Vaccination with SIVmac239Δnef activates CD4+ T cells in the absence of CD4+ T-cell loss

Background  Pathogenic HIV and SIV infections characteristically deplete central memory CD4⁺ T cells and induce chronic immune activation, but it is controversial whether this also occurs after vaccination with attenuated SIVs and whether depletion or activation of CD4⁺ T-cell play roles in protection against wild-type virus challenge.
Methods  Rhesus macaques were vaccinated with SIVmac239Δnef and quantitative and phenotypic polychromatic flow cytometry analyses were performed on mononuclear cells from blood, lymph nodes and rectal biopsies.
Results  Animals vaccinated with SIVmac239Δnef demonstrated no loss of CD4⁺ T cells in any tissue, and in fact CCR5⁺ and CD28⁺CD95⁺ central memory CD4⁺ T cells were significantly increased. In contrast, CD4⁺ T-cell numbers and CCR5 expression significantly declined in unvaccinated controls challenged with SIVmac239. Also, intracellular Ki67 increased acutely as much as 3-fold over baseline in all tissues after SIVmac239Δnef vaccination then declined following primary infection.
Conclusion  We demonstrated in this study that SIVmac239Δnef vaccination did not deplete CD4⁺ T cells but transiently activated and expanded the memory cell population. However, increases in numbers and activation of memory CD4⁺ T cells did not appear to influence protective immunity.     

Ex vivo expansion and lentiviral transduction of Macaca nemestrina CD4+ T cells

Background  Macaca nemestrina is a nonhuman primate used as a model in preclinical studies of hematopoietic stem cell transplantation and adoptive transfer of T cells. Adoptive T cell transfer studies typically require ex vivo expansion of substantial numbers of T cells prior to their reinfusion into the subject.
Methods  Pigtailed macaque peripheral blood CD4⁺ cells were expanded using CD3 and CD28 antibody-coated beads. These cells were transformed using Herpesvirus saimiri and were also transduced with HIV-1 based lentiviral vectors.
Results  We report an efficient method for the ex vivo expansion of CD4⁺ T cells from Macaca nemestrina peripheral blood. With this protocol, primary CD4⁺ T cells can be expanded between 300- to 6000-fold during 24-day period and can be efficiently transduced with lentiviral vectors. Furthermore, these T cells can be transformed by Herpesvirus saimiri and maintained in culture for several months. The transformed T cell lines can be productively infected with the simian immunodeficiency virus (SIV) strain SIV_mac239.
Conclusions  We have established methods for the expansion and transformation of primary M. nemestrina CD4⁺ T cells and demonstrated the utility of these methods for several applications.     

Uncommon endocytic and trafficking pathway of the natural killer cell CD94/NKG2A inhibitory receptor

The CD94/NKG2A inhibitory receptor, expressed by natural killer and T cells, is constantly exposed to its HLA-E ligand expressed by surrounding cells. Ligand exposure often induces receptor downregulation. For CD94/NKG2A, this could potentiate activation receptor(s) induced responses to normal bystander cells. We investigated CD94/NKG2A endocytosis and found that it occurs by an amiloride-sensitive, Rac1-dependent macropinocytic- like process; however, it does not require clathrin, dynamin, ADP ribosylation factor-6, phosphoinositide-3 kinase or the actin cytoskeleton. Once endocytosed, CD94/NKG2A traffics to early endosomal antigen 1⁺, Rab5⁺ early endosomes. It does appear in Rab4⁺ early/sorting endosome, but, in the time period examined, fails to reach Rab11⁺ recycling or Rab7⁺ late endosomes or lysosome-associated membrane protein-1⁺ lysosomes. These results indicate that CD94/NKG2A utilizes a previously undescribed endocytic mechanism coupled with an abbreviated trafficking pattern, perhaps to insure surface expression.     

