Purton, Louise E., Irwin D. Bernstein und Steven J. Collins. „All-Trans Retinoic Acid Delays the Differentiation of Primitive Hematopoietic Precursors (lin−c-kit+Sca-1+) While Enhancing the Terminal Maturation of Committed Granulocyte/Monocyte Progenitors“. Blood 94, Nr. 2 (15.07.1999): 483–95. http://dx.doi.org/10.1182/blood.v94.2.483.
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Abstract All-trans retinoic acid (ATRA) is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. In this study, we investigated the effect of ATRA on highly enriched murine hematopoietic precursor cells (lin−c-kit+Sca-1+) grown in liquid suspension culture for 28 days. ATRA initially slowed the growth of these hematopoietic precursors but prolonged and markedly enhanced their colony-forming cell production compared with the hematopoietic precursors cultured in its absence. At 7 and 14 days of culture, a substantially greater percentage of cells cultured with ATRA did not express lineage-associated antigens (55.4% at day 7 and 68.6% at day 14) and retained expression of Sca-1 (44.7% at day 7 and 79.9% at day 14) compared with cells grown in its absence (lin−cells: 31.5% at day 7 and 4% at day 14; Sca-1+: 10.4% at day 7 and 0.7% at day 14). Moreover, a marked inhibition of granulocyte production was observed in cultures continuously incubated with ATRA. Significantly, ATRA markedly prolonged and enhanced the production of transplantable colony-forming unit-spleen (CFU-S) during 14 days of liquid suspension culture. In contrast with its effects on primitive lin−c-kit+Sca-1+hematopoietic precursors, ATRA did not exert the same effects on the more committed lin−c-kit+Sca-1−progenitor cells. Moreover, the late addition of ATRA (7 days post-culture initiation) to cultures of primitive hematopoietic precursors resulted in a marked decrease in colony-forming cell production in these cultures, which was associated with enhanced granulocyte differentiation. These observations indicate that ATRA has different effects on hematopoietic cells depending on their maturational state, preventing and/or delaying the differentiation of primitive hematopoietic precursors while enhancing the terminal differentiation of committed granulocyte/monocyte progenitors.
5
Purton, Louise E., Irwin D. Bernstein und Steven J. Collins. „All-Trans Retinoic Acid Delays the Differentiation of Primitive Hematopoietic Precursors (lin−c-kit+Sca-1+) While Enhancing the Terminal Maturation of Committed Granulocyte/Monocyte Progenitors“. Blood 94, Nr. 2 (15.07.1999): 483–95. http://dx.doi.org/10.1182/blood.v94.2.483.414k12_483_495.
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All-trans retinoic acid (ATRA) is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. In this study, we investigated the effect of ATRA on highly enriched murine hematopoietic precursor cells (lin−c-kit+Sca-1+) grown in liquid suspension culture for 28 days. ATRA initially slowed the growth of these hematopoietic precursors but prolonged and markedly enhanced their colony-forming cell production compared with the hematopoietic precursors cultured in its absence. At 7 and 14 days of culture, a substantially greater percentage of cells cultured with ATRA did not express lineage-associated antigens (55.4% at day 7 and 68.6% at day 14) and retained expression of Sca-1 (44.7% at day 7 and 79.9% at day 14) compared with cells grown in its absence (lin−cells: 31.5% at day 7 and 4% at day 14; Sca-1+: 10.4% at day 7 and 0.7% at day 14). Moreover, a marked inhibition of granulocyte production was observed in cultures continuously incubated with ATRA. Significantly, ATRA markedly prolonged and enhanced the production of transplantable colony-forming unit-spleen (CFU-S) during 14 days of liquid suspension culture. In contrast with its effects on primitive lin−c-kit+Sca-1+hematopoietic precursors, ATRA did not exert the same effects on the more committed lin−c-kit+Sca-1−progenitor cells. Moreover, the late addition of ATRA (7 days post-culture initiation) to cultures of primitive hematopoietic precursors resulted in a marked decrease in colony-forming cell production in these cultures, which was associated with enhanced granulocyte differentiation. These observations indicate that ATRA has different effects on hematopoietic cells depending on their maturational state, preventing and/or delaying the differentiation of primitive hematopoietic precursors while enhancing the terminal differentiation of committed granulocyte/monocyte progenitors.
6
Girlich, Delphine, Liliana Mihaila, Vincent Cattoir, Frédéric Laurent, Christine Begasse, Florence David, Carole-Ann Metro und Laurent Dortet. „Evaluation of CHROMagar™ LIN-R for the Screening of Linezolid Resistant Staphylococci from Positive Blood Cultures and Nasal Swab Screening Samples“. Antibiotics 11, Nr. 3 (25.02.2022): 313. http://dx.doi.org/10.3390/antibiotics11030313.
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The increasing number of nosocomial pathogens with resistances towards last resort antibiotics, like linezolid for gram positive bacteria, leads to a pressing need for screening and, consequently, suitable screening media. Some national guidelines on infection prevention (e.g., in Germany) have already recommended screening for linezolid-resistant bacteria, despite an accurate screening medium that was not available yet. In this study, we analyzed the performance and reliability of the first commercial chromogenic medium, CHOMagar™ LIN-R, for screening of linezolid-resistant gram-positive isolates. Thirty-four pure bacterial cultures, 18 positive blood cultures, and 358 nasal swab screening samples were tested. This medium efficiently detected linezolid-resistant S. epidermidis isolates from pure bacterial cultures and from positive blood cultures with a high sensitivity (100%) and specificity (100%). Among the 358 nasal swab screening samples prospectively tested, 10.9% were cultured with linezolid-resistant isolates (mostly S. epidermidis). Of note, slight growth was observed for 7.5% samples with linezolid-susceptible isolates of S. epidermidis (n = 1), S. aureus (n = 1), Enterococcus faecalis (n = 4), Lactobacillus spp. (n = 3), gram negatives (n = 18). Moreover, few Candida spp. also cultured on this medium (1.4%).
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Young, JC, E. Bruno, KM Luens, S. Wu, M. Backer und LJ Murray. „Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells“. Blood 88, Nr. 5 (01.09.1996): 1619–31. http://dx.doi.org/10.1182/blood.v88.5.1619.1619.
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Abstract Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy- 1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200–10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony- forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.
8
Young, JC, E. Bruno, KM Luens, S. Wu, M. Backer und LJ Murray. „Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells“. Blood 88, Nr. 5 (01.09.1996): 1619–31. http://dx.doi.org/10.1182/blood.v88.5.1619.bloodjournal8851619.
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Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy- 1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200–10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony- forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.
9
Luens, Karin M., Marilyn A. Travis, Ben P. Chen, Beth L. Hill, Roland Scollay und Lesley J. Murray. „Thrombopoietin, kit Ligand, and flk2/flt3 Ligand Together Induce Increased Numbers of Primitive Hematopoietic Progenitors From Human CD34+Thy-1+Lin− Cells With Preserved Ability to Engraft SCID-hu Bone“. Blood 91, Nr. 4 (15.02.1998): 1206–15. http://dx.doi.org/10.1182/blood.v91.4.1206.
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Abstract CD34+Thy-1+Lin− cells are enriched for primitive hematopoietic progenitor cells (PHP), as defined by the cobblestone area-forming cell (CAFC) assay, and for bone marrow (BM) repopulating hematopoietic stem cells (HSC), as defined by the in vivo SCID-hu bone assay. We evaluated the effects of different cytokine combinations on BM-derived PKH26-labeled CD34+Thy-1+Lin− cells in 6-day stroma-free cultures. Nearly all (>95%) of the CD34+Thy-1+Lin− cells divided by day 6 when cultured in thrombopoietin (TPO), c-kit ligand (KL), and flk2/flt3 ligand (FL). The resulting CD34hiPKHlo (postdivision) cell population retained a high CAFC frequency, a mean 3.2-fold increase of CAFC numbers, as well as a capacity for in vivo marrow repopulation similar to freshly isolated CD34+Thy-1+Lin− cells. Initial cell division of the majority of cells occurred between day 2 and day 4, with minimal loss of CD34 and Thy-1 expression. In contrast, cultures containing interleukin-3 (IL-3), IL-6, and leukemia inhibitory factor contained a mean of 75% of undivided cells at day 6. These CD34hi PKHhi cells retained a high frequency of CAFC, whereas the small population of CD34hiPKHlo postdivision cells contained a decreased frequency of CAFC. These data suggest that use of a combination of TPO, KL, and FL for short-term culture of CD34+Thy-1+Lin− cells increases the number of postdivision PHP, measured as CAFC, while preserving the capacity for in vivo engraftment.
10
Muench, MO, MG Roncarolo, S. Menon, Y. Xu, R. Kastelein, S. Zurawski, CH Hannum, J. Culpepper, F. Lee und R. Namikawa. „FLK-2/FLT-3 ligand regulates the growth of early myeloid progenitors isolated from human fetal liver“. Blood 85, Nr. 4 (15.02.1995): 963–72. http://dx.doi.org/10.1182/blood.v85.4.963.bloodjournal854963.
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The effects of the recently identified FLK-2/FLT-3 ligand (FL) on the growth of purified human fetal liver progenitors were investigated under serum-deprived culture conditions. FL alone was found to stimulate modest proliferation in short-term cultures of CD34++ CD38+ lineage (Lin)- light-density fetal liver (LDFL) cells and the more primitive CD34++ CD38- Lin- LDFL cells. However, the low levels of growth induced by FL were insufficient for colony formation in clonal cultures. Synergism between FL and either granulocyte-macrophage colony- stimulating factor (GM-CSF), interleukin-3 (IL-3) or KIT ligand (KL) was observed in promoting the growth of high-proliferative potential (HPP) colony-forming cells (CF) and/or low-proliferative potential (LPP)-CFC in cultures of CD34++ CD38+ Lin- and CD34++ CD38- Lin- LDFL- cells. FL, alone or in combination with other cytokines, was not found to affect the growth of CD34+ Lin- LDFL cells, the most mature subpopulation of fetal liver progenitors investigated. The growth of the most primitive subset of progenitors studied, CD34++ CD38- Lin- LDFL cells, required the interactions of at least two cytokines, because only very low levels of growth were observed in response to either FL, GM-CSF, IL-3 or KL alone. However, the results of delayed cytokine-addition experiments suggested that individually these cytokines did promote the survival of this early population of progenitors. Although two-factor combinations of FL, KL, and GM-CSF were observed to promote the growth of early progenitors in a synergistic manner, neither of these factors was found to make fetal liver progenitors more responsive to suboptimal concentrations of a second cytokine. Only myeloid cells were recovered from liquid cultures of CD34++ CD38- Lin- LDFL cells grown in the presence of combinations of FL, KL, and GM-CSF. These results indicate that FL is part of a network of growth factors that regulate the growth and survival of early hematopoietic progenitors.
