首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epidermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immunohistology and RT-PCR were conducted to identify the expression of specific markers (β1m α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epidermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being cocultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas. Supported in part by Hi-tech Research and Development Program of China (Grant No. 2003AA205005), the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP, No.20030558074), the Key Technologies Research and Development Programme of the Tenth Five-Year Plan (Grant No. 2004BA720A15), Scientific and Technological Program (Grant Nos. A3020101 and 2003A3020401) of Guangdong Province  相似文献   

2.
Adult stem cells are important cell sources in regenerative medicine, but isolating them is technically challenging. This study employed a novel strategy to generate stem-like corneal epithelial cells and promote the functional properties of these cells by coculture with embryonic stem cells. The primary corneal epithelial cells were labelled with GFP and cocultured with embryonic stem cells in a transwell or by direct cell-cell contact. The embryonic stem cells were pre-transfected with HSV-tk-puro plasmids and became sensitive to ganciclovir. After 10 days of coculture, the corneal epithelial cells were isolated by treating the cultures with ganciclovir to kill the embryonic stem cells. The expression of stem cell-associated markers (ABCG2, p63) increased whereas the differentiation mark (Keratin 3) decreased in corneal epithelial cells isolated from the cocultures as evaluated by RT-PCR and flow cytometry. Their functional properties of corneal epithelial cells, including cell adhesion, migration and proliferation, were also enhanced. These cells could regenerate a functional stratified corneal epithelial equivalent but did not form tumors. Integrin β1, phosphorylated focal adhesion kinase and Akt were significantly upregulated in corneal epithelial cells. FAK Inhibitor 14 that suppressed the expression of phosphorylated focal adhesion kinase and Akt inhibited cell adhesion, migration and proliferation. LY294002 that suppressed phosphorylated Akt but not phosphorylated focal adhesion kinase inhibited cell proliferation but had no effect on cell adhesion or migration. These findings demonstrated that the functional properties of stem-like corneal epithelial cells were enhanced by cocultured embryonic stem cells via activation of the integrin β1-FAK-PI3K/Akt signalling pathway.  相似文献   

3.
Stem/progenitor cells of the human corneal epithelium are present in the human corneal limbus, and several corneal epithelial stem/progenitor cell markers have been reported. Recently, the neurotrophin family receptors were reported to be useful markers of corneal epithelial stem/progenitor cells. Therefore, we examined an enzymatic separation method for obtaining corneal epithelial stem/progenitor cells and measuring the change in the expression of low-affinity neurotrophin receptor p75 (p75NTR), a receptor belonging to the neurotrophin family. As a result, it was found that our separation method preserved cell viability. Furthermore, p75NTR was mainly observed in epithelial basal cells as were the corneal epithelial stem/progenitor markers p63 and integrin β1. p75NTR was also observed in the cultured cells, but its frequency decreased with passage. In conclusion, we propose that our culture method will enable the culture of corneal stem cells and that it is a useful tool for elucidating the molecular basis of the niche that is necessary for the maintenance of epithelial stem cells in the corneal limbus. Furthermore, we conclude that p75NTR is a useful cell marker for evaluating the characteristics of stem/progenitor cells in culture.  相似文献   

4.
Several types of adult stem cells are capable of transdifferentiaton into other types of tissues. The hair follicle bulge area is an abundant and easily accessible source of pluripotent adult stem cells. We demonstrate that the bulge KSCs have the potential for transdifferentiation into corneal epithelial-like cells. Bulge KSCs isolated by collagen type IV adhesiveness possessed the highest colony formation efficiency (CFE), and expressed specific markers (CD34 and α6-integrin). The isolated cells transdifferentiate into corneal epithelial-like cells in conditioned medium containing corneal limbus soluble factors, including their specific marker, keratin12. The transdifferentiation depends on upregulation of pax6 and downregulation of β-catenin and Lef-1. Furthermore, overexpression of pax6 in bulge KSCs induced their expression of k12. The expressions of β-catenin and Lef-1 were not suppressed in the pax6-transfected bulge KSCs, but which were downregulated pax6-transfected cells cultured in the conditioned medium. Bulge KSCs may have potential therapeutic application as cell source for the construction of bioengineered corneas.  相似文献   

