Sengupta A et al. (JUN 2011)
Proceedings of the National Academy of Sciences of the United States of America 108 24 9957--62
Atypical protein kinase C (aPKCzeta and aPKClambda) is dispensable for mammalian hematopoietic stem cell activity and blood formation.
The stem-cell pool is considered to be maintained by a balance between symmetric and asymmetric division of stem cells. The cell polarity model proposes that the facultative use of symmetric and asymmetric cell division is orchestrated by a polarity complex consisting of partitioning-defective proteins Par3 and Par6,and atypical protein kinase C (aPKCζ and aPKCλ),which regulates planar symmetry of dividing stem cells with respect to the signaling microenvironment. However,the role of the polarity complex is unexplored in mammalian adult stem-cell functions. Here we report that,in contrast to accepted paradigms,polarization and activity of adult hematopoietic stem cell (HSC) do not depend on either aPKCζ or aPKCλ or both in vivo. Mice,having constitutive and hematopoietic-specific (Vav1-Cre) deletion of aPKCζ and aPKCλ,respectively,have normal hematopoiesis,including normal HSC self-renewal,engraftment,differentiation,and interaction with the bone marrow microenvironment. Furthermore,inducible complete deletion of aPKCλ (Mx1-Cre) in aPKCζ(-/-) HSC does not affect HSC polarization,self-renewal,engraftment,or lineage repopulation. In addition,aPKCζ- and aPKCλ-deficient HSCs elicited a normal pattern of hematopoietic recovery secondary to myeloablative stress. Taken together,the expression of aPKCζ,aPKCλ,or both are dispensable for primitive and adult HSC fate determination in steady-state and stress hematopoiesis,contrary to the hypothesis of a unique,evolutionary conserved aPKCζ/λ-directed cell polarity signaling mechanism in mammalian HSC fate determination.
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Zhou H et al. (SEP 2010)
The Journal of biological chemistry 285 39 29676--80
Conversion of mouse epiblast stem cells to an earlier pluripotency state by small molecules.
Epiblast stem cells (EpiSCs) are pluripotent cells derived from post-implantation late epiblasts in vitro. EpiSCs are incapable of contributing to chimerism,indicating that EpiSCs are less pluripotent and represent a later developmental pluripotency state compared with inner cell mass stage murine embryonic stem cells (mESCs). Using a chemical approach,we found that blockage of the TGFβ pathway or inhibition of histone demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes in EpiSCs toward mESC phenotypes with simultaneous activation of inner cell mass-specific gene expression. However,full conversion of EpiSCs to the mESC-like state with chimerism competence could be readily generated only with the combination of LSD1,ALK5,MEK,FGFR,and GSK3 inhibitors. Our results demonstrate that appropriate synergy of epigenetic and signaling modulations could convert cells at the later developmental pluripotency state to the earlier mESC-like pluripotency state,providing new insights into pluripotency regulation.
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Canonical Wnt/β-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here,we show that SB-216763,a glycogen synthase kinase-3 (GSK3) inhibitor,can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4,Sox2,and Nanog. Furthermore,Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers,and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge,SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months.
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产品类型：
产品号#：
72872
72874
产品名：
SB216763
Lu M et al. (AUG 2009)
Experimental hematology 37 8 924--36
Enhanced generation of hematopoietic cells from human hepatocarcinoma cell-stimulated human embryonic and induced pluripotent stem cells
Objective: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) constitute unique sources of pluripotent cells,although the molecular mechanisms involved in their differentiation into specific lineages are just beginning to be defined. Here we evaluated the ability of MEDII (medium conditioned by HepG2 cells,a human hepatocarcinoma cell line) to selectively enhance generation of mesodermal derivatives,including hematopoietic cells,from hESCs and hiPSCs. Materials and Methods: Test cells were exposed to MEDII prior to being placed in conditions that promote embryoid body (EB) formation. Hematopoietic activity was measured by clonogenic assays,flow cytometry,quantitative real-time polymerase chain reaction of specific transcript complementary DNAs and the ability of cells to repopulate sublethally irradiated nonobese diabetic/severe combined immunodeficient interleukin-2 receptor ??-chain-null mice for almost 1 year. Results: Exposure of both hESCs and hiPSCs to MEDII induced a rapid and preferential differentiation of hESCs into mesodermal elements. Subsequently produced EBs showed a further enhanced expression of transcripts characteristic of multiple mesodermal lineages,and a concurrent decrease in endodermal and ectodermal cell transcripts. Frequency of all types of clonogenic hematopoietic progenitors in subsequently derived EBs was also increased. In vivo assays of MEDII-treated hESC-derived EBs also showed they contained cells able to undertake low-level but longterm multilineage repopulation of primary and secondary nonobese diabetic/severe combined immunodeficient interleukin-2 receptor ??-chain-null mice. Conclusions: MEDII treatment of hESCs and hiPSCs alike selectively enhances their differentiation into mesodermal cells and allows subsequent generation of detectable levels of hematopoietic progenitors with in vitro and in vivo differentiating activity. ?? 2009 ISEH - Society for Hematology and Stem Cells.