11
Dohnal, Barbara. „Investigations on some metabolites of Tecoma stans Juss. callus tissue. Part I. Tissue culture“. Acta Societatis Botanicorum Poloniae 45, Nr. 1–2 (2015): 93–100. http://dx.doi.org/10.5586/asbp.1976.008.
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Callus tissue satisfactorily growing was established from <i>Tecoma stans</i> Juss. seedlings in static and suspension cultures on Murashige medium modified by Mei-Lie-Lin (M-L) and on Murashige-Skoog Revised Tobacco Medium supplemented with 0.3 ppm kinetin (RT-k). Faster growth, better growth efficiency and higher anatomical organization of the cultured tissue were observed on RT-k medium.
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Doan, Ton C., Brian M. Jeong, Mackenzie Coden, Hiam Abdala-Valencia und Sergejs Berdnikovs. „Matrix protein Tenascin-C expands and reversibly blocks maturation of eosinophil progenitors“. Journal of Immunology 200, Nr. 1_Supplement (01.05.2018): 103.9. http://dx.doi.org/10.4049/jimmunol.200.supp.103.9.
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Abstract While eosinophil depletion is a sought after strategy in treating allergic disease, little is known about the local tissue factors regulating eosinophils. In disease, eosinophils encounter provisional extracellular matrix (ECM) glycoproteins such as Tenascin-C (TNC), which strongly correlates with eosinophil expansion in tissues. While restricted in healthy adult lungs, TNC is a hematopoietic niche component in bone marrow stroma and is expressed de novo during wound healing or in pathologic epithelial remodeling in asthma. RNA-Seq shows that exposing naïve murine eosinophils to TNC upregulates immaturity markers (CD34, CD117, and Sca-1) and suppresses IL-5Rα. Thus, we hypothesized that TNC represents a factor in the provisional allergic tissue microenvironment that could support in situ eosinophil progenitor expansion. In murine bone marrow-derived cultures, TNC: 1) 3-fold expanded the available Lin−Sca1+ early precursor pool; 2) downregulated IL-5Rα expression on Lin− CD45+ c-kit+ cells during the eosinophil lineage commitment phase; 3) served as a reversible eosinophil maturation block, as TNC withdrawal rescued maturation and increased final eosinophil yield. Moreover, TNC knockout mice lack in situ lung expansion of Lin− c-kit+ CD34+ common myeloid progenitors and Lin− Siglec-F+ Sca-1+ cells in an allergic inflammation model as well as exhibit accelerated lung eosinophil maturation ex vivo. Adding TNC to lung homogenates cultured in IL-5 ex vivo suppressed eosinophil maturation in a manner consistent with TNC block of eosinophil maturation in bone marrow-derived cultures. Together, our results stress the local tissue factor potential to promote in situ expansion and eosinophil persistence in allergic disease.
13
Nakamura, Koji, Taku Kouro, Paul W. Kincade, Alexander Malykhin, Kazuhiko Maeda und K. Mark Coggeshall. „Src Homology 2–containing 5-Inositol Phosphatase (SHIP) Suppresses an Early Stage of Lymphoid Cell Development through Elevated Interleukin-6 Production by Myeloid Cells in Bone Marrow“. Journal of Experimental Medicine 199, Nr. 2 (12.01.2004): 243–54. http://dx.doi.org/10.1084/jem.20031193.
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The Src homology (SH)2–containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP−/− animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP−/− mice. Lineage-negative marrow (Lin−) cells isolated from wild-type mice failed to differentiate into B cells when cocultured with those of SHIP−/− mice. Furthermore, culture supernatants of SHIP−/− Lin− cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP−/− Lin− cells contained more IL-6 transcripts than wild-type Lin− cells, and neutralizing anti–IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP−/− Lin− cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin− bone marrow cells reproduced the phenotype of SHIP−/− bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow.
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Ramanauskaitė, Giedrė, Dovilė Žalalytė, Vytautas Kašėta, Aida Vaitkuvienė, Lilija Kalėdienė und Genė Biziulevičienė. „Skin extracellular matrix components accelerate the regenerative potential of Lin− cells“. Open Life Sciences 9, Nr. 4 (01.04.2014): 367–73. http://dx.doi.org/10.2478/s11535-013-0283-9.
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AbstractDue to their unique properties, bone marrow-derived Lin− cells can be used to regenerate damaged tissues, including skin. The objective of our study was to determine the influence of the skin tissue-specific microenvironment on mouse Lin− cell proliferation and migration in vitro. Cells were analyzed for the expression of stem/progenitor surface markers by flow cytometry. Proliferation of MACS-purified cells in 3D cultures was investigated by WST-8 assay. Lin− cell migration was evaluated by in vitro scratch assay. The results obtained show that basement membrane matrix is more effective for Lin− cell proliferation in vitro. However, type I collagen matrix better enhances the re-epithelization process, that depends on the cell migratory properties. These studies are important for preparing cells to be used in transplantation.
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Jiang, Xiaoyan, Yun Zhao, Wing Yiu Chan, Emily Pang, Allen Eaves und Connie Eaves. „Leukemic Stem Cells of Chronic Phase CML Patients Consistently Display Very High BCR-ABL Transcript Levels and Reduced Responsiveness to Imatinib Mesylate in Addition to Generating a Rare Subset That Produce Imatinib Mesylate-Resistant Differentiated Progeny.“ Blood 104, Nr. 11 (16.11.2004): 711. http://dx.doi.org/10.1182/blood.v104.11.711.711.
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Abstract Imatinib mesylate (IM) is an inhibitor of the BCR-ABL oncoprotein associated with human chronic myeloid leukemia (CML). IM therapy has shown remarkable effects in initial clinical trials, but both clinical and laboratory studies increasingly suggest that, on its own, IM may have limited curative potential, due to a reduced IM sensitivity of the more primitive, slowly proliferating CD34+ CML cells thought to be responsible for sustaining the disease in vivo. To investigate the basis of this unresponsiveness, we compared the IM sensitivity and BCR-ABL expression of FACS-purified subsets of lin−CD34+ cells from 4 CML chronic phase patients. None of these had been treated with IM and their cells at all stages of differentiation were exclusively leukemic; i.e., >95% of the lin−CD34+CD38−, lin−CD34+CD38+ and lin+CD34− cells were BCR-ABL+ (by direct FISH) and all longterm culture-initiating cell (LTC-IC) -derived CFCs were Ph+. In the absence of IM, suspension cultures initiated with these lin−CD34+CD38− CML cells (0.5–5% of the lin−CD34+ cells) showed a net expansion of viable cells after 3 weeks; 100x with and 10x without added growth factors (GFs). Addition of 0.1–10 μM/ml IM reduced the yield of viable cells in a dose-dependent fashion, particularly when GFs were not added (100-fold decrease with 10 μM/ml IM). Parallel cultures of the corresponding lin−CD34+CD38+ CML cells showed these did not expanded as much (~8x +GFs, 2x -GFs) and were more sensitive to IM (1000-fold decrease after 3 weeks in 10 μM/ml IM -GFs). Quantitative real-time RT-PCR analysis revealed BCR-ABL transcripts to be present in the most primitive, freshly isolated lin−CD34+CD38− cells (n=12) at >300-fold higher levels than in the terminally differentiating lin+CD34− CML cells (n=21), at >10-fold higher levels than the normal BCR transcripts in the same lin−CD34+CD38− cells, and at 40-fold higher levels than in the less primitive lin−CD34+CD38+ cells (n=12), indicating a correlation between decreasing BCR-ABL transcripts and increasing IM sensitivity during CML stem cell differentiation in vivo. Interestingly, maintenance of the lin−CD34+CD38− CML cells for 3 weeks in vitro with 10 μM/ml IM (±GFs) consistently selected for a subset of leukemic cells (80–100% BCR-ABL+ by FISH) that showed complete resistance to 5 μM/ml IM in CFC assays, in marked contrast to the CFCs in the starting lin−CD34+CD38− cells that were inhibited 5–10-fold by 5 μM/ml IM. Moreover, although the Ph was the sole abnormality present in all direct metaphases, initial CFCs and LTC-IC-derived CFCs from all samples, a 17p+ abnormality was seen in 4/4 metaphases obtained from one colony generated from the cells present in one of the 3-week IM-containing cultures, suggesting the selective survival of differentiating progeny of rare, pre-existing, IM-resistant stem cells. Consistent with this possibility was the finding that BCR-ABL transcript levels in the cells present in the 3 week cultures were reduced 50-fold relative to the input lin−CD34+CD38− cells. Taken together, these findings suggest a previously undescribed epigenetic mechanism of IM unresponsiveness characteristic of chronic phase CML stem cells, in addition to the silent accumulation of genetically-determined IM-resistant members as the CML stem cell population expands during the development of the chronic phase of the disease.
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DiGiusto, DL, R. Lee, J. Moon, K. Moss, T. O'Toole, A. Voytovich, D. Webster und JJ Mule. „Hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood progenitor cells evaluated in vitro and in vivo“. Blood 87, Nr. 4 (15.02.1996): 1261–71. http://dx.doi.org/10.1182/blood.v87.4.1261.bloodjournal8741261.