5.
Corneal epithelium transdifferentiation into a hair-bearing epidermis provides a particularly useful system for studying the possibility that transient amplifying (TA) cells are able to activate different genetic programs in response to a change in their fibroblast environment, as well as to follow the different steps of rebuilding an epidermis from induced stem cells. Corneal stem and TA cells are found in different locations - stem cells at the periphery, in the limbus, and TA cells more central. Moreover, the TA cells already express the differentiating corneal-type keratin pair K3/K12, whereas the limbal keratinocytes express the basal keratin pair K5/K14. In contrast, suprabasal epidermal keratinocytes express keratin pair K1-2/K10, and basal keratinocytes the keratin pair K5/K14. The results of tissue recombination experiments show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. First, the cells located at the base of the corneal epithelium show a decrease in expression of K12 keratin, followed by an increase in K5 expression; they then proliferate and form hair follicles. The first K10 expressing cells appear at the junction of the new hair follicles and the covering corneal epithelium. Their expansion finally gives rise to epidermal strata, which displace the corneal suprabasal keratinocytes. Corneal TA cells can thus be reprogrammed to form epidermal cells, first by reverting to a basal epithelial-type, then to hair pegs and probably concomitantly to hair stem cells. This confirms the role of the hair as the main reservoir of epidermal stem cells and raises the question of the nature of the dermal messages which are both involved in hair induction and stem cell specification.  相似文献   

6.
Regulation and clinical implications of corneal epithelial stem cells   总被引:7,自引:0,他引:7  
The corneal epithelium is known to have a rapid self-renewing capacity. The major advance in the field of cornead epithelial cell biology in the last decade is the establishment of the location of corneal epithelial stem cells at the limbus, i.e., the junctional zone between the cornea and the conjunctiva. This concept has helped explain several experimental and clinical paradoxes, produced a number of important clinical applications, and spawned many other research studies. This unique enrichment of epithelial stem cells at a site anatomically separated from their transient amplifying cells makes the ocular surface an ideal model to study the regulation of epithelial stem cells. The present review includes data from more recent studies and lays out other areas for future investigation, especially with respect to the role of apoptosis and cytokine dialogue between limbal epithelial stem cells and their stromal microenvironment.Abbreviations EGF epidermal growth factor - EGFR epidermal growth factor receptor - bFGF basic fibroblast growth factor - HGF hepatocyte growth factor - IGF-I insulin-like growth factor type I - IL-1 interleukin 1 - K3 or K12 keratin type 3 or 12 - KGF keratinocyte growth factor - LIF leukemia inhibitory factor - PDGF platelet-derived growth factor - PKC protein kinase C - TGF- transforming growth factor- - TGF- transforming growth factor- - TPA phorbol ester tumor promoting agents  相似文献   

7.
目的:观察小鼠角膜上皮祖细胞系TKE2在扩增以及分化状态下的角蛋白及干细胞标志物的表达情况。方法小鼠角膜上皮祖细胞系TKE2在无血清培养基Keratinocyte-SFM (KSFM)以及含10﹪胎牛血清(FBS)的DMEM培养基中培养,约70﹪融合时进行角蛋白10、12、14、15、16(K10、K12、K14、K15、K16)以及Connexin43、ABCG2的免疫荧光染色,以及Ki67、P63、PCNA的免疫细胞化学染色。结果无血清培养状态下的TKE2细胞呈克隆样生长,克隆内所有细胞呈ABCG2、K14、Ki67、PCNA以及P63阳性,K15阳性细胞散在分布,K16阳性细胞呈片状分布于克隆中央区,K10、K12以及Connexin43染色为阴性。在含有10﹪胎牛血清的DMEM中培养2 d后,细胞明显增大, ABCG2、K15、P63、Ki67以及PCNA转为阴性,克隆内只有少量细胞呈K16、K14阳性染色, K10、K12、Connexin43仍为阴性。结论 TKE2细胞具有角膜上皮干细胞特性,可以作为角膜缘上皮干细胞表型维持和分化诱导研究的良好工具。  相似文献   

