Asherman syndrome and insufficient endometrial thickness: A hypothesis of integrated approach to restore the endometrium.

Asherman syndrome consists in an acquired condition characterized by the development of fibrous intrauterine adhesions involving until two-thirds of the uterine cavity. Common signs of the syndrome are represented by alterations of regular menses, hypomenorrhea and amenorrhea. Moreover, women affected by Asherman syndrome, often struggle with fertility problems such as difficulty in spontaneous conceiving as well as complications including recurrent pregnancy loss and invasive placentation. The abnormality of the endometrial line consisting in insufficient thickness and/or endometrial trauma damaging the decidua basalis, are characteristic elements of the disease. Several studies have been conducted during the last ten years to find a solution restoring the regular endometrial line solving the fertility issue in Asherman women. Hormonal therapy as well as the use of stem cells seem to represent valid options to regenerate the endometrium opening a new scenario in the fertility treatment of these women. In this context, the presented study proposes an integrated approach to reach an adequate endometrial reconstitution and consequentially optimal fertility outcomes.

Steady state peripheral blood provides cells with functional and metabolic characteristics of real hematopoietic stem cells.

Hematopoietic stem cells (HSCs), which are located in the bone marrow, also circulate in cord and peripheral blood. Despite high availability, HSCs from steady state peripheral blood (SSPB) are little known and not used for research or cell therapy. We thus aimed to characterize and select HSCs from SSPB by a direct approach with a view to delineating their main functional and metabolic properties and the mechanisms responsible for their maintenance. We chose to work on Side Population (SP) cells which are highly enriched in HSCs in mouse, human bone marrow, and cord blood. However, no SP cells from SSBP have as yet been characterized. Here we showed that SP cells from SSPB exhibited a higher proliferative capacity and generated more clonogenic progenitors than non-SP cells in vitro. Furthermore, xenotransplantation studies on immunodeficient mice demonstrated that SP cells are up to 45 times more enriched in cells with engraftment capacity than non-SP cells. From a cell regulation point of view, we showed that SP activity depended on O2 concentrations close to those found in HSC niches, an effect which is dependent on both hypoxia-induced factors HIF-1α and HIF-2α. Moreover SP cells displayed a reduced mitochondrial mass and, in particular, a lower mitochondrial activity compared to non-SP cells, while they exhibited a similar level of glucose incorporation. These results provided evidence that SP cells from SSPB displayed properties of very primitive cells and HSC, thus rendering them an interesting model for research and cell therapy.

Establishment and Analysis of Cancer Stem-Like and Non-Cancer Stem-Like Clone Cells from the Human Colon Cancer Cell Line SW480.

Human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be isolated as side population (SP) cells, aldehyde dehydrogenase high (ALDHhigh) cells or cell surface marker-positive cells including CD44+ cells and CD133+ cells. CSCs/CICs and non-CSCs/CICs are unstable in in vitro culture, and CSCs/CICs can differentiate into non-CSCs/CICs and some non-CSCs/CICs can dedifferentiate into CSCs/CICs. Therefore, experiments using a large amount of CSCs/CICs are technically very difficult. In this study, we isolated single cell clones from SP cells and main population (MP) cells derived from the human colon cancer cell line SW480. SP analysis revealed that SP clone cells had relatively high percentages of SP cells, whereas MP clone cells showed very few SP cells, and the phenotypes were sustainable for more than 2 months of in vitro culture. Xenograft transplantation revealed that SP clone cells have higher tumor-initiating ability than that of MP clone cells and SP clone cell showed higher chemo-resistance compared with MP clone cells. These results indicate that SP clone cells derived from SW480 cells are enriched with CSCs/CICs, whereas MP clone cells are pure non-CSCs/CICs. SP clone cells and MP clone cells are a very stable in vitro CSC/CIC-enriched and non-CSC/CIC model for further analysis.

Cell therapy in muscular dystrophies: many promises in mice and dogs, few facts in patients.

