Effects of Apatite Nanocrystals on Proliferation, Subcellular Structure, Protein Synthesis Activity of Human Periodontal Ligament Cells

To determine the cellular events occurring in the presence of yttrtum/ hyclroxyapatite（Y / HA ） or HA nanocrystals, human periodontal ligament （HPDL） cells were isolated and maintained in culture. The specificity of the cells was evidenced by their proliferation, subcellular structure, and deposition of extracellular matrix components. The presence of nanocrystals was significantly related to an increase in the proliferation. Moreover, the presence of Y/HA nanocrystals was significantly related to an increase in the proliferation with HA nanocrystals. Transmission electron microscopy （TEM） demonstrated the phagocytotic process of HPDL cells toward Y/ HA or HA nanocrystuls . The presence of Y/ HA or HA nanocrystuls was significantly related to an increase in the protein synthesis activity of HPDL cells.

Characterization of the osteogenic potential of mesenchymal stem cells from human periodontal ligament based on cell surface markers

Mesenchymal stem cell （MSC）-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations： CD51/CD140a, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells （DMSCs） from heterogeneous periodontal ligament cells （PDLCs）. Fluorescence-activated cell sorting analysis revealed that 24% of PDLCs were CD51＋/CD140a＋, 0.8% were CD271＋, and 2.4% were STRO-1＋/CD146＋. Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD271＋ DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.

Overview of noncoding RNAs involved in the osteogenic differentiation of periodontal ligament stem cells

Periodontal diseases are infectious diseases that are characterized by progressive damage to dental support tissue.The major goal of periodontal therapy is to regenerate the periodontium destroyed by periodontal diseases.Human periodontal ligament(PDL)tissue possesses periodontal regenerative properties,and periodontal ligament stem cells(PDLSCs)with the capacity for osteogenic differentiation show strong potential in clinical application for periodontium repair and regeneration.Noncoding RNAs(ncRNAs),which include a substantial portion of poly-A tail mature RNAs,are considered“transcriptional noise.”Recent studies show that ncRNAs play a major role in PDLSC differentiation;therefore,exploring how ncRNAs participate in the osteogenic differentiation of PDLSCs may help to elucidate the underlying mechanism of the osteogenic differentiation of PDLSCs and further shed light on the potential of stem cell transplantation for periodontium regeneration.In this review paper,we discuss the history of PDLSC research and highlight the regulatory mechanism of ncRNAs in the osteogenic differentiation of PDLSCs.

Low-power laser irradiation promotes the proliferation and osteogenic differentiation of human periodontal ligament cells via cyclic adenosine monophosphate

Retaining or improving periodontal ligament （PDL） function is crucial for restoring periodontal defects. The aim of this study was to evaluate the physiological effects of low-power laser irradiation （LPLI） on the proliferation and osteogenic differentiation of human PDL （hPDL） cells. Cultured hPDL cel Is were irradiated （660 nm） daily with doses of O, 1, 2 or 4 J .cm-2. Cell proliferation was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide （MTT） assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase （ALP） activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription-polymerase chain reaction （RT-PCR）. Our data showed that LPLI at a dose of 2 J.cm-2 significantly promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J.cm-2 showed potential osteogenic capacity, as it stimulated ALP activity, calcium deposition, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate （cAMP） is a critical regulator of the LPLI-mediated effects on hPDL cells. This study shows that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. These results suggest the potential use of LPLI in clinical applications for periodontal tissue regeneration.

Therapeutic potential of periodontal ligament stem cells

Inflammatory periodontal disease known as periodontitis is one of the most common conditions that affect human teeth and often leads to tooth loss.Due to the complexity of the periodontium,which is composed of several tissues,its regeneration and subsequent return to a homeostatic state is challenging with the therapies currently available.Cellular therapy is increasingly becoming an alternative in regenerative medicine/dentistry,especially therapies using mesenchymal stem cells,as they can be isolated from a myriad of tissues.Periodontal ligament stem cells(PDLSCs)are probably the most adequate to be used as a cell source with the aim of regenerating the periodontium.Biological insights have also highlighted PDLSCs as promising immunomodulator agents.In this review,we explore the state of knowledge regarding the properties of PDLSCs,as well as their therapeutic potential,describing current and future clinical applications based on tissue engineering techniques.

Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Enhance the Osteoblastic Differentiation of Periodontal Ligament Stem Cells Under High Glucose Conditions Through the PI3K/AKT Signaling Pathway

Objective High glucose(HG)can influence the osteogenic differentiation ability of periodontal ligament stem cells(PDLSCs).Human umbilical cord mesenchymal stem cell-derived exosomes(hUCMSC-exo)have broad application prospects in tissue healing.The current study aimed to explore whether hUCMSC-exo could promote the osteogenic differentiation of hPDLSCs under HG conditions and the underlying mechanism.Methods We used a 30 mmol/L glucose concentration to simulate HG conditions.CCK-8 assay was performed to evaluate the effect of hUCMSC-exo on the proliferation of hPDLSCs.Alkaline phosphatase(ALP)staining,ALP activity,and qRT-PCR were performed to evaluate the pro-osteogenic effect of hUCMSC-exo on hPDLSCs.Western blot analysis was conducted to evaluate the underlying mechanism.Results The results of the CCK-8 assay,ALP staining,ALP activity,and qRT-PCR assay showed that hUCMSC-exo significantly promoted cell proliferation and osteogenic differentiation in a dosedependent manner.The Western blot results revealed that hUCMSC-exo significantly increased the levels of p-PI3K and p-AKT in cells,and the effect was inhibited by LY294002(PI3K inhibitor)or MK2206(AKT inhibitor),respectively.Moreover,the increases in osteogenic indicators induced by hUCMSC-exo were significantly suppressed by LY294002 and MK2206.Conclusion hUCMSC-exo promote the osteogenic differentiation of hPDLSCs under HG conditions through the PI3K/AKT signaling pathway.

Effects of Aging on the Proliferation and Differentiation Capacity of Human Periodontal Ligament Stem Cells

periodontal ligament stem cells; aging; proliferation; osteogenic differentiation Objective The aim of this study is to investigate the proliferation, differentiation and apoptosis of periodontal ligament stem cells （PDLSC） derived from different aged donors, and to evaluate the effects of aging on the biological characteristics of PDLSC. Methods Periodontal ligament tissues were obtained from 24 surgically extracted human premolars during orthodontics therapy. The specimens were divided into three groups according to the donor’s age. Group A： 18-20 years, group B： 30-35 years, group C： 45-50 years. PDLSC were isolated and cultured using a tissue-block-based enzymolytic method by limiting dilution assay. The colony forming efficiency of PDLSC for three experimental groups was determined. Senescence-Associated β-Galactosidase （SA-β-G） expression in the three groups was examined using β-galactosidase staining working solution. Cell cycle and apoptosis of the PDLSC were examined by the flow cytometry. Alkaline phosphatase （ALP） activity was evaluated by ALP staining. The expression of osteoplastic differentiation related genes Runt-related transcription factor-2 （Runx-2）, Collagen Type 1 （col-1）, and ALP of PDLSC were examined by quantitative real-time RT-PCR. Results The colony forming efficiency of PDLSC in Group A, B and C was 36.67%, 22.67% and 9.33%, respectively, which decreased with donors’ age （P〈0.05）. SA-β-G expression of the senescent PDLSC in group A, B and C were 4.14%, 16.39%, 50.38%, respectively （P〈0.05）. Cells in G2/S phase was 38.73%, 29.88%, 18.25% （P〈0.05）, and the apoptosis rate was 1.57%, 4.56%, 5.84% （P〈0.05）, in group A, B and C respectively. The ALP staining in the three groups decreased with the increase of donors’ ages, and the expression of Runx-2, col-1 and ALP decreased gradually from group A to group C （all P〈0.05）, which indicated the osteogenic differentiation capacity of PDLSC decreased while donor aging. Conclusion Human PDLSC could be successfully isolated from periodontal ligament tissues of different aged donors. However, the proliferation and osteogenic differentiation capacity of PDLSC decreased while donor aging.

The Effects of Dense/Nanometer Hydroxyapatite on Proliferation and Osteogenetic Differentiation of Periodontal Ligament Cells

The objective of this study is to investigate possible effects of nanometer powder of hydroxyapatite on proliferation of periodontal ligament cells. With sol-gel method, the nanometer hydroxyapatite powder were fabricated. The primary periodontal ligcament cells were caltured on dense particle hydroxyapatite and nanometer particle hydroxyapatite. The effects on proliferation of periodontal ligament cell were examined in vitro with MTT （ methyl thiazolil tetracolium） test. The intercellalar effects were observed with scanning electron microscopy and energy dispersive X-ray analyzer. In addition, the influence of two materials on osteogenetic differentiation was determined with measurement of ALP （ alkaline phosphatase ） activity. It is concluded that nanometer hydroxyapatite can promote proliferation and osteogenetic differeraiation of periodontal ligament cells and it may become absorbable agent in osseous restoration.

Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells

Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.