8.
The corneal epithelial stem cell   总被引:4,自引:0,他引:4  
The aim of this paper was to develop a GFP-expressing transgenic mouse model for the keratoepithelioplasty and to use this to follow the outcome of this form of graft, when placed on an inflamed corneal surface. Further aims were to characterize both the graft and the epithelial surface of the mouse and rat cornea using putative stem cell markers (P63 and Telomerase) and marker of cell differentiation (14-3-3 sigma). Keratepithelioplasty was carried out using a GFP transgenic mouse cornea as donor tissue. Fluorescent epithelial outgrowth from each keratepithelioplasty was scored and quantified. Donor corneal graft tissue was obtained from the paracentral region or the anatomical limbal region of murine corneas. Paracentral donor grafts (n = 20) consistently demonstrated a significant increase in proliferative potential compared to grafts obtained from the anatomical limbal region of the mouse cornea (n = 25) (P = 0.000, Mann-Whitney U). Correspondingly, P63 expression was maximal in the paracentral region of the mouse cornea, in keeping with the demonstrated increased proliferative potential of donor grafts harvested from this region of the cornea. The murine corneal epithelium demonstrated decreased rather than increased cellular layers at the limbal region, in contrast to that of the rat or human epithelium. In addition, as a general finding in all species tested, there was an apparent increase noted in P63 expression in basal corneal epithelial cells in regions that had increased cellular layers (limbus in humans and rats and the paracentral corneal region in the mouse). Epithelium, which had migrated from donor grafts onto recipient corneas, retained P63 expression for the period of time examined (up to 3 days postengraftment). In addition, the conjunctival surface of an injured conjunctivalized displayed an abnormal pattern of P63 expression. Telomerase expression was widespread throughout many layers of both the murine and rat corneal epithelium. In the mouse and rat corneal epithelium P63 expression was maximal in areas of increased proliferative potential. Its expression, however, was not confined to stem cells alone. Migrating cells from transplanted keratoepithelial grafts retained P63 expression at least in the early stages post-transplantation. Finally, damaged conjunctivalized corneas displayed an abnormal P63 expression pattern when compared to either normal conjunctiva or normal cornea.  相似文献   

9.
In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. Epidermal stem cells represent a promising source of stem cells, and their culture has great potential in scientific research and clinical application. However, no single method has been universally adopted for identifying and isolating epidermal stem cells. Here, we reported the isolation and characterization of putative epidermal stem cells from newborn mouse skin. The keratinocytes were separated enzymatically. Putative epidermal stem cells were selected by rapid adherence on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded after 10 min, and the attached cells were cultured in a defined culture medium. The isolated cells showed the typical epidermal stem cell morphology. Immunofluorescence indicated that the cells were strongly stained for β1 integrin family of extracellular matrix receptors. In conclusion, mouse putative epidermal stem cells were successfully isolated from newborn mouse epidermis on the basis of high rapid adhesion to extracellular matrix proteins and cultured in vitro.  相似文献   

