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Background Retinal pigment epithelium (RPE) cell transplantation is the primary means of human trial for the treatment of retinal degeneration.Induced pluripotent stem cells (iPSCs) will become an important source for cell transplantation.In addition,iPS-RPE cells may provide a personalized treatment platform for the patient's own cells treatment.Objective This study was to evaluate the feasibility of human fibroblasts differentiate toward iPSCs from retinitis pigmentosa (RP) patients and toward iPSC-RPE cells from non-RP individual by retroviral transfection of Oct4,Sox2,c-Myc and KLF4 genes.Methods Human thigh skin tissues were obtained from a RP patient with hotspot mutation of SNRNP200 p.S1087L and individual without SNRNP200 p.S1087L mutation,respectively,with the size 2 c m×3 cm.Human dermal fibroblasts were isolated and cultured by trypsin digestion and explant method.The fibroblasts were transfected by a series of retrovirus and cultured by human embyonic cellconditioned medium to generate and induce iPSCs,and then the iPSCs were identified by morphology,alkaline phosphatase (AP) staining and immunofluorescence assay of specific markers of pluripotent stem cells.iPSCs suspension were injected into SCID mouse to observe the tumorigenesis.The iPSCs from non-RP subject were induced to differentiate toward iPS-RPE cells by embryonic body (EB) inducing method,and iPS-RPE cells were identified by detecting the expression of RPE65,zonula occludens protein 1 (ZO-1) and lecithin retinol acyltransferase (LRAT).Results Cultured human dermal fibroblasts showed fusiform or polygon shape and intercellular close arrangement,and Vimentin was positively expressed in the cells.Small cell colonies were harvested 5-7 days after infected by retroviruses,and the morphology changed from spindle into round mass.The hESC-like iPSCs clonies appeared 20 days after cultivation,and the positive expressions of hESC-specific surface antigens including SSEA3,SSEA4,TRA-1-60,TRA-1-81 and Nanog were found in the cells 25-30 days after cultivation,and the positive staining of AP was obtained 12 weeks after cultivation.A teratoma was formed at the injection site of iPSCs suspension in SCID mouse.Immunofluorescence technique showed that RPE cell-specific proteins including RPE65,ZO-1 and LRAT proteins were positively expressed in iPS-RPE cells at 30 days after differentiation.Conclusions Mutation SNRNP200 p.S1087L of RP patient-specific iPSCs can be induced from human dermal fibroblast by retrovirus infection method.The function and morphology of the iPSCs were similar to hESCs.Human iPSCs cell line generated from the dermal fibroblasts of non-RP individuals can differentiate into iPS-RPE cells.
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Keratoplasty is a choice for the treatment of ocular surface diseases caused by corneal limbal stem cells (LSCs) deficiency.The application of traditional keratoplasty is limited by avaibility of donor corneas and allograft rejection.Constructruction of tissue engineering corneal epithelium provides an important and effective approach to the transplantation of cornea,because it can solve the lack of donor corneas and avoid allograft rejection following keratoplasty.However,the selection of the seed cells is crucial to corneal tissue engineering.What is more,the research of seed cells is becoming more and more widespread,just like embryonic stem cells (ESCs) and LSCs.This article summarized the selection of seed cells and the progress of tissue-engineered human corneal epithelium.
