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

Studying ciliary genes in the context of the human central nervous system is crucial for understanding the underlying causes of neurodevelopmental ciliopathies. Here,we use pluripotent stem cell-derived spinal organoids to reveal distinct functions of the ciliopathy gene RPGRIP1L in humans and mice,and uncover an unexplored role for cilia in human axial patterning. Previous research has emphasized Rpgrip1l critical functions in mouse brain and spinal cord development through the regulation of SHH/GLI pathway. Here,we show that RPGRIP1L is not required for SHH activation or motoneuron lineage commitment in human spinal progenitors and that this feature is shared by another ciliopathy gene,TMEM67 . Furthermore,human RPGRIP1L -mutant motoneurons adopt hindbrain and cervical identities instead of caudal brachial identity. Temporal transcriptome analysis reveals that this antero-posterior patterning defect originates in early axial progenitors and correlates with cilia loss. These findings provide important insights into the role of cilia in human neural development. Subject terms: Ciliogenesis,Pattern formation,Pluripotent stem cells,Neurogenesis View Publication

Here,we present a protocol for isolating and culturing mouse photoreceptors in a minimal,chemically defined medium free from serum. We describe steps for retina dissection,enzymatic dissociation,photoreceptor enrichment,cell culture,extracellular vesicles (EVs) enrichment,and EV ultrastructural analysis. This protocol,which has been verified for cultured cells derived from multiple murine strains,allows for the study of several aspects of photoreceptor biology,including EV isolation and nanotube formation. For complete details on the use and execution of this protocol,please refer to Kalargyrou et al. (2021). 1 Subject areas: Cell Biology,Molecular Biology,Neuroscience View Publication

To search for genes that promote hematopoietic development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs),we overexpressed several known hematopoietic regulator genes in hESC/iPSC-derived CD34(+)CD43(-) endothelial cells (ECs) enriched in hemogenic endothelium (HE). Among the genes tested,only Sox17,a gene encoding a transcription factor of the SOX family,promoted cell growth and supported expansion of CD34(+)CD43(+)CD45(-/low) cells expressing the HE marker VE-cadherin. SOX17 was expressed at high levels in CD34(+)CD43(-) ECs compared with low levels in CD34(+)CD43(+)CD45(-) pre-hematopoietic progenitor cells (pre-HPCs) and CD34(+)CD43(+)CD45(+) HPCs. Sox17-overexpressing cells formed semiadherent cell aggregates and generated few hematopoietic progenies. However,they retained hemogenic potential and gave rise to hematopoietic progenies on inactivation of Sox17. Global gene-expression analyses revealed that the CD34(+)CD43(+)CD45(-/low) cells expanded on overexpression of Sox17 are HE-like cells developmentally placed between ECs and pre-HPCs. Sox17 overexpression also reprogrammed both pre-HPCs and HPCs into HE-like cells. Genome-wide mapping of Sox17-binding sites revealed that Sox17 activates the transcription of key regulator genes for vasculogenesis,hematopoiesis,and erythrocyte differentiation directly. Depletion of SOX17 in CD34(+)CD43(-) ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a key role in priming hemogenic potential in ECs,thereby regulating hematopoietic development from hESCs/iPSCs. View Publication

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. View Publication

Induced pluripotent stem cell (iPSC) therapeutics are a promising treatment for genetic and infectious diseases. To assess engraftment,risk of neoplastic formation,and therapeutic benefit in an autologous setting,testing iPSC therapeutics in an appropriate model,such as the pigtail macaque (Macaca nemestrina; Mn),is crucial. Here,we developed a chemically defined,scalable,and reproducible specification protocol with bone morphogenetic protein 4,prostaglandin-E2 (PGE2),and StemRegenin 1 (SR1) for hematopoietic differentiation of Mn iPSCs. Sequential coculture with bone morphogenetic protein 4,PGE2,and SR1 led to robust Mn iPSC hematopoietic progenitor cell formation. The combination of PGE2 and SR1 increased CD34(+)CD38(-)Thy1(+)CD45RA(-)CD49f(+) cell yield by 6-fold. CD34(+)CD38(-)Thy1(+)CD45RA(-)CD49f(+) cells isolated on the basis of CD34 expression and cultured in SR1 expanded 3-fold and maintained this long-term repopulating HSC phenotype. Purified CD34(high) cells exhibited 4-fold greater hematopoietic colony-forming potential compared with unsorted hematopoietic progenitors and had bilineage differentiation potential. On the basis of these studies,we calculated the cell yields that must be achieved at each stage to meet a threshold CD34(+) cell dose that is required for engraftment in the pigtail macaque. Our protocol will support scale-up and testing of iPSC-derived CD34(high) cell therapies in a clinically relevant nonhuman primate model. View Publication

It is often predicted that stem cells divide asymmetrically,creating a daughter cell that maintains the stem-cell capacity,and 1 daughter cell committed to differentiation. While asymmetric stem-cell divisions have been proven to occur in model organisms (eg,in Drosophila),it remains illusive whether primitive hematopoietic cells in mammals actually can divide asymmetrically. In our experiments we have challenged this question and analyzed the developmental capacity of separated offspring of primitive human hematopoietic cells at a single-cell level. We show for the first time that the vast majority of the most primitive,in vitro-detectable human hematopoietic cells give rise to daughter cells adopting different cell fates; 1 inheriting the developmental capacity of the mother cell,and 1 becoming more specified. In contrast,approximately half of the committed progenitor cells studied gave rise to daughter cells,both of which adopted the cell fate of their mother. Although our data are compatible with the model of asymmetric cell division,other mechanisms of cell fate specification are discussed. In addition,we describe a novel human hematopoietic progenitor cell that has the capacity to form natural killer (NK) cells as well as macrophages,but not cells of other myeloid lineages. View Publication

