Retinal degeneration diseases,such as age-related macular de-generation(AMD)and retinitis pigmentosa(RP),initially mani-fest as dysfunction or death of the retinal pigment epithelium(RPE).Subretinal transplantation of...Retinal degeneration diseases,such as age-related macular de-generation(AMD)and retinitis pigmentosa(RP),initially mani-fest as dysfunction or death of the retinal pigment epithelium(RPE).Subretinal transplantation of human pluripotent stem cell(hPSC)-derived RPE cells has emerged as a potential therapy for retinal degeneration.However,RPE cells differentiated from hPSCs using current protocols are xeno-containing and are rarely applied in clinical trials.The development of hPSC-derived RPE cell differentiation protocols using xeno-free bio-materials is urgently needed for clinical applications.In this study,two protocols(the activin A and NIC84 protocols)were selected for modification and use in the differentiation of hiPSCs into RPE cells;the chetomin concentration was gradually increased to achieve high differentiation efficiency of RPE cells.The xeno-free extracellular matrix(ECM)proteins,laminin-511,laminin-521 and recombinant vitronectin,were selected as plate-coating substrates,and a Matrigel(xeno-containing ECM)-coated surface was used as a positive control.Healthy,mature hPSC-derived RPE cells were transplanted into 21-day-old Royal College of Surgeons(RCS)rats,a model of retinal degeneration disease.The visual func-tion of RCS rats was evaluated by optomotor response(qOMR)and electroretinography after transplantation of hPSC-derived RPE cells.Our study demonstrated that hPSCs can be efficiently differentiated into RPE cells on LN521-coated dishes using the NIC84 pro-tocol,and that subretinal transplantation of the cell suspensions can delay the progression of vision loss in RCS rats.展开更多
Circulating cell-free DNAs(cfDNAs)are fragmented DNA molecules released into the blood by cells.Previous studies have suggested that mitochondria-originated cfDNA fragments(mt-cfDNAs)in cancer patients are more fragme...Circulating cell-free DNAs(cfDNAs)are fragmented DNA molecules released into the blood by cells.Previous studies have suggested that mitochondria-originated cfDNA fragments(mt-cfDNAs)in cancer patients are more fragmented than those from healthy controls.However,it is still unknown where these short mtcfDNAs originate,and whether the length of mt-cfDNAs can be correlated with tumor burden and cancer progression.In this study,we first performed whole-genome sequencing analysis(WGS)of cfDNAs from a human tumor cell line-xenotransplantation mouse model and found that mt-cfDNAs released from transplanted tumor cells were shorter than the mouse counterpart.We next analyzed blood cfDNA samples from hepatocellular carcinoma and prostate cancer patients and found that mt-cfDNA lengths were inversely related to tumor size as well as the concentration of circulating tumor DNA.Our study suggested that monitoring the size of mt-cfDNAs in cancer patients would be a useful way to estimate tumor burden and cancer progression.展开更多
Fragile X syndrome(FXS) patients carry the expansion of over 200 CGG repeats at the promoter of fragile X mental retardation 1(FMR1), leading to decreased or absent expression of its encoded fragile X mental retardati...Fragile X syndrome(FXS) patients carry the expansion of over 200 CGG repeats at the promoter of fragile X mental retardation 1(FMR1), leading to decreased or absent expression of its encoded fragile X mental retardation protein(FMRP). However, the global transcriptional alteration by FMRP deficiency has not been well characterized at single nucleotide resolution, i.e., RNA-seq. Here,we performed in-vitro neuronal differentiation of human induced pluripotent stem(iPS) cells that were derived from fibroblasts of a FXS patient(FXS-iPSC). We then performed RNA-seq and examined the transcriptional misregulation at each intermediate stage during in-vitro differentiation of FXS-iPSC into neurons. After thoroughly analyzing the transcriptomic data and integrating