INTRODUCTION: Muscular dystrophies (MDs) are genetic diseases that produce progressive loss of skeletal muscle fibers. Cell therapy (CT) is an experimental approach to treat MD. The first clinical trials of CT in MD conducted in the 1990s were based on myoblast transplantation (MT). Since they did not yield the expected results, several researchers sought to discover other cells with more advantageous properties than myoblasts whereas others sought to improve MT. AREAS COVERED: We explain the properties that are required for a cell to be used in CT of MD. We briefly review most of the cells that were proposed for this CT, and to what extent these properties were met not only in laboratory animals but also in clinical trials. EXPERT OPINION: Although the repertoire of cells proposed for CT of MD has been expanded since the 1990s, only myoblasts have currently demonstrated unequivocally to significantly engraft in humans. Indeed, MT for MD involves significant technical challenges that need be solved. While it would be ideal to find cells involving less technical challenges for CT of MD, there is so far no clinical evidence that this is possible and therefore the work to improve MT should continue.

CXCL14 and MCP1 are potent trophic factors associated with cell migration and angiogenesis leading to higher regenerative potential of dental pulp side population cells.

INTRODUCTION: The release of trophic factors from mesenchymal stem cells (MSCs) is critical for tissue regeneration. A systematic investigation of the regenerative potential of trophic factors from different MSCs, however, has not been performed. Thus, in the present study, the regenerative potential of conditioned medium (CM) from dental pulp, bone marrow, and adipose tissue-derived CD31(-) side population (SP) cells from an individual source was compared in an ectopic tooth transplantation model. METHODS: The tooth root transplantation in an ectopic site model was used for investigation of the regenerative potential and trophic effects in vivo. Either pulp CD31(-) SP cell populations (1×10(6) cells) at the third to fourth passage or 5 µg/ml of CM from dental pulp, bone marrow, and adipose stem cells from four different individuals were injected into the root with collagen TE. Each root was transplanted subcutaneously in 5-week-old severe combined immunodeficiency mice. Each root with surrounding tissue was harvested for histology on days 7, 21, and 28 and for Western blot analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis on day 28. Furthermore, the trophic factors responsible for the regenerative potential were identified as the upregulated genes present in pulp CD31(-) SP cells when compared with the genes in both bone marrow and adipose CD31(-) SP cells by using microarray analysis, real-time RT-PCR, and Western blot analysis. RESULTS: Transplantation of pulp CM yielded increased volume of pulp regeneration, more bromodeoxyuridine (BrdU)-positive migrated cells, and fewer caspase 3-positive cells in the regenerated pulp compared with the others. Pulp CM also demonstrated significantly increased cell migration, anti-apoptosis, and angiogenesis in C2C12 cells. Higher expression of CXCL14 and MCP1 in pulp SP cells suggested candidate trophic factors. The stimulatory effects on both migration and angiogenesis of CXCL14 and MCP1 were demonstrated in vitro. In the regenerated tissue, BrdU-positive migrated cells expressed CXCR4 and CCR2, receptors for CXCL14 and MCP1, respectively. CONCLUSIONS: The higher regenerative potential of pulp SP cells may be due to potent trophic factors, including CXCL14 and MCP1, which promote migration and angiogenesis.

Nuclear Localization Signal-Enhanced Polyurethane-Short Branch Polyethylenimine-Mediated Delivery of Let-7a Inhibited Cancer Stem-Like Properties by Targeting the 3'-UTR of HMGA2 in Anaplastic Astrocytoma.