Derivation and characterization of a highly pathogenic isolate of human immunodeficiency virus type 2 that causes rapid CD4+ cell depletion in Macaca nemestrina

With few exceptions, humans are the only species known to develop acquired immunodeficiency syndrome (AIDS) after human immunodeficiency virus (HIV) infection. We report here that an isolate of HIV type 2, EHO, readily established persistent infection in 100% of Macaca nemestrina in three consecutive transmission studies. Of the eight infected animals, five showed persistently high virus load and six developed AIDS-like diseases or CD4⁺ cell depletion within 4 years of infection. The pathology and clinical signs closely parallel those of HIV-1 infection of humans, including lymphadenopathy, anemia, CD4⁺ cell depletion, and opportunistic infections. A cell-free virus stock was established from the lymph nodes of an animal that developed AIDS-like diseases. This virus, HIV-2/287, was highly pathogenic in M. nemestrina, causing CD4⁺ cell depletion within 2–8 weeks post-infection. While both HIV-2 EHO and HIV-2/287 use predominantly CXCR4, the latter shows greatly enhanced replicative capacity in macaque peripheral blood mononuclear cells (PBMCs). The establishment of a human immunodeficiency virus that causes rapid and reproducible CD4⁺ cell depletion in macaques could facilitate the study of HIV pathogenesis and the development of effective vaccines and therapy against AIDS.     

Analysis of splenic Gr-1int immature myeloid cells in tumor-bearing mice

It is known that the number of ImC, expressing myeloid markers, CD11b and Gr-1, increase with tumor growth and ImC play a role in the escape of tumor cells from immunosurveillance in tumor-bearing mice and cancer patients. However, the mechanisms by which ImC suppress immune responses in tumor-bearing mice have not been completely elucidated. In the present study, we investigated the function of splenic ImC freshly isolated from tumor-bearing mice and splenic ImC differentiated in vitro by GM-CSF. Freshly isolated splenic ImC were divided into two groups depending on Gr-1 expression, Gr-1 high (Gr-1^hi) and intermediate (Gr-1^int). Freshly isolated splenic Gr-1^int ImC, but not Gr-1^hi ImC, from tumor-bearing mice reduced production of IFN-γ in CD8⁺ T cells, but neither splenic Gr-1^int ImC nor Gr-1^hi ImC isolated from naive mice did. Both Gr-1^int and Gr-1^hi ImC differentiated in vitro by GM-CSF inhibited production of IFN-γ in both CD8⁺ and CD4⁺ T cells. In addition, the differentiated Gr-1^int ImC, one-third of which were CD11c⁺F4/80⁺ cells, and their culture supernatants suppressed proliferative responses of T cells stimulated by CD3 ligation, but the differentiated Gr-1^hi ImC and their culture supernatants did not. These results suggest that Gr-1^int ImC are altered to immune-suppressive cells in tumor circumstances and that they are differentiated by GM-CSF progressively into CD11c⁺F4/80⁺ cells with further suppressive activity against T cells.     

Interaction between superantigen and T-cell receptor Vβ element determines levels of superantigen-dependent cell-mediated cytotoxicity of CD8+ T cells in induction and effector phases

Specific superantigens activate different T-cell fractions with distinct TCR Vβ elements in association with MHC class II molecules and also induce SDCC against MHC class II⁺ target cells. In the present study, to determine whether the responsiveness of each CD8⁺ T-cell fraction expressing a different TCR Vβ element is primarily determined by the TCR Vβ, we compared the levels of proliferation and SDCC in Vβ3⁺ and Vβ11⁺ T cells upon stimulation with SEA. Upon stimulation with SEA_wt, the levels of proliferation were higher in Vβ3⁺ T cells than in Vβ11⁺ T cells. The levels of SDCC were also higher for the combination of Vβ3⁺ T cells and SEA_wt than for the combination of Vβ11⁺ T cells and SEA_wt during both the induction phase and the effector phase. In addition, upon stimulation with SEA_m, the levels of proliferation were higher in Vβ11⁺ T cells than in Vβ3⁺ T cells. And then, the levels of SDCC were also higher for the combination of Vβ11⁺ T cells and SEA_m than for the combination of Vβ3⁺ T cells and SEA_m during both the induction phase and the effector phase. These results suggest that the SAG-responsiveness of each CD8⁺ T-cell fraction expressing a different TCR Vβ element is primarily determined by the interaction between the TCR Vβ element and the SAG.     