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The hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood (UCB) samples was evaluated in vitro and in vivo. Phenotypic analysis shows that approximately 1% of cord blood mononuclear cells express high levels of CD34 antigen on their surface (CD34hi), but none of a panel of lineage antigens (Lin-), suggesting that they are hematopoietic progenitor cells that have not yet committed to a specific lineage. Approximately 1% of CD34hi/Lin- cells are primitive hematopoietic progenitors that produce B lymphoid and multiple myeloid progeny for up to 7 weeks in stromal cell cultures. Twenty-one percent (+/- 13%) of CD34hi/Lin- cells also express low levels of the Thy-1 antigen and are threefold to fourfold enriched over CD34hi/Lin- cells in primitive hematopoietic potential as measured by long-term culture and phenotypic analysis. One-week liquid cultures of CD34-enriched UCB progenitor cells in the presence of interleukin (IL)- 3, IL-6, and stem cell factor (SCF) results in a two-fold to threefold expansion of progenitors capable of reinitiating long-term stromal cell cultures. Only the CD34hi/Thy-1+/Lin- cell population was capable of maintaining progenitors with secondary transfer potential in long-term stromal cell cultures and is thus postulated to contain all of the primitive hematopoietic stem cells in UCB. The in vivo transplantation potential of UCB was also measured. Ex vivo manipulated UCB progenitor cells were used to engraft irradiated human thymus fragments implanted in severe combined immunodeficiency (SCID) mice. Thymic engraftment with >5% donor-derived cells and a normal CD4/CD8 distribution was observed in 19 of 23 tissues tested. UCB cells from in vitro expansion cultures engrafted with efficiencies comparable to nonexpanded cells. Similar results were obtained for UCB engraftment of human bone fragments implanted in SCID mice. In all cases, engraftment was achieved in competition with endogenous competitor stem cells and across major histocompatibility barriers. Taken together, this data demonstrates that human UCB is a rich source of multipotent hematopoietic progenitors that can be cryopreserved, enriched by physical methods, and expanded in a limited fashion without measurable loss of long-term culture or in vivo engrafting potential as measured in these assays.
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Bernstein, ID, RG Andrews und KM Zsebo. „Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor“. Blood 77, Nr. 11 (01.06.1991): 2316–21. http://dx.doi.org/10.1182/blood.v77.11.2316.2316.
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Abstract We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.
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Bernstein, ID, RG Andrews und KM Zsebo. „Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor“. Blood 77, Nr. 11 (01.06.1991): 2316–21. http://dx.doi.org/10.1182/blood.v77.11.2316.bloodjournal77112316.
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We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.
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Peled, Tony, Ela Glukhman, Sophie Adi, Efrat Landau, Nira Hasson, Chana Lador, Julie Mandel und Eitan Fibach. „Antagonists to Retinoid Receptors Down-Regulate CD38 Expression and Inhibit In Vitro Differentiation of Cord Blood Derived CD34+ Cells.“ Blood 108, Nr. 11 (16.11.2006): 3652. http://dx.doi.org/10.1182/blood.v108.11.3652.3652.
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Abstract CD38, originally described as a differentiation marker, has emerged as an important multifunctional protein. Its most well-characterized function is the ability to catalyze the synthesis of cyclic ADP-ribose (cADPR) from NAD. However, its major enzymatic activity is the hydrolysis of NAD (NADase) implicating it as the major regulator of cellular NAD levels. CD38 expression increases with commitment and differentiation. It is not clear, however, whether such changes in CD38 are merely phenotypic, or reflect an active role for CD38 in the regulation of cell differentiation. The regulation of CD38 gene expression is under the direct control of retinoid receptors (RAR). Antagonists to RAR abolish up-regulation of CD38 gene expression as well as RA induction of granulocytic differentiation down-stream of the myeloid compartment. In the present study we evaluated the involvement of CD38 in the regulation of HPC differentiation by treatment of ex-vivo cultures with LMW antagonists, targeted to either CD38 expression or to its biological activities. CB derived CD34+ cells were cultured with cytokines (S,T,F,6). Treatment of these cultures with an RAR-antagonist (AGN194310) abolished the expression of surface CD38. After 3 weeks in culture, the content of CFC was 3 ±1.1-fold higher, the content of CD34+ cells was 2.4 ± 0.24-fold higher and percentage CD34+ cells displaying CD34+Lin− phenotype was by 35 ± 10-fold higher (p<0.05, n= 14) in RAR-antagonist (10−6M) compared to cytokines-only treated cultures. Colonies derived from RAR-antagonist treated cultures sustained high re-plating capacity, a property that was lost during the first 3-weeks of expansion with cytokines only. In long-term cultures, the peak of CFUc and CD34+ cell expansion of RAR-antagonist treated cultures was 6–10 weeks later than control cultures. At the peak of expansion, cumulative numbers of CD34+ and CFUc were by 130- and 512-fold higher (p<0.05, n=4), respectively, in treated than in control cultures. CFU-MIX colonies were exclusively observed in RAR-antagonist treated cultures (between weeks 7–10). Interestingly, limited (1 week) exposure to the RAR-antagonist was sufficient for this long-term effect. Similarly, we tested the effect of an RXR antagonist (LGN 100754) (10−9 – 10−5 M) on short- and long-term cultures. Treatment with the RXR-antagonist did not down-regulate CD38 expression and only slightly improved ex-vivo expansion parameters over cytokines-only treated cultures. We next evaluated whether inhibition of CD38 enzymatic activities will also modulate in-vitro differentiation of cultured cells. To this end, CD34+ cell cultures were treated with nicotinamide (NA), a non-competitive inhibitor of CD38 NADase, previously demonstrated to abolish its enzymatic activities. 3-week treatment with NA (5mM) resulted in a marked decrease in CD38 expression and a marked increase in the fraction of CD34+Lin− cells as compared to cytokines-only treated cultures (48.0 ± 3.7% vs. 2.8 ± 0.7% and 18.6 ± 3% vs. 0.7 ± 0.06%, n=6, p<0.05, respectively). As with the RAR-antagonist, long-term expansion potential, as determined by CFC and CD34+ cell content, was significantly higher in cultures treated with NA relative to cytokines-only treated cultures. These results demonstrate that both a pan-RAR antagonist and NA inhibit differentiation and promote ex-vivo expansion of progenitor cells, suggesting the possible involvement of CD38 protein in these processes.
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Fujisaki, Tomoaki, Marc G. Berger, Stefan Rose-John und Connie J. Eaves. „Rapid Differentiation of a Rare Subset of Adult Human Lin−CD34−CD38− Cells Stimulated by Multiple Growth Factors In Vitro“. Blood 94, Nr. 6 (15.09.1999): 1926–32. http://dx.doi.org/10.1182/blood.v94.6.1926.
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Abstract Recently, several reports of lineage-negative (lin−) CD34− cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin−CD34−CD38− cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)–mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin−CD34−CD38− cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin−CD34−CD38− cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper–IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% ± 2% were CD34+ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 ± 7 CFC per lin−CD34−CD38− pre-CFC). In contrast to lin−CD34+CD38−cells, this response of lin−CD34−CD38− cells required exposure to all of the 5 growth factors used. Up to 1.7 × 105 lin−CD34− adult marrow cells failed to engraft sublethally irradiated NOD/SCID-β2M−/− mice. These studies demonstrate unique properties of a rare subset of lin−CD34−CD38− cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.
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Fujisaki, Tomoaki, Marc G. Berger, Stefan Rose-John und Connie J. Eaves. „Rapid Differentiation of a Rare Subset of Adult Human Lin−CD34−CD38− Cells Stimulated by Multiple Growth Factors In Vitro“. Blood 94, Nr. 6 (15.09.1999): 1926–32. http://dx.doi.org/10.1182/blood.v94.6.1926.418k14_1926_1932.
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Recently, several reports of lineage-negative (lin−) CD34− cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin−CD34−CD38− cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)–mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin−CD34−CD38− cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin−CD34−CD38− cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper–IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% ± 2% were CD34+ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 ± 7 CFC per lin−CD34−CD38− pre-CFC). In contrast to lin−CD34+CD38−cells, this response of lin−CD34−CD38− cells required exposure to all of the 5 growth factors used. Up to 1.7 × 105 lin−CD34− adult marrow cells failed to engraft sublethally irradiated NOD/SCID-β2M−/− mice. These studies demonstrate unique properties of a rare subset of lin−CD34−CD38− cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.
22
Filipczuk, Magdalena. „Introducing Chinese Philosophy to Western Readers – Lin Yutang as a Cross-cultural Interpreter“. Perspektywy Kultury 35, Nr. 4 (30.12.2021): 87–104. http://dx.doi.org/10.35765/pk.2021.3504.06.
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The article reconstructs selected motifs in the philosophy of Lin Yutang, a twentieth-century Chinese thinker, translator and editor, especially popular in the West, undertaken, as it were, on the margins of his work to explain and popularize Chinese culture and philosophy in the West. Lin reflects on issues such as how to effectively and accurately explain a radically alien civilization to the Western-educated reader, in his or her own language, and who can appoint himself as the representative of Chinese culture at all? As a bilingual author, Lin very accurately shows the state of suspension between two cultures, characteristic of an intercultural interpreter who attempts to simultaneously move within two disproportionate, culturally determined conceptual schemes.
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Young, JC, A. Varma, D. DiGiusto und MP Backer. „Retention of quiescent hematopoietic cells with high proliferative potential during ex vivo stem cell culture“. Blood 87, Nr. 2 (15.01.1996): 545–56. http://dx.doi.org/10.1182/blood.v87.2.545.bloodjournal872545.