Cell Attachment of Periodontal Ligament Cells on Commercially Pure Titanium at the Early Stage

Summary: In order to study the character of periodontal ligament cells (PDLCs) attaching on commercially pure titanium (cpTi) by morphology and metrology on the early stage (24 h), 1×105/ml PDLCs in 2 ml culture medium were seeded on cpTi discs fixed in 24-well culture plates. Morphology of cell attachment was observed by contrast phase microscope, scanning electron microscope (SEM) and fluroscence microscopy. Cell adhesion was analyzed by MTT at 0.5, 1, 2, 4 h respectively. PDLCs could attach and spread on cpTi discs. SEM showed that PDLCs had pseudopod-like protuberance. PDLCs showed different attaching phases and reached saturation in cell number at 2 h. It was concluded that PDLCs had good biocompatibility with cpTi, and showed a regular and dynamic pattern in the process of attaching to cpTi.

Influence of sodium fluoride on alkaline phosphatase activity and bone gla protein synthesis in human yellow ligament cells in vitro

Objective To investigate the influence of sodium fluoride(NaF)on alkaline phosphatase(ALP)activity and bone gla protein(BGP)synthesis in yellow ligament cells from different surgical simples in vitro.Methods The human ligament cells

Exposure to Hyaluronan and Radon-Containing Water during the Treatment of Periodontal Pockets

Hyaluronic acid (HA) preparations have emerged as pivotal components in contemporary dentistry, gaining widespread recognition for their multifaceted roles in various biological functions. Extensive literature underscores the significance of HA in maintaining tissue water balance, fostering cell proliferation, promoting rapid cell migration, influencing cell differentiation during organism development, and facilitating tissue regeneration. Notably, HA’s interactions with cell surface receptors contribute to the viscosity of synovial fluid, activate the immune system, and enhance cartilage elasticity. Beyond these established functions, HA has also been investigated for its potential involvement in determining and studying the hormetic effects of radon water, adding a novel dimension to its applications in dental research. A thorough exploration of existing studies reveals a nuanced understanding of how HA interventions impact the outcomes of dental procedures. The comprehensive scope of these investigations allows for a more accurate assessment of the potential effectiveness of specific interventions and provides valuable insights into post-procedural prognoses for individual patients. This synthesis of literature serves as the foundation for elucidating the intricate interplay between HA, radon exposure, and their relevance in modern dental practices.

Effects of lysophosphatidic acid on human periodontal ligament stem cells from teeth extracted from dental patients

Despite their potential applications in future regenerative medicine, periodontal ligament stem cells(PDLSCs) are difficult to obtain in large amounts from patients. Therefore, maintaining sternness while expanding the cell numbers for medical use is the key to transitioning PDLSCs from the bench to the clinic. Lysophosphatidic acid(LPA), which is present in the human body and saliva, is a signaling molecule derived from phospholipids. In this study, we examined the effects of LPA on sternness maintenance in human PDLSCs. Several spindle-shaped and fibroblast-like periodontal ligament stem-like cell lines were established from PDLSC isolation. Among these cell lines, the most morphologically appropriate cell line was characterized. The expression levels of OCT4, NANOG(a stem cell marker), and CD90(a mesenchymal stem cell marker) were high. However, CD73(a negative marker of mesenchymal stem cells) expression was not observed. Notably, immunofluorescence analysis identified the expression of STRO-1, CD146(a mesenchymal stem cell marker), and sex determining region Y-box 2 at the protein level. In addition, lipid droplets were stained by Oil red O after the induction of adipogenesis for 21 days, and mineralized nodules were stained by Alizarin Red S after the induction of osteogenesis for 14 days. Alkaline phosphate staining also demonstrated the occurrence of osteogenesis. In summary, we established a human PDLSC line, which could be applied as a cell source for tissue regeneration in dental patients. However, further studies are needed to determine the detailed effects of LPA on PDLSCs.

Mass acquisition of human periodontal ligament stem cells

The periodontal ligament(PDL)is an essential fibrous tissue for tooth retention in the alveolar bone socket.PDL tissue further functions to cushion occlusal force,maintain alveolar bone height,allow orthodontic tooth movement,and connect tooth roots with bone.Severe periodontitis,deep caries,and trauma cause irreversible damage to this tissue,eventually leading to tooth loss through the destruction of tooth retention.Many patients suffer from these diseases worldwide,and its prevalence increases with age.To address this issue,regenerative medicine for damaged PDL tissue as well as the surrounding tissues has been extensively investigated regarding the potential and effectiveness of stem cells,scaffolds,and cytokines as well as their combined applications.In particular,PDL stem cells(PDLSCs)have been well studied.In this review,I discuss comprehensive studies on PDLSCs performed in vivo and contemporary reports focusing on the acquisition of large numbers of PDLSCs for therapeutic applications because of the very small number of PDLSCs available in vivo.