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood,initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however,the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome,RNA-seq),Colony forming assay and xenograft studies,we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones,both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells,JMML-PCs are not always restricted to this compartment,highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML,and the identity of JMML-PCs,and provides robust models to monitor the disease and test novel therapeutic approaches. View Publication

We measured the free concentration of 1,25-dihydroxyvitamin D (1,25[OH]2D) using centrifugal ultrafiltration,and the level of vitamin D-binding protein (DBP) in 24 normal subjects,17 pregnant subjects,and 25 alcoholic subjects with liver disease. Our objective was to determine whether the increase in total 1,25(OH)2D levels in pregnant women and the reduction in total 1,25(OH)2D levels in subjects with liver disease reflected a true difference in free 1,25(OH)2D levels or whether such differences were due solely to the variations in DBP levels (and thus,the amount of 1,25[OH]2D bound) in these groups. In subjects with liver disease the mean total 1,25(OH)2D concentration (22.6 +/- 12.5 pg/ml) and the mean DBP concentration (188 +/- 105 micrograms/dl) were nearly half the normal values (41.5 +/- 11.5 pg/ml and 404 +/- 124 micrograms/dl,respectively,P less than 0.001),whereas the mean free 1,25(OH)2D level was similar to normal values (209 +/- 91 fg/ml and 174 +/- 46 fg/ml,respectively). In contrast,in pregnant subjects the mean total 1,25(OH)2D level (82 +/- 21 pg/ml) and mean DBP level (576 +/- 128 micrograms/dl) were significantly higher than normal (P less than 0.001). Although the mean percent free 1,25(OH)2D level in pregnant subjects was below normal (0.359 +/- 0.07% vs. 0.424 +/- 0.07%,P less than 0.001),the mean free 1,25(OH)2D level was 69% higher than normal (294 +/- 98 fg/ml vs. 174 +/- 46 fg/ml,P less than 0.001). When data from all three groups were combined,there was a linear correlation between total 1,25(OH)2D and DBP levels but not between DBP and percent free 1,25(OH)2D levels; the increased DBP levels in the pregnant subjects were associated with less of an effect on percent free 1,25(OH)2D than were the reduced DBP levels in the subjects with liver disease. Our data suggest that (a) free 1,25(OH)2D levels appear to be well maintained even in subjects with liver disease and reduced DBP levels,(b) free 1,25(OH)2D levels are increased during pregnancy despite the increase in DBP levels,and (c) free 1,25(OH)2D levels cannot be inferred accurately from measurements of total 1,25(OH)2D and DBP levels alone in subjects with various physiologic and pathophysiologic conditions. View Publication

BACKGROUND Human rhinoviruses (HRVs) are a major trigger of asthma exacerbations,with the bronchial epithelium being the major site of HRV infection and replication. Mast cells (MCs) play a key role in asthma where their numbers are increased in the bronchial epithelium with increasing disease severity. OBJECTIVE In view of the emerging role of MCs in innate immunity and increased localization to the asthmatic bronchial epithelium,we investigated whether HRV infection of MCs generated innate immune responses which were protective against infection. METHODS The LAD2 MC line or primary human cord blood-derived MCs (CBMCs) were infected with HRV or UV-irradiated HRV at increasing multiplicities of infection (MOI) without or with IFN-β or IFN-λ. After 24 h,innate immune responses were assessed by RT-qPCR and IFN protein release by ELISA. Viral replication was determined by RT-qPCR and virion release by TCID50 assay. RESULTS HRV infection of LAD2 MCs induced expression of IFN-β,IFN-λ and IFN-stimulated genes. However,LAD2 MCs were permissive for HRV replication and release of infectious HRV particles. Similar findings were observed with CBMCs. Neutralization of the type I IFN receptor had minimal effects on viral shedding,suggesting that endogenous type I IFN signalling offered limited protection against HRV. However,augmentation of these responses by exogenous IFN-β,but not IFN-λ,protected MCs against HRV infection. CONCLUSION AND CLINICAL RELEVANCE MCs are permissive for the replication and release of HRV,which is prevented by exogenous IFN-β treatment. Taken together,these findings suggest a novel mechanism whereby MCs may contribute to HRV-induced asthma exacerbations. View Publication

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However,the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound,vitamin C (Vc),enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence,a recently identified roadblock for reprogramming. In addition,Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process. View Publication

Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates,specifically,hematopoietic,mesodermal,and neurectodermal. In this study,we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore,we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells,expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo,and potentially for understanding and treating neurodegenerative and psychiatric diseases. View Publication

Neurogenesis decreases during aging causing a progressive cognitive decline but it is still controversial whether proliferation defects in neurogenic niches result from a loss of neural stem cells or from an impairment of their progression through the cell cycle. Using an accurate fluorescence-activated cell sorting technique,we show that the pool of neural stem cells is maintained in the subventricular zone of middle-aged mice while they have a reduced proliferative potential eventually leading to the subsequent decrease of their progeny. In addition,we demonstrate that the G1 phase is lengthened during aging specifically in activated stem cells,but not in transit-amplifying cells,and directly impacts on neurogenesis. Finally,we report that inhibition of TGFβ signaling restores cell cycle progression defects in stem cells. Our data highlight the significance of cell cycle dysregulation in stem cells in the aged brain and provide an attractive foundation for the development of anti-TGFβ regenerative therapies based on stimulating endogenous neural stem cells. View Publication