Although widely applied in treating hematopoietic malignancies,transplantation of hematopoietic stem/progenitor cells(HSPCs)is impeded by HSPC shortage.Whether circulating HSPCs(cHSPCs)in steady-state blood could be u...Although widely applied in treating hematopoietic malignancies,transplantation of hematopoietic stem/progenitor cells(HSPCs)is impeded by HSPC shortage.Whether circulating HSPCs(cHSPCs)in steady-state blood could be used as an alternative source remains largely elusive.Here we develop a three-dimensional culture system(3DCS)including arginine,glycine,aspartate,and a series of factors.Fourteen-day culture of peripheral blood mononuclear cells(PBMNCs)in 3DCS led to 125-and 70-fold increase of the frequency and number of CD34+cells.Further,3DCS-expanded cHSPCs exhibited the similar reconstitution rate com-pared to CD34+HSPCs in bone marrow.Mechanistically,3DCS fabricated an immunomodulatory niche,secreting cytokines as TNF to support cHSPC survival and proliferation.Finally,3DCS could also promote the expansion of cHSPCs in patients who failed in HSPC mobilization.Our 3DCS successfully expands rare cHSPCs,providing an alternative source for the HSPC therapy,particularly for the patients/donors who have failed in HSPC mobilization.展开更多
Beyond glycemic control, applications of glucagon-like peptide-1 receptor (GLP-1r) agonists (GLP-1 RAs) inhibit inflammationand plaque development in murine atherosclerotic models. However, whether they modulate hemat...Beyond glycemic control, applications of glucagon-like peptide-1 receptor (GLP-1r) agonists (GLP-1 RAs) inhibit inflammationand plaque development in murine atherosclerotic models. However, whether they modulate hematopoietic stem/progenitor cells(HSPCs)to prohibit skewed myelopoiesis in hypercholesteremia remains unknown. In this study, GLP-1r expression in fluorescenceactivated cell sorting (FACS)-sorted wild-type HSPCs was determined by capillary western blotting. Bone marrow cells (BMCs)of wild-type or GLP-1r−/− mice were transplanted into lethally irradiated low-density lipoprotein receptor deficient (LDLr−/−)recipients followed by high-fat diet (HFD) for chimerism analysis by FACS. In parallel, LDLr−/− mice were placed on HFD for 6weeks and then treated with saline or Exendin-4 (Ex-4) for another 6 weeks. HSPC frequency and cell cycle were analyzed byFACS, and intracellular metabolite levels were assessed by targeted metabolomics. The results demonstrated that HSPCs expressedGLP-1r and transplantation of GLP-1r−/− BMCs resulted in skewed myelopoiesis in hypercholesterolemic LDLr−/− recipients.In vitro, Ex-4 treatment of FACS-purified HSPCs suppressed cell expansion and granulocyte production induced by LDL. In vivo, Ex-4treatment inhibited plaque progression, suppressed HSPC proliferation, and modified glycolytic and lipid metabolism in HSPCs ofhypercholesteremic LDLr−/− mice. In conclusion, Ex-4 could directly inhibit HSPC proliferation induced by hypercholesteremia.展开更多
The effects of hematopoietic stem/progenitor cells(HSPCs)expanded in the two step coculture with human bone marrow mesenchymal stem cells(hMSCs)on the hematopoietic reconstruction of irradiated NOD/SCID mice were stud...The effects of hematopoietic stem/progenitor cells(HSPCs)expanded in the two step coculture with human bone marrow mesenchymal stem cells(hMSCs)on the hematopoietic reconstruction of irradiated NOD/SCID mice were studied.Mononuclear cells(MNCs)were isolated from human umbilical cord blood(UCB)and cultured in the non-coculture scheme of rhSCF+rhG−CSF+rhMDGF combination and the coculture scheme of rhSCF+rhG−CSF+rhMDGF+hMSCs.Sublethally-irradiated NOD/SCID mice were transplanted with ex vivo expanded HSPCs with the dose of 8.5×10^(6) cells per mouse.After transplantation,the dynamics of WBC in the transplanted mice was measured periodically,and the Alu sequence fragment special for human in the transplanted mice was inspected by PCR.Results showed that the coculture scheme increased proliferation of UCB-derived HSPCs.After transplantation with expanded HSPCs,the population of WBC in the transplanted mice increased in 12 d and reached the first peak in 25 d,then showed the second increasing of WBC in 45~55 d.Expanded cells from the coculture scheme appeared to be favorable for the second increasing of WBC in the transplanted mice.After 85 d,the Alu sequence fragment was detected in the probability of 87.5%(7/8)for the non-coculture scheme and 88.9%(8/9)for the coculture scheme.展开更多