10.
Location and phenotype of human adult keratinocyte stem cells of the skin   总被引:32,自引:0,他引:32  
The location and identity of interfollicular epidermal stem cells of adult human skin remain undefined. Based on our previous work in both adult murine and neonatal human foreskin, we demonstrate that cell surface levels of the alpha6 integrin and the transferrin receptor (CD71) are valid markers for resolving a putative stem cell, transit amplifying and differentiating compartment in adult human skin by flow cytometry. Specifically, epidermal cells expressing high levels of alpha6 integrin and low levels of the transferrin receptor CD71 (phenotype alpha6 (bri)CD71(dim)) exhibit several stem cell characteristics, comprising a minor population (2%-5%) of the K14(bri) fraction, enriched for quiescent and small blast-like cells with high clonogenic capacity, lacking the differentiation marker K10. Conversely, the majority of K14(bri) K10(neg) epidermal cells express high levels of CD71 (phenotype alpha6 (bri)CD71(bri)), and represent the actively cycling fraction of keratinocytes displaying greater cell size due to an increase in cytoplasmic area, consistent with their being transient amplifying cells. The alpha6 (bri)CD71(bri) population exhibited intermediate clonogenic capacity. A third population of K14(dim) but K10 positive epidermal cells could be identified by their low levels of alpha6 integrin expression (i.e. alpha6 (dim) cells), representing the differentiation compartment; predictably, this subpopulation exhibited poor clonogenic efficiency. Flow cytometric analysis for the hair follicle bulge region (stem cell) marker K15 revealed preferential expression of this keratin in alpha6 (bri) cells (i.e., both stem and transient amplifying fractions), but not the alpha6 (dim) population. Given that K15 positive cells could only be detected in the deep rete ridges of adult skin in situ, we conclude that stem and transient amplifying cells reside in this location, while differentiating (K15 negative) cells are found in the shallow rete ridges.  相似文献   

11.
Corneal transplantation is the primary treatment option to restore vision for patients with corneal endothelial blindness. Although the success rate of treatment is high, limited availability of transplant grade corneas is a major obstacle. Tissue-engineered corneal endothelial grafts constructed using cultivated human corneal endothelial cells (hCENC) isolated from cadaveric corneas may serve as a potential graft source. Currently, tools for the characterization of cultured hCENC and enrichment of hCENC from potential contaminating cells such as stromal fibroblasts are lacking. In this study, we describe the generation and characterization of novel cell surface monoclonal antibodies (mAbs) specific for hCENC. These mAbs could be used for enrichment and characterization of hCENC. Out of a total of 389 hybridomas, TAG-1A3 and TAG-2A12 were found to be specific to the corneal endothelial monolayer by immunostaining of frozen tissue sections. Both mAbs were able to clearly identify hCENC with good ‘cobblestone-like’ morphology from multiple donors. The antigen targets for TAG-1A3 and TAG-2A12 were found to be CD166/ALCAM and Peroxiredoxin-6 (Prdx-6), respectively, both of which have not been previously described as markers of hCENC. Additionally, unlike other Prdx-6 mAbs, TAG-2A12 was found to specifically bind cell surface Prdx-6, which was only expressed on hCENC and not on other cell types screened such as human corneal stromal fibroblasts (hCSF) and human pluripotent stem cells (hPSC). From our studies, we conclude that TAG-1A3 and TAG-2A12 are promising tools to quantitatively assess hCENC quality. It is also noteworthy that the binding specificity of TAG-2A12 could be used for the enrichment of hCENC from cell mixtures of hCSF and hPSC.  相似文献   

12.
Ning J  Li C  Li H  Chang J 《Cytotechnology》2011,63(5):531-539
To determine the ability of cultured bone marrow-derived mesenchymal stem cells (BMSCs) to differentiate into functional urothelium. BMSCs were isolated from the long bones of aborted fetal limbs by Percoll density gradient centrifugation and characterized by flow cytometry. Human fetal urinary bladders were cut into small pieces and cultured for 3–5 days until the growth of urothelial cells was established. BMSCs were then cocultured with neonatal urothelial cells and subsequently evaluated for antigen expression and ultramicrostructure, by immunocytochemistry and electron microscopy, respectively. A subset of BMSCs expressed the differentiation marker CD71. The BMSC markers CD34, CD45, and HLA-DR were barely detectable, confirming that these cells were not derived from hematopoietic stem cells or differentiated cells. In contrast, the stem cell markers CD29, CD44, CD105, and CD90 were highly expressed. BMSCs possessed the ability to differentiate into a variety of cellular subtypes, including osteocytes, adipocytes, and chondrocytes. The shapes of BMSCs changed, and the size of the cells increased, following in vitro coculture with urothelial cells. After 2 weeks of coculture, immunostaining of the newly differentiated BMSCs positively displayed the urothelial-specific keratin marker. Electron microscopy revealed that the cocultured BMSCs had microstructural features characteristic of epithelial cells. Pluripotent BMSCs can transdifferentiate into urothelial cells in response to an environment conditioned by neonatal urothelial cells, providing a means for the time-, labor- and cost-effective reconstruction of urinary bladder mucosa.  相似文献   

