Human mesenchymal stem cells differentiate into osteoblasts

AIM:To investigate the differentiation from human mesenchymal stem cells (hMSC) into osteoblasts. METHODS:MSC were separated from human marrow with Ficoll-Paque reagent and expanded in cuture medium. To detect the surface antigens, The labeled cells were analysed on a FACScan flow cytometer. hMSC were induced to differentiate from mesenchymal stem cells into osteoblasts with dexamethasone, vitamin C, β-GP. Cell morphology、AP activity、calcium deposition and osteopontin were detected. P10 MSC were compared to P3 MSC in the tendency of osteoblastic differentiation. RESULTS:The cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. hMSC showed a strong self-renewal capacity. After primary culture, approximately (5-6)×10⁵ cells were obtained. These expanded attached MSC were uniformaly positive for CD29,CD44,CD59,CD105,CD166 and didn’t express CD11a, CD14, CD33, CD34, CD45, CD38, CD80, CD86, CD117. After osteoblasts induction, the cells changed from spindle-shape to cuboidal and polygonal in cell morphology. The AP activity increased gradually and many scattered calcium nodes were observed. The expression of osteopontin was positive. CONCLUSION:hMSC can be induced to differentiate into osteoblasts.     

The ex vivo expansion characteristic of endothelial progenitor cells

AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34⁺ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34⁺ and CD34^- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34⁺ and CD34^-,total MNC led to a significant increase in the expansion of CD34⁺ cells compared with CD34 enrichment (P＜0.05). There was a trend toward decreased apoptosis in cultures when early passage was performed once the linear cord like structures appeared. There was no significant effect on apoptosis between with VEGF and without VEGF group (P＞0.05). These differentiated EPCs were stained positive for CD34⁺, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34⁺ and AC133⁺cells accounted for 68.2%±6.3% (n=6) and 57.2%±9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34⁺ and CD34^- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs.     

High-efficient genetic transfection of CD41+,UT7, U937 and MDA-MB-435 cells with a recombined murine stem cell retroviral vector

AIM: Gene transduction with a recombined murine stem cell retroviral vector has been investigated to find an effective way of gene transduction and to offer theory and experimental basis for the recombined murine stem cell retroviral vector used for gene transduction. METHODS: 1. Construction of retrovirus vector: EC1-4 gene (repeats 1-4 of cadher in-5 extracellular domain) and mutant (Ser 222A) MEK1 gene were cloned into retrovirus vector pMSCV after cut by Bgl Ⅱ and EcoR 1 restriction endonuclease. 2. Obtaining CD41⁺ cells and cell culture: Cells expressing CD34⁺ from cord blood were isolated. The inducement of cells expressing CD41 from CD34⁺ cells was performed by using TPO and cells CD41⁺ were selected by FACS. NIH 3T3 cells were cultured in high sugar DMEM medium and U937 in RPMI 1640 medium. UT7 cells which is cytokine-dependent cell line were grown in Iscove's modified Dulbeco's medium supplemented by GM-CSF. 3. Determination of viral titers: Retroviral vectors were transferred to packaging cell line 293. Retroviral containing supenatant was collected after transfection. The viral titers was tested on infection of NIH 3T3 cells by FACS analysis. 4. Western blot: Transduced CD41⁺, UT7, U937 and MDA-MB-435 cells were analyzed by western blot to examine expression of transduced genes. RESULTS: A packaging cell line 293 produces high-titer MEK1 pMSCV retroviruses (3.1×10⁷) and EC1-4 pMSCV retroviruses (1.0×10⁸). With 8-folds dilution retroviruses, 60.73% GFP positive cells have been obtained in MEK1 pMSCV transduced UT7 cells, 72.56% in U937 cells and 30.57% in CD41⁺ cells, respectively. GFP positive cells have reached up 97.54 % in EC1-4 pMSCV transducted MDA-MB-435 cells. Phosphorylated MEK1 has been decreased in experