Human mesenchymal stem cells,such as human adipose-derived stem cells(hASCs),are typically cultured on a two-dimensional(2 D)monolayer material surface,on which 2 D culturing methods are easily performed and time-savi...Human mesenchymal stem cells,such as human adipose-derived stem cells(hASCs),are typically cultured on a two-dimensional(2 D)monolayer material surface,on which 2 D culturing methods are easily performed and time-saving.However,hASCs usually suffer from decreased pluripotency and differentiation ability when cultured with a 2 D monolayer culturing method compared to hASCs cultured with a three-dimensional(3 D)culturing method,such as suspension culture.In this study,we evaluated whether the pluripotency and differentiation ability of hASCs can be reversibly changed during sequential cultivation with 2 D and 3 D culturing processes.The hASCs cultivated with a 3 D culturing process after 2 D culture showed at least 2-fold enhanced pluripotency(Sox2,Nanog,and OCT4)compared with that of hASCs cultured with the 2 D culture process alone.Furthermore,hASCs obtained from the 3 D culture process expressed increased levels of differentiation markers of chondrocytes and osteoblasts compared with hASCs obtained from the 2 D culture process when hASCs were induced to differentiate.However,their pluripotency and differentiation ability were extensively reduced when hASCs were shifted from 3 D culture to 2 D culture and vice versa,which indicates that hASCs show reversibility in terms of their pluripotency and differentiation ability depending on their environment in 2 D and 3 D culture.The reversibility of pluripotency and differentiation ability were found to last for at least 5 passages in culture during the alternative and sequential culture of cells with 2 D and 3 D culturing processes.Our study revealed the importance of the culture microenvironment in maintaining the pluripotency and differentiation ability of hASCs,which may reduce the effects of the aging process in hASCs.We discuss whether the environment of stem cell culture(i.e.,2 D or 3 D cultivation)can affect stem cell fate in terms of pluripotency and differentiation reversibility.展开更多
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
Fibroblast growth factor-2(FGF-2)is a critical molecule for sustaining the pluripotency of human pluripotent stem(PS)cells.However,FGF-2 is extremely unstable and cannot be stored long periods at room temperature.Ther...Fibroblast growth factor-2(FGF-2)is a critical molecule for sustaining the pluripotency of human pluripotent stem(PS)cells.However,FGF-2 is extremely unstable and cannot be stored long periods at room temperature.Therefore,the following FGF-2-conjugated cell culture materials were developed to stabilize FGF-2:FGF-2-conjugated polyvinyl alcohol(PVAI-C-FGF)hydro-gels and FGF-2-conjugated carboxymethyl cellulose-coated(CMC-C-FGF)dishes.Human induced pluripotent stem(iPS)cells were proliferated on recombinant vitronectin(rVN)-coated PVAI-C-FGF hydrogels and CMC-C-FGF dishes in medium without FGF-2.Human iPS cells could not be cultivated on rVN-coated PVAI-C-FGF hydrogels for more than two passages but could proliferate on rVN-coated CMC-C-FGF dishes.These results indicated that the amount of immobilized FGF-2 and the base cell materials are important,including the amount of immobilized rVN and the conformation of FGF-2 on the surfaces.When human iPS cells were pro-liferated on rVN-coated CMC-C-FGF surfaces in medium containing no FGF-2 for 10 passages,their pluripotency and potential to differen-tiate into cells originating from three germ layers were maintained in vivo and in vitro.Furthermore,the cells could extensively differentiate into cardiomyocytes,which can be used for cardiac infarction treatment in future and retinal pigment epithe-lium for retinal pigmentosa treatment in future.The FGF-2-immobilized surface could enable human PS cell culture in medium that does not need to contain unstable FGF-2.The amount of FGF-2 immobilization on the rVN-coated CMC-C-5FGF and CMC-C-20FGF dishes was reduced to 93.6 and 52.2 times,respectively,which is less than the conventional amount of FGF-2 used in culture medium for one passage(6 days)of human iPS cell culture.This reduction resulted from the stabilization of unstable FGF-2 by the immobilization of FGF-2,which was achieved by utilizing optimal base materials(CMC),coating materials(rVN)and long-joint segment(PEG4-SPDP)design.展开更多
Lipid nanoparticles(LNPs)are critical for the delivery of drugs and nucleic acids.However,current mRNA-LNP formulations require stringent freezing for storage,which limits their global distribution.Our previous studie...Lipid nanoparticles(LNPs)are critical for the delivery of drugs and nucleic acids.However,current mRNA-LNP formulations require stringent freezing for storage,which limits their global distribution.Our previous studies demonstrated that optimizing the lipid type or molar ratio of Comirnaty-type mRNA-LNPs could enhance their lyophilization stability,thus improving their long-term storage stability under mild conditions.This study aims to enhance the storage stability of Spikevax-type mRNA-LNPs by optimizing lipid compositions and utilizing lyophilization for storage at 4�C.Fifteen mRNA-LNP formulations were evaluated for their physicochemical properties and transfection efficiency(TE)in human embryonic kidney(HEK)-293T cells using the I-optimal design of mixture experiments.Mathematical models were developed to predict the relationships among encapsulation efficiency,transfection performance and lipid ratios.The optimized mRNA-LNP formulation(N4),with a 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)-to-cholesterol ratio of 0.36,exhibited superior stability and TE after lyophilization.N4 outperformed the original Spikevax formulation in several cell lines,including eye-derived ARPE-19 cells and lung-derived A549 cells.In vivo,N4 demonstrated high TE in the spleen of C57BL/6 mice both before and after lyophilization,with no signals observed in the kidneys,heart or eyes.These findings suggest that the optimized N4 formulation offers a robust,stable and efficient delivery system for gene therapy and vaccines,potentially overcoming the storage limitations of current Spikevax-type mRNA-LNPs and broadening their therapeutic applications.展开更多
Due to the limitations in autogenous nerve grafting or Schwann cell transplantation,large gap peripheral nerve injuries require a bridging strategy supported by nerve conduit.Cell based therapies provide a novel treat...Due to the limitations in autogenous nerve grafting or Schwann cell transplantation,large gap peripheral nerve injuries require a bridging strategy supported by nerve conduit.Cell based therapies provide a novel treatment for peripheral nerve injuries.In this study,we first experimented an optimal scaffold material synthesis protocol,from where we selected the 10%GFD formula(10%GelMA hydrogel,recombinant human basic fibroblast growth factor and dental pulp stem cells(DPSCs))to fill a cellulose/soy protein isolate composite membrane(CSM)tube to construct a third generation of nerve regeneration conduit,CSM-GFD.Then this CSM-GFD conduit was applied to repair a 15-mm long defect of sciatic nerve in a rat model.After 12 week post implant surgery,at histologic level,we found CSM-GFD conduit could regenerate nerve tissue like neuron and Schwann like nerve cells and myelinated nerve fibers.At physical level,CSM-GFD achieved functional recovery assessed by a sciatic functional index study.In both levels,CSM-GFD performed like what gold standard,the nerve autograft,could do.Further,we unveiled that almost all newly formed nerve tissue at defect site was originated from the direct differentiation of exogeneous DPSCs in CSM-GFD.In conclusion,we claimed that this third-generation nerve regeneration conduit,CSM-GFD,could be a promising tissue engineering approach to replace the conventional nerve autograft to treat the large gap defect in peripheral nerve injuries.展开更多
The authors regret that the author(A.H.)was removed as an agreement could not be reached between the two affiliations of the author.The authors would like to apologise for any inconvenience caused.