Change of specific T cells in an emerging neonatal infectious disease induced by a bacterial superantigen

A new epidemic, NTED, has recently occurred in Japan. The cause of NTED is a bacterial superantigen, TSST-1. The aim of the present study was to analyze the change in Vβ2⁺ T cells reactive to TSST-1 in NTED in order to establish T-cell-targeted diagnostic criteria for NTED. Blood samples from 75 patients with clinically diagnosed NTED were collected from 13 neonatal intensive care units throughout Japan. We investigated the percentages of Vβ2⁺, Vβ3⁺ and Vβ12⁺ T cells and their CD45RO expressions in the samples using flow cytometry. In 18 of the 75 patients, we conducted multiple examinations of the T cells and monitored serial changes. The Vβ2⁺ T-cell population rapidly changed over three phases of the disease. Whereas the percentage of Vβ2⁺ T cells was widely distributed over the entire control range, CD45RO expression on Vβ2⁺ T cells in CD4⁺ in all 75 patients was consistently higher than the control range. Patients cannot necessarily be diagnosed as having NTED based on expansion of Vβ2⁺ T cells alone in the early acute phase. Instead, CD45RO expression on specific Vβ2⁺ cells is a potential diagnostic marker for a rapid diagnosis of NTED. We present three diagnostic categories of NTED. Fifty patients (66.7%) were included in the category 'definitive NTED'. It is important to demonstrate an increase of Vβ2⁺ T cells in the following phase in cases of 'probable NTED' or 'possible NTED'.     

OK-432 and IL-2-augmental cytotoxicity of human natural killer cells and cytotoxic T lymphocytes at the clonal level

Abstract The biology response modifiers OK-432 and interleukin-2 (IL-2) were found to enhance the lytic capacity of cloned CD3⁻ natural killer (NK) cells and CD3⁺ T cells. With respect to NK cells, only those clones with a high proliferative capacity and cultured without phytohaemagglutinin (PHA) responded with enhaced lytic capacity to OK-432. OK-432, but not IL-2, was found to augment the antibody-dependent cellular cytotoxicity of cloned NK cells. With T-cell clones, OK-432 augmented the cellular cytotoxicity of CD3⁺8⁺ but not that of CD3⁺4⁺ cytotoxic T lymphocytes, while IL-2 augmented the cytotoxicity of both types of clone. Taken together, these results indicate that OK-432-augmented lytic capacity is not restricted to NK cells and its pathway of action may be independent of IL-2.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Mechanisms of cobalt uptake in plants: 60CO uptake and distribution in Chara

The mechanism of cobalt uptake was investigated using cells of the giant alga Chara corallina in which it is possible to resolve separately uptake by the cell wall and actual influx across the cell membrane. The absorption of ⁶⁰Co by Chara cells appeared to saturate within 2 h, but this was mainly due to rapid uptake into the cell wall which accounted for 87–92% of the total activity. Even after prolonged desorption most of the cell‐associated ⁶⁰Co was found on the cell wall. The intracellular distribution of absorbed ⁶⁰Co was investigated by fractionating the cell into cytoplasm and vacuole. It was shown that ⁶⁰Co influx to the vacuole occurs simultaneously with influx to the cytoplasm. The transported species appears to be Co²⁺ rather than the less charged Co(OH)⁺ or Co(OH)₂. ⁶⁰Co influx is pH dependent (optimum pH 7–9), and is sensitive to some other divalent metals. Influx from solutions containing 1 µ M ⁶⁰Co was inhibited by 5 µ M Cd²⁺, Cu²⁺, and Zn²⁺, but Mn²⁺ and Ni²⁺ had no significant effect. The sensitivity of Co uptake to N ‐ethyl maleimide (NEM) and cysteine suggests that the transport system involves direct binding of CO²⁺ to ‐SH groups.     