Anaplastic astrocytoma (AA) is a grade III glioma that often occurs in middle-aged patients and presents a uniformly poor prognosis. A small subpopulation of cancer stem cells (CSCs) possessing a self-renewing capacity is reported to be responsible for tumor recurrence and therapeutic resistance. An accumulating amount of microRNAs (miRNA) were found aberrantly expressed in human cancers and regulate CSCs. Efforts have been made to couple miRNAs with nonviral gene delivery approaches to target specific genes in cancer cells. However, the efficiency of delivery of miRNAs to AA-derived CSCs is still an applicability hurdle. The present study aimed to investigate the effectiveness and applicability of nonviral vector-mediated delivery of Let-7a with regard to eradication of AA and AA-derived CSC cells. Herein, our miRNA/mRNA microarray and RT-PCR analysis showed that the expression of Let-7a, a tumor-suppressive miRNA, is inversely correlated with the levels of HMGA2 and Sox2 in the AA side population (SP(+)) cells. Luciferase reporter assay showed that Let-7a directly targets the 3'-UTRs of HMGA2 in AA-SP(+) cells. Knockdown of HMGA2 significantly suppressed the protein expression of Sox2 in AA-SP(+) cells, whereas overexpression of HMGA2 upregulated Sox2 expression in AA-SP(-). Nuclear localization signal (NLS) peptides can facilitate nuclear targeting of DNA and are used to improve gene delivery. Using polyurethane-short branch polyethylenimine (PU-PEI) as a therapeutic delivery vehicle, we conjugated NLS with Let-7 and successfully delivered it to AA-SP(+) cells, resulting in significantly suppressed expression of HMGA2 and Sox2, tumorigenicity, and CSC-like abilities. This treatment facilitated the differentiation of AA-SP(+) cells into non-SP CSCs. Furthermore, PU-PEI-mediated delivery of NLS-conjugated Let-7a in AA-SP(+) cells suppressed the expression of drug-resistant and antiapoptotic genes, and increased cell sensitivity to radiation. Finally, the in vivo delivery of PU-PEI-NLS-Let-7a significantly suppressed the tumorigenesis of AA-SP(+) cells and synergistically improved the survival rate of orthotopically AA-SP(+)-transplanted immunocompromised mice when combined with radiotherapy. Therefore, PU-PEI-NLS-Let-7a is a potential novel therapeutic approach for AA.

Enhanced bone-forming activity of side population cells in the periodontal ligament.

Regeneration of alveolar bone is critical for the successful treatment of periodontal diseases. The periodontal ligament (PDL) has been widely investigated as a source of cells for the regeneration of periodontal tissues. In the present study where we attempted to develop an effective strategy for alveolar bone regeneration, we examined the osteogenic potential of side population (SP) cells, a stem cell-containing population that has been shown to be highly abundant in several kinds of tissues, in PDL cells. Isolated SP cells from the rat PDL exhibited a superior ability to differentiate into osteoblastic cells compared with non-SP (NSP) and unsorted PDL cells in vitro. The mRNA expressions of osteoblast markers and bone morphogenetic protein (BMP) 2 were significantly upregulated in SP cells and were further increased by osteogenic induction. To examine the bone-forming activity of SP cells in vivo, PDL SP cells isolated from green fluorescent protein (GFP)-transgenic rats were transplanted with hydroxyapatite (HA) disks into wild-type animals. SP cells exhibited a high ability to induce the mineralized matrix compared with NSP and unsorted PDL cells. At 12 weeks after the implantation, some of the pores in the HA disks with SP cells were filled with mineralized matrices, which were positive for bone matrix proteins, such as osteopontin, bone sialoprotein, and osteocalcin. Furthermore, osteoblast- and osteocyte-like cells on and in the bone-like mineralized matrices were GFP positive, suggesting that the matrices were directly formed by the transplanted cells. These results suggest that PDL SP cells possess enhanced osteogenic potential and could be a potential source for cell-based regenerative therapy for alveolar bone.

Vasculogenesis of decidua side population cells of first-trimester pregnancy.