13.
Corneal opacities are a leading cause of global blindness. They are conventionally treated by the transplantation of donor corneal tissue, which is, restricted by a worldwide donor material shortage and allograft rejection. Autologous adult stem cells with a potential to differentiate into corneal stromal keratocytes (CSKs) could offer a suitable choice of cells for regenerative cell therapy. Postnatal periodontal ligament (PDL) contains a population of adult stem cells, which has a similar embryological origin as CSK, that is cranial neural crest. We harvested PDL cells from young adult teeth extracted because of non‐functional or orthodontic reason and differentiated them towards CSK phenotype using a two‐step protocol with spheroid formation followed by growth factor and cytokine induction in a stromal environment (human amnion stroma and porcine corneal stroma). Our results showed that the PDL‐differentiated CSK‐like cells expressed CSK markers (CD34, ALDH3A1, keratocan, lumican, CHST6, B3GNT7 and Col8A2) and had minimal expression of genes related to fibrosis and other lineages (vasculogenesis, adipogenesis, myogenesis, epitheliogenesis, neurogenesis and hematogenesis). Introduction of PDL spheroids into the stroma of porcine corneas resulted in extensive migration of cells inside the host stroma after 14‐day organ culture. Their quiescent nature and uniform cell distribution resembled to that of mature CSKs inside the native stroma. Our results demonstrated the potential translation of PDL cells for regenerative corneal cell therapy for corneal opacities.  相似文献   

14.
Summary The specific performance of the adult hepatic parenchymal cell is maintained and controlled by factors deriving from the stromal bed; the chemical nature of these factors is unknown. This study aimed to develop a serum-free hierarchical hepatocyte-nonparenchymal (stromal) cell coculture system. Hepatic stromal cells proliferated on crosslinked collagen in serum-free medium with epidermal growth factor, basic fibroblast growth factor, and hepatocyte-conditioned medium; cell type composition changed during the 2-wk culture period. During the first wk, the culture consisted of proliferating sinusoidal endothelial cells with well-preserved sieve plates, proliferating hepatic stellate cells, and partially activated Kupffer cells. The number of endothelial cells declined thereafter; stellate cells and Kupffer cells became the prominent cell types after 8 d. Hepatocytes were seeded onto stromal cells precultured for 4–14 d; they adhered to stellate and Kupffer cells, but spared the islands of endothelial cells. Stellate cells spread out on top of the hepatocytes; Kupffer cell extensions established multiple contacts to hepatocytes and stellate cells. Hepatocyte viability was maintained by coculture; the positive influence of stromal cell signals on hepatocyte differentiation became evident after 48 h; a strong improvement of cell responsiveness toward hormones could be observed in cocultured hepatocytes. Hierarchial hepatocyte coculture enhanced the glucagon-dependent increases in phosphoenolpyruvate carboxykinase activity and messenger ribonucleic acid (mRNA) content three- and twofold, respectively; glucagon-activated urea production was elevated twofold. Coculturing also stimulated glycogen deposition; basal synthesis was increased by 30% and the responsiveness toward insulin and glucose was elevated by 100 and 55%, respectively. The insulin-dependent rise in the glucokinase mRNA content was increased twofold in cocultured hepatocytes. It can be concluded that long-term signals from stromal cells maintain hepatocyte differentiation. This coculture model should, therefore, provide the technical basis for the investigation of stroma-derived differentiation factors.  相似文献   