Effects of Tension Force on Proliferation and Differentiation of Human Periodontal Ligament Cells Induced by Lipopolysaccharides

Human periodontal ligament cells (hPDLCs), with the potential for multi-directional differentiation and reproduction, are the target cells of orthodontic tooth movement. The aim of this study was to examine the effect of mechanical tension force and lipopolysaccharides (LPS) on hPDLCs and whether they induce proliferative and differentiated characters in vitro. Tension force was applied to hPDLCs stimulated with and without LPS for 24 hrs. Real-time polymerase chain reaction (qPCR) was carried out to analyze the mRNA expression of Cyclin 2 (CCND2), WNT1 inducible signaling pathway protein 1 (WISP1), runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP). Analysis of variance (ANOVA) was used for statistical analysis. Significant differences were indicated by P < 0.05. The results showed that tension force promoted the mRNA expression of both the proliferation-related genes (CCND2 and WISP1) and differentiation-related genes (RUNX2 and ALP), and that both were enhanced by the simulation of LPS. In addition, the relative expression ratios CCND2/RUNX2 and CCND2/ALP both increased significantly after the application of tension, and this effect was further enhanced by LPS. All results indicated that with the assessed level of mechanical force loading, tension could promote both the proliferation and differentiation of hPDLCs, which could be enhanced by LPS, and that proliferation is promoted to a greater extent than differentiation. These findings may be valuable for understanding the importance of the application of suitable mechanical force in periodontal remodeling, especially in the process of orthodontic tooth movement with inflammation.

Activation of cannabinoid receptor CB2 regulates LPS-induced pro-inflammatory cytokine production and osteoclastogenic gene expression in human periodontal ligament cells

Background and Objective: It has been found that human periodontal ligament (hPDL) cells express cannabinoid receptor CB2. However, the functional importance of CB2 in hPDL cells exposed to bacterial endotoxins is not known. Here we investigate if the inflammation promoter lipopolysaccharide (LPS) affects CB2 expression and if activation of CB2 regulates LPS-induced pro-inflammatory cytokine production and osteoclastogenic gene expression in hPDL cells. Methods: The hPDL cells were obtained from extracted teeth of periodontally healthy subjects. CB2 expression in hPDL cells exposed to LPS was deter- mined by quantitative real-time PCR analysis. Then, the cells were incubated with or without CB2-specific agonist HU-308 before further stimulation with LPS. In some experiments, the cells were pre-treated with CB2-specific antagonist SR144528. The production of pro-inflammatory cytokines interleukin-1 beta (IL- 1β), interleukin-6 (IL-6) and tumor necrosis factoralpha (TNF-α) was assessed by enzyme-linked immunosorbent assay (ELISA). The mRNA expression of osteoclastogenic genes osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) was examined using quantitative real-time PCR analysis. Results: CB2 expression in hPDL cells was markedly enhanced by LPS. HU-308 significantly suppressed the production of IL-1β, IL-6 and TNF-α exposed to LPS, whereas SR144528 attenuated this effect. The OPG/RANKL ratio decreased when exposed to LPS, furthermore increased significantly with the addition of HU-308 and finally decreased markedly after pretreatment with SR144528. Conclusion: Our study demonstrated that activation of CB2 had anti-inflammatory and anti-resorptive effects on LPS-stimulated hPDL cells. These findings suggest that activation of CB2 might be an effective therapeutic strategy for the treatment of inflammation and alveolar bone resorption in periodontitis.