INTRODUCTION: Sufficient uterine blood supply is essential for the fetus to develop normally in the uterus. Several mechanisms are involved in the process of vessel development in deciduas and villus. We focus on whether first-trimester decidua side population (SP) cells contain cells capable of differentiating into endothelial cells. METHODS: Eight decidua samples were collected from healthy women, 22- to 30-years old, undergoing elective terminations of early pregnancy (six to eight gestational weeks). The cell suspensions from human deciduas were stained by Hoechst 33342 and sorted by flow cytometry, further cultured under differentiation conditions and analyzed for specific markers. These cells were implanted into ischemic limbs of nude mice to test the capacity of angiogenesis in vivo by DiI tracers and immunohistochemistry. RESULTS: Decidua CD31(-)CD146(-) SP cells of first-trimester human pregnancy can differentiate into endothelial cells, express the corresponding specific markers of endothelial cells, such as CD31 and CD146, and form tube-like structures on Matrigel and part of newly formed vessels in the ischemic limbs of nude mice. Vascular endothelial growth factor was more effective in promoting proliferation of CD31(-)CD146(-)SP cells compared with other growth factors, and estrogen and progesterone at a final concentration of 10 µmol/L and 30 µmol/L, respectively, promoted the migration of CD31()-CD146(-)SP cells in a dose-dependent manner. CONCLUSIONS: CD31(-)CD146(-) SP cells may be involved in the formation of new vessels in the maternal aspect of the placenta in the first trimester.

Stimulation of angiogenesis, neurogenesis and regeneration by side population cells from dental pulp.

Mesenchymal stem cells (MSCs) have been used for cell therapy in various experimental disease models. However, the regenerative potential of MSCs from different tissue sources and the influence of the tissue niche have not been investigated. In this study, we compared the regenerative potential of dental pulp, bone marrow and adipose tissue-derived CD31(-) side population (SP) cells isolated from an individual porcine source. Pulp CD31(-) SP cells expressed the highest levels of angiogenic/neurotrophic factors and had the highest migration activity. Conditioned medium from pulp CD31(-) SP cells produced potent anti-apoptotic activity and neurite outgrowth, compared to those from bone marrow and adipose CD31(-) SP cells. Transplantation of pulp CD31(-) SP cells in a mouse hindlimb ischemia model produced higher blood flow and capillary density than transplantation of bone marrow and adipose CD31(-) SP cells. Motor function recovery and infarct size reduction were greater with pulp CD31(-) SP cells. Pulp CD31(-) SP cells induced maximal angiogenesis, neurogenesis and pulp regeneration in ectopic transplantation models compared to other tissue sources. These results demonstrate that pulp stem cells have higher angiogenic, neurogenic and regenerative potential and may therefore be superior to bone marrow and adipose stem cells for cell therapy.

Transplantation of side population cells restores the function of damaged exocrine glands through clusterin.

Stem cell-based therapy has been proposed as a promising strategy for regenerating tissues lost through incurable diseases. Side population (SP) cells have been identified as putative stem cells in various organs. To examine therapeutic potential of SP cells in hypofunction of exocrine glands, SP cells isolated from mouse exocrine glands, namely, lacrimal and salivary glands, were transplanted into mice with irradiation-induced hypofunction of the respective glands. The secretions from both glands in the recipient mice were restored within 2 months of transplantation, although the transplanted cells were only sparsely distributed and produced no outgrowths. Consistent with this, most SP cells were shown to be CD31-positive endothelial-like cells. In addition, we clarified that endothelial cell-derived clusterin, a secretory protein, was an essential factor for SP cell-mediated recovery of the hypofunctioning glands because SP cells isolated from salivary glands of clusterin-deficient mice had no therapeutic potential, whereas lentiviral transduction of clusterin restored the hypofunction. In vitro and in vivo studies showed that clusterin had an ability to directly inhibit oxidative stress and oxidative stress-induced cell damage. Thus, endothelial cell-derived clusterin possibly inhibit oxidative stress-induced hypofunction of these glands.

[Isolation and characterization of side population cells in human gastric cancer cell line BGC-823].

