Multipotent mesenchymal stem cells of desquamated endometrium: Isolation,characterization, and application as a feeder layer for maintenance of human embryonic stem cells

In this study, we characterize new multipotent human mesenchymal stem cell lines (MSCs) derived from desquamated (shedding) endometrium of menstrual blood. The isolated endometrial MSC (eMSC) is an adhesive to plastic heterogeneous population composed mainly of endometrial glandular and stromal cells. The established cell lines meet the criteria of the International Society for Cellular Therapy for defining multipotent human MSCs of any origin. The eMSCs have positive expression of CD13, CD29, CD44, CD73, CD90, and CD105 markers and lack hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130, and HLA-DR (class II). Multipotency of the established eMSCs is confirmed by their ability to differentiate into other mesodermal lineages, such as osteocytes and adipocytes. In addition, the isolated eMSCs partially (over 50%) express the pluripotency marker SSEA-4. However, they do not express Oct-4. Immunofluorescent analysis of the derived cells revealed the expression of the neural precursor markers nestin and β-III-tubulin. This suggests a neural predisposition of the established eMSCs. These cells are characterized by a high proliferation rate (doubling time 22–23 h) and a high colony-forming efficiency (about 60%). In vitro, the eMSCs undergo more than 45 population doublings without karyotypic abnormalities. We demonstrate that mitotically inactivated eMSCs are perfect feeder cells for maintenance of human embryonic stem cell lines (hESCs) C612 and C910. The eMSCs, being a feeder culture, sustain the hESC pluripotent status that verified by expression of Oct-4, alkaline phosphatase and SSEA-4 markers. The hESCs cocultured with the eMSCs retain their morphology and proliferative rate for more than 40 passages and exhibit the capability for spontaneous differentiation into embryoid bodies comprising three embryonic germ layers. Thus, an easy and noninvasive isolation of the eMSCs from menstrual blood, their multipotency and high proliferative activity in vitro without karyotypic abnormalities demonstrate the potential of use of these stem cells in regenerative medicine. Using the derived eMSCs as the feeder culture eliminates the risks associated with animal cells while transferring hESCs to clinical setting.     

Fetal mesenchymal stem cells derived from human umbilical cord sustain primitive characteristics during extensive expansion

Stem cells of fetal origin lie between embryonic and adult stem cells in terms of potentiality. Because of the ethical controversy surrounding embryonic stem cells and the relatively inferior quality of adult stem cells, the use of fetal stem cells would be an attractive option in future therapeutic applications. Here, we have investigated primitive characteristics of human umbilical-cord-derived fetal mesenchymal stem cells (UC fMSCs) during extensive expansion. We have successfully isolated and cultured UC fMSCs from all UC samples, but with two early fungal contaminations. UC fMSCs proliferated without significant evidence of morphological changes, and the average cumulative population-doubling level was over 25 for about 3 months. UC fMSCs showed the positive expression of several CD markers, known to be related to MSCs, including CD73 (SH-3, 4), CD90 (Thy-1), CD105 (SH-2), CD117 (c-kit), and CD166 (ALCAM). They demonstrated primitive properties throughout the expansion period: multilineage differentiation potentials examined by functional assays, a variety of pluripotent stem cell markers including Nanog, Oct-4, Sox-2, Rex-1, SSEA-3, SSEA-4, Tra-1–60, and Tra-1–81, minimal evidence of senescence as shown by β-galactosidase staining, and the consistent expression of telomerase activity. These results suggest that UC fMSCs have more primitive properties than adult MSCs, which might make them a useful source of MSCs for clinical applications. This work was supported by the Seoul R&BD Program (10548).     

Amnion Epithelial Cells of Buffalo (Bubalus bubalis) Term Placenta Expressed Embryonic Stem Cells Markers and Differentiated into Cells of Neurogenic Lineage In Vitro

Aim of the present study was the isolation, culture, and characterization of amniotic membrane-derived epithelial cells (AE) from term placenta collected postpartum in buffalo. We found that cultured cells were of polygonal in shape, resistance to trypsin digestion and expressed cytokeratin-18 indicating that they were of epithelial origin. These cells have negative expression of mesenchymal stem cell markers (CD29, CD44, and CD105) and positive for pluripotency marker (OCT4) genes indicated that cultured cells were not contaminated with mesenchymal stem cells. Immunofluorescence staining with pluripotent stem cell surface markers, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81 indicated that these cells may retain pluripotent stem cell characteristics even after long period of differentiation. Differentiation potential of these cells was determined by their potential to differentiate into cells of neurogenic lineages using retinoic acid. In conclusion, we demonstrate that AE cells expressed pluripotent stem cell markers and have propensity to differentiate into cells of neurogenic lineage upon directed differentiation in vitro.     