The Use of Zein and Shuanghuangbu for Periodontal Tissue Engineering

Aim Tissue engineering is a promising area with a broad range of applications in the fields of regenerative medicine and human health. The emergence of periodontal tissue engineering for clinical treatment of periodontal disease has opened a new therapeutic avenue. The choice of scaffold is crucial. This study was conducted to prepare zein scaffold and explore the suitability of zein and Shuanghuangbu for periodontal tissue engineering.Methodology A zein scaffold was made using the solvent casting/particulate leaching method with sodium chloride （NaC1） particles as the porogen. The physical properties of the zein scaffold were evaluated by observing its shape and determining its pore structure and porosity. Cytotoxicity testing of the scaffold was carried out via in vitro cell culture experiments, including a liquid extraction experi- ment and the direct contact assay. Also, the Chinese medicine Shuanghuangbu, as a growth factor, was diluted by scaffold extract into different concentrations. This Shuanghuangbu-scaffold extract was then added to periodontal ligament cells （PDLCs） in order to determineits effect on cell proliferation. Results The zein scaffold displayed a sponge-like structure with a high porosity and sufficient thickness. The porosity and pore size of the zein scaffold can be controlled by changing the porogen particles dosage and size. The porosity was up to 64.1%-78.0%. The pores were well-distributed, interconnected, and porous. The toxicity of the zein scaffold was graded as 0-1. Furthermore, PDLCs displayed full stretching and vigorous growth under scanning electronic microscope （SEM）. Shuanghuangbu-scaffold extract could reinforce proliferation activity of PDLCs compared to the control group, especially at 100 μg.mL^-1 （P〈0.01）. Conclusion A zein scaffold with high porosity, open pore wall structure, and good biocompatibility is conducive to the growth of PDLCs. Zein could be used as scaffold to repair periodontal tissue defects. Also, Shuanghuangbuscaffold extract can enhance the proliferation activity of PDLCs. Altogether, these findings provide the basis for in vivo testing on animals.

Human periodontal ligament stem cells repair mental nerve injury

Human periodontal ligament stem cells are easily accessible and can differentiate into Schwann cells. We hypothesized that human periodontal ligament stem cells can be used as an alternative source for the autologous Schwann cells in promoting the regeneration of injured peripheral nerve. To validate this hypothesis, human periodontal ligament stem cells （1 × 106） were injected into the crush-injured left mental nerve in rats. Simultaneously, autologous Schwann cells （1 × 106） and PBS were also injected as controls. Real-time reverse transcriptase polymerase chain reaction showed that at 5 days after injection, mRNA expression of low affinity nerve growth factor receptor was sig-nificantaly increased in the left trigeminal ganglion of rats with mental nerve injury. Sensory tests, histomorphometric evaluation and retrograde labeling demonstrated that at 2 and 4 weeks after in-jection, sensory function was significantly improved, the numbers of retrograde labeled sensory neurons and myelinated axons were significantly increased, and human periodontal ligament stem cells and autologous Schwann cells exhibited similar therapeutic effects. These findings suggest that transplantation of human periodontal ligament stem cells show a potential value in repair of mental nerve injury.

Overview of the main biological mechanisms linked to changes in periodontal ligament stem cells and the inflammatory microenvironment

Periodontitis is a complex chronic inflammatory disease.The invasion of pathogens induces the inflammatory microenvironment in periodontitis.Cell behavior changes in response to changes in the microenvironment,which in turn alters the local inflammatory microenvironment of the periodontium through factors secreted by cells.It has been confirmed that periodontal ligament stem cells(PDLSCs)are vital in the development of periodontal disease.Moreover,PDLSCs are the most effective cell type to be used for periodontium regeneration.This review focuses on changes in PDLSCs,their basic biological behavior,osteogenic differentiation,and drug effects caused by the inflammatory microenvironment,to provide a better understanding of the influence of these factors on periodontal tissue homeostasis.In addition,we discuss the underlying mechanism in detail behind the reciprocal responses of PDLSCs that affect the microenvironment.

Comparative analysis of different feeder layers with 3T3 fibroblasts for culturing rabbits limbal stem cells

AIM： To explore the possibility of human umbilical cord mesenchymal stem cells（h UCMSCs）, human umbilical vein endothelial cells（h UVECs）, human dental pulp stem cells（h DPSCs） and human periodontal ligament stem cells（h PDLSCs） serving as feeder cells in co-culture systems for the cultivation of limbal stem cells.METHODS： Different feeder layers were cultured in Dulbecco＇s modified Eagle＇s medium（DMEM）/F12 and were treated with mitomycin C. Rabbits limbal stem cells（LSCs） were co-cultured on h UCMSCs, h UVECs, h DPSCs, h PDLSCs and NIH-3T3, and then comparative analysis were made between each group to see their respective colony-forming efficiency（CFE） assay and immunofluorescence（IPO13,CK3/12）.RESULTS： The efficiency of the four type cells in supporting the LSCs morphology and its cellular differentiation was similar to that of NIH-3T3 fibroblasts as demonstrated by the immunostaining properties analysis, with each group exhibiting a similar strong expression pattern of IPO13, but lacking CK3 and CK12 expression in terms of immunostaining. But h UCMSCs, h DPSCs and h PDLSCs feeder layers were superior in promoting colony formation potential of cells when compared to h UVECs and feedercell-free culture.CONCLUSION： hUCMSCs, hDPSCs and hPDLSCs can be a suitable alternative to conventional mouse NIH-3T3 feeder cells, so that risk of zoonotic infection can be diminished.