OBJECTIVE: Isolate and characterize the side population (SP) cells with potency of stem cells from human gastric carcinoma cell line BGC-823. METHODS: SP and non-SP cells were sorted from BGC-823 cells by fluorescence-activated cell sorting (FACS) using Hoechst33342 staining. The tumorigenic ability of the SP cells was assessed by in vivo transplantation into non-obese diabetic/severe combined immunodeficiency mice. RESULTS: SP cells were isolated from BGC-823 cells in a proportion of 0.9% to 2.1% with respect to the whole cell population. The colony formation assay showed that the colony formation rate of the SP cells was significantly higher than that of the non-SP cells (72.56% vs. 49.00%, P < 0.01). The drug sensitivity test showed that the SP cells showed stronger drug resistance to 5-Fu than the non-SP cells. The in vivo transplantation of SP cells in mice showed that the tumor weight was (0.176 ± 0.034) g, significantly higher than that of non-SP cells (0.045 ± 0.046) g (P < 0.01). CONCLUSIONS: The results of this study indicate the existence of cancer stem-like SP cells in the human gastric cancer line BGC-823 cells. Further characterization of this SP cell population may provide new insights for diagnosis and treatment of gastric cancer.

Regeneration of dental pulp by stem cells.

Angiogenesis/vasculogenesis and neurogenesis are essential for pulp regeneration. Two subfractions of side-population (SP) cells, CD31(-)/CD146(-) SP cells and CD105(+) cells with angiogenic and neurogenic potential, were isolated by flow cytometry from canine dental pulp. In an experimental model of mouse hindlimb ischemia, transplantation of these cell populations resulted in an increase in blood flow, including high-density capillary formation. In a model of rat cerebral ischemia, stem cell transplantations enhanced neuronal regeneration and recovery from motor disability. Autologous transplantation of the CD31(-)/CD146(-) SP cells into an in vivo model of amputated pulp resulted in complete regeneration of pulp tissue with vascular and neuronal processes within 14 days. The transplanted cells expressed pro-angiogenic factors, implying trophic action on endothelial cells. Autologous transplantation of CD31(-)/CD146(-) SP cells or CD105(+) cells with stromal-cell-derived factor-1 (SDF-1) into root canals after whole pulp removal of mature teeth resulted in complete regeneration of pulp replete with nerves and vasculature by day 14, followed by dentin formation along the dentinal wall by day 35. Therefore, the potential utility of fractionated SP cells and CD105(+) cells in angiogenesis and neurogenesis was demonstrated by treatment of limb and cerebral ischemia following pulpotomy and pulpectomy.

Biologic characteristics of the side population of human small cell lung cancer cell line H446.

BACKGROUND AND OBJECTIVE: Recently, the theory of cancer stem cells (CSCs) has presented new targets and orientations for tumor therapy. The major difficulties in researching CSCs include their isolation and purification. The aim of this study is to identify and characterize the side population (SP) cells in small cell lung cancer (SCLC) cell line H446, which lays the foundation for the isolation and purification of CSCs. METHODS: Fluorescence-activated cell sorting (FACS) was used to sort SP and non-SP (NSP) cells from H446. Both subgroups were cultivated to survey the capacity to form into suspended tumor cell spheres. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR were used to evaluate the expression levels of the mRNA of CD133, ABCG2, and nucleostemin in both subgroups. The capacity of proliferation and the differences in drug resistance of both subgroups and unsorted cells were tested by the MTT method. The differentiation ability of both subgroups was determined by FACS. Proliferation was determined by subcutaneous tumor formation in nude mice. RESULTS: The percent of Hoechst 33342 negative cells was about (5.1 +/- 0.2)% in H446 by fluorescence microscopy. The percent of SP cells was (6.3 +/- 0.1)% by flow cytometry. SP cells had a stronger capability of forming into tumor spheres than NSP cells. The mRNA expression levels of ABCG2, CD133, and nucleostemin in SP cells were 21.60 +/- 0.26, 7.10 +/- 0.14, and 1.02 +/- 0.08 folds higher than that in NSP cells (P < 0.01, P < 0.01, and P > 0.05, respectively). In vivo, SP cells showed better proliferative ability and tougher viability when treated with drugs. SP cells can differentiate into NSP cells, but NSP cells cannot differentiate into SP cells. SP cells had a greater ability to form tumors. CONCLUSION: The H446 cell line contained some SP cells with stem cell properties. CD133 and ABCG2 may be cancer stem cell markers of SCLC.