15.
Corneal lymphangiogenesis plays a key role in diverse pathological conditions of the eye. Here, we demonstrate that a versatile extracellular matrix protein, transforming growth factor‐β induced protein (TGFBIp), promotes lymphatic sprouting in corneal lymphangiogenesis. TGFBIp is highly up‐regulated in inflamed mouse corneas. Immunolocalization of TGFBIp is detected in infiltrating macrophages in inflamed mouse corneas. Subconjunctival injection of liposomal clodronate can significantly reduce macrophage infiltration in inflamed mouse cornea, and decrease the expression of TGFBIp and areas of corneal lymphangiogenesis and angiogenesis after corneal suture placement. In brief, these results indicate that the up‐regulation of TGFBIp in sutured cornea correlates with macrophage infiltration. Although TGFBIp alone cannot significantly stimulate corneal lymph vessel ingrowth in vivo, it can enhance the effect of vascular endothelial growth factor‐C in promoting corneal lymphangiogenesis. The in vitro results show that TGFBIp promotes migration, tube formation and adhesion of human lymphatic endothelial cells (HLECs), but it has no effect on HLECs' proliferation. We also find that the in vitro effect of TGFBIp is mediated by the integrin α5β1‐FAK pathway. Additionally, integrin α5β1 blockade can significantly inhibit lymphatic sprouting induced by TGFBIp. Taken together, these findings reveal a new molecular mechanism of lymphangiogenesis in which the TGFBIp‐integrin pathways plays a pivotal role in lymphatic sprouting.  相似文献   

16.
Zhang C  Chen P  Fei Y  Liu B  Ma K  Fu X  Zhao Z  Sun T  Sheng Z 《Aging cell》2012,11(1):14-23
Aged epidermal cells have the capacity to dedifferentiate into stem cell-like cells. However, the signals that regulate the dedifferentiation of aged epidermal cells remain unclear. Here, we provide evidence that Wnt/β-catenin is critical for aged epidermal cell dedifferentiation in vivo and in vitro. Some aged epidermal cells in human ultrathin epidermal sheets lacking basal stem cells transplanted onto wounds dedifferentiated into stem cell-like cells that were positive for CK19 and β1 integrin but negative for CK10. In addition, Wnt/β-catenin pathway was activated during this process. There was increased expression of Wnt-1, Wnt-4, Wnt-7a, β-catenin, cyclin D1, and c-myc. Secreted frizzled-related protein 1, a Wnt/β-catenin pathway inhibitor, blocked dedifferentiation in vivo. Then, the activator, a highly specific glycogen synthase kinase (GSK)-3β inhibitor, of Wnt/β-catenin pathway was added to the culture medium of aged epidermal cells. Surprisingly, we found that the activator induced higher expression of CK19, β1 integrin, Oct4, and Nanog proteins. The induced aged epidermal cells exhibited high colony-forming efficiency, long-term proliferative potential and could regenerate a skin equivalent (as do epidermal stem cells). These results suggested that activation of Wnt/β-catenin pathway induced the dedifferentiation of aged epidermal cells, which suggest a new approach to generate epidermal stem cell-like cells.  相似文献   

17.
18.
Plasticity of epidermal adult stem cells derived from adult goat ear skin   总被引:3,自引:0,他引:3  
Here we report the isolation and characterization of pluripotent stem cells from adult goat skin. We found that these primary cells have the properties of embryonic stem cells (ESC), including the expression of appropriate immunological markers and the capability of forming embryoid bodies. The subcultured cells also show the characteristics of stem cells, such as the expression of CK19, beta(1-)integrin, P63, and formation of holo-clones in culture. Therefore, we termed these cells epidermal adult stem cells (EpiASC), although their origin was not identified. We have shown that clones of individual EpiASC proliferate and differentiate in culture to produce neurons, cardiomyocytes, osteoblasts, and occytes. Further, we cultivated EpiASC on bioengineered dermis and denuded human amniotic membrane (HAM), to reconstruct artificial skin and corneal epithelium. We successfully transplanted those artificial tissues in goats with acute full-thickness skin defect (AFTSD) and limbal stem cell deficiency (LSCD), respectively. Our results showed that indeed EpiASC reconstructed the skin (hair was observed in restored areas), and repaired the damaged cornea of goats with total LSCD. These data confirm that EpiASC can differentiate into different functional cell types in vivo or in vitro. Due to their high degree of inherent plasticity, and to their easy accessibility for collection from the skin, EpiASC are excellent candidate sources for diverse cell therapies.  相似文献   