Generation and Characterization of an Immortalized Human Esophageal Myofibroblast Line

Stromal cells with a myofibroblast phenotype present in the normal human esophagus are increased in individuals with gastro-esophageal reflux disease (GERD). We have previously demonstrated that myofibroblasts stimulated with acid and TLR4 agonists increase IL-6 and IL-8 secretion using primary cultures of myofibroblasts established from normal human esophagus. While primary cultures have the advantage of reflecting the in vivo environment, a short life span and unavoidable heterogeneity limits the usefulness of this model in larger scale in vitro cellular signaling studies. The major aim of this paper therefore was to generate a human esophageal myofibroblast line with an extended lifespan. In the work presented here we have generated and characterized an immortalized human esophageal myofibroblast line by transfection with a commercially available GFP-hTERT lentivirus. Immortalized human esophageal myofibroblasts demonstrate phenotypic, genotypic and functional similarity to primary cultures of esophageal myofibroblasts we have previously described. We found that immortalized esophageal myofibroblasts retain myofibroblast spindle-shaped morphology at low and high confluence beyond passage 80, and express α-SMA, vimentin, and CD90 myofibroblast markers. Immortalized human esophageal myofibroblasts also express the putative acid receptor TRPV1 and TLR4 and retain the functional capacity to respond to stimuli encountered in GERD with secretion of IL-6. Finally, immortalized human esophageal myofibroblasts also support the stratified growth of squamous esophageal epithelial cells in 3D organotypic cultures. This newly characterized immortalized human esophageal myofibroblast cell line can be used in future cellular signaling and co-culture studies.     

Characterization of novel clonal murine endothelial cell lines with an extended life span

Summary A murine endothelial cell line was recently established from microvessels that had invaded a subcutaneous sponge implant (Dong, Q. G.; Bernasconi, S.; Lostaglio, S., et al. Arterioscl. Thromb. Vasc. Biol. 17:1599–1604; 1997). From these sponge-induced endothelial (SIE) cells, we have isolated two subpopulations endowed with different phenotypic properties. Clone SIE-F consists of large, highly spread cells that have a relatively slow growth rate, form contact-inhibited monolayers, do not grow under anchorage-independent conditions, express elevated levels of thrombospondin-1 (TSP-1) and are not tumorigenic in vivo. In contrast, clone SIE-S2 consists of small, spindle-shaped cells that have a high proliferation rate, do not show contact-inhibition, grow under anchorage-independent conditions, express very low levels of TSP-1 and are tumorigenic in vivo. Both clones express the endothelial markers vascular endothelial-cadherin and vascular intercellular adhesion molecule-1, but do not express CD31 and E-selectin. In addition, SIE-S2 cells, but not SIE-F cells, express the α-smooth muscle actin isoform. SIE-S2 cells, but not SIE-F cells, are able to form branching tubes in fibrin gels. The SIE-F and SIE-S2 clones, which have properties of nontransformed and transformed cells, respectively, should provide useful tools to investigate physiological and pathological processes involving vascular endothelium.     

Evidence that osteogenic progenitor cells in the human tunica albuginea may originate from stem cells: implications for peyronie disease

Tissue ossification in Peyronie disease (commonly known as Peyronie's disease [PD]), a localized fibrotic lesion within the tunica albuginea (TA) of the penis, may result from osteogenic differentiation of fibroblasts, myofibroblasts, and/or adult stem cells in the TA, and may be triggered by chronic inflammation, oxidative stress, and profibrotic factors like transforming growth factor beta 1 (TGFB1). In this study, we have investigated whether cultures of cells from normal TA and PD plaques undergo osteogenesis, express markers for stem cells, and originate other cell lineages via processes modulated by TGFB1. We found that TA and PD cells in osteogenic medium (OM) expressed osteogenic markers, alkaline phosphatase, and osteopontin and underwent calcification. PD cells, but not TA cells, formed foci in soft agar that were positive for alkaline phosphatase and calcification and expressed the mRNAs for osteoblast-specific factors pleiotrophin and periostin and bone morphogenic protein 2. Both cultures expressed stem cell marker CD34 antigen but not protein tyrosine phosphatase, receptor type c. TA and PD cells expressed smooth-muscle cell markers smoothelin and transgelin. None of the cultures underwent adipogenesis in adipogenic medium. Incubation with TGFB1 increased osteogenesis and myofibroblast differentiation and reduced CD34 antigen expression in both cultures. TA and PD cells modulated the differentiation of the multipotent C3H 10T(1/2) cells in dual cultures, into osteoblasts and myofibroblasts. In conclusion, both TA and PD cultures contain cells, presumably stem cells, that undergo osteogenic and myofibroblast differentiation, and may induce these processes by paracrine interactions. This may explain progression of fibrosis in the PD plaque and its eventual calcification.     