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Human CD34+/Thy-1+/Lin- hematopoietic cells purified from bone marrow (BM) or mobilized peripheral blood (MPB) are highly enriched for pluripotent stem cells. Ex vivo expansion of this population is proposed as a means of providing accelerated short-term, as well as long-term, engraftment after myeloablative therapy. Here we demonstrate that primitive quiescent cells are retained in bulk expansion cultures of CD34+/Thy-1+/Lin- cells and that the cell production capacity of the expanded cell product can largely be attributed to cells exhibiting quiescent behavior during culture. CD34+/Thy-1+/Lin- cells from adult BM or MPB were labeled with the fluorescent membrane dye PKH26, followed by in vitro culture of 10(4) cells on a murine stromal layer in the presence of interleukin (IL)-3, IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF). With each subsequent cell division, PKH26 fluorescence is reduced by roughly half, which allows tracking of the number of cell divisions. Progenitor cells present after a 2-week expansion period were sorted into CD34+/Lin-/dyebright and CD34+/Lin- /dyedim fractions and then cultured in a 4-week single-cell proliferation assay to characterize the proliferative capacity of each group. Fifty-nine percent of progenitors remaining dyebright after bulk culture (four or fewer cell divisions) were observed to proliferate in single cell culture, and produced an average of 1,780 cells per plated cell. In contrast, only 26% of dyedim (more than four divisions) progenitors were observed to proliferate and displayed a lower average proliferative capacity of 225 cells per plated cell. Similar behaviors were observed after a second consecutive cycle of bulk culture, indicating that quiescent cells with high proliferative capacity existed in culture for at least 4 weeks. Single CD34+/Lin-/dyebright progenitors purified from bulk cultures were observed to produce as many as 1,000 CD34 positive progeny during single cell culture, and these progeny included multilineage colony forming cells. These data demonstrate that among CD34 positive cells recovered after in vitro bulk culture, a higher proliferative capacity correlated with quiescent behavior. The described culture method provides quantitation of the cell producing capacity of individual cells in hematopoietic cell mixtures and may prove useful for predicting engrafting potential in products intended for cellular therapy.
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Storms, Robert W., Margaret A. Goodell, Alan Fisher, Richard C. Mulligan und Clay Smith. „Hoechst dye efflux reveals a novel CD7+CD34− lymphoid progenitor in human umbilical cord blood“. Blood 96, Nr. 6 (15.09.2000): 2125–33. http://dx.doi.org/10.1182/blood.v96.6.2125.
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Abstract A novel Hoechst 33342 dye efflux assay was recently developed that identifies a population of hematopoietic cells termed side population (SP) cells. In the bone marrow of multiple species, including mice and primates, the SP is composed primarily of CD34−cells, yet has many of the functional properties of hematopoietic stem cells (HSCs). This report characterizes SP cells from human umbilical cord blood (UCB). The SP in unfractionated UCB was enriched for CD34+ cells but also contained a large population of CD34− cells, many of which were mature lymphocytes. SP cells isolated from UCB that had been depleted of lineage-committed cells (Lin− UCB) contained CD34+ and CD34− cells in approximately equivalent proportions. Similar to previous descriptions of human HSCs, the CD34+Lin− SP cells were CD38dimHLA-DRdimThy-1dimCD45RA−CD71−and were enriched for myelo-erythroid precursors. In contrast, the CD34−Lin− SP cells were CD38−HLA-DR−Thy-1−CD71−and failed to generate myelo-erythroid progeny in vitro. The majority of these cells were CD7+CD11b+CD45RA+, as might be expected of early lymphoid cells, but did not express other lymphoid markers. The CD7+CD34−Lin− UCB SP cells did not proliferate in simple suspension cultures but did differentiate into natural killer cells when cultured on stroma with various cytokines. In conclusion, the human Lin− UCB SP contains both CD34+ multipotential stem cells and a novel CD7+CD34−Lin− lymphoid progenitor. This observation adds to the growing body of evidence that CD34− progenitors exist in humans.
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Storms, Robert W., Margaret A. Goodell, Alan Fisher, Richard C. Mulligan und Clay Smith. „Hoechst dye efflux reveals a novel CD7+CD34− lymphoid progenitor in human umbilical cord blood“. Blood 96, Nr. 6 (15.09.2000): 2125–33. http://dx.doi.org/10.1182/blood.v96.6.2125.h8002125_2125_2133.
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A novel Hoechst 33342 dye efflux assay was recently developed that identifies a population of hematopoietic cells termed side population (SP) cells. In the bone marrow of multiple species, including mice and primates, the SP is composed primarily of CD34−cells, yet has many of the functional properties of hematopoietic stem cells (HSCs). This report characterizes SP cells from human umbilical cord blood (UCB). The SP in unfractionated UCB was enriched for CD34+ cells but also contained a large population of CD34− cells, many of which were mature lymphocytes. SP cells isolated from UCB that had been depleted of lineage-committed cells (Lin− UCB) contained CD34+ and CD34− cells in approximately equivalent proportions. Similar to previous descriptions of human HSCs, the CD34+Lin− SP cells were CD38dimHLA-DRdimThy-1dimCD45RA−CD71−and were enriched for myelo-erythroid precursors. In contrast, the CD34−Lin− SP cells were CD38−HLA-DR−Thy-1−CD71−and failed to generate myelo-erythroid progeny in vitro. The majority of these cells were CD7+CD11b+CD45RA+, as might be expected of early lymphoid cells, but did not express other lymphoid markers. The CD7+CD34−Lin− UCB SP cells did not proliferate in simple suspension cultures but did differentiate into natural killer cells when cultured on stroma with various cytokines. In conclusion, the human Lin− UCB SP contains both CD34+ multipotential stem cells and a novel CD7+CD34−Lin− lymphoid progenitor. This observation adds to the growing body of evidence that CD34− progenitors exist in humans.
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Schwieterman, W. D., G. M. Wood, D. E. Scott und A. D. Steinberg. „Studies of bone marrow progenitor cells in lupus-prone mice. I. NZB marrow cells demonstrate increased growth in Whitlock-Witte culture and increased splenic colony-forming unit activity in the Thy-1-, lineage- population.“ Journal of Immunology 148, Nr. 8 (15.04.1992): 2405–10. http://dx.doi.org/10.4049/jimmunol.148.8.2405.
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Abstract Previous studies have demonstrated that NZB marrow can transfer features of autoimmunity. Therefore, we undertook a study of NZB marrow to determine whether it demonstrated any phenotypic abnormalities. In Whitlock-Witte cultures, NZB marrow cells generated nonadherent cells at low seeding densities, densities at which marrow from other strains did not generate nonadherent cells. In contrast, NZB marrow grew less well than controls in Dexter cultures. Inasmuch as the latter favor growth of granulocyte-macrophage precursors and the former B cells, these results suggest a possible skewing of NZB marrow cells toward lymphocyte production. Unfractionated marrow cells from NZB mice were found to produce 10-fold more splenic colonies in lethally irradiated recipients than marrow cells from control mice. This result was independent of the genotype of the recipient. When the progenitor Thy-1lo, Lin- marrow subpopulation was studied, NZB mice did not differ substantially from controls regarding splenic CFU. Therefore, Thy-1-, Lin- marrow cells were studied as a possible source of the excess splenic CFU in NZB mice. Indeed, the NZB Thy-1-, Lin- population contained 30-fold more splenic CFU than did the Thy-1-, Lin- population from control mice. These results suggest that NZB mice have unusual marrow progenitor cells; such cells may play a role in their autoimmune disease.
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Chitteti, Brahmananda Reddy, Yinghua Cheng, Melissa A. Kacena und Edward F. Srour. „Hierarchical Organization of Osteoblast Reveals the Significant Role of CD166 in Hematopoietic Stem Cell Maintenance and Function“. Blood 120, Nr. 21 (16.11.2012): 215. http://dx.doi.org/10.1182/blood.v120.21.215.215.
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Abstract Abstract 215 The role of osteoblasts (OB) in maintaining hematopoietic stem cells (HSC) in their niche is well elucidated, but the exact definition, both phenotypically and hierarchically of OB responsible for these functions is not clearly known. We previously demonstrated (Chitteti et al. Blood 115(16):3239–48, 2010) that OB identified by their expression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) or CD166, represent a class of OB capable of mediating high levels of hematopoiesis enhancing activity (HEA). We also demonstrated that OB maturational status influences HSC function whereby immature OB with high Runx2 expression promote hematopoietic expansion. Here, we show that CD166 expression tracks the maturational status of OB and is directly correlated with Runx2 expression and high HEA. Fractionation of 2d calvariae-derived OB with lineage markers (CD45, CD31, and Ter119), Sca1, osteopontin (OPN), CD166, CD44, and CD90 revealed that Lin-Sca1-OPN+CD166+ cells (CD166+) and their subpopulations fractionated with CD44 and CD90 expressed high levels of Runx2 and low levels of osteocalcin (OC) demonstrating the relatively immature status of these cells. Conversely, the majority of the Lin-Sca1-OPN+CD166- cells (CD166-) expressed high levels of OC suggesting that CD166- OB are more mature. We then used a co-culture system previously described by our group to assess the potential of different groups of OB to mediate HEA and sustain the expansion of clonogenic cells in culture. In vitro hematopoietic potential of bone marrow-derived Lineage-Sca1+ckit+ (LSK) cells co-cultured for 7 days with fresh OB or OB pre-cultured for 0, 1, 2, or 3 weeks (followed by an additional 1 week of co-culture with LSK cells) declined precipitously with increasing culture duration concomitant with loss of CD166 expression. Percentage of cells expressing CD166 dropped from 63.5% at week 1 to 2.5% at week 4 (measured as total culture duration). During the same time period, the number of total colony forming units per culture dropped from 34,300 ± 4,000 (at week 1) to 1,800 ± 800 (at week 4) reflecting also the drop in the plating efficiency of cultured hematopoietic cells (22.9% ± 1.6% at week 1 versus 5.5% ± 0.5% at week 4). To assess the ability of OB to sustain stem cell function in vitro, we transplanted the progeny of LSK cells maintained in co-culture with OB for 7 days. Levels of chimerism 4mo post-transplantation in primary recipients of LSK progeny harvested from co-cultures containing Lin-Sca1-OPN+CD166+CD90+CD44+ OB were not significantly different from those in recipients of fresh LSK cells 71.8% ± 7.4% vs 86.1% ± 5.2%, respectively. Interestingly, when cells from primary recipients were transplanted into secondary recipients (4mo post primary transplantation), chimerism in mice receiving LSK progeny harvested from co-cultures containing Lin-Sca1-OPN+CD166+CD90+CD44+ OB increased to 95.8% ± 1.2% suggesting that these cells maintained the full potential of their self-renewal capacity. Chimerism levels in secondary recipients of LSK progeny harvested from co-cultures containing other fractions of OB were significantly lower than those observed for cells co-cultured with Lin-Sca1-OPN+CD166+CD90+CD44+ OB. These data suggest that robust HEA activity is best mediated by immature CD166+ OB with high Runx2 and low OC expression. Furthermore, these studies begin to define the hierarchical organization of osteoblastic cells and provide a more refined phenotypic definition of OB that can mediate HEA and maintain stem cell function. Disclosures: No relevant conflicts of interest to declare.