T cells play essential roles in antitumor therapy.Via gene engineering technique to enhance tumor-antigen specificity,patient peripheral blood-derived T cells(PBT)show encouraging clinical outcomes in treating certain...T cells play essential roles in antitumor therapy.Via gene engineering technique to enhance tumor-antigen specificity,patient peripheral blood-derived T cells(PBT)show encouraging clinical outcomes in treating certain blood malignancies.However,the high costs,functionality exhaustion,and disease-condition-dependent availability of PBT prompt the attempts of exploring alternative T cell sources.Theoretically,induced T cells from pluripotent stem cells(PSC)are ideal candidates that integrate plenty of advantages that primary T cells lack,including unlimited off-the-shelf cell source and precision gene editing feasibility.However,researchers are still struggling with developing a straightforward protocol to induce functional and immunocompetent human T cells from PSC.Based on stromal cell-expressing or biomaterial-presenting Notch ligands DLL1 or DLL4,natural and induced blood progenitors can differentiate further toward T lineage commitment.However,none of the reported T induction protocols has yet translated into any clinical application,signaling the existence of numerous technical barriers for regenerating T cells functionally matching their natural PBT counterparts.Alternatively,new approaches have been developed to repopulate induced T lymphopoiesis via in vivo reprogramming or transplanting induced T cell precursors.Here,we review the most recent progress in the T cell regeneration field,and the remaining challenges dragging their clinical applications.展开更多
Single-cell RNA-seq data analysis generally requires quality control,normalization,highly variable genes screening,dimensionality reduction and clustering.Among these processes,downstream analysis including dimensiona...Single-cell RNA-seq data analysis generally requires quality control,normalization,highly variable genes screening,dimensionality reduction and clustering.Among these processes,downstream analysis including dimensionality reduction and clustering are sensitive to the selection of highly variable genes.Though increasing number of tools for selecting the highly variable genes have been developed,an evaluation of theirperformances and a general strategy are lack.Here,wecompare the performance of nine commonly usedmethods for screening variable genes by using single-cell RNA-seq data from hematopoietic stem/progenitor cells and mature blood cells,and find that SCHS outperforms other methods regarding to reproducibility and accuracy.However,this method prefers the selection of highly expressed genes.We further propose a new strategy SIEVE(SIngle-cEll Variable gEnes)bymultiple rounds of randomsampling,therefore minimizing the stochastic noise and identifying a robust set of variable genes.Moreover,SIEVE recovers lowly expressed genes as variable genes and substantially improves the accuracy of single cell classification,especially for the methods with lower reproducibility.The SIEVE software is freely available at https://github.com/YinanZhang522/SIEVE.展开更多
基金Data and materials availability:Processed and raw data can be downloaded from NCBI GEO(#GSE122682,and#GSE153421).
文摘Although widely applied in treating hematopoietic malignancies,transplantation of hematopoietic stem/progenitor cells(HSPCs)is impeded by HSPC shortage.Whether circulating HSPCs(cHSPCs)in steady-state blood could be used as an alternative source remains largely elusive.Here we develop a three-dimensional culture system(3DCS)including arginine,glycine,aspartate,and a series of factors.Fourteen-day culture of peripheral blood mononuclear cells(PBMNCs)in 3DCS led to 125-and 70-fold increase of the frequency and number of CD34+cells.Further,3DCS-expanded cHSPCs exhibited the similar reconstitution rate com-pared to CD34+HSPCs in bone marrow.Mechanistically,3DCS fabricated an immunomodulatory niche,secreting cytokines as TNF to support cHSPC survival and proliferation.Finally,3DCS could also promote the expansion of cHSPCs in patients who failed in HSPC mobilization.Our 3DCS successfully expands rare cHSPCs,providing an alternative source for the HSPC therapy,particularly for the patients/donors who have failed in HSPC mobilization.
基金supported by grants from the National Natural Science Foundation of China(81670765 and 82070841)to Y.F.
文摘Beyond glycemic control, applications of glucagon-like peptide-1 receptor (GLP-1r) agonists (GLP-1 RAs) inhibit inflammationand plaque development in murine atherosclerotic models. However, whether they modulate hematopoietic stem/progenitor cells(HSPCs)to prohibit skewed myelopoiesis in hypercholesteremia remains unknown. In this study, GLP-1r expression in fluorescenceactivated cell sorting (FACS)-sorted wild-type HSPCs was determined by capillary western blotting. Bone marrow cells (BMCs)of wild-type or GLP-1r−/− mice were transplanted into lethally irradiated low-density lipoprotein receptor deficient (LDLr−/−)recipients followed by high-fat diet (HFD) for chimerism analysis by FACS. In parallel, LDLr−/− mice were placed on HFD for 6weeks and then treated with saline or Exendin-4 (Ex-4) for another 6 weeks. HSPC frequency and cell cycle were analyzed byFACS, and intracellular metabolite levels were assessed by targeted metabolomics. The results demonstrated that HSPCs expressedGLP-1r and transplantation of GLP-1r−/− BMCs resulted in skewed myelopoiesis in hypercholesterolemic LDLr−/− recipients.In vitro, Ex-4 treatment of FACS-purified HSPCs suppressed cell expansion and granulocyte production induced by LDL. In vivo, Ex-4treatment inhibited plaque progression, suppressed HSPC proliferation, and modified glycolytic and lipid metabolism in HSPCs ofhypercholesteremic LDLr−/− mice. In conclusion, Ex-4 could directly inhibit HSPC proliferation induced by hypercholesteremia.