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产品类型：
产品号#：
04230
05850
05857
05870
05875
36254
85850
85857
85870
85875
产品名：
MethoCult™ H4230
DMEM/F-12 with 15 mM HEPES
mTeSR™1
mTeSR™1
Kang S et al. (APR 2009)
Molecular and cellular biology 29 8 2105--17
Fibroblast growth factor receptor 3 associates with and tyrosine phosphorylates p90 RSK2, leading to RSK2 activation that mediates hematopoietic transformation.
Dysregulation of the receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) plays a pathogenic role in a number of human hematopoietic malignancies and solid tumors. These include t(4;14) multiple myeloma associated with ectopic expression of FGFR3 and t(4;12)(p16;p13) acute myeloid leukemia associated with expression of a constitutively activated fusion tyrosine kinase,TEL-FGFR3. We recently reported that FGFR3 directly tyrosine phosphorylates RSK2 at Y529,which consequently regulates RSK2 activation. Here we identified Y707 as an additional tyrosine in RSK2 that is phosphorylated by FGFR3. Phosphorylation at Y707 contributes to RSK2 activation,through a putative disruption of the autoinhibitory alphaL-helix on the C terminus of RSK2,unlike Y529 phosphorylation,which facilitates ERK binding. Moreover,we found that FGFR3 interacts with RSK2 through residue W332 in the linker region of RSK2 and that this association is required for FGFR3-dependent phosphorylation of RSK2 at Y529 and Y707,as well as the subsequent RSK2 activation. Furthermore,in a murine bone marrow transplant assay,genetic deficiency in RSK2 resulted in a significantly delayed and attenuated myeloproliferative syndrome induced by TEL-FGFR3 as compared with wild-type cells,suggesting a critical role of RSK2 in FGFR3-induced hematopoietic transformation. Our current and previous findings represent a paradigm for tyrosine phosphorylation-dependent regulation of serine-threonine kinases.
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Optimization of seeding density of OP9 cells to improve hematopoietic differentiation efficiency
BackgroundOP9 mouse stromal cell line has been widely used to induce differentiation of human embryonic stem cells (hESCs) into hematopoietic stem/progenitor cells (HSPCs). However,the whole co-culture procedure usually needs 14–18 days,including preparing OP9 cells at least 4 days. Therefore,the inefficient differentiation system is not appreciated. We aimed to optimize the culture conditions to improve differentiation efficiency.MethodsIn the experimental group,we set six different densities of OP9 cells and just cultured them for 24 h before co-culture,and in the control group,OP9 cells were cultured for 4 days to reach an overgrown state before co-culture. Then we compared the hematopoietic differentiation efficiency among them.ResultsOP9 cells were randomly assigned into two groups. In the experimental group,six different plated numbers of OP9 cells were cultured for 1 day before co-culture with hESCs. In contrast,in the control group,OP9 cells were cultured for 4 days at a total number of 3.1 × 104 cells/cm2 in a 6-well plate to reach an overgrown state before co-culture. Hematopoietic differentiation was evaluated with CD34 immunostaining,and compared between these two groups. We could not influence the differentiation efficiency of OP9 cells with a total number of 10.4 × 104 cells/cm2 in a 6-well plate which was cultured just for 1 day,followed by co-culture with hESCs. It reached the same differentiation efficiency 5 days earlier than the control group.ConclusionThe peak of CD34 + cells appeared 2 days earlier compared to the control group. A total number of 1.0 × 106 cells in a 6-well plate for OP9 cells was appropriate to have high differentiation efficiency.
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产品类型：
产品号#：
85850
85857
产品名：
mTeSR™1
mTeSR™1
M. C. Czarnog\'orski et al. (nov 2022)
Immunity & ageing : I & A 19 1 51
Ageing-resembling phenotype of long-term allogeneic hematopoietic cells recipients compared to their donors.
BACKGROUND Ageing is a complex phenomenon that leads to decreased proliferative activity,loss of function of the cells,and cellular senescence. Senescence of the immune system exacerbates individual's immune response,both humoral and cellular but increases the frequency of infections. We hypothesized that physiological ageing of adaptive immune system occurs in recipients of allogeneic hematopoietic cells transplant (allo-HCT) at faster rate when compared to their respective donors since the small number of donor cells undergo immense proliferative stress restoring recipients hematopoiesis. We compared molecular characterizations of ageing between recipients and donors of allo-HCT: telomeric length and immunophenotypic changes in main lymphocyte subsets - CD4+,CD8+,CD19+,CD56+. RESULTS Median telomeric length (TL) of CD8+ lymphocytes was significantly longer in donors compared to recipients (on average 2,1 kb and 1,7 kb respectively,p??=??0,02). Similar trends were observed for CD4+ and CD19+ although the results did not reach statistical significance. We have also found trends in the immunophenotype between recipients and donors in the subpopulations of CD4+ (na{\{i}}ve and effector memory) CD8+ Eomes+ and B-lymphocytes (B1 and B2). Lower infection risk recipients had also a significantly greater percentage of NK cells (22 3%) than high-risk patients (9 3%) p??=??0 04. CONCLUSION Our data do not support the initial hypothesis of accelerated aging in the long term all-HCT recipients with the exception of the recipients lymphocytes (mainly CD8+) which present some molecular features characteristic for physiological ageing (telomeric shortening immunophenotype) when compared to their respective donors. However a history of lower infection numbers in HCT recipients seems to be associated with increased percentage of NK cells. The history of GVHD seems not to affect the rate of ageing. Therefore it is safe to conclude that the observed subtle differences between recipients' and donors' cells result mainly from the proliferative stress in the early period after allo-HCT and the difference between hosts' and recipients' microenvironments."