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Li, CL, und GR Johnson. „Stem cell factor enhances the survival but not the self-renewal of murine hematopoietic long-term repopulating cells“. Blood 84, Nr. 2 (15.07.1994): 408–14. http://dx.doi.org/10.1182/blood.v84.2.408.408.
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Abstract The effects of stem cell factor (SCF) have been tested on a murine bone marrow subpopulation (RH123lo, Lin-, Ly6A/E+) that is highly enriched for long-term hematopoietic repopulating cells. SCF maintained cells from this population with long-term repopulating ability for up to 10 days in vitro. However, compared with freshly isolated cells, the level of engraftment in vivo by the cultured cells declined during the in vitro culture period, suggesting that SCF alone was unable to stimulate the self-renewal of long-term repopulating cells. By direct visualization of cultures, only small numbers of cells survived and rarely underwent cell division. However, SCF did directly stimulate proliferation of a population (Rh123med/hi,Lin-,Ly6A/E+) enriched for short-term repopulating cells. These data suggest that stem cell differentiation is associated with the development of mitogenic activity by SCF at least in some progenitor cell populations.
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Li, CL, und GR Johnson. „Stem cell factor enhances the survival but not the self-renewal of murine hematopoietic long-term repopulating cells“. Blood 84, Nr. 2 (15.07.1994): 408–14. http://dx.doi.org/10.1182/blood.v84.2.408.bloodjournal842408.
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The effects of stem cell factor (SCF) have been tested on a murine bone marrow subpopulation (RH123lo, Lin-, Ly6A/E+) that is highly enriched for long-term hematopoietic repopulating cells. SCF maintained cells from this population with long-term repopulating ability for up to 10 days in vitro. However, compared with freshly isolated cells, the level of engraftment in vivo by the cultured cells declined during the in vitro culture period, suggesting that SCF alone was unable to stimulate the self-renewal of long-term repopulating cells. By direct visualization of cultures, only small numbers of cells survived and rarely underwent cell division. However, SCF did directly stimulate proliferation of a population (Rh123med/hi,Lin-,Ly6A/E+) enriched for short-term repopulating cells. These data suggest that stem cell differentiation is associated with the development of mitogenic activity by SCF at least in some progenitor cell populations.
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Grundt, Inger K., und Harald Nyland. „Effects of Polyunsaturated Fatty Acids on Glial Cell Activation. A Study Using Primary Cultures“. Alternatives to Laboratory Animals 22, Nr. 3 (Mai 1994): 201–6. http://dx.doi.org/10.1177/026119299402200311.
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The aim of this study was to examine the effects of essential fatty acids (gammalinolenic acid [18:3 n-6; GLA] and alpha-linolenic acid [18:3 n-3; Lin]) on the activation of glial cells, using lipopolysaccharides as the activating agent. Primary cultures of mixed glial cells from rat brain were used as the model. The morphological activation of microglia was the most significant response to the exposure. This activation was followed by an increase in 5’-nucleotidase (5’-NT) activity. The 5’-NT activity was increased by GLA or Lin alone to 250–350% of the control value and further increased by co-incubation with lipoteichoic acid (a lipopolysaccharide) to 500–600% of the control value. The lipopolysaccharide-induced activation of glial cells was also followed by an augmented release of prostaglandin E2. GLA increased the release of prostaglandin E2 in a dose-dependent manner, whereas Lin had no effect on its release. The results show that this model system is useful for studies on factors affecting the activation of glial cells. GLA and Lin did not reverse glial activation induced by lipopolysaccharides under these experimental conditions.
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Gupta, Rashmi, Simon Karpatkin und Ross S. Basch. „Hematopoiesis and stem cell renewal in long-term bone marrow cultures containing catalase“. Blood 107, Nr. 5 (01.03.2006): 1837–46. http://dx.doi.org/10.1182/blood-2005-03-1180.
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Culturing mouse bone marrow in the presence of catalase dramatically alters hematopoiesis. Granulocyte output is initially increased 4- to 5-fold. This increase is transient and granulocyte production declines as immature (Sca-1+/LIN-) cells accumulate. One third of these immature cells have a phenotype (Sca-1+/c-Kit+) characteristic of hematopoietic stem cells. At 2 to 3 weeks there are greater than 200-fold more Sca-1+/c-Kit+/LIN- cells in treated cultures than in controls. This population contains functional stem cells with both short-term and long-term bone marrow repopulating activity. In addition to myeloid progenitors, this Sca-1+/LIN- population contains a large number of cells that express CD31 and CD34 and have an active Tie-2 promoter, indicating that they are in the endothelial lineage. After 3 to 4 weeks hematopoiesis in treated cultures wanes but if catalase is removed, hematopoiesis resumes. After 7 to 10 days the cultures are indistinguishable from untreated controls. Thus, protected from H2O2, hematopoietic progenitors multiply and become quiescent. This sequence resembles in vivo development in normal marrow. These results make it clear that peroxide-sensitive regulatory mechanisms play an important role in controlling hematopoiesis ex vivo and presumably in vivo as well. They also indicate that manipulation of the peroxide levels can be used to enhance the growth of hematopoietic stem cells in culture.
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Rebel, VI, W. Dragowska, CJ Eaves, RK Humphries und PM Lansdorp. „Amplification of Sca-1+ Lin-WGA+ cells in serum-free cultures containing steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential“. Blood 83, Nr. 1 (01.01.1994): 128–36. http://dx.doi.org/10.1182/blood.v83.1.128.128.
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Abstract Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties, Sca-1 expression (Sca-1+), lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-), and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/-0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in > or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients, respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor, interleukin-6 (IL-6), and erythropoietin (with or without IL-3), a large increase in total cell number, including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly, this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo, as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum-and stroma cell-free culture, providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype.
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Rebel, VI, W. Dragowska, CJ Eaves, RK Humphries und PM Lansdorp. „Amplification of Sca-1+ Lin-WGA+ cells in serum-free cultures containing steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential“. Blood 83, Nr. 1 (01.01.1994): 128–36. http://dx.doi.org/10.1182/blood.v83.1.128.bloodjournal831128.
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Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties, Sca-1 expression (Sca-1+), lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-), and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/-0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in > or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients, respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor, interleukin-6 (IL-6), and erythropoietin (with or without IL-3), a large increase in total cell number, including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly, this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo, as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum-and stroma cell-free culture, providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype.
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Miller, Jeffrey S., Valarie McCullar und Catherine M. Verfaillie. „Ex Vivo Culture of CD34+/Lin−/DR− Cells in Stroma-Derived Soluble Factors, Interleukin-3, and Macrophage Inflammatory Protein-1α Maintains Not Only Myeloid But Also Lymphoid Progenitors in a Novel Switch Culture Assay“. Blood 91, Nr. 12 (15.06.1998): 4516–22. http://dx.doi.org/10.1182/blood.v91.12.4516.
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Abstract We have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1α (MIP-1α). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin−/DR−cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin−/DR− cells assayed in SNC cultures supplemented with IL-3 and MIP-1α. When CD34+/Lin−/DR− progeny from the SNC culture were plated sequentially into “NK cell progenitor switch” conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by “NK cell differentiation” conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3− NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1α directly to “NK cell differentiation” conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33− cells present after SNC cultures with IL-3 and MIP-1α, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33− population similar to fresh sorted CD34+/Lin−/DR− cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1α, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.
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Miller, Jeffrey S., Valarie McCullar und Catherine M. Verfaillie. „Ex Vivo Culture of CD34+/Lin−/DR− Cells in Stroma-Derived Soluble Factors, Interleukin-3, and Macrophage Inflammatory Protein-1α Maintains Not Only Myeloid But Also Lymphoid Progenitors in a Novel Switch Culture Assay“. Blood 91, Nr. 12 (15.06.1998): 4516–22. http://dx.doi.org/10.1182/blood.v91.12.4516.412k05_4516_4522.
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We have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1α (MIP-1α). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin−/DR−cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin−/DR− cells assayed in SNC cultures supplemented with IL-3 and MIP-1α. When CD34+/Lin−/DR− progeny from the SNC culture were plated sequentially into “NK cell progenitor switch” conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by “NK cell differentiation” conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3− NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1α directly to “NK cell differentiation” conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33− cells present after SNC cultures with IL-3 and MIP-1α, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33− population similar to fresh sorted CD34+/Lin−/DR− cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1α, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.
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SUN, Yihe. „The Compound Cultural Identity of Lin Yutang and his Intercultural Communication of Chinese Ethnic Culture“. International Journal of Translation and Interpretation Studies 2, Nr. 2 (18.09.2022): 50–58. http://dx.doi.org/10.32996/ijtis.2022.2.2.5.
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This paper analyzes the construction of Lin Yutang's compound cultural identity and the strategies employed in his English translation of Chinese culture with Lin Yutang's literary translations as the case. Lin Yutang prides himself on mastering both Chinese and western cultures. With his Chinese and western cultural backgrounds, he promoted the spreading of Chinese culture around the world. On the one hand, he maintains the characteristics of the source text, thereby introducing Chinese thinking to enhance the influence of traditional Chinese culture. On the other hand, in the face of cultural differences in translation and the needs of readers, he, as the main body of the translation process, must be responsible for readers who used western culture to interpret Chinese thoughts or change the forms of the source text. In translation, Lin Yutang flexibly used two cultural identities to construct a unique compound cultural identity, with Chinese as the center and western as the media, to introduce the Chinese culture to the world.