基金the Researchers supporting Project number(RSP-2019/98)King Saud University,Riyadh,Saudi Arabia。
文摘Human mesenchymal stem cells,such as human adipose-derived stem cells(hASCs),are typically cultured on a two-dimensional(2 D)monolayer material surface,on which 2 D culturing methods are easily performed and time-saving.However,hASCs usually suffer from decreased pluripotency and differentiation ability when cultured with a 2 D monolayer culturing method compared to hASCs cultured with a three-dimensional(3 D)culturing method,such as suspension culture.In this study,we evaluated whether the pluripotency and differentiation ability of hASCs can be reversibly changed during sequential cultivation with 2 D and 3 D culturing processes.The hASCs cultivated with a 3 D culturing process after 2 D culture showed at least 2-fold enhanced pluripotency(Sox2,Nanog,and OCT4)compared with that of hASCs cultured with the 2 D culture process alone.Furthermore,hASCs obtained from the 3 D culture process expressed increased levels of differentiation markers of chondrocytes and osteoblasts compared with hASCs obtained from the 2 D culture process when hASCs were induced to differentiate.However,their pluripotency and differentiation ability were extensively reduced when hASCs were shifted from 3 D culture to 2 D culture and vice versa,which indicates that hASCs show reversibility in terms of their pluripotency and differentiation ability depending on their environment in 2 D and 3 D culture.The reversibility of pluripotency and differentiation ability were found to last for at least 5 passages in culture during the alternative and sequential culture of cells with 2 D and 3 D culturing processes.Our study revealed the importance of the culture microenvironment in maintaining the pluripotency and differentiation ability of hASCs,which may reduce the effects of the aging process in hASCs.We discuss whether the environment of stem cell culture(i.e.,2 D or 3 D cultivation)can affect stem cell fate in terms of pluripotency and differentiation reversibility.
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 under grant numbers 112-2923-E-008-005-MY3,111-2221-E-008-007 and 111-2923-E-008-005-MY3.
文摘Fibroblast growth factor-2(FGF-2)is a critical molecule for sustaining the pluripotency of human pluripotent stem(PS)cells.However,FGF-2 is extremely unstable and cannot be stored long periods at room temperature.Therefore,the following FGF-2-conjugated cell culture materials were developed to stabilize FGF-2:FGF-2-conjugated polyvinyl alcohol(PVAI-C-FGF)hydro-gels and FGF-2-conjugated carboxymethyl cellulose-coated(CMC-C-FGF)dishes.Human induced pluripotent stem(iPS)cells were proliferated on recombinant vitronectin(rVN)-coated PVAI-C-FGF hydrogels and CMC-C-FGF dishes in medium without FGF-2.Human iPS cells could not be cultivated on rVN-coated PVAI-C-FGF hydrogels for more than two passages but could proliferate on rVN-coated CMC-C-FGF dishes.These results indicated that the amount of immobilized FGF-2 and the base cell materials are important,including the amount of immobilized rVN and the conformation of FGF-2 on the surfaces.When human iPS cells were pro-liferated on rVN-coated CMC-C-FGF surfaces in medium containing no FGF-2 for 10 passages,their pluripotency and potential to differen-tiate into cells originating from three germ layers were maintained in vivo and in vitro.Furthermore,the cells could extensively differentiate into cardiomyocytes,which can be used for cardiac infarction treatment in future and retinal pigment epithe-lium for retinal pigmentosa treatment in future.The FGF-2-immobilized surface could enable human PS cell culture in medium that does not need to contain unstable FGF-2.The amount of FGF-2 immobilization on the rVN-coated CMC-C-5FGF and CMC-C-20FGF dishes was reduced to 93.6 and 52.2 times,respectively,which is less than the conventional amount of FGF-2 used in culture medium for one passage(6 days)of human iPS cell culture.This reduction resulted from the stabilization of unstable FGF-2 by the immobilization of FGF-2,which was achieved by utilizing optimal base materials(CMC),coating materials(rVN)and long-joint segment(PEG4-SPDP)design.
