中国药理学与毒理学杂志2025，Vol.39Issue(7)：500-510,11.DOI:10.3867/j.issn.1000-3002.2025.08289
人胚胎干细胞来源巨核细胞衍生微囊泡促进血管新生
Microparticles from human embryonic stem cell-derived megakaryocytes promote angiogenesis
摘要
Abstract
OBJECTIVE To establish a preparation system for megakaryocytes(MKs)derived from human embryonic stem cells(hESCs)and MK microparticles(MKMPs),and to assess the pro-angio-genic efficiency of these microparticles.METHODS ①hESCs were induced to mesodermal progenitor cells via monolayer culture with the first-stage induction medium for 2 days before the cells were induced to hemogenic endothelial/hematopoietic progenitor cells by culturing with the second-stage induction medium for another 3 days.Then,the cells were dissociated into single cells,seeded into the third-stage induction medium,and cultured using the suspension method for 8 days to obtain MKs.The specific characters of differentiated cells were identified through morphological observation and flow cytometry before stage-specific marker proteins in different periods were analyzed[hESCs:TRA-1-60,sialyl glycolipid stage-specific embryonic antigen4(SSEA4)];mesodermal progenitor cells:brachyury;hemogenic endothelial/hematopoietic progenitor cells:CD34,CD43;MKs:CD41a,CD42b),and immu-nofluorescence staining[β1-tubulin,von Willebrand factor(VWF)],[friend leukemia integration 1(FLI1),CD42].② MKMP collection and verification:MKMPs were collected via differential centrifugation.The concentration and size of these MKMPs were determined by nanoparticle tracking analysis(NTA),and both the morphology and ultrastructure were examined by transmission electron microscopy(TEM).Besides,the MKMPs-specific proteins[CD41,tumor susceptibility gene 101(TSG101)and CD9]were detected by Western blotting analysis.③ Biological function of MKMPs:MKMPs were stained with CD41a-PE antibodies and co-cultured with human umbilical veinvascular endothelial cells(HUVECs)labeled by CD34-APC for 3 h.Live-cell immunofluorescence was employed to find out whether HUVECs could absorb MKMPs.To find out whether MKMPs could affect the role of HUVECs in angio-genesis and cell migration,platelet microvesicles(PMPs)were used as positive controls.The experi-mental groups were added with different concentrations of microparticles(1,5,10 and 20 mg·L-1)while the control group was given no microparticles(0 mg·L-1).The number of nodes that formed the lumen after 5 h of incubation in Matrigel was counted,and the size of healing of the scratch area was analyzed after 6 h.To elucidate the mechanism through which MKMPs impacted angiogenesis,ELISA was used out to quantitatively detect the concentration of proteins in microparticles.RESULTS ① A three-stage differentiation cultural system was established to develop hESCs into MKs.Flow cytometry revealed progressive loss of pluripotency markers SSEA4 and TRA-1-60,while the mesodermal progenitor marker brachyury peaked at d 2.Subsequently,hemogenic endothelial/hematopoietic progenitor markers CD34 and CD43 emerged at d 5,followed by megakaryocytic markers CD41a and CD42b at d 13.Immunofluorescent images further demonstrated that MKs expressed specific proteins CD42,β1-tubulin,von VWF and FLI1 at d 13.②Microparticles were collected via differential centrifuga-tion.Transmission electron microscopy revealed that their substructure exhibited a typical double-layered membrane.Nanoparticle tracking analysis indicated that the size was(164.3±14.0)nm.The result of WB demonstrated that the microparticles expressed specific markers,including TSG101,CD9 and CD41.③ MKMPs were absorbed after being co-cultured with HUVECs for 3 h and enhanced the ability of HUVECs to form tubes and migrate.Notably,the treatment of 5 mg·L-1 MKMPs was more effective than 5 mg·L-1 PMPs treatment.The results of ELISA showed that the content of VEGF from MKMPs was higher than from PMPs,which may be the key factor in regulating endothelial biological function.CONCLUSION MKs derived from hESCs can generate functional microparticles which can promote angiogenesis.关键词
人胚胎干细胞/巨核细胞/巨核细胞微囊泡/人脐静脉内皮细胞/血管新生Key words
human embryonic stem cells/megakaryocytes/megakaryocyte microparticles/human umbilical veinvascular endothelial cells/angiogenesis分类
医药卫生引用本文复制引用
唐轩,吴旭敏,陈可一,胡亮,李基晟,刘传丽,覃金华,张博文,李艳华..人胚胎干细胞来源巨核细胞衍生微囊泡促进血管新生[J].中国药理学与毒理学杂志,2025,39(7):500-510,11.基金项目
国家自然科学基金(82270132) （82270132）
国家自然科学基金(32470788) Nature Science Foundation of China(82270132) （32470788）
and Nature Science Foundation of China(32470788) （32470788）