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Povinelli, Benjamin, Michael Baranello, Kathleen Kokolus und Michael Nemeth. „Balanced Wnt5a-Mediated Signaling Is Necessary for Normal Proliferation of Primitive Hematopoietic Cells.“ Blood 114, Nr. 22 (20.11.2009): 2533. http://dx.doi.org/10.1182/blood.v114.22.2533.2533.
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Abstract Abstract 2533 Poster Board II-510 Multiple members of the Wnt family of ligands have been implicated in the regulation of self-renewal and proliferation of hematopoietic stem cells (HSCs). Previously, we have observed that ex vivo expansion of HSCs in the presence of recombinant murine Wnt5a (rmWnt5a) resulted in increased hematopoietic repopulation. Based on these data, we hypothesized that Wnt5a is necessary for normal function of HSCs and hematopoietic progenitors (HPCs). Since Wnt5a deficiency (Wnt5a−/−) is perinatal lethal in vivo, we tested this hypothesis using in vitro Dexter stroma cultures established using whole bone marrow. To determine the ability of Wnt5a to support hematopoiesis in the context of the adult hematopoietic microenvironment, we cultured lineage-negative (lin−) HPCs on irradiated bone marrow stroma in the presence of 5 μg/ml Wnt5a-neutralizing antibody (Wnt5a-Ab). After two weeks, we observed that hematopoietic cells cultured on untreated stroma contained 4.8-fold more myeloid CFU (33.1 ± 12.3 CFU/104 cells) than cells cultured on Wnt5a-Ab stroma (6.9 ± 0.7 CFU; n = 3, p < .01). A similar difference was observed after 4 weeks (control: 16.0 ± 7.2 CFU/104 cells; Wnt5a-Ab: 1.3 ± 2.3 CFU; n = 3; p = .03). In the converse experiment, lin− HPCs were cultured on stroma in the presence of Wnt5a conditioned medium (Wnt5a-CM). We observed after two weeks that hematopoietic cells cultured on stroma with control-CM contained 4.4-fold more myeloid CFU (29.8 ± 13.5 CFU/104 cells) than cells cultured on Wnt5a-CM stroma (6.8 ± 2.2 CFU; n = 4, p = .02). Together, these data indicate that Wnt5a-mediated signaling must be balanced in order for normal hematopoiesis to occur. To determine if the effects of Wnt5a required the presence of developmental stage-specific factors, we established stroma cultures from fetal spleens harvested from E17.5 Wnt5a−/− mice and littermate controls and seeded them with lin− HPCs. We observed that hematopoietic cells cultured on control fetal spleen stroma contained 12.5-fold more myeloid CFU (72.6 ± 21.6 CFU/104 cells) than cells cultured on Wnt5a−/− fetal spleen stroma (5.8 ± 5.8; n = 6, p < .001) after two weeks. These data indicate that the effect of Wnt5a on hematopoietic cells is independent of the developmental stage of the surrounding microenvironment. To determine if the effects of Wnt5a were due to regulation of proliferation or differentiation of hematopoietic cells, we cultured bone marrow cells in cytokine-supplemented methylcellulose in the presence of 300 ng/ml rmWnt5a. We observed a 5.5-fold decrease in the number of myeloid CFU formed in cultures with rmWnt5a (14.1 ± 3.8/104 cells) compared to control (77.6 ± 5.1 CFU; n = 3; p < .001), suggesting that Wnt5a could be regulating both processes. In the converse experiment, we cultured bone marrow cells for 4 days in cytokine-supplemented serum-free media with the same dose of rmWnt5a after which equal numbers of cells were plated in rmWnt5a-free methylcellulose. We did not observe any difference in CFU frequency between control (19.3 ± 4.2 CFU/104 cells) and rmWnt5a (24.0 ± 2.6 CFU; n = 3) cultures, indicating that treatment with rmWnt5a inhibited hematopoietic proliferation but not differentiation. To identify the mechanism by which Wnt5a regulates HSC and HPC proliferation, we analyzed potential Wnt5a-mediated signaling pathways. We observed that Wnt5a induced intracellular Ca2+ (iCa2+) flux in HSCs (defined as lin−, Sca-1HI, c-kitHI; LSK). Previous studies have shown that Wnt5a-mediated induction of iCa2+ can result in activation of the NFAT family of transcription factors. Since NFATc1 promotes quiescence of hair follicle stem cells and is expressed in HSCs, we hypothesized that the effects of Wnt5a required activation of NFAT family members. We cultured bone marrow cells with Wnt5a-CM in the presence of cyclosporine A (CsA), which inhibits activation of NFAT factors. In agreement with our earlier findings, we observed that culturing bone marrow cells in Wnt5a-CM increased the percentage of quiescent (defined as Ki-67-) LSKCD34− HSCs (79.7 ± 3.3%) compared to control-CM (55.0 ± 1.6%; n = 3; p < .001). This increase was inhibited by CsA (69.4 ± 2.6%; n = 3; p = .01 compared to Wnt5a-CM alone). In conclusion, our data point to a role for Wnt5a in regulating HSC and HPC proliferation and that this function may require the activation of NFAT transcription factors. Disclosures: No relevant conflicts of interest to declare.
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Miller, Jeffrey S., Valarie McCullar, Michael Punzel, Ihor R. Lemischka und Kateri A. Moore. „Single Adult Human CD34+/Lin−/CD38− Progenitors Give Rise to Natural Killer Cells, B-Lineage Cells, Dendritic Cells, and Myeloid Cells“. Blood 93, Nr. 1 (01.01.1999): 96–106. http://dx.doi.org/10.1182/blood.v93.1.96.
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Abstract Marrow stromal cultures support adult CD34+/Lin−/HLA-DR− or CD34+/Lin−/CD38− cell differentiation into natural killer (NK) or myeloid cells, but unlike committed lymphoid progenitors (CD34+/Lin−/CD45RA+/CD10+), no B cells are generated. We tested whether different microenvironments could establish a developmental link between the NK and B-cell lineages. Progenitors were cultured in limiting dilutions with interleukin-7 (IL-7), flt3 ligand (FL), c-kit ligand (KL), IL-3, IL-2, and AFT024, a murine fetal liver line, which supports culture of transplantable murine stem cells. NK cells, CD10+/CD19+ B-lineage cells and dendritic cells (DC) developed from the same starting population and IL-7, FL, and KL were required in this process. Single cell deposition of 3,872 CD34+/Lin−/CD38− cells onto AFT024 with IL-7, FL, KL, IL-2, and IL-3 showed that a one time addition of IL-3 at culture initiation was essential for multilineage differentiation from single cells. Single and double lineage progeny were frequently detected, but more importantly, 2% of single cells could give rise to at least three lineages (NK cells, B-lineage cells, and DC or myeloid cells) providing direct evidence that NK and B-lineage differentiation derive from a common lymphomyeloid hematopoietic progenitor under the same conditions. This study provides new insights into the role of the microenvironment niche, which governs the earliest events in lymphoid development.
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Miller, Jeffrey S., Valarie McCullar, Michael Punzel, Ihor R. Lemischka und Kateri A. Moore. „Single Adult Human CD34+/Lin−/CD38− Progenitors Give Rise to Natural Killer Cells, B-Lineage Cells, Dendritic Cells, and Myeloid Cells“. Blood 93, Nr. 1 (01.01.1999): 96–106. http://dx.doi.org/10.1182/blood.v93.1.96.401k13_96_106.
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Marrow stromal cultures support adult CD34+/Lin−/HLA-DR− or CD34+/Lin−/CD38− cell differentiation into natural killer (NK) or myeloid cells, but unlike committed lymphoid progenitors (CD34+/Lin−/CD45RA+/CD10+), no B cells are generated. We tested whether different microenvironments could establish a developmental link between the NK and B-cell lineages. Progenitors were cultured in limiting dilutions with interleukin-7 (IL-7), flt3 ligand (FL), c-kit ligand (KL), IL-3, IL-2, and AFT024, a murine fetal liver line, which supports culture of transplantable murine stem cells. NK cells, CD10+/CD19+ B-lineage cells and dendritic cells (DC) developed from the same starting population and IL-7, FL, and KL were required in this process. Single cell deposition of 3,872 CD34+/Lin−/CD38− cells onto AFT024 with IL-7, FL, KL, IL-2, and IL-3 showed that a one time addition of IL-3 at culture initiation was essential for multilineage differentiation from single cells. Single and double lineage progeny were frequently detected, but more importantly, 2% of single cells could give rise to at least three lineages (NK cells, B-lineage cells, and DC or myeloid cells) providing direct evidence that NK and B-lineage differentiation derive from a common lymphomyeloid hematopoietic progenitor under the same conditions. This study provides new insights into the role of the microenvironment niche, which governs the earliest events in lymphoid development.
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Grundt, Inger K., Harald Nyland und S. Mørk. „Protein Kinase C and Phagocytic Activity in Amitriptyline-exposed Primary Cultures of Glial Cells“. Alternatives to Laboratory Animals 21, Nr. 2 (April 1993): 181–86. http://dx.doi.org/10.1177/026119299302100210.
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Factors which can have either an aggravating or a protective effect on glial cell activation, as found in the early stages of multiple sclerosis and other neurological disorders, are not well known. Enzyme analyses and time-lapse video film were used to study the mechanisms underlying glial cell activation as induced by exposure to amitriptyline (AT). When the effects on the two enzymes protein kinase C (PKC) and 2'3’-cyclic nucleotide 3’-phosphodiesterase (CNP) were compared, PKC activity was increased by 49% and CNP activity was not affected. The addition of the essential fatty acids arachidonic acid (Ara) and alpha-linolenic acid (Lin), revealed that Lin alone activated PKC by 59%, and when Lin was co-exposed with AT, by 67%. The activation of astroglial and microglial cells and phagocytosis of oligodendroglial cells in an AT-exposed culture was recorded by video film. Further studies on AT-induced events in primary cultures of glial cells and the modulating effects of fatty acids, are in progress.