Multipotent mesenchymal stem cells of desquamated endometrium: isolation, characterization and use as feeder layer for maintenance of human embryonic stem cell lines

In this study, we characterize new multipotent human mesenchymal stem cell (MSC) lines derived from desquamated (shedding) endometrium in menstrual blood. The isolated endometrial MSC (eMSC) is an adhesive to plastic heterogeneous population composed mainly of endometrial glandular and stromal cells. The established cell lines meet the criteria of the International Society for Cellular Therapy for defining multipotent human MSC of any origin. The eMSCs have positive expression of CD73, CD90, CD105, CD13, CD29, CD44 markers and the absence of expression of the hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130 and HLA-DR (class II). Multipotency of the established eMSC is confirmed by their ability to differentiate into other mesodermal cell types such as osteocytes and adipocytes. Besides, the isolated eMSC lines partially (over 50%) express the pluripotency marker SSEA-4, but do not express Oct-4. Immunofluorescent analysis of the derived cells revealed the expression of the neural precursor markers nestin and beta-III-tubulin. This suggests a neural predisposition of the established eMSC. These cells are characterized by high rate of cell proliferation (doubling time 22-23 h) and high cloning efficiency (about 60%). In vitro the eMSCs undergo more than 45 population doublings revealing normal karyotype without karyotipic abnormalilies. We demonstrate, that the mititotically inactivated eMSCs are perfect feeder cells for human embryonic stem cell lines (hESC) C612 and C910. The eMSC being a feeder culture maintain the pluripotent status of the hESC, which is revealed by the expression of Oct-4, alkaline phosphatase and SSEA-4. When co-culturing, hESC retain their morphology, proliferative rate for more than 40 passages and capability for spontaneous differentiation into embryoid bodies comprising the three embryonic germ layers. Thus, an easy and non-invasive extraction of the eMSC in menstrual blood, their multipotency and high proliferative activity in vitro without karyotypic abnormalities demonstrate the potential of use of these stem cells in regenerative medicine. Using the derived eMSCs as the feeder culture eliminates the risks associated with animal cells while transferring hESC to clinical setting.     

Differential responses to retinoic acid and endocrine disruptor compounds of subpopulations within human embryonic stem cell lines

The heterogeneous nature of stem cells is an important issue in both research and therapeutic use in terms of directing cell lineage differentiation pathways, as well as self-renewal properties. Using flow cytometry we have identified two distinct subpopulations by size, large and small, within cultures of human embryonic stem (hES) cell lines. These two cell populations respond differentially to retinoic acid (RA) differentiation and several endocrine disruptor compounds (EDC). The large cell population responds to retinoic acid differentiation with greater than a 50% reduction in cell number and loss of Oct-4 expression, whereas the number of the small cell population does not change and Oct-4 protein expression is maintained. In addition, four estrogenic compounds altered SSEA-3 expression differentially between the two cell subpopulations changing their ratios relative to each other. Both populations express stem cell markers Oct-4, Nanog, Tra-1–60, Tra-1–80 and SSEA-4, but express low levels of differentiation markers common to the three germ layers. Cloning studies indicate that both populations can revive the parental population. Furthermore, whole genome microarray identified approximately 400 genes with significantly different expression between the two populations (p<0.01). We propose the differential response to RA in these populations is due to differential gene expression of Notch signaling members, CoupTF1 and CoupTF2, chromatin remodeling and histone modifying genes that render the small population resistant to RA differentiation. The findings that hES cells exist as heterogeneous populations with distinct responses to differentiation signals and environmental stimuli will be relevant for their use for drug discovery and disease therapy.     

Novel human embryonic stem cell lines C612 and C910

Novel human embryonic stem cell lines C612 and C910 have been established from atching blastocytes. Cells were cultivated in mTeSR medium on a mouse fibroblast feeder layer; they exhibit common pluripotent markers, such as alkaline phosphatase, Oct 3/4, SSEA-4, Nanog, Rex1. The immunophenotyping of these cells by flow cytometry revealed CD90 (Thy-1) and CD117 (c-kit) antigens and showed weak or no expression of CD13, CD34, CD45, CD130, and HLA class I and II antigens, which is typical for human embryonic stem cells. Karyotypic structure of C612 and C910 assayed by the G-banding of metaphase plates is normal in both chromosome number and structure. The cells generate embryoid bodies, undergo spontaneous differentiation, and express three germ-layer markers (nestin, keratin, vimentin ectoderm), α-fetoprotein (entoderm), muscle α-actinin (mesoderm), i.e., possess pluripotent potency. Thus, C612 and C910 display accepted human embryonic stem cell properties, including unlimited self-renewal, expression of pluripotent markers, ability to differentiate into three germ layers, and are diploid; therefore, they may be of potential use for fundamental research, as well as for replacement therapy studies.     

Recapitulation of fibromatosis nodule by multipotential stem cells in immunodeficient mice

Musculoskeletal fibromatosis remains a disease of unknown etiology. Surgical excision is the standard of care, but the recurrence rate remains high. Superficial fibromatosis typically presents as subcutaneous nodules caused by rapid myofibroblast proliferation followed by slow involution to dense acellular fibrosis. In this study, we demonstrate that fibromatosis stem cells (FSCs) can be isolated from palmar nodules but not from cord or normal palm tissues. We found that FSCs express surface markers such as CD29, CD44, CD73, CD90, CD105, and CD166 but do not express CD34, CD45, or CD133. We also found that FSCs are capable of expanding up to 20 passages, that these cells include myofibroblasts, osteoblasts, adipocytes, chondrocytes, hepatocytes, and neural cells, and that these cells possess multipotentiality to develop into the three germ layer cells. When implanted beneath the dorsal skin of nude mice, FSCs recapitulated human fibromatosis nodules. Two weeks after implantation, the cells expressed immunodiagnostic markers for myofibroblasts such as α-smooth muscle actin and type III collagen. Two months after implantation, there were fewer myofibroblasts and type I collagen became evident. Treatment with the antifibrogenic compound Trichostatin A (TSA) inhibited the proliferation and differentiation of FSCs in vitro. Treatment with TSA before or after implantation blocked formation of fibromatosis nodules. These results suggest that FSCs are the cellular origin of fibromatosis and that these cells may provide a promising model for developing new therapeutic interventions.     

PECAM1+/Sca1+/CD38+ Vascular Cells Transform into Myofibroblast-Like Cells in Skin Wound Repair

Skin injury induces the formation of new blood vessels by activating the vasculature in order to restore tissue homeostasis. Vascular cells may also differentiate into matrix-secreting contractile myofibroblasts to promote wound closure. Here, we characterize a PECAM1⁺/Sca1⁺ vascular cell population in mouse skin, which is highly enriched in wounds at the peak of neoangiogenesis and myofibroblast formation. These cells express endothelial and perivascular markers and present the receptor CD38 on their surface. PECAM1⁺/Sca1⁺/CD38⁺ cells proliferate upon wounding and could give rise to α-SMA⁺ myofibroblast-like cells. CD38 stimulation in immunodeficient mice reduced the wound size at the peak of neoangiogenesis and myofibroblast formation. In humans a corresponding cell population was identified, which was enriched in sprouting vessels of basal cell carcinoma biopsies. The results indicate that PECAM1⁺/Sca1⁺/CD38⁺ vascular cells could proliferate and differentiate into myofibroblast-like cells in wound repair. Moreover, CD38 signaling modulates PECAM1⁺/Sca1⁺/CD38⁺ cell activation in the healing process implying CD38 as a target for anti-angiogenic therapies in human basal cell carcinoma.     