them with those from other platforms, we found up-regulation of many genes encoding TFs for neuronal differentiation(WNT1, BMP4,POU3F4, TFAP2 C, and PAX3), down-regulation of potassium channels(KCNA1, KCNC3, KCNG2, KCNIP4, KCNJ3, KCNK9,and KCNT1) and altered temporal regulation of SHANK1 and NNAT in FXS-iPSC derived neurons, indicating impaired neuronal differentiation and function in FXS patients. In conclusion, we demonstrated that the FMRP deficiency in FXS patients has significant impact on the gene expression patterns during development, which will help to discover potential targeting candidates for the cure of FXS symptoms.展开更多
The newly developed next-generation sequencing platforms, in combination with gcnome-scale amplification methods, provide a powerful tool to study genomics from a single cell. This mini-review summarizes the technolog...The newly developed next-generation sequencing platforms, in combination with gcnome-scale amplification methods, provide a powerful tool to study genomics from a single cell. This mini-review summarizes the technologies of single cell genomics and their applications in several areas of biomedical research including stem cells, cancer biology and reproductive medicine. Particularly, it highlights recent advances in single cell exome sequencing, RNA-seq, and genome sequencing. The application of these powerful techniques will shed new light on the fundamental principles of gene transcription and genome organization at single-cell level and improve our understanding of cellular heterogeneity and diversity in multicellular organisms展开更多
基金supported by the National Natural Science Foundation of China(52250710155)the National Key Research and Development Program of China(2021YFA1101200,2022YFA1105501)+6 种基金the Project of Zhejiang Provincial Natural Science Foundation of China(LQ24H120006)supported by the State Key Laboratory of Ophthalmology,Optometry and Vision Science,Wenzhou Medical University(J02-20210201)the Wenzhou Municipal Science and Technology Bureau(Y20240054,Y2023799)supported by the Veteran Hospital Project(VGHUST113-G4-4-1 and VGHUSTll2-G4-3-l)the National Defense Medical Center Project(112-NCU-TRI-01,113-NCU-TRI-01 and TSGH-A-112003)the Department of Science and Technology/Global Innovation and Technology Alliance(DST/GITA)(GITA/DST/TWN/P-105/2022)the National Science and Technology Council(112-2923-E-008-005-MY3,111-2221-E-008-007,111-2923-E-008-005-MY3).
文摘Retinal degeneration diseases,such as age-related macular de-generation(AMD)and retinitis pigmentosa(RP),initially mani-fest as dysfunction or death of the retinal pigment epithelium(RPE).Subretinal transplantation of human pluripotent stem cell(hPSC)-derived RPE cells has emerged as a potential therapy for retinal degeneration.However,RPE cells differentiated from hPSCs using current protocols are xeno-containing and are rarely applied in clinical trials.The development of hPSC-derived RPE cell differentiation protocols using xeno-free bio-materials is urgently needed for clinical applications.In this study,two protocols(the activin A and NIC84 protocols)were selected for modification and use in the differentiation of hiPSCs into RPE cells;the chetomin concentration was gradually increased to achieve high differentiation efficiency of RPE cells.The xeno-free extracellular matrix(ECM)proteins,laminin-511,laminin-521 and recombinant vitronectin,were selected as plate-coating substrates,and a Matrigel(xeno-containing ECM)-coated surface was used as a positive control.Healthy,mature hPSC-derived RPE cells were transplanted into 21-day-old Royal College of Surgeons(RCS)rats,a model of retinal degeneration disease.The visual func-tion of RCS rats was evaluated by optomotor response(qOMR)and electroretinography after transplantation of hPSC-derived RPE cells.Our study demonstrated that hPSCs can be efficiently differentiated into RPE cells on LN521-coated dishes using the NIC84 pro-tocol,and that subretinal transplantation of the cell suspensions can delay the progression of vision loss in RCS rats.