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Gupta, Rashmi, Simon Karpatkin und Ross Basch. „Hematopoiesis and Stem Cell Renewal in Long-Term Bone Marrow Cultures Containing Catalase.“ Blood 104, Nr. 11 (16.11.2004): 371. http://dx.doi.org/10.1182/blood.v104.11.371.371.
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Abstract Many of the events that occur within the bone marrow can be modeled in long-term bone marrow cultures (LTBMC), which are capable of producing stem cells. Although the cultures faithfully replicate the differentiation of many hematopoietic lineages, they are relatively short-lived. The stem cell compartment is rapidly depleted and attempts to achieve expansion of hematopoietic cells in culture have met with limited success. These cultures accumulate large numbers of granulocytes and monocytes capable of producing significant levels of reactive oxygen species (ROS). It has recently become clear that some ROS, including H2O2 can play a critical role in intracellular signalling induced by various growth factors and cytokines. We therefore elected to test the effect of 2 different H2O2 scavenger catalases, (bovine or aspergillosis added on alternate days) on LTBMC hematopoiesis of mouse low density bone marrow cells on irradiated adherent preformed stromal monolayers. Dramatic alterations were noted with either catalase, whereas heat-inactivated catalase had no effect. Initially there is a 5–10 fold increase in the non-adherent granulocytes and their precursors. The increase is relatively short-lived at 3–4 weeks when catalase cultures contain 1/5 as many hematopoietic cells as controls. However these cells contain 5 times the number of myeloid clonal progenitors (CFU-c) than controls. After 4–5 weeks the catalase treated cells become quiescent. When catalase is removed hematopoiesis returns promptly, ruling out a catalase-induced toxic effect. By the 3rd week of catalase treatment >90% of non-adherent cells are Sca-1+ and 36% of them are Lin−. In absolute numbers the Sca-1+ and Lin− population increase 80 fold at 3 weeks. If losses induced by removal of half of the non-adherent cells with each weekly feeding are considered, the absolute increase is >500 fold. Virtually all of the Sca-1+, Lin− cells express C-Kit+. At 2–3 weeks, approximately 15% of cells recovered from the catalase cultures have this stem cell phenotype described for murine cells, which represents a 200 fold increase in stem cells compared to controls. These cells (20,000 Ly 5.1 cells) were then tested for their ability to sustain both short- and long-term hematopoiesis in lethally irradiated Ly 5.2 mice along with 30,000 freshly isolated Ly 5.2 bone marrow cells. The catalase-treated cells showed both short- and long-term repopulating activity. At 3,6 and 10 weeks sorted Sca-1+, Lin− catalase-treated Ly 5.1 cells were 14,20 and 39% respectively of host cells, compared to 1,3 and 5% of cells cultured without catalase. These catalase-treated cells underwent multilinege repopulation granulocytes (Gr-1+), monocytes (mac-1+), T-cells (CD3+) and B− cells (B-220+) in the Ly 5.2 host. Thus, peroxide-sensitive regulatory mechanisms play an important role in regulating hematopoietic stem cell renewal and differentiation. Protected from H2O2, hematopoietic progenitors multiply and become quiescent. These cells are 200–500 fold enriched with functional stem cells. Manipulation of peroxide levels in vitro can dramatically enhance the growth of self-renewing hematopoietic stem cells and may provide a unique source of undifferentiated hematopoietic progenitors.
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Rowley, SD, C. Brashem-Stein, R. Andrews und ID Bernstein. „Hematopoietic precursors resistant to treatment with 4- hydroperoxycyclophosphamide: requirement for an interaction with marrow stroma in addition to hematopoietic growth factors for maximal generation of colony-forming activity“. Blood 82, Nr. 1 (01.07.1993): 60–65. http://dx.doi.org/10.1182/blood.v82.1.60.bloodjournal82160.
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We tested the ability of CD34+lin- precursor cells isolated from marrow after treatment with 4-hydroperoxycyclophosphamide (4HC) to generate colony-forming cells (CFC). In liquid cultures, recombinant human stem cell factor (SCF), in combination with interleukin-1 (IL-1), IL-3, IL- 6, granulocyte-macrophage colony-stimulating factor, or granulocyte colony-stimulating factor caused untreated, but not 4HC-treated, CD34+lin- cells to form CFC. However, generation of CFC from CD34+lin- cells treated with 60 micrograms/mL of 4HC was possible in the presence of an irradiated allogeneic stromal cell layer. This generation was increased when combinations of hematopoietic growth factors including SCF and IL-3 were added. Maximal generation of CFC was seen after 11 to 21 days of culture. At that time, generation of CFC from CD34+lin- 4HC- treated cells equalled that from untreated cells. The phenotype of these 4HC-resistant CD34+lin- precursors was also further defined as CD38-. These studies show that the generation of CFC from the 4HC- resistant, highly immature population of CD34+lin- cells requires an as yet undefined interaction with marrow stroma in addition to known hematopoietic growth factors.
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Peled, Tony, Sophie Adi, Iddo Peleg, Noga G. Rosenheimer, Yaron Daniely, Arnon Nagler, Eitan Fibach und Amnon Peled. „Nicotinamide Modulates Ex-Vivo Expansion of Cord Blood Derived CD34+ Cells Cultured with Cytokines and Promotes Their Homing and Engraftment in SCID Mice.“ Blood 108, Nr. 11 (16.11.2006): 725. http://dx.doi.org/10.1182/blood.v108.11.725.725.
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Abstract Nicotinamide (NA) is a non-competitive inhibitor of NAD(+)-dependent ADP-ribosyl transferases, of CD38 NADase (a major regulator of cellular NAD levels) and of Sir2 histone-deacetylase. These enzymes are playing a pivotal role in regulation of signal transduction pathways and gene expression. In the present study, we evaluated the effect of NA on the ex-vivo expansion of cord-blood (CB) derived CD34+ cells and their bone-marrow (BM) homing and engraftment potential. Culturing of CD34+ cells for three weeks in the presence of cytokines (SCF, TPO, IL-6, FLT3-ligand) only or cytokines + NA (5mM) resulted in similar expansion of CD34+ cells (40-fold relative to input). However, a remarkable increase in the fraction of CD34+ cells displaying an early progenitor cell phenotype (CD34+Lin−) was observed in the NA-treated cultures as compared with cytokines-only treated cultures (18.6 ± 3% and 0.7 ± 0.06%, n=6, p<0.05, respectively). Tracking the cell-cycle history by PKH2 staining showed fewer division cycles of CD34+ cells cultured with NA. These results may suggest a direct correlation between the rate of proliferation and expansion of CD34+Lin− cells. NA-treated CD34+ cells express similar levels of CXCR4 but display increased migratory activity in response to CXCL12 over CD34+ cells treated with cytokines only (36 ± 19% and 11 ± 4%, n=4, p<0.05, respectively). In order to test their homing potential, similar number of mononuclear cells (MNC), before or following expansion with or without NA, were labeled with CFSE and transplanted into irradiated NOD/SCID mice. Twenty-four hours later the numbers of human cells (CD45+CFSE+) and human progenitor cells (CD34+CFSE+) in the BM were counted. Homing of CD45+CFSE+ cells was comparable in the three groups tested. However, CD34+CFSE+ cells with BM homing potential were 3-fold more numerous in NA-treated cultures relative to cytokines-treated cultures, and 6-fold more than in non-cultured CB cells (n=14, p<0.05). To evaluate engraftment, SCID mice were transplanted with 3x103, 6x103 and 12x103 non-cultured CD34+ cells or their entire progeny following 3-week expansion with cytokines only or cytokines + NA (n = 63). The frequency of SCID repopulating cells (SRC) was estimated by limiting dilution analysis as 1/ 36,756 (non-cultured), 1/19,982 (cytokines), 1/ 2,620 (NA) (SCID engraftment was considered as ≥0.5% human CD45+ cells). We found that, in correlation with homing, NA-treated cells have a 14- and 7.6-fold more SRC than non-cultured cells or cytokine-treated cells, respectively. The marked increase in SCID engraftment potential following culturing with NA may be attributed to both improved homing of CD34+ cells as well as higher proportion of early progenitor cells within the CD34+ cell compartment. Despite their numerical expansion, progenitor cells generated in cytokine-supplemented cultures have reduced homing and engraftment capacity. Our study demonstrates that NA modulates in-vitro expansion and augments the in-vivo homing and engraftment of CB-derived CD34+ cells cultured with cytokines.
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SUN, Yihe, und Hai WANG. „The Translation Gap in Moment in Peking and its Metaphor of Identities“. International Journal of Linguistics and Translation Studies 4, Nr. 1 (31.01.2023): 1–12. http://dx.doi.org/10.36892/ijlts.v4i1.285.
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Lin Yutang adopted two translation methods at the same time, including foreignizing and domesticating, to demonstrate the names of the female roles in Moment in Peking, forming an obvious translation differences in the text. As the subject of the writing and translation process, Lin Yutang took use of this language difference to suggest the identity symbols behind the characters, implies the social and cultural identities of them. Combining their words and deeds, personalities in the life and their destinies, Lin Yutang tried to reveal the social reality of modern China , showing the fusion and conflict of Chinese and Western cultures in the wave of cultural change, calling for the transformation of social thought so as to realize his literary intentions.
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Vodyanik, Maxim A., James A. Thomson und Igor I. Slukvin. „Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures“. Blood 108, Nr. 6 (15.09.2006): 2095–105. http://dx.doi.org/10.1182/blood-2006-02-003327.
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AbstractDuring hematopoietic differentiation of human embryonic stem cells (hESCs), early hematopoietic progenitors arise along with endothelial cells within the CD34+ population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays, their phenotype has not been defined. Here, using hESC differentiation in coculture with OP9 stromal cells, we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45, persisted on differentiating hematopoietic cells, and reliably separated the hematopoietic CD34+ population from CD34+CD43–CD31+KDR+ endothelial and CD34+CD43–CD31–KDR– mesenchymal cells. Furthermore, we demonstrated that the first-appearing CD34+CD43+CD235a+CD41a+/–CD45– cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34+CD43+CD41a–CD235a–CD45– cells. These cells were negative for lineage-specific markers (Lin–), expressed KDR, VE-cadherin, and CD105 endothelial proteins, and expressed GATA-2, GATA-3, RUNX1, C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34+CD43+CD45–Lin– cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34+CD43+CD45+Lin– cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3highGATA3lowRUNX1lowPU1highMPOhighIL7RAhigh gene expression profile.