基金supplying HUCB and irradiating mice.This project was supported by project grant From Zhejiang Science Foundation (No.2006C23027).
文摘The effects of hematopoietic stem/progenitor cells(HSPCs)expanded in the two step coculture with human bone marrow mesenchymal stem cells(hMSCs)on the hematopoietic reconstruction of irradiated NOD/SCID mice were studied.Mononuclear cells(MNCs)were isolated from human umbilical cord blood(UCB)and cultured in the non-coculture scheme of rhSCF+rhG−CSF+rhMDGF combination and the coculture scheme of rhSCF+rhG−CSF+rhMDGF+hMSCs.Sublethally-irradiated NOD/SCID mice were transplanted with ex vivo expanded HSPCs with the dose of 8.5×10^(6) cells per mouse.After transplantation,the dynamics of WBC in the transplanted mice was measured periodically,and the Alu sequence fragment special for human in the transplanted mice was inspected by PCR.Results showed that the coculture scheme increased proliferation of UCB-derived HSPCs.After transplantation with expanded HSPCs,the population of WBC in the transplanted mice increased in 12 d and reached the first peak in 25 d,then showed the second increasing of WBC in 45~55 d.Expanded cells from the coculture scheme appeared to be favorable for the second increasing of WBC in the transplanted mice.After 85 d,the Alu sequence fragment was detected in the probability of 87.5%(7/8)for the non-coculture scheme and 88.9%(8/9)for the coculture scheme.
基金supported by the Key R&D program from Ministry of Science and Technology of China(2019YFA0110203)Major Research and Development Project of Guangzhou Regenerative Medicine and Health Guangdong Laboratory(2018GZR110104006)+2 种基金the Health and Medical Care Collaborative Innovation Program of Guangzhou Scientific and Technology(201803040017)the Science and Technology Planning Project of Guangdong Province(2017B030314056)the National Natural Science Foundation of China(31471117,81470281,31600948).
文摘T cells play essential roles in antitumor therapy.Via gene engineering technique to enhance tumor-antigen specificity,patient peripheral blood-derived T cells(PBT)show encouraging clinical outcomes in treating certain blood malignancies.However,the high costs,functionality exhaustion,and disease-condition-dependent availability of PBT prompt the attempts of exploring alternative T cell sources.Theoretically,induced T cells from pluripotent stem cells(PSC)are ideal candidates that integrate plenty of advantages that primary T cells lack,including unlimited off-the-shelf cell source and precision gene editing feasibility.However,researchers are still struggling with developing a straightforward protocol to induce functional and immunocompetent human T cells from PSC.Based on stromal cell-expressing or biomaterial-presenting Notch ligands DLL1 or DLL4,natural and induced blood progenitors can differentiate further toward T lineage commitment.However,none of the reported T induction protocols has yet translated into any clinical application,signaling the existence of numerous technical barriers for regenerating T cells functionally matching their natural PBT counterparts.Alternatively,new approaches have been developed to repopulate induced T lymphopoiesis via in vivo reprogramming or transplanting induced T cell precursors.Here,we review the most recent progress in the T cell regeneration field,and the remaining challenges dragging their clinical applications.
基金This work has been supported by the National Natural Science Foundation of China(82022002,81900117,81890993,81890990,32000803)National Key Research and Development Program of China(2018YFA0107804)+1 种基金CAMS Initiative for Innovative Medicine(2017-I2M-1-015,2017-I2M-3-009,2019-I2M-2-001)Fundamental Research Funds for the Central Research Institutes(2020-RC310-005).
文摘Single-cell RNA-seq data analysis generally requires quality control,normalization,highly variable genes screening,dimensionality reduction and clustering.Among these processes,downstream analysis including dimensionality reduction and clustering are sensitive to the selection of highly variable genes.Though increasing number of tools for selecting the highly variable genes have been developed,an evaluation of theirperformances and a general strategy are lack.Here,wecompare the performance of nine commonly usedmethods for screening variable genes by using single-cell RNA-seq data from hematopoietic stem/progenitor cells and mature blood cells,and find that SCHS outperforms other methods regarding to reproducibility and accuracy.However,this method prefers the selection of highly expressed genes.We further propose a new strategy SIEVE(SIngle-cEll Variable gEnes)bymultiple rounds of randomsampling,therefore minimizing the stochastic noise and identifying a robust set of variable genes.Moreover,SIEVE recovers lowly expressed genes as variable genes and substantially improves the accuracy of single cell classification,especially for the methods with lower reproducibility.The SIEVE software is freely available at https://github.com/YinanZhang522/SIEVE.