基金We would like to thank Dr.Anthony E.Sisk Jr.at UCLA Genitourinary Pathology Unit for histological analysis.This study is funded by National Institutes of Health(Grant No:RO1 DE DE025474,P50CA092131,and P50CA211015),and CIRM Stem Cell Genomics Centers of Excellence Award.
文摘Circulating cell-free DNAs(cfDNAs)are fragmented DNA molecules released into the blood by cells.Previous studies have suggested that mitochondria-originated cfDNA fragments(mt-cfDNAs)in cancer patients are more fragmented than those from healthy controls.However,it is still unknown where these short mtcfDNAs originate,and whether the length of mt-cfDNAs can be correlated with tumor burden and cancer progression.In this study,we first performed whole-genome sequencing analysis(WGS)of cfDNAs from a human tumor cell line-xenotransplantation mouse model and found that mt-cfDNAs released from transplanted tumor cells were shorter than the mouse counterpart.We next analyzed blood cfDNA samples from hepatocellular carcinoma and prostate cancer patients and found that mt-cfDNA lengths were inversely related to tumor size as well as the concentration of circulating tumor DNA.Our study suggested that monitoring the size of mt-cfDNAs in cancer patients would be a useful way to estimate tumor burden and cancer progression.
基金supported by National Program on Key Basic Research Project(2015CB964601,2015CB964702)Joint Research Fund for Overseas Chinese,Hong Kong and Macao Young Scholars(31428016)+3 种基金National Natural Science Foundation of China(Key Program 81430026)Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry(Xianmin Zhu)Shanghai Municipal Commission of Health and Family Planning(XBR2013094)Jiangsu Science and Technology Planning Project(BM2014052)
文摘Fragile X syndrome(FXS) patients carry the expansion of over 200 CGG repeats at the promoter of fragile X mental retardation 1(FMR1), leading to decreased or absent expression of its encoded fragile X mental retardation protein(FMRP). However, the global transcriptional alteration by FMRP deficiency has not been well characterized at single nucleotide resolution, i.e., RNA-seq. Here,we performed in-vitro neuronal differentiation of human induced pluripotent stem(iPS) cells that were derived from fibroblasts of a FXS patient(FXS-iPSC). We then performed RNA-seq and examined the transcriptional misregulation at each intermediate stage during in-vitro differentiation of FXS-iPSC into neurons. After thoroughly analyzing the transcriptomic data and integrating them with those from other platforms, we found up-regulation of many genes encoding TFs for neuronal differentiation(WNT1, BMP4,POU3F4, TFAP2 C, and PAX3), down-regulation of potassium channels(KCNA1, KCNC3, KCNG2, KCNIP4, KCNJ3, KCNK9,and KCNT1) and altered temporal regulation of SHANK1 and NNAT in FXS-iPSC derived neurons, indicating impaired neuronal differentiation and function in FXS patients. In conclusion, we demonstrated that the FMRP deficiency in FXS patients has significant impact on the gene expression patterns during development, which will help to discover potential targeting candidates for the cure of FXS symptoms.
基金This work was supported by ＂973＂ Programs （Nos. 2012CB966300, 2011CB966204 and 2011CB965102） from the Ministry of Science and Technology in China, the International Science and Technology Cooperation Program of China （No. 2011DFB30010）, the National Natural Science Foundation of China （Grant Nos. 81271258, 31301184）, and the Natural Science Foundation of Jiangsu Province of China （No. DK2011321）.
文摘The newly developed next-generation sequencing platforms, in combination with gcnome-scale amplification methods, provide a powerful tool to study genomics from a single cell. This mini-review summarizes the technologies of single cell genomics and their applications in several areas of biomedical research including stem cells, cancer biology and reproductive medicine. Particularly, it highlights recent advances in single cell exome sequencing, RNA-seq, and genome sequencing. The application of these powerful techniques will shed new light on the fundamental principles of gene transcription and genome organization at single-cell level and improve our understanding of cellular heterogeneity and diversity in multicellular organisms