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Hess, David A., Krysta D. Levac, Francis N. Karanu, Michael Rosu-Myles, Martin J. White, Lisa Gallacher, Barbara Murdoch et al. „Functional analysis of human hematopoietic repopulating cells mobilized with granulocyte colony-stimulating factor alone versus granulocyte colony-stimulating factor in combination with stem cell factor“. Blood 100, Nr. 3 (01.08.2002): 869–78. http://dx.doi.org/10.1182/blood.v100.3.869.
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Abstract Using in vitro progenitor assays, serum-free in vitro cultures, and the nonobese diabetic/severe combined immune-deficient (NOD/SCID) ecotropic murine virus knockout xenotransplantation model to detect human SCID repopulating cells (SRCs) with multilineage reconstituting function, we have characterized and compared purified subpopulations harvested from the peripheral blood (PB) of patients receiving granulocyte colony-stimulating factor (G-CSF) alone or in combination with stem cell factor (SCF). Mobilized G-CSF plus SCF PB showed a 2-fold increase in total mononuclear cell content and a 5-fold increase in CD34-expressing cells depleted for lineage-marker expression (CD34+Lin−) as compared with patients treated with G-CSF alone. Functionally, G-CSF plus SCF–mobilized CD34+CD38−Lin−cells contained a 2-fold enhancement in progenitor frequency as compared with G-CSF–mobilized subsets. Despite enhanced cellularity and progenitor capacity, G-CSF plus SCF mobilization did not increase the frequency of SRCs as determined by limiting dilution analysis by means of unfractionated PB cells. Purification of SRCs from these sources demonstrated that as few as 1000 CD34+CD38−Lin− cells from G-CSF–mobilized PB contained SRC capacity while G-CSF plus SCF–mobilized CD34+CD38−Lin−cells failed to repopulate at doses up to 500 000 cells. In addition, primitive CD34−CD38−AC133+Lin−cells derived from G-CSF plus SCF–mobilized PB were capable of differentiation into CD34-expressing cells, while the identical subfractions from G-CSF PB were unable to produce CD34+cells in serum-free cultures. Our study defines qualitative and quantitative distinctions among subsets of primitive cells mobilized by means of G-CSF plus SCF versus G-CSF alone, and therefore has implications for the utility of purified repopulating cells from these sources.
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Sinha, Pinaki, und Miskat Ara Akhter Jahan. „Clonal Propagation of Rhynchostylis retusa (Lin.) Blume through in vitro Culture and their Establishment in the Nursery“. Plant Tissue Culture and Biotechnology 22, Nr. 1 (01.07.2012): 1–11. http://dx.doi.org/10.3329/ptcb.v22i1.11242.
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For high frequency regeneration of Rhyncnostylis retusa (Lin.) Blume apical nodal segments were used. Half strength MS + 2% sucrose + 1.5 mg/l BA + 0.5 mg/l NAA + 2 g/l peptone + 10% (v/v) coconut water (CW) + 0.5 g/l activated charcoal (AC) was the best nutrient medium, on which 89% cultures induced 8 microshoots per culture. Subculture of microshoots for further 8 weeks on the same nutrient medium enhanced the number of microshoots up to 95. For further proliferation of microshoots, their development into shoots as well as formation of secondary microshoots from the base of the old ones, the best medium was half strength of MS + 2% sucrose + 2 g/l peptone + 10% (v/v) CW + 0.5 g/l AC + 150 mg/l L-glutamine. Plantlets with roots were obtained in half strength of MS + 2% sucrose + 2 g/l peptone + 10% (v/v) CW + 0.5 g/l AC + 5.0 g/l banana powder, on which cent per cent shoots rooted within eight weeks. The pH of all the categories of cultures were maintained at 5.6 before adding 2.2 g/l gelrite and autoclaving, and the cultures were incubated at 2000 - 3000 lux for 16/8 hrs light/dark at 24 ± 2ºC. Regeneration of plantlets continued due to repeated subculture of microshoots and regenerants were acclimatized and established in the nursery. Plant Tissue Cult. & Biotech. 22(1): 1-11, 2012 (June) DOI: http://dx.doi.org/10.3329/ptcb.v22i1.11242
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Jacobsen, FW, T. Stokke und SE Jacobsen. „Transforming growth factor-beta potently inhibits the viability- promoting activity of stem cell factor and other cytokines and induces apoptosis of primitive murine hematopoietic progenitor cells“. Blood 86, Nr. 8 (15.10.1995): 2957–66. http://dx.doi.org/10.1182/blood.v86.8.2957.2957.
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Abstract In contrast with the extensively characterized effects of transforming growth factor-beta (TGF-beta) on proliferation and differentiation of hematopoietic progenitors, little is known about the effects of TGF-beta on viability of normal hematopoietic progenitors. In the present report, we demonstrate that TGF-beta potently counteracts hematopoietic growth factor (HGF)-induced survival of individually cultured primitive Lin-Sca-1+ bone marrow progenitors. Specifically, 74% of single Lin-Sca- 1+ cells cultured for 40 hours in the presence of stem cell factor (SCF) survived, whereas only 16% survived in the presence of SCF plus TGF-beta. Similarly, the enhanced survival of primitive hematopoietic progenitors in response to granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-1, IL-6, or IL-11 was also potently opposed by TGF-beta. Furthermore, it is demonstrated that neutralization of endogenous TGF-beta present in the cultures enhances survival of Lin- Sca-1+ progenitors in the absence, as well as in the presence, of HGFs such as SCF and IL-6. The reduced HGF-induced survival of primitive hematopoietic progenitors in the presence of TGF-beta was associated with increased apoptosis, as detected by an in situ terminal deoxynucleotidyl transferase (TdT) assay. After 16 hours of incubation in the absence of HGFs, 61% +/- 6% of the hematopoietic progenitors had DNA strand breaks characteristic of apoptosis. The presence of SCF reduced the frequency of apoptic cells to 27% +/- 5%, whereas 55% +/- 3% of the cells had signs of apoptosis in the presence of SCF plus TGF- beta.
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Jacobsen, FW, T. Stokke und SE Jacobsen. „Transforming growth factor-beta potently inhibits the viability- promoting activity of stem cell factor and other cytokines and induces apoptosis of primitive murine hematopoietic progenitor cells“. Blood 86, Nr. 8 (15.10.1995): 2957–66. http://dx.doi.org/10.1182/blood.v86.8.2957.bloodjournal8682957.
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In contrast with the extensively characterized effects of transforming growth factor-beta (TGF-beta) on proliferation and differentiation of hematopoietic progenitors, little is known about the effects of TGF-beta on viability of normal hematopoietic progenitors. In the present report, we demonstrate that TGF-beta potently counteracts hematopoietic growth factor (HGF)-induced survival of individually cultured primitive Lin-Sca-1+ bone marrow progenitors. Specifically, 74% of single Lin-Sca- 1+ cells cultured for 40 hours in the presence of stem cell factor (SCF) survived, whereas only 16% survived in the presence of SCF plus TGF-beta. Similarly, the enhanced survival of primitive hematopoietic progenitors in response to granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-1, IL-6, or IL-11 was also potently opposed by TGF-beta. Furthermore, it is demonstrated that neutralization of endogenous TGF-beta present in the cultures enhances survival of Lin- Sca-1+ progenitors in the absence, as well as in the presence, of HGFs such as SCF and IL-6. The reduced HGF-induced survival of primitive hematopoietic progenitors in the presence of TGF-beta was associated with increased apoptosis, as detected by an in situ terminal deoxynucleotidyl transferase (TdT) assay. After 16 hours of incubation in the absence of HGFs, 61% +/- 6% of the hematopoietic progenitors had DNA strand breaks characteristic of apoptosis. The presence of SCF reduced the frequency of apoptic cells to 27% +/- 5%, whereas 55% +/- 3% of the cells had signs of apoptosis in the presence of SCF plus TGF- beta.
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Purton, Louise E., Irwin D. Bernstein und Steven J. Collins. „All-trans retinoic acid enhances the long-term repopulating activity of cultured hematopoietic stem cells“. Blood 95, Nr. 2 (15.01.2000): 470–77. http://dx.doi.org/10.1182/blood.v95.2.470.
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The retinoic acid receptor (RAR) agonist, all-trans retinoic acid (ATRA), is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. We previously reported that pharmacologic levels (1 μmol) of ATRA enhanced the generation of colony-forming cell (CFC) and colony-forming unit-spleen (CFU-S) in liquid suspension cultures of lin− c-kit+ Sca-1+ murine hematopoietic precursors. In this study, we further investigated the effects of ATRA as well as an RAR antagonist, AGN 193109, on the generation of transplantable cells, including pre–CFU-S, short-term repopulating stem cells (STRCs), and long-term repopulating stem cells (LTRCs). ATRA enhanced the ex vivo maintenance and production of competitive repopulating STRCs and LTRCs from lin− c-kit+ Sca-1+ cells cultured in liquid suspension for 14 days. In addition, ATRA prevented the differentiation of these primitive stem cells into more mature pre–CFU-S during the 14 days of culture. In marked contrast, lin− c-kit+ Sca-1+ cells cultured with AGN 193109 for 7 days had virtually no short- or long-term repopulating ability, but displayed an approximately 6-fold increase in the pre–CFU-S population. The data suggest that the RAR agonist ATRA enhances the maintenance and self-renewal of short- and long-term repopulating stem cells. In contrast, the RAR antagonist AGN 193109 abrogates reconstituting ability, most likely by promoting the differentiation of the primitive stem cells. These results imply an important and unexpected role of retinoids in regulating hematopoietic stem cell differentiation.