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

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

The long-term maintenance of hematopoietic stem cells (HSCs) relies on the regulation of endoplasmic reticulum (ER) stress at a low level,but the underlying mechanism remains poorly understood. Here,we demonstrate that suppression of ER stress improves the functions of HSCs and protects HSCs against ionizing radiation (IR)-induced injury. We identify epithelial membrane protein 1 (EMP1) as a key regulator that mitigates ER stress in HSCs. Emp1 deficiency leads to the accumulation of protein aggregates and elevated ER stress,ultimately resulting in impaired HSC maintenance and self-renewal. Mechanistically,EMP1 is located within the ER and interacts with ceramide synthase 2 (CERS2) to limit the production of a class of sphingolipids,dihydroceramides (dhCers). DhCers accumulate in Emp1-deficient HSCs and induce protein aggregation. Furthermore,Emp1 deficiency renders HSCs more susceptible to IR,while overexpression of Emp1 or inhibition of CERS2 protects HSCs against IR-induced injury. These findings highlight the critical role played by the EMP1-CERS2-dhCers axis in constraining ER stress and preserving HSC potential. A new study shows EMP1 protects hematopoietic stem cells by suppressing sphingolipid metabolism and ER stress. EMP1 interacts with CERS2 to limit dihydroceramide production,which causes protein aggregation when elevated. View Publication

The hematopoietic stem cell and multipotent progenitor (HSC/MPP) pool dynamically responds to stress to adapt blood output to specific physiological demands. In β-thalassemia (Bthal),severe anemia and ineffective erythropoiesis generate expansion of erythroid precursors and a chronic stress status in the bone marrow (BM) microenvironment. However,the response to the BM altered status at the level of the HSC/MPP compartment in terms of lineage commitment has not been investigated. Bulk and single-cell RNA-sequencing reveal that Bthal HSCs/MPPs are expanded and activated with enhanced priming along the whole Ery differentiation trajectory. Consistently,HSC/MPP showed an altered TGFβ expression and autophagy transcriptional signatures along with a declined dormancy state. We discovered that the altered TGFβ signaling fosters the Ery potential of HSCs by reducing their autophagic levels,and in vivo stimulation of autophagy is sufficient to rescue the imbalance of the HSC compartment. Our findings identify the interplay between TGFβ and HSC autophagy as a key driver in the context of non-malignant hematopoiesis. Subject terms: Haematopoietic stem cells,Haematological diseases,Autophagy View Publication

Various growth factors are known to stimulate both early and late stages of human hematopoietic cell development in semisolid assay systems,but their role as microenvironmental regulators is poorly understood. To address this problem,we developed a novel coculture system in which highly purified primitive human hematopoietic cells were seeded onto an irradiated feeder layer of cells from a murine marrow-derived stromal cell line (M2-10B4) previously engineered by retroviral-mediated gene transfer to produce specific human factors. Effects on cells at very early,intermediate,and late stages of hematopoiesis were then evaluated by assessing the number of clonogenic cell precursors (long-term culture initiating cells [LTC-IC]),clonogenic cells,and mature granulocyte and macrophage progeny present in the cultures after 5 weeks. In the absence of any feeders,cells at all stages of hematopoiesis decreased to very low levels. In contrast,maintenance of LTC-IC was found to be supported by control murine stromal cells as effectively as by standard human marrow adherent layers. The presence of granulocyte colony-stimulating factor (G-CSF) and interleukin-3-producing M2-10B4 cells in combination was able to further enhance the maintenance and early differentiation of these cells without a decline in their proliferative potential as measured by the clonogenic output per LTC-IC. However,this effect was lost if granulocyte-macrophage CSF (GM-CSF)-producing feeders were also present. On the other hand,in the presence of GM-CSF-producing feeders,the output of mature granulocytes and macrophages increased 20-fold. These findings show that it is possible to selectively improve the maintenance of very primitive human hematopoietic cells in vitro or their output of mature progeny by appropriate manipulation of the long-term marrow culture system. Further exploitation of this approach should facilitate investigation of the mechanisms operative within the human marrow microenvironment in vivo and the design of protocols for in vitro manipulation of human marrow for future therapeutic applications. View Publication

The continued success of pluripotent stem cell research is ultimately dependent on access to reliable and defined reagents for the consistent culture and cryopreservation of undifferentiated,pluripotent cells. The development of defined and feeder-independent culture media has provided a platform for greater reproducibility and standardization in this field. Here we provide detailed protocols for the use of mTeSR™1 and TeSR™2 with various cell culture matrices as well as defined cryopreservation protocols for human embryonic and human induced pluripotent stem cells. View Publication

Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling,we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor,p21Cip1/Waf1 (p21),have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling,we tested whether reduction of p21 alone in human adult-derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control,manipulated cells. Further,we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore,lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion. View Publication

Transient lymphopenia is a hallmark of measles virus (MV)-induced immunosuppression. To address to what extent replenishment of the peripheral lymphocyte compartment from bone marrow (BM) progenitor/stem cells might be affected,we analyzed the interaction of wild-type MV with hematopoietic stem and progenitor cells (HS/PCs) and stroma cells in vitro. Infection of human CD34(+) HS/PCs or stroma cells with wild-type MV is highly inefficient yet noncytolytic. It occurs independently of CD150 in stroma cells but also in HS/PCs,where infection is established in CD34(+) CD150(-) and CD34(+) CD150(+) (in humans representing HS/PC oligopotent precursors) subsets. Stroma cells and HS/PCs can mutually transmit MV and may thereby create a possible niche for continuous viral exchange in the BM. Infected lymphocytes homing to this compartment may serve as sources for HS/PC or stroma cell infection,as reflected by highly efficient transmission of MV from both populations in cocultures with MV-infected B or T cells. Though MV exposure does not detectably affect the viability,expansion,and colony-forming activity of either CD150(+) or CD150(-) HS/PCs in vitro,it efficiently interferes with short- but not long-term hematopoietic reconstitution in NOD/SCID mice. Altogether,these findings support the hypothesis that MV accession of the BM compartment by infected lymphocytes may contribute to peripheral blood mononuclear cell lymphopenia at the level of BM suppression. View Publication

Innate lymphoid cells 2 (ILC2) play a significant role in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). An important aspect of ILC2-mediated tumorigenesis is the expansion of the resident ILC2 and simultaneous recruitment of the peripheral ILC2. Here,we describe a protocol for isolation,enrichment,and DiD labeling of ILC2 for in vivo tracking of ILC2s in the mouse. Further,we describe steps for the adoptive transfer of ILC2 to a recipient mouse model of PDAC. For complete details on the use and execution of this protocol,please refer to Alam et al. (2022). View Publication

The classical model of hematopoiesis predicts a dichotomous lineage restriction of multipotent hematopoietic progenitors (MPPs) into common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). However,this idea has been challenged by the identification of lymphoid progenitors retaining partial myeloid potential (e.g.,LMPPs),implying that granulocytes can arise within both the classical lymphoid and the myeloid branches. Here,we resolve this issue by using cell-surface CD133 expression to discriminate functional progenitor populations. We show that eosinophilic and basophilic granulocytes as well as erythrocytes and megakaryocytes derive from a common erythro-myeloid progenitor (EMP),whereas neutrophilic granulocytes arise independently within a lympho-myeloid branch with long-term progenitor function. These findings challenge the concept of a CMP and restore dichotomy to the classical hematopoietic model. 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

To exploit the full potential of human pluripotent stem cells for regenerative medicine,developmental biology and drug discovery,defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed,but finding chemically defined,robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides,which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells. View Publication

Coronary heart disease (CHD) is a leading cause of death globally,while its current management is limited to reducing the myocardial infarction area without actually replacing dead cardiomyocytes. Direct cell reprogramming is a method of cellular cardiomyoplasty which aims for myocardial tissue regeneration,and CD34+ cells are one of the potential sources due to their shared embryonic origin with cardiomyocytes. However,the isolation and culture of non-adherent CD34+ cells is crucial to obtain adequate cells for high-efficiency genetic modification. This study aimed to investigate the optimal method for isolation and culture of CD34+ peripheral blood cells using certain culture media. A peripheral blood sample was obtained from a healthy subject and underwent pre-enrichment,isolation,and expansion. The culture was subsequently observed for their viability,adherence,and confluence. Day 0 observation of the culture showed a healthy CD34+ cell with a round cell shape,without any adherent cells present yet. Day 4 of observation showed that CD34+ cells within the blood plasma medium became adherent,indicated by their transformations into spindle or oval morphologies. Meanwhile,CD34+ cells in vitronectin and fibronectin media showed no adherent cells and many of them died. Day 7 observation revealed more adherent CD34+ cells in blood plasma medium,and which had 75% of confluence. In conclusion,the CD34+ cells that were isolated using a combination of density and magnetic methods may be viable and adequately adhere in culture using blood plasma medium,but not in cultures using fibronectin and vitronectin. View Publication