Effect of K+ and H+ stress and role of Ca2+ in the regulation of intracellular K+ concentration in mung bean roots

The effects of external K⁺, H⁺ and Ca2⁺ concentrations on the intracellular K+ concentration, [K⁺]i, and the K⁺-ATPase activity in 2-day-old mung bean roots [ Vigna mungo (L.) Hepper] were investigated. [K⁺]i, in mung bean roots was markedly decreased by external K⁺ or H⁺ stress and did not recover the initial value even after the stress was removed. This decrease in [K⁺]i, gradually disappeared with the addition of (Ca2⁺. Ca2⁺ may offset the harmful effects of ion stress. Ca2⁺ seems to have two effects on K+ transport; control of K⁺ permeability and activation of K⁺ uptake, although K⁺-ATPase activity was inhibited by Ca2⁺ concentrations higher than 10–4 M. We suggest that Ca2⁺ activates K⁺ uptake indirectly through the acidification of the cytoplasm.     

Source of cell injected is a critical factors for short and long engraftment in xeno-transplantation

Abstract. This study aims to investigate engraftment of human cord blood and foetal bone marrow stem cells after in utero transplantation via the intracoelomic route in the sheep. Here, we performed transplantation in 14 single and 1 twin sheep foetuses at 40–47 days of development, using a novel schedule for injection. (i) Single injection of CD34⁺ human cord blood stem cells via the coelomic route (from 10 to 50 × 10⁴) in seven single foetuses. (ii) Single injection of CD34⁺ foetal bone marrow stem cells via the intracoelomic route with further numbers of cells (20 × 10⁵ and 8 × 10⁵, respectively) in three single and in one twin foetuses. (iii) Double fractioned injection (20–30 × 10⁶) via the coelomic route and 20 × 10⁶ postnatally, intravenously, shortly after birth of CD3-depleted cord blood stem cells in four single foetuses. In the first group, three single foetuses showed human/sheep chimaerism at 1, 8 and 14 months after birth. In the second group, the twin foetuses showed human/sheep chimaerism at 1 month after birth. In the third group, only two out of four single foetuses that underwent transplantation showed chimaerism at 1 month. While foetal bone marrow stem cells showed good short-term engraftment (1 month after birth), cord blood stem cells were able to persist longer in the ovine recipients (at 1, 8 and 14 months after birth).     

Lysis of uninfected HIV-1 gp120-coated peripheral blood-derived T lymphocytes by monocyte-mediated antibody-dependent cellular cytotoxicity

Abstract Previous reports from our laboratory have demonstrated that peripheral blood monocytes (PBM) from HIV-1 infected individuals are de novo activated and are cytotoxic in vitro. Significant monocyte-antibody-dependent cellular cytotoxicity (ADCC) was obtained against HIV-1 inactivated CD4⁺ CEM target cells coated with HIV-1 in the presence of autologous seropositive serum. Based on these findings, we hypothesized that in HIV-seropositive individuals the monocytes may play an important role in vivo in the autodestruction of non-infected CD4⁺ T lymphocytes. The present study was designed to test this hypothesis. Monocytes from normal donors activated with M-CSF lysed CD4+ T cells (CEM) coated with gp120 sensitized by plasma from asymptomatic HIV-1+ individuals in a 8 h ⁵¹Cr release assay. ADCC cytotoxic activity varied from one individual to another and was a function of the dilution of the individual seropositive plasma used. We then used circulating CD3+ T lymphocytes as targets for ADCC following treatment with actinomycin D to facilitate the release of radioactive ⁵¹Cr. Like CEM, ADCC was obtained with CD3⁺ T cells coated with gp120 in the presence of HIV seropositive plasma and monocytes. Lysis was specific as T cells that were not coated with gp120 were not destroyed. These findings demonstrate that activated peripheral blood derived monocytes can destroy non-infected gp120-coated circulating T lymphocytes by an ADCC-mediated mechanism. Thus, these findings suggest that ADCC may be one mechanism operating in vivo for the destruction of non-infected CD4⁺ T lymphocytes.     

