Rac1 is essential for intraembryonic hematopoiesis and for the initial seeding of fetal liver with definitive hematopoietic progenitor cells.
Definitive hematopoietic stem and progenitor cells (HSCs/Ps) originating from the yolk sac and/or para-aorta-splanchno-pleura/aorta-gonad-mesonephros are hypothesized to colonize the fetal liver,but mechanisms involved are poorly defined. The Rac subfamily of Rho GTPases has been shown to play essential roles in HSC/P localization to the bone marrow following transplantation. Here,we study the role of Rac1 in HSC/P migration during ontogeny and seeding of fetal liver. Using a triple-transgenic approach,we have deleted Rac1 in HSCs/Ps during very early embryonic development. Without Rac1,there was a decrease in circulating HSCs/Ps in the blood of embryonic day (E) 10.5 embryos,while yolk sac definitive hematopoiesis was quantitatively normal. Intraembryonic hematopoiesis was significantly impaired in Rac1-deficient embryos,culminating with absence of intra-aortic clusters and fetal liver hematopoiesis. At E10.5,Rac1-deficient HSCs/Ps displayed decreased transwell migration and impaired inter-action with the microenvironment in migration-dependent assays. These data suggest that Rac1 plays an important role in HSC/P migration during embryonic development and is essential for the emergence of intraembryonic hematopoiesis.
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产品类型：
产品号#：
03134
09600
09650
产品名：
MethoCult™M3134
StemSpan™ SFEM
StemSpan™ SFEM
Heckl D et al. (APR 2011)
Blood 117 14 3737--47
Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia.
Thpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet,it is unknown whether this loss of HSCs is an irreversible process. In this study,we used the Mpl knockout (Mpl(-/-)) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations,namely,HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors,we performed bone marrow transplantation of transduced Mpl(-/-) bone marrow cells into Mpl(-/-) mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression,replenishes the HSC pool,restores stem cell properties,and corrects platelet production. In some mice,megakaryocyte counts were atypically high,accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl(-/-) cells had increased long-term repopulating potential,with a marked increase in lineage(-)Sca1(+)cKit(+) cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage(-)Sca1(+)cKit(+) cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.
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产品类型：
产品号#：
09600
09650
产品名：
StemSpan™ SFEM
StemSpan™ SFEM
M. Tarunina et al. ( 2016)
Stem cells and development 25 22 1709--1720
A Novel High-Throughput Screening Platform Reveals an Optimized Cytokine Formulation for Human Hematopoietic Progenitor Cell Expansion.
The main limitations of hematopoietic cord blood (CB) transplantation,viz,low cell dosage and delayed reconstitution,can be overcome by ex vivo expansion. CB expansion under conventional culture causes rapid cell differentiation and depletion of hematopoietic stem and progenitor cells (HSPCs) responsible for engraftment. In this study,we use combinatorial cell culture technology (CombiCult{\textregistered}) to identify medium formulations that promote CD133+ CB HSPC proliferation while maintaining their phenotypic characteristics. We employed second-generation CombiCult screens that use electrospraying technology to encapsulate CB cells in alginate beads. Our results suggest that not only the combination but also the order of addition of individual components has a profound influence on expansion of specific HSPC populations. Top protocols identified by the CombiCult screen were used to culture human CD133+ CB HSPCs on nanofiber scaffolds and validate the expansion of the phenotypically defined CD34+CD38lo/-CD45RA-CD90+CD49f+ population of hematopoietic stem cells and their differentiation into defined progeny.
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Silencing p21(Waf1/Cip1/Sdi1) expression increases gene transduction efficiency in primitive human hematopoietic cells.
Adult hematopoietic and other tissue stem cells have highly constrained cell cycling that limits their susceptibility to standard gene therapy vectors,which depend upon chromosomal integration. Using cytokine cocktails to increase transduction efficiency often compromises subsequent stem cell function in vivo. We previously showed that p21(Waf1/Cip1/Sdi1) (p21) mediates stem cell quiescence in vivo and decreasing its expression ex vivo leads to an expansion of stem cell pool in vivo. Here,we report that application of p21 specific siRNA increased the gene transduction efficiency in hematopoietic stem cells while preserving cell multipotentiality. Both types of siRNA,synthesized siRNA and transcribed shRNA,reduced p21 expression in target cells by 85-98%. The effect of RNAi in these cells was transient and the level of p21 mRNA returned to base line 14-28 days after siRNA treatment. This brief interval of reduction,however,was sufficient to increase transduction efficiency to two- to four-fold in cell cultures,and followed by a seven- to eight-fold increase in mice. The RNAi treated,lentivector-transduced CD34+ cells retained multipotentiality as assessed in vitro by colony formation assay and in vivo by NOD/SCID mouse transplantation assay. Reduction of p21 resulted in an increased chromosomal integration of lentivector into target cellular DNA. Taken together,both synthesized and transcribed siRNA knocked down p21 expression in human CD34+ hematopoietic stem/progenitor cells. Silencing p21 expression increased gene transduction efficiency and vector integration while retaining stem cell multipotentiality. Thus,RNAi targeting of p21 is a useful strategy to increase stem cell gene transfer efficiency. Decreasing p21 expression transiently while increasing gene-transfer vector integration may ultimately facilitate clinical applications of gene therapy.
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产品类型：
产品号#：
09850
产品名：
Leung CG et al. (JUL 2007)
The Journal of experimental medicine 204 7 1603--11
Requirements for survivin in terminal differentiation of erythroid cells and maintenance of hematopoietic stem and progenitor cells.