19.
TNFα-matured dendritic cells (DCs) pulsed with tumor antigens are being evaluated as cancer vaccines. It has been shown that DCs produce IL12 during a limited time span and subsequently enter a stage of IL12 exhaustion. If DCs are generated ex vivo, the patient could receive IL12-exhausted DCs which may be detrimental for stimulating anti-tumor Th1 responses. Furthermore, many cancer patients exhibit a cytokine profile skewed toward IL10 and TGFβ. This immunological profile, called the Tr1/Th3 response, is associated with the presence of regulatory T-cells. Tr1/Th3 responses potently inhibit DC maturation, thereby regulating Th1 responses. In the present study, we produced genetically engineered DCs that continuously express Th1-related cytokines such as IL12, and resist negative signals from Tr1/Th3-dominated bladder carcinoma cells. Human immature DCs were genetically engineered by adenoviral vectors to express CD40L, or were treated with TNFα as a positive control for maturation. The expression of different Th1/Th3 and inflammatory cytokines was monitored. IL12 and IFNγ were expressed by CD40L-engineered DCs, while TNFα-matured DCs lacked IFNγ and exhibited low IL12 expression. The addition of recombinant IL10 to genetically engineered DCs did not abolish their Th1 profile. Likewise, coculture with tumor cell lines expressing TGFβ with or without recombinant IL10 did not revert to the engineered DCs. We further demonstrate that the resistance of CD40L-expressing DCs to TGFβ and IL10 may be due to decreased levels of TGFβ and IL10 receptors. Thus, CD40L-engineered DCs are robust Th1-promoting ones that are resistant to Tr1/Th3-signaling via IL10 and TGFβ.  相似文献   

20.
Differentiated epidermal cells can dedifferentiate into stem cells or stem cell‐like cells in vivo. In this study, we report the isolation and characterization of dedifferentiation‐derived cells. Epidermal sheets eliminated of basal stem cells were transplanted onto the skin wounds in 47 nude athymic (BALB/c‐nu/nu) mice. After 5 days, cells negative for CK10 but positive for CK19 and β1‐integrin emerged at the wound‐neighbouring side of the epidermal sheets. Furthermore, the percentages of CK19 and β1‐integrin+ cells detected by flow cytometric analysis were increased after grafting (P < 0.01) and CK10+ cells in grafted sheets decreased (P < 0.01). Then we isolated these cells on the basis of rapid adhesion to type IV collagen and found that there were 4.56% adhering cells (dedifferentiation‐derived cells) in the grafting group within 10 min. The in vitro phenotypic assays showed that the expressions of CK19, β1‐integrin, Oct4 and Nanog in dedifferentiation‐derived cells were remarkably higher than those in the control group (differentiated epidermal cells) (P < 0.01). In addition, the results of the functional investigation of dedifferentiation‐derived cells demonstrated: (1) the numbers of colonies consisting of 5–10 cells and greater than 10 cells were increased 5.9‐fold and 6.7‐fold, respectively, as compared with that in the control (P < 0.01); (2) more cells were in S phase and G2/M phase of the cell cycle (proliferation index values were 21.02% in control group, 45.08% in group of dedifferentiation); (3) the total days of culture (28 days versus 130 days), the passage number of cells (3 passages versus 20 passages) and assumptive total cell output (1 × 105 cells versus 1 × 1012 cells) were all significantly increased and (4) dedifferentiation‐derived cells, as well as epidermal stem cells, were capable of regenerating a skin equivalent, but differentiated epidermal cells could not. These results suggested that the characteristics of dedifferentiation‐derived cells cultured in vitro were similar to epidermal stem cells. This study may also offer a new approach to yield epidermal stem cells for wound repair and regeneration.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号