Actual escape area and lateral escape from the xylem of the alkali ions Na+, K+, Rb+ and Cs+ in tomato

Approximation of the total escape area of the xylem in an inbred line of tomato (Ly-copersicon escutentum Mill. cv. Tiny Tim) with help of the frequency distribution of xylem vessel radii provides the possibility to calculate realistic escape constant values from uptake experiments of several elements into tomato stem segments. Comparison of the lateral escape rates of ²⁴Na⁺, ⁴²K⁺, ⁸⁶Rb⁺ and ¹³⁴Cs⁺ indicate that Na⁺ escape is rate-limited by its uptake into a rather constant number of surrounding cells, regardless of changes in the total escape area of the xylem vessels. The escape of K⁺, Rb⁺ and Cs⁺ seems to be proportional to the surface area of the xylem vessels and their escape is apparently controlled by their transport across the cell walls of the transport channels. The calculated small values for the escape rate constants (apparent permeability of the xylem cell walls, ca 2–3 · 10−⁹ m s−⁷) are probably due to the presence of lignin in the xylem cell walls, the discrimination between ions as a result of differing affinities and selectivities and the presence of other solutes in the applied solution.     

Efficient in vitro generation of adult multipotent cells from mobilized peripheral blood CD133+ cells

Abstract. Objectives : To generate non-haematopoietic tissues from mobilized haematopoietic CD133⁺ stem cells. Materials and methods : Mobilized peripheral blood CD133⁺ cells from adult healthy donors were used. In vitro ability of highly enriched CD133⁺ cells from mobilized peripheral blood to generate multipotent cells, and their potential to give rise to cells with characteristics of neuroectoderm, endoderm and mesoderm layers was investigated. Results : We found that a recently identified population of CD45⁺ adherent cells generated in vitro after culture of highly purified CD133⁺ cells for 3–5 weeks with Flt3/Flk2 ligand and interleukin-6 can, in presence of the appropriate microenvironmental cues, differentiate into neural progenitor-like cells (NPLCs), hepatocyte-like cells and skeletal muscle-like cells. We have termed them to be adult multipotent haematopoietic cells (AMHCs). AMHC-derived NPLCs expressed morphological, phenotypic and molecular markers associated with primary neural progenitor cells. They can differentiate into astrocyte-like cells, neuronal-like cells and oligodendrocyte-like cells. Moreover, AMHC-derived NPLCs produced 3,4-dihydrophenylalanine and dopamine and expressed voltage-activated ion channels, suggesting their functional maturation. In addition, AMHC-derived hepatocyte-like cells and skeletal muscle-like cells, showed typical morphological features and expressed primary tissue-associated proteins. Conclusion : Our data demonstrate that AMHCs may therefore serve as a novel source of adult multipotent cells for autologous replacement cell therapies.     

Prospective isolation and characterization of mesenchymal stem cells from human placenta using a frizzled-9-specific monoclonal antibody