Survivin,which is the smallest member of the inhibitor of apoptosis protein (IAP) family,is a chromosomal passenger protein that mediates the spindle assembly checkpoint and cytokinesis,and also functions as an inhibitor of apoptosis. Frequently overexpressed in human cancers and not expressed in most adult tissues,survivin has been proposed as an attractive target for anticancer therapies and,in some cases,has even been touted as a cancer-specific gene. Survivin is,however,expressed in proliferating adult cells,including human hematopoietic stem cells,T-lymphocytes,and erythroid cells throughout their maturation. Therefore,it is unclear how survivin-targeted anticancer therapies would impact steady-state blood development. To address this question,we used a conditional gene-targeting strategy and abolished survivin expression from the hematopoietic compartment of mice. We show that inducible deletion of survivin leads to ablation of the bone marrow,with widespread loss of hematopoietic progenitors and rapid mortality. Surprisingly,heterozygous deletion of survivin causes defects in erythropoiesis in a subset of the animals,with a dramatic reduction in enucleated erythrocytes and the presence of immature megaloblastic erythroblasts. Our studies demonstrate that survivin is essential for steady-state hematopoiesis and survival of the adult,and further,that a high level of survivin expression is critical for proper erythroid differentiation.
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Zhang L et al. (APR 2016)
Human Reproduction 31 4 832--843
Protein kinase A inhibitor, H89, enhances survival and clonogenicity of dissociated human embryonic stem cells through Rho-associated coiled-coil containing protein kinase (ROCK) inhibition
H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK),myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1),significantly increases cell survival and colony formation,and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro.
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Valamehr B et al. (SEP 2011)
Regenerative medicine 6 5 623--34
Developing defined culture systems for human pluripotent stem cells.
Human pluripotent stem cells hold promising potential in many therapeutics applications including regenerative medicine and drug discovery. Over the past three decades,embryonic stem cell research has illustrated that embryonic stem cells possess two important and distinct properties: the ability to continuously self-renew and the ability to differentiate into all specialized cell types. In this article,we will discuss the continuing evolution of human pluripotent stem cell culture by examining requirements needed for the maintenance of self-renewal in vitro. We will also elaborate on the future direction of the field toward generating a robust and completely defined culture system,which has brought forth collaborations amongst biologists and engineers. As human pluripotent stem cell research progresses towards identifying solutions for debilitating diseases,it will be critical to establish a defined,reproducible and scalable culture system to meet the requirements of these clinical applications.
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产品类型：
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Kim H-R et al. ( 2016)
Cell & bioscience 6 1 50
Improved hematopoietic differentiation of human pluripotent stem cells via estrogen receptor signaling pathway.
BACKGROUND Aside from its importance in reproduction,estrogen (E2) is known to regulate the proliferation and differentiation of hematopoietic stem cells in rodents. However,the regulatory role of E2 in human hematopoietic system has not been investigated. The purpose of this study is to investigate the effect of E2 on hematopoietic differentiation using human pluripotent stem cells (hPSCs). RESULTS E2 improved hematopoietic differentiation of hPSCs via estrogen receptor alpha (ER-$$)-dependent pathway. During hematopoietic differentiation of hPSCs,ER-$$ is persistently maintained and hematopoietic phenotypes (CD34 and CD45) were exclusively detected in ER-$$ positive cells. Interestingly,continuous E2 signaling is required to promote hematopoietic output from hPSCs. Supplementation of E2 or an ER-$$ selective agonist significantly increased the number of hemangioblasts and hematopoietic progenitors,and subsequent erythropoiesis,whereas ER-$$ selective agonist did not. Furthermore,ICI 182,780 (ER antagonist) completely abrogated the E2-induced hematopoietic augmentation. Not only from hPSCs but also from human umbilical cord bloods,does E2 signaling potentiate hematopoietic development,suggesting universal function of E2 on hematopoiesis. CONCLUSIONS Our study identifies E2 as positive regulator of human hematopoiesis and suggests that endocrine factors such as E2 influence the behavior of hematopoietic stem cells in various physiological conditions.
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产品类型：
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Kobune M et al. (AUG 2004)
Blood 104 4 1002--9
Indian hedgehog gene transfer augments hematopoietic support of human stromal cells including NOD/SCID-beta2m-/- repopulating cells.
Hematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of giving rise to all types of blood cells. However,the mechanisms involved in controlling the number and abilities of HSCs remain largely unknown. The Indian hedgehog (Ihh) signal has an essential role in inducing hematopoietic tissue during embryogenesis. We investigated the roles of the Ihh in coculture with CD34+ cells and human stromal cells. Ihh mRNA was expressed in primary and telomerized human (hTERT) stromal cells,and its receptor molecules were detected in CD34+ cells. Ihh gene transfer into hTERT stromal cells enhanced their hematopoietic supporting potential,which was elevated compared with control stromal cells,as indicated by the colony-forming units in culture (CFU-Cs) (26-fold +/- 2-fold versus 59-fold +/- 3-fold of the initial cell number; mixed colony-forming units [CFU-Mix's],63-fold +/- 37-fold versus 349-fold +/- 116-fold). Engraftments of nonobese diabetic/severe combined immunodeficiency-beta2m-/- (NOD/SCID-beta2-/-) repopulating cells (RCs) expanded on Ihh stromal cells were significantly higher compared with control coculture results,and engraftment was neutralized by addition of an antihedgehog antibody. Limiting dilution analysis indicated that NOD/SCID-beta2m-/- RCs proliferated efficiently on Ihh stromal cells,compared with control stromal cells. These results indicate that Ihh gene transfer could enhance the primitive hematopoietic support ability of human stromal cells.
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EasySep™小鼠TIL（CD45）正选试剂盒
产品手册STEMvision™ Automated and Standardized Counting of CFU Assays of Human Hematopoietic Cells
产品手册TeSR™-E8™: Feeder-Free Culture Medium for Human Pluripotent Stem Cells
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