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Background Development of corneal tissue engineering creates a new therapeutic method for severe corneal diseases.However,ideal seed cells and scaffold for corneal surface reconstruction have not yet been investigated well.Adipose-derived stem cells (ADSCs) are varified to have a self-renewal ability and epithelioid features,and temperature-responsive scaffolds (TRSs) can offer technical support for stem cell sheet.Objective This study was to investigate the characteristics of ADSCs cultured on TRSs and compare these features to typical oral mucosal epithelial cells (OMECs),and therefore to explore the feasibility of reconstruction of ocular surface with ADSCs as seed cells.Methods Self-made TRSs were prepared by adding isopropyl alcohol dissolved poly-Nisopropylacrylamide (PNIPAAm) to each polystyrene tissue culture dish and then irradiating using an election beam.Subcutaneious fatty tissue of rabbit neck was obtained to culture ADSCs,and 4 pieces of oral cavity mucosal tissue were digested and cultured to obtain OMECs.Then the ADSCs and OMECs were incubated on TRSs,and cell morphology,growth rate,detached duration and survival counts were compared between ADSCs and OMECs.The ADSCs sheet and OMECs sheet were stained with hematoxylin and eosin for morphological examination.Immunochemistry was used to observe the expressions of stem-cell biomakers and epithelioid-cell biomakers in the cells.The ultrastructure of cell surface was observed under the scanning electron microscope.Results Self-made TRSs were similar to ordinary culture dish in the transparancy and smoothness.The water contact angle of 4 in 5 samples were ＞10° with the effective rate upto 80％.A DSCs showed the elongated fusiform in shape,while OMECs showed a cobblestone appearance.The growth cycle,detached duration and cell number of ADSCs were 12-14 days,(46.0 ±9.6) minutes and (7.9 ±1.1)×105/sheet,and those of OMECs were 14-16 days,(91.9 ±10.9) minutes and (45.8 ±26.5)×105/sheet,respectively,showing statistically significant differences in the detached duration and cell counts between ADSCs and OMECs (P=0.002,0.028).Hematoxylin and eosin staining showed that ADSCs sheet comprised only 1-3 layer cells,while OMECs showed 4-5 layer cells.ATP-binding cassette superfamily G member 2 (ABCG2),p63 and cytokeratin 12 (CK12) were positively expressed in both ADSCs sheet and OMECs sheet.Closely packed cells and typical eithelial microvilli in the cell surface were exhibited in both ADSCs sheet and OMECs sheet under the scanning electron microscope.Conclusions Self-made TRSs can be used as scaffold of ADSCs.The ADSCs sheet on the TRSs appears to have a good cell vitality and therefore is a new seed source of ocular surface reconstruction.
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Objective To evaluate the feasibility of transfection of Sonic hedgehog gene (SHH)into bone marrow mesenchymal stem cells(BMMSC).Methods After the SHH gene was transfected into BMMSC by electroporation apparatus,the transfection rate was evaluated by fluorescence inverted microscope.The growth curves of untransfected and transfected BMMSC were drawn,respectively,to observe the influence of transfection on cells.The expression of SHH gene in the BMMSC was detected by PCR,RT-PCR,Western-blot analyses.Results Through fluorescence inverted microscope,the observed transfection rate was appropriately 30％,PCR showed a obvious increase of SHH expression in transfected cells than that in untransfected cells,and it is quantified by qPCR for appropriately 7 times.Western-blot further demonstrated that the SHH protein expression in transfected cells had a distinct increase.However,it was observed that the exponential phase of BMMSCSHH growth curve delayed.The growth curves of both overlap 12 days after transfection.Conclusions This electroporation method can transfect exogenous SHH gene into BMMSC sufficiently with the effective protein expression in BMMSCSHH.It is the foundation of further research of genetic therapy for ischemic heart disease.
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Objective To explore the experimental conditions of labeling rabbit bone marrow mesenchymal stem cells (Rb-MSCs) with 5,6-carboxyfluorescein diacetic succinimidyl ester (CFSE) and to investigate the impact on the biological characteristics of Rb-MSCs in vitro.Methods Rb-MSCs were separated and purified by whole bone marrow adherent culture and then were identified by morphology and surface markers.Rb-MSCs were labeled with CFSE and the labeling effect was measured by flow cytometer.The proliferation capacity of labeled cells was detected with CCK-8.The differentiation capacity of labeled cells was investigated by being induced to osteoblasts and lipoblasts.The capacity of labeled cells to secret vascular endothelial growth factor (VEGF) was detected by VEGF ELISA kits.Results The primary Rb-MSCs adhered in 48 h,being fusiform and colony-like growth.Subculture cells became fibroblast-like cells in order with uniform configuration.Most (above 98％) cultured cells expressed the surface markers CD29 and CD44 except for CD45.Compared with other labeling conditions,10μmol/L final concentration of CFSE and 10 min was the best one with a 100％ labeling rate and high fluorescence intensity.Compared with unlabeled cells,the ability of the labeled cells to proliferate and to secrete VEGF was not significantly decreased (P ＞ 0.05).Moreover,the labeled cells had osteogenic and adipogenic differentiation capacity.Conclusions It was a simple and efficient method to label Rb-MSCs with CFSE,especially in a short-term.The capacity of cell proliferation,differentiation,and secretion were not affected.