Abstract We have recently shown that frizzled-9 (FZD9, CD349) is expressed on the cell surface of cultured mesenchymal stromal cells (MSC) derived from the human bone marrow (BM) and chorionic placenta (PL). To study whether FZD9 is also a marker for naive mesenchymal stem cells (MSC), we analyzed the expression pattern of FZD9 on freshly isolated PL cells and determined the clonogenic potential of isolated FZD9⁺ cells using the colony-forming units-fibroblastic (CFU-F) assay. About 0.2% of isolated PL cells were positive for FZD9. Two-color analysis revealed that FZD9⁺ PL cells uniformly express CD9, CD63, and CD90, but are heterogeneous for CD10, CD13, and CD26 expression. In contrast to BM-derived MSC, PL-derived MSC expressed only low levels of CD271. Colony assays of sorted cells showed that clonogenic CFU-F reside exclusively in the FZD9⁺ but not in the FZD9⁻ fraction. Further analysis revealed that CFU-F were enriched by 60-fold in the FZD9⁺CD10⁺CD26⁺ fraction but were absent in the FZD9⁺CD10⁻CD26⁻ population. Cultured FZD9⁺ cells expressed the embryonic stem cell makers Oct-4 and nanog as well as SSEA-4 and TRA1-2-49/6E. In addition, they could be differentiated into functional adipocytes and osteoblasts. This report describes for the first time that FZD9 is a novel and specific marker for the prospective isolation of MSC from human term PL.     

In Human Fetal Astrocytes Exposure to Interleukin-1β Stimulates Acquisition of the GD3+ Phenotype and Inhibits Cell Division

Abstract: In human astrocyte cultures established from second-trimester fetal brain tissue, ∼5–10% of total astrocyte population in unstimulated cultures were GD3⁺/glial fibrillary acidic protein (GFAP)⁺. The GD3⁺ cells were always GFAP⁺ and grew as flat, highly spread cells but changed to process-bearing cells after interleukin-1β (IL-1β) stimulation. It is interesting that IL-1β, a known mitogen for rat astrocytes, suppressed human fetal astrocyte proliferation as determined by [³H]thymidine incorporation, bromodeoxyuridine (BrdU) labeling, and cell counting. The GD3⁺ population, however, consistently increased in absolute number after IL-1β stimulation, in a dose- and time-dependent manner. The IL-1β-mediated increase in number of GD3⁺ astrocytes was independent of initial cell density or serum concentration. By flow cytometry, IL-1β enhanced both the mean fluorescence intensity and the percentage of GD3⁺ cells. To investigate whether the increase in GD3⁺ astrocyte cell number was due to proliferation of preexisting GD3⁺ astrocytes or due to conversion of GD3⁻ to GD3⁺ cells, we performed BrdU/GD3 double immunocytochemistry. BrdU/GD3 double-positive cells were extremely rare in both control and IL-1β-stimulated cultures. Moreover, an increase in number of GD3⁺ astrocytes was still observed in control and IL-1β-stimulated cultures where GD3⁺ cells had been initially eliminated by cell sorting. These results indicate that GD3⁺ astrocytes in human fetal culture may represent a postmitotic, differentiated, distinct phenotype.     

Ca2+-Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin-Dependent Kinases

Abstract: We studied effects of Ca²⁺ in the incubation medium on [³H]dopamine ([³H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca²⁺ (0–30 min) and Ca²⁺ concentration (0–10 m M ) in Krebs-Ringer medium affected [³H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca²⁺ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V _max of the [³H]DA uptake process. On the other hand, [³H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca²⁺. The Ca²⁺-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca²⁺-free medium following preincubation with 1 m M Ca²⁺. Protein kinase C inhibitors did not affect apparently Ca²⁺-dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca²⁺/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca²⁺-dependent enhancement of [³H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.     

Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells

Abstract Identification of mesenchymal stem cells (MSCs) derived from alternative sources has provided an exciting prospect for intensive investigation. This work focused on characterizing a new source of MSCs from stromal cells from human eye conjunctiva. In this study, after conjunctiva biopsies and culture of stromal segment of this tissue, fibroblast-like (SH2⁺, SH3⁺, CD29⁺, CD44⁺, CD166⁺, CD13⁺) human stromal cells, which can be differentiated toward the osteogenic, adipogenic, chondrogenic, and neurogenic lineages, were obtained. These cells expressed Oct-4, Nanog, Rex-1 genes, and some lineage-specific markers like cardiac actin and Keratin. Taken together, the results indicate that conjunctiva stromal-derived cells are a new source of multipotent MSCs and despite originating from an adult source, they express undifferentiated stem cell markers.