Human placenta and bone marrow derived MSC cultured in serum-free, b-FGF-containing medium express cell surface frizzled-9 and SSEA-4 and give rise to multilineage differentiation

Conventionally, mesenchymal stem cells (MSC) are generated by plating cells from bone marrow (BM) or other sources into culture flasks and selecting plastic-adherent cells with fibroblastoid morphology. These cells express CD9, CD10, CD13, CD73, CD105, CD166, and other markers but show only a weak or no expression of the embryonic markers stage-specific embryonic antigen-4 (SSEA-4), Oct-4 and nanog-3. Using a novel protocol we prepared MSC from BM and non-amniotic placenta (PL) by culture of Ficoll-selected cells in gelatin-coated flasks in the presence of a serum-free, basic fibroblast growth factor (b-FGF)-containing medium that was originally designed for the expansion of human embryonic stem cells (ESC). MSC generated in gelatin-coated flasks in the presence of ESC medium revealed a four-to fivefold higher proliferation rate than conventionally prepared MSC which were grown in uncoated flasks in serum-containing medium. In contrast, the colony forming unit fibroblast number was only 1.5- to twofold increased in PL-MSC and not affected in BM-MSC. PL-MSC grown in ESC medium showed an increased surface expression of SSEA-4 and frizzled-9 (FZD-9), an increased Oct-4 and nestin mRNA expression, and an induced expression of nanog-3. BM-MSC showed an induced expression of FZD-9, nanog-3, and Oct-4. In contrast to PL-MSC, only BM-MSC expressed the MSC-specific W8B2 antigen. When cultured under appropriate conditions, these MSC gave rise to functional adipocytes and osteoblast-like cells (mesoderm), glucagon and insulin expressing pancreatic-like cells (endoderm), as well as cells expressing the neuronal markers neuron-specific enolase, glutamic acid decarboxylase-67 (GAD), or class III beta-tubulin, and the astrocyte marker glial fibrillary acidic protein (ectoderm). In conclusion, using a novel protocol we demonstrate that adult BM-and neonatal PL-derived MSC can be induced to express high levels of FZD-9, Oct-4, nanog-3, and nestin and are able of multi-lineage differentiation.     

Beta2-integrins contribute to skeletal muscle hypertrophy in mice

We tested the contribution of β₂-integrins, which are important for normal function of neutrophils and macrophages, to skeletal muscle hypertrophy after mechanical loading. Using the synergist ablation model of hypertrophy and mice deficient in the common β-subunit of β₂-integrins (CD18^–/–), we found that overloaded muscles of wild-type mice had greater myofiber size, dry muscle mass, and total protein content compared with CD18^–/– mice. The hypertrophy in wild-type mice was preceded by elevations in neutrophils, macrophages, satellite cell/myoblast proliferation (5'-bromo-2'-deoxyuridine- and desmin-positive cells), markers of muscle differentiation (MyoD1 and myogenin gene expression and formation and size of regenerating myofibers), signaling for protein synthesis [phosphorylation of Akt and 70-kDa ribosomal protein S6 kinase (p70S6k)], and reduced signaling for protein degradation (decreased gene expression of muscle atrophy F box/atrogin-1). The deficiency in β₂-integrins, however, altered the accumulation profile of neutrophils and macrophages, disrupted the temporal profile of satellite cell/myoblast proliferation, reduced the markers of muscle differentiation, and impaired the p70S6k signaling, all of which could serve as mechanisms for the impaired hypertrophy in overloaded CD18^–/– mice. In conclusion, our findings indicate that β₂-integrins contribute to the hypertrophic response to muscle overload by temporally regulating satellite cells/myoblast proliferation and by enhancing muscle differentiation and p70S6k signaling. skeletal muscle growth; neutrophils; macrophages; compensatory hypertrophy     

Stromal cells cultured from omentum express pluripotent markers,produce high amounts of VEGF,and engraft to injured sites

When rat omentum becomes activated by intraperitoneal injection of inert polydextran particles, these particles are rapidly surrounded by cells that express markers of adult stem cells (SDF–1α, CXCR4, WT–1) and of embryonic pluripotent cells (Oct–4, Nanog, SSEA–1). We have cultured such cells, because they may offer a convenient source of adult stem cells, and have found that they retain stem cell markers and produce high levels of vascular endothelial growth factor for up to ten passages. After systemic or local injection of these cultured cells into rats with acute injury of various organs, the cells specifically engraft at the injured sites. Thus, our experiments show that omental stromal cells can be cultured from activated omentum, and that these cells exhibit stem cell properties enabling them to be used for repair and possibly for the regeneration of damaged tissues.     

