搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Xenopus in vitro studies have implicated both transforming growth factor beta (TGF-beta) and fibroblast growth factor (FGF) families in mesoderm induction. Although members of both families are present during mouse mesoderm formation,there is little evidence for their functional role in mesoderm induction. We show that mouse embryonic stem cells,which resemble primitive ectoderm,can differentiate to mesoderm in vitro in a chemically defined medium (CDM) in the absence of fetal bovine serum. In CDM,this differentiation is responsive to TGF-beta family members in a concentration-dependent manner,with activin A mediating the formation of dorsoanterior-like mesoderm and bone morphogenetic protein 4 mediating the formation of ventral mesoderm,including hematopoietic precursors. These effects are not observed in CDM alone or when TGF-beta 1,-beta 2,or -beta 3,acid FGF,or basic FGF is added individually to CDM. In vivo,at day 6.5 of mouse development,activin beta A RNA is detectable in the decidua and bone morphogenetic protein 4 RNA is detectable in the egg cylinder. Together,our data strongly implicate the TGF-beta family in mammalian mesoderm development and hematopoietic cell formation. View Publication

Generating engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is an ideal approach for obtaining induced HSCs for cell therapy. However,the path from PSCs to robustly induced HSCs (iHSCs) in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study,we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2) accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly,the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore,the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo. View Publication

Clinical hematopoietic transplantation outcomes are strongly correlated with the numbers of cells infused. Anticipated novel therapeutic implementations of hematopoietic stem cells (HSCs) and their derivatives further increase interest in strategies to expand HSCs ex vivo. A fundamental limitation in all HSC-driven culture systems is the rapid generation of differentiating cells and their secreted inhibitory feedback signals. Herein we describe an integrated computational and experimental strategy that enables a tunable reduction in the global levels and impact of paracrine signaling factors in an automated closed-system process by employing a controlled fed-batch media dilution approach. Application of this system to human cord blood cells yielded a rapid (12-day) 11-fold increase of HSCs with self-renewing,multilineage repopulating ability. These results highlight the marked improvements that control of feedback signaling can offer primary stem cell culture and demonstrate a clinically relevant rapid and relatively low culture volume strategy for ex vivo HSC expansion. View Publication

Autografts using untreated or in vitro manipulated bone marrow and peripheral blood stem cells represent promising approaches to the treatment of malignant diseases. In this work,the collagen gel culture technique was compared with agar and methylcellulose for its capacity to permit the growth of human granulomonocytic (day 14 CFU-GM; collagen vs agar or MTC) or erythroblastic (day 7 CFU-E and day 14 BFU-E; collagen versus methylcellulose) colonies in autologous transplantation products. Our results show that the collagen culture system always gave as many or more colonies than the other techniques. It also allowed harvesting of gels onto glass slides and subsequent May-Grünwald-Giemsa,cytochemical or immunocytochemical staining. We suggest that the collagen assay represents an interesting alternative to the widely used agar or methylcellulose systems for the culture of hematopoietic progenitors because of the equal or higher number of colonies detected,the easy phenotypical identification of colonies in stained gels,and the ability to store high-quality documentation. This technique is particularly attractive for use in the quality control of autologous bone marrow transplantation procedures. View Publication

Chimerism testing after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represents a promising tool for predicting disease relapse,although its precise role in this setting remains unclear. We investigated the predictive value of T lymphocyte chimerism analysis at 90 to 120 days after allo-HSCT in 378 patients with AML/MDS who underwent busulfan/fludarabine-based myeloablative preparative regimens. Of 265 (70%) patients with available T lymphocyte chimerism data,43% of patients in first or second complete remission (CR1/CR2) at the time of transplantation had complete (100%) donor T lymphocytes at day +90 to +120 compared with 60% of patients in the non-CR1/CR2 cohort (P = .005). In CR1/CR2 patients,donor T lymphocyte chimerism ?85% at day +90 to +120 was associated with a higher frequency of 3-year disease progression (29%; 95% confidence interval [CI],18% to 46% versus 15%; 95% CI,9% to 23%; hazard ratio [HR],2.1; P = .04). However,in the more advanced,non-CR1/CR2 cohort,mixed T lymphocyte chimerism was not associated with relapse (37%; 95% CI,20% to 66% versus 34%; 95% CI,25% to 47%; HR,1.3; P = .60). These findings demonstrate that early T lymphocyte chimerism testing at day +90 to +120 is a useful approach for predicting AML/MDS disease recurrence in patients in CR1/CR2 at the time of transplantation. View Publication

Clonal hematopoiesis (CH),a prevalent and premalignant state in the elderly,has been detected in young individuals under selective pressures such as hematopoietic cell transplantation (HCT). However,the origin of CH and mutational processes underlying CH driver mutations in young blood systems remain unclear. Here,we used genome‐wide somatic mutation profiles to retrospectively trace the origin of DNMT3A‐mutant CH in three individuals,14–41 years after childhood HCT. Both the rate and spectrum of somatic mutations in individuals with posttransplant CH were consistent with normal age‐associated mutagenesis. Phylogenetic analysis revealed that DNMT3A‐mutant HSPCs were present in the donor before 6.8 years of age,including during fetal development,despite being undetectable with a limit of detection of variant allele frequency of 0.001 at the time of transplantation. These findings were validated by comparing the observed mutations to expected age‐dependent mutational signatures. Our results reveal that undetectable DNMT3A‐mutant clones in young donors can expand into significant CH clones within decades upon transplantation. The rapid expansion of these clones in this context indicates that specific environmental pressures,rather than solely mutation acquisition,drive the development of CH. View Publication

Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties,and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4,Klf4,and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3beta (GSK3beta) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin,a protein kinase A pathway agonist which can induce Klf4 and Klf2 expression,transiently substitutes for the requirement for ectopic transgene expression. In contrast to conventional human ESCs,these epigenetically converted cells have growth properties,an X-chromosome activation state (XaXa),a gene expression profile,and a signaling pathway dependence that are highly similar to those of mouse ESCs. Finally,the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of validated naïve" human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans and may open up new opportunities for patient-specific� View Publication

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34(+) cells from the human umbilical cord blood (hUCB),cocultured with neonatal mouse cardiomyocytes,acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34(+) cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days,mononucleated EGFP(+) cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca(2+)] ([Ca(2+)](i)) homeostasis by [Ca(2+)](i) imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP(+) cells were connected to surrounding cells by gap junctions,acquired electrophysiological properties similar to those of cardiomyocytes,and showed action potential-associated [Ca(2+)](i) transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca(2+)](i) oscillations and the associated membrane potential depolarization. However,RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion,under our experimental conditions,hUCB CD34(+) cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However,the expression of human-specific cardiac genes was undetectable by RT-PCR. View Publication

Elucidation of mechanisms that regulate hematopoietic stem cell self-renewal and differentiation would be facilitated by the identification of defined culture conditions that allow these cells to be amplified. We now demonstrate a significant net increase (3-fold,P textless 0.001) in vitro of cells that are individually able to permanently and competitively reconstitute the lymphoid and myeloid systems of syngeneic recipient mice when Sca-1(+)lin- adult marrow cells are incubated for 10 days in serum-free medium with interleukin 11,flt3-ligand,and Steel factor. Moreover,the culture-derived repopulating cells continued to expand their numbers in the primary hosts at the same rate seen in recipients of noncultured stem cells. In the expansion cultures,long-term culture-initiating cells increased 7- +/- 2-fold,myeloid colony-forming cells increased 140- +/- 36-fold,and total nucleated cells increased 230- +/- 62-fold. Twenty-seven of 100 cultures initiated with 15 Sca-1(+)lin- marrow cells were found to contain transplantable stem cells 10 days later. This frequency of positive cultures is the same as the frequency of transplantable stem cells in the original input suspension,suggesting that most had undergone at least one self-renewal division in vitro. No expansion of stem cells was seen when Sca-1+TER119- CD34+ day 14.5 fetal liver cells were cultured under the same conditions. These findings set the stage for further investigations of the mechanisms by which cytokine stimulation may elicit different outcomes in mitotically activated hematopoietic stem cells during ontogeny and in the adult. View Publication

In mouse,a subset of dendritic cells (DCs) known as CD8alpha+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However,translation into clinical protocols has been hampered by the failure to identify CD8alpha+ DCs in humans. Here,we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8alpha+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8alpha+ DCs,human DNGR-1+ BDCA3hi DCs express Necl2,CD207,BATF3,IRF8,and TLR3,but not CD11b,IRF4,TLR7,or (unlike CD8alpha+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8,but not of TLR7,and produce interleukin (IL)-12 when given innate and T cell-derived signals. Notably,DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy. View Publication

SummaryInducible loss-of-function strategies are crucial for understanding gene function. However,creating inducible,multiple-gene knockout models is challenging and time-consuming. Here,we present a protocol for establishing a doxycycline-inducible CRISPR interference (CRISPRi) system to concurrently silence multiple genes in human induced pluripotent stem cells (hPSCs). We describe the steps for establishing host CRISPRi hPSCs,designing and cloning single-guide RNAs (sgRNAs) into a lentivirus plasmid,and establishing monoclonal CRISPRi hPSC lines transduced with sgRNAs. We also detail the procedures for selecting effective CRISPRi clones.For complete details on the use and execution of this protocol,please refer to Matsui et al.1 Graphical abstract Highlights•Dox-inducible CRISPRi system to silence multiple genes concurrently•Instructions for generating CRISPRi hPSCs transduced with four sgRNAs•FOXA1/A2/A3-CRISPRi system represses expression of all three FOXA genes by 95% Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Inducible loss-of-function strategies are crucial for understanding gene function. However,creating inducible,multiple-gene knockout models is challenging and time-consuming. Here,we present a protocol for establishing a doxycycline-inducible CRISPR interference (CRISPRi) system to concurrently silence multiple genes in human induced pluripotent stem cells (hPSCs). We describe the steps for establishing host CRISPRi hPSCs,designing and cloning single-guide RNAs (sgRNAs) into a lentivirus plasmid,and establishing monoclonal CRISPRi hPSC lines transduced with sgRNAs. We also detail the procedures for selecting effective CRISPRi clones. View Publication

The efficacy of donor HSCT is partly reduced as a result of slow post-transplantation immune recovery. In particular,T cell regeneration is generally delayed,resulting in high infection-related mortality in the first years post-transplantation. Adoptive transfer of in vitro-generated human T cell progenitors seems a promising approach to accelerate T cell recovery in immunocompromised patients. AA may enhance T cell proliferation and differentiation in a controlled,feeder-free environment containing Notch ligands and defined growth factors. Our experiments show a pivotal role for AA during human in vitro T cell development. The blocking of NOS diminished this effect,indicating a role for the citrulline/NO cycle. AA promotes the transition of proT1 to proT2 cells and of preT to DP T cells. Furthermore,the addition of AA to feeder cocultures resulted in development of DP and SP T cells,whereas without AA,a preT cell-stage arrest occurred. We conclude that neither DLL4-expressing feeder cells nor feeder cell conditioned media are required for generating DP T cells from CB and G-CSF-mobilized HSCs and that generation and proliferation of proT and DP T cells are greatly improved by AA. This technology could potentially be used to generate T cell progenitors for adoptive therapy. View Publication