Successful hematopoietic stem cell (HSC) transplantation is often limited by the numbers of HSCs, and robust methods to expand HSCs ex vivo are needed. We previously showed that angiopoietin-like proteins (Angptls), a group of growth factors isolated from a fetal liver HSC-supportive cell population, improved ex vivo expansion of HSCs. Here, we demonstrate that insulin-like growth factor-binding protein 2 (IGFBP2), secreted by a tumorigenic cell line, also enhanced ex vivo expansion of mouse HSCs. On the basis of these findings, we established a completely defined, serumfree culture system for mouse HSCs, containing SCF, thrombopoietin, fibroblast growth factor 1, Angptl3, and IGFBP2. As measured by competitive repopulation analyses, there was a 48-fold increase in numbers of long-term repopulating mouse HSCs after 21 days of culture. This is the first demonstration that IGFBP2 stimulates expansion or proliferation of murine stem cells. Our finding also suggests that certain cancer cells synthesize proteins that can stimulate HSC expansion.
The role of IGF binding protein 2 (IGFBP2) in cell growth is intriguing and largely undefined. Previously we identified IGFBP2 as an extrinsic factor that supports ex vivo expansion of hematopoietic stem cells (HSCs). Here we showed that IGFBP2-null mice have fewer HSCs than wild-type mice. While IGFBP2 has little cell-autonomous effect on HSC function, we found decreased in vivo repopulation of HSCs in primary and secondary transplanted IGFBP2-null recipients. Importantly, bone marrow stromal cells that are deficient for IGFBP2 have significantly decreased ability to support the expansion of repopulating HSCs. To investigate the mechanism by which IGFBP2 supports HSC activity, we demonstrated that HSCs in IGFBP2-null mice had decreased survival and cycling, down-regulated expression of antiapoptotic factor Bcl-2, and up-regulated expression of cell cycle inhibitors p21, p16, p19, p57, and PTEN. Moreover, we found that the C-terminus, IntroductionThe number of hematopoietic stem cells (HSCs) is determined by the balance among different cell fates-self-renewal, differentiation, apoptosis, and migration-which are regulated by the intrinsic factors and environmental cues in vivo or in vitro. 1,2 We have identified several growth factors and secreted proteins that support the repopulation of HSCs and have developed an efficient serumfree system to support ex vivo expansion of mouse and human HSCs. [3][4][5] Insulin-like growth factor binding protein 2 (IGFBP2) is one of these secreted proteins; we isolated IGFBP2 from a cancer line that supports ex vivo expansion of HSCs. 6,7 IGFBP2 is a member of the IGFBP family that is found in all vertebrates; it modulates the biologic effects of IGFs by controlling the distribution, function, and activity of IGF-1 and IGF-2. 8 IGFBP2 is expressed in the fetus and in several adult tissues and biologic fluids. It is also overexpressed in many tumors and in some cases its expression level correlates with grade of malignancy. [9][10][11] The level of IGFBP2 appears to be low in well-differentiated tumors but high in poorly differentiated tumors. 12 The known functions of IGFBP2 are very interesting. IGFBP2 displays IGF-dependent inhibitory effects on normal somatic cell growth. However, several studies demonstrated that IGFBP2 has intrinsic bioactivities that are independent of IGF-1 or IGF-2. IGFBP2 stimulates proliferation, survival, differentiation, and motility of various types of cells. 9,[13][14][15][16][17][18][19][20] Multiple mechanisms for these IGF-independent actions of IGFBP2 have been proposed. One line of studies supported the concept that intracellular IGFBP2 binds integrin and supports cell survival. 13 A second line of studies suggested that IGFBP2 acts as secreted proteins and binds to cell surface receptors. For example, when bound to the cell surface integrin, extrinsic IGFBP2 influences cell mobility and proliferation. [9][10][11]21 IGFBP2 also binds to Frizzled 8 and LDL receptorrelated protein 6 and is proposed to antagonize Wnt signaling in heart cells. 22...
Natural killer (NK) cells belong to an important lymphocyte population that eliminates transformed cells and invading pathogens without any prior sensitization. NK cells possess not only natural killing activity against non-self and altered-self cells but also exhibit cytokine production and antibody-dependent cell-mediated cytotoxicity (ADCC). Despite their important roles in the innate immune system, little is known about the details of NK cell biology. In spite of that several murine NK cell clones have been established, studies have mainly focused on their natural killing activity but not their cytokine production or ADCC. In this study, we established and characterized eight novel, immortalized murine NK cell clones derived from a temperature-sensitive SV40 large-T antigen transgenic mouse. These NK cell lines continuously proliferated for more than 30 months in a culture medium supplemented with interleukin 2. All cell lines contained azurophilic granules in the cytoplasm, and a few clones retained the NK cell functions, such as natural killing activity, cytokine production, and ADCC. In addition, one clone could serve as a host for transient as well as stable gene transfection. Taken together, these findings indicate that the cell lines could constitute useful tools for detailed analysis of murine NK cell biology.
An intermittent supply of ozonated water to underground roots revealed a promotive effect on the growth of komatsuna (Brassica rapa var. perviridis). Although no clear d after germination. A remarkable difference was observed in growth rate after 49 d, whereas growth rate in the control was nearly saturated. Plant weight after 49 d increased by 2-3 times, compared to that of the control. An intermittent supply of a suitable amount of ozonated water to the underground soil increased plant growth promotion.
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