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Objective To observe the effects of subretinal transplantation of rat mesenchymal stem cells (rMSCs) on Sodium Iodate (SI)-induced retinal degeneration.Methods One hundred and twenty Brown-Norway (BN) rats were divided into three groups including SI injection group,rMSCs transplantation group and normal control group,each with 40 rats.The retinal degeneration was induced by caudal vein injection of SI.The retinal pigment epithelium(RPE)and neural retinal were evaluated by ocular fundus photograph,fluorescein fundus angiography (FFA),electroretinogram (ERG) and histological approach,and TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling).CM-Dilprelabeled primary rMSCs were transplanted into the subretinal space of Sl-induced rats.The survival,integration,and differentiation of rMSCs were observed between 14 day to 60 day after the transplantation.Results The rat retinal function was gradually reduced afterl4 days of SI injection,with a time-dependent manner.After the RPE cells were damaged,the outer segments of photoreceptors became disrupted and shortened until karyopyknosis.The nuclear morphology and positive TUNEL labeling indicated that the death of photoreceptor cells was apoptosis.After rMSCs transplantation,CM-DiI labeled donor cells were observed to be scattered in the subretinal space and expressed RPE cell markers.Average amplitude of bwave and Ops (oscillation potential) in ERG improved 27.80%,59.38% respectively after rMSCs transplantation.Conclusions Transplanted rMSCs can survive in subretinal space and differentiate into RPE cells,thus cure SI- induced retinal degeneration.
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Replacement therapy of stem cells transplantation represents a potential treatment for neural retinal diseases.Despite the encouraging results in laboratory,the clinical application of cells replacement therapy is still difficult because the limitation of seed cells,immunologic rejection,oncogenicity and ethical problems,etc.Recent breakthrough in somatic reprogramming provides a promising solution overcoming these obstacles.Further researches on virus-free reprogramming will make the clinical application of stem cell replacement therapy possible.
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Objective To investigate the feasibility of differentiation of invitro induced rat bone marrow-derived mesenchymal stem cells(rMSCs) into retinal pigment epithelial (RPE) cells.Methods The rMSCs from Brwon-Norway (BN) rats were isolated and cultured by adherent screening method.RPE cells lysate made by repeated freeze-thawing was put into the rMSCs culture system to identify whether the induced cells could express characteristic label cytokeratin(CK)and S-100 simultaneously or not.Results The growth rate of rMSCs induced by RPE cells lysate was slower and protuberant burr surrounded the fusiform cells.The results of immunoblotting and double immunofluorescence showed that partial induced cells expressed CK and S-100 simultaneously.The result of flow cytometry indicated that 14.1% induced cells expressed CK and S-100 simultaneously.Conclusion Induced by RPE cells lysate,rMSCs can differentiate into RPE cells.
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Objective To observe the survival of human umbilical cord derived mesenchymal stem cells (hUC-MSCs) after injection into the vitreous of rabbits,and the animal safety under those procedures.Methods Twenty-seven pigmented rabbits were randomly divided into 3 groups (intravitreal injection 1 week group,2 weeks group and 4 weeks group),each with 9 rabbits.For each animal the right eye was the experimental eye receiving hUC-MSCs injection,while the left eye was the control eye receiving cuhure medium.The rabbit eyes were examined by slit-lamp microscope,indirect ophthalmoscopy,fundus photography,fundus fluorescence angiography(FFA)and Tono-pen tonometer before and after injection.hUC-MSCs were labeled by CM-Dil in vitro,and their survival status was measured by eonfocal fluorescence microscopy,light microscope and transmission electron microscope at 4 weeks after injection.Results Four weeks after injection,a large number of the hUC-MSCs were still alive in the vitreous cavity.The overall condition of those rabbits was good.The anterior segment and retina of experimental eyes were normal,without hyperfluorescence,hypofluorescenee and leakage in the retina at 1,2 and 4 weeks after injection.There was no significant difference on lOP before and after injection at different time points (P>0.05),and no obvious changes at cornea,anterior chamber angle,lens,retinal structure by.light microscope and transmission electron microscope examination.Conclusion hUC-MSCs can survive in the rabbit vitreous for four weeks;intravitreal injection of hUC-MSCs was safe and feasible.