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产品类型：
产品号#：
19655
19655RF
产品名：
EasySep™ Direct人总淋巴细胞分选试剂盒
RoboSep™ Direct人总淋巴细胞分选试剂盒
D. M. Gravano et al. (DEC 2016)
Journal of autoimmunity 75 58--67
CD8+ T cells drive autoimmune hematopoietic stem cell dysfunction and bone marrow failure.
Bone marrow (BM) failure syndrome encompasses a group of disorders characterized by BM stem cell dysfunction,resulting in varying degrees of hypoplasia and blood pancytopenia,and in many patients is autoimmune and inflammatory in nature. The important role of T helper 1 (Th1) polarized CD4+ T cells in driving BM failure has been clearly established in several models. However,animal model data demonstrating a functional role for CD8+ T cells in BM dysfunction is largely lacking and our objective was to test the hypothesis that CD8+ T cells play a non-redundant role in driving BM failure. Clinical evidence implicates a detrimental role for CD8+ T cells in BM failure and a beneficial role for Foxp3+ regulatory T cells (Tregs) in maintaining immune tolerance in the BM. We demonstrate that IL-2-deficient mice,which have a deficit in functional Tregs,develop spontaneous BM failure. Furthermore,we demonstrate a critical role for CD8+ T cells in the development of BM failure,which is dependent on the cytokine,IFNgamma$. CD8+ T cells promote hematopoietic stem cell dysfunction and depletion of myeloid lineage progenitor cells,resulting in anemia. Adoptive transfer experiments demonstrate that CD8+ T cells dramatically expedite disease progression and promote CD4+ T cell accumulation in the BM. Thus,BM dysregulation in IL-2-deficient mice is mediated by a Th1 and IFNgamma$-producing CD8+ T cell (Tc1) response.
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产品类型：
产品号#：
18556
18556RF
产品名：
Christ O et al. (SEP 2007)
Haematologica 92 9 1165--72
Improved purification of hematopoietic stem cells based on their elevated aldehyde dehydrogenase activity.
BACKGROUND AND OBJECTIVES: Primitive human hematopoietic cells contain higher levels of aldehyde dehydrogenase (ALDH) activity than their terminally differentiating progeny but the particular stages when ALDH levels change have not been well defined. The objective of this study was to compare ALDH levels among the earliest stages of hematopoietic cell differentiation and to determine whether these could be exploited to obtain improved purity of human cord blood cells with long-term lympho-myeloid repopulating activity in vivo. DESIGN AND METHODS: ALDEFLUOR-stained human cord blood cells displaying different levels of ALDH activity were first analyzed for co-expression of various surface markers. Subsets of these cells were then isolated by multi-parameter flow cytometry and assessed for short-and long-term repopulating activity in sublethally irradiated immunodeficient mice. RESULTS: Most short-term myeloid repopulating cells (STRC-M) and all long-term lympho-myeloid repopulating cells (LTRC-ML) stained selectively as ALDH+. Limiting dilution analysis of the frequencies of both STRC-M and LTRC-ML showed that they were similarly and most highly enriched in the 10% top ALDH+ cells. Removal of cells expressing CD2,CD3,CD7,CD14,CD16,CD24,CD36,CD38,CD56,CD66b,or glycophorin A from the ALDH+ low-density fraction of human cord blood cells with low light side-scattering properties yielded a population containing LTRC-ML at a frequency of 1/360. INTERPRETATION AND CONCLUSION: Elevated ALDH activity is a broadly inclusive property of primitive human cord blood cells that,in combination with other markers,allows easy isolation of the stem cell fraction at unprecedented purities.
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EasySep™小鼠TIL（CD45）正选试剂盒
科学海报Immunomagnetic Cell Isolation of Highly Purified Mouse Naïve CD4+ T Cells in 15 Minutes
科学海报Fully Automated Magnetic Labeling and Separation of Hematopoietic Cells from Multiple Samples
产品手册STEMvision™ Automated and Standardized Counting of CFU Assays of Human Hematopoietic Cells
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