Tricyclic antidepressants prevent the differentiation of monocytes into macrophage-like cells in vitro

The investigation was designed to determine whether the two tricyclic antidepressant agents (TCAs) clomipramine and imipramine and the selective reuptake inhibitor citalopram affect differentiation of human monocytes to macrophage-like cells (MAC-LCs). We established primary adherent cultures of peripheral blood monocytes and monitored their morphology, capacity for phagocytosis and antigen expression during transformation to MAC-LCs. As expected, maturation of monocytes to MACs is accompanied by changes in morphology, elevated expression of the antigens CD16 and CD51 and an increase in the percentage of phagocytic cells. Treatment of cells with the TCAs clomipramine (40 μmol/L) or imipramine (100 μmol/L) and with citalopram (100 μmol/L), for 11 days resulted in the following observations: (1) monocytes treated with TCAs never developed the morphology characteristic of the MAC-LCs; (2) TCAs reduced the percentage of phagocytic cells; (3) TCAs had little influence on the expression of CD14, CD16, CD51, and HLA-DR. However, when added after monocyte differentiation into MAC-LCs, citalopram and clomipramine no longer reduced the percentage of phagocytic cells and these effects were not simply due to irreversible cytotoxicity. Thus clomipramine, imipramine, and citalopram inhibit differentiation of human monocytes into MAC-LCs in vitro, but in a reversible manner. This revised version was published online in July 2006 with corrections to the Cover Date.     

Multidimensional flow-cytometric analysis of dendritic cells in peripheral blood of normal donors and cancer patients

 We studied the potential of multidimensional flow cytometry to evaluate the frequency and maturation/activation status of dendritic cells in minimally manipulated peripheral blood mononuclear cell preparations (i.e., only separated on Ficoll-Hypaque) of normal donors and cancer patients. A rare subset of HLA-DR⁺ leukocytes (less than 1% mononuclear cells) was detected in blood of normal donors that displayed all the features of dendritic cells: these cells had high forward-light-scatter characteristics and coexpressed CD4, CD86 and CD54 surface antigens, but lacked the lineage-associated surface markers of T cells, B cells, monocytes, granulocytes or NK i.e. they were CD3^–, CD19^–, CD20^–, CD14^–, CD11b^–, CD16^–, CD56^–). These physical and phenotypic properties were virtually identical to those of immunomagnetically sorted leukocytes characterized as dendritic-cells on the basis of morphology, phenotype and high stimulatory activity in allogeneic mixed-lymphocyte cultures. Using this flow-cytometric approach we observed that the frequency of dendritic cell-like cells in peripheral blood mononuclear cell specimens of cancer patients receiving chemotherapy alone or those recovering from stem cell transplantation was significantly lower than that of normal individuals (mean ± SE: 0.36 ± 0.05%, 0.14 ± 0.06%, and 0.75 ± 0.04% respectively). Multidimensional flow-cytometric analysis of dendritic cells might represent an important new tool for assessing immunocompetence, and for monitoring the effects of therapeutic regimens on the immune system. Received: 20 June 1997 / Accepted: 14 August 1997     

Isolation,characterization and differentiation of cells expressing pluripotent/multipotent markers from adult human ovaries

Pluripotent stem cells are still generally accepted not to exist in adult human ovaries, although increasing studies confirm the presence of pluripotent/multipotent stem cells in adult mammalian ovaries, including those of humans. The aim of this study is to isolate, characterize and differentiate in vitro stem cells that originate from the adult human ovarian cortex and that express markers of pluripotency/multipotency. After enzymatic degradation of small ovarian cortex biopsies retrieved from 18 women, ovarian cell cultures were successfully established from 17 and the formation of cell colonies was observed. The presence of cells/colonies expressing some markers of pluripotency (alkaline phosphatase, surface antigen SSEA-4, OCT4, SOX-2, NANOG, LIN28, STELLA), germinal lineage (DDX4/VASA) and multipotency (M-CAM/CD146, Thy-1/CD90, STRO-1) was confirmed by various methods. Stem cells from the cultures, including small round SSEA-4-positive cells with diameters of up to 4 μm, showed a relatively high degree of plasticity. We were able to differentiate them in vitro into various types of somatic cells of all three germ layers. However, these cells did not form teratoma when injected into immunodeficient mice. Our results thus show that ovarian tissue is a potential source of stem cells with a pluripotent/multipotent character for safe application in regenerative medicine.