基金supported by the Zhejiang Provincial Natural Science Foundation of China(LQ24H120006)the Wenzhou Municipal Science and Technology Bureau(Y2023799)+3 种基金the Startup Foundation for Scientific Research,Eye Hospital,Wenzhou Medical University(KYQD20211204)the National Natural Science Foundation of China(52250710155)the National Key Research and Development Program of China(2021YFA1101200,2022YFA1105501)the Project of State Key Laboratory of Ophthalmology,Optometry and Visual Science,the Wenzhou Medical University(J02-20210201).
文摘Lipid nanoparticles(LNPs)are critical for the delivery of drugs and nucleic acids.However,current mRNA-LNP formulations require stringent freezing for storage,which limits their global distribution.Our previous studies demonstrated that optimizing the lipid type or molar ratio of Comirnaty-type mRNA-LNPs could enhance their lyophilization stability,thus improving their long-term storage stability under mild conditions.This study aims to enhance the storage stability of Spikevax-type mRNA-LNPs by optimizing lipid compositions and utilizing lyophilization for storage at 4�C.Fifteen mRNA-LNP formulations were evaluated for their physicochemical properties and transfection efficiency(TE)in human embryonic kidney(HEK)-293T cells using the I-optimal design of mixture experiments.Mathematical models were developed to predict the relationships among encapsulation efficiency,transfection performance and lipid ratios.The optimized mRNA-LNP formulation(N4),with a 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)-to-cholesterol ratio of 0.36,exhibited superior stability and TE after lyophilization.N4 outperformed the original Spikevax formulation in several cell lines,including eye-derived ARPE-19 cells and lung-derived A549 cells.In vivo,N4 demonstrated high TE in the spleen of C57BL/6 mice both before and after lyophilization,with no signals observed in the kidneys,heart or eyes.These findings suggest that the optimized N4 formulation offers a robust,stable and efficient delivery system for gene therapy and vaccines,potentially overcoming the storage limitations of current Spikevax-type mRNA-LNPs and broadening their therapeutic applications.
基金supported by the National Natural Science Funding of China(81701032,81871503)the Wenzhou Science and Technology Association Project,the Wenzhou Major Scientific and Technological Innovation Key Medical and Health Project(ZY2019010)+4 种基金the Wenzhou Medical University grant(QTJ16026)Wenzhou Science and Technology Association Project,Wenzhou Basic Research Project(Y20180131)Zhejiang Province Program of the Medical and Health Science and Technology(2018KY537)Zhejiang Natural Science Foundation(LGF18C100002)Zhejiang Xinmiao Talents Program(2018R413186).
文摘Due to the limitations in autogenous nerve grafting or Schwann cell transplantation,large gap peripheral nerve injuries require a bridging strategy supported by nerve conduit.Cell based therapies provide a novel treatment for peripheral nerve injuries.In this study,we first experimented an optimal scaffold material synthesis protocol,from where we selected the 10%GFD formula(10%GelMA hydrogel,recombinant human basic fibroblast growth factor and dental pulp stem cells(DPSCs))to fill a cellulose/soy protein isolate composite membrane(CSM)tube to construct a third generation of nerve regeneration conduit,CSM-GFD.Then this CSM-GFD conduit was applied to repair a 15-mm long defect of sciatic nerve in a rat model.After 12 week post implant surgery,at histologic level,we found CSM-GFD conduit could regenerate nerve tissue like neuron and Schwann like nerve cells and myelinated nerve fibers.At physical level,CSM-GFD achieved functional recovery assessed by a sciatic functional index study.In both levels,CSM-GFD performed like what gold standard,the nerve autograft,could do.Further,we unveiled that almost all newly formed nerve tissue at defect site was originated from the direct differentiation of exogeneous DPSCs in CSM-GFD.In conclusion,we claimed that this third-generation nerve regeneration conduit,CSM-GFD,could be a promising tissue engineering approach to replace the conventional nerve autograft to treat the large gap defect in peripheral nerve injuries.
文摘The authors regret that the author(A.H.)was removed as an agreement could not be reached between the two affiliations of the author.The authors would like to apologise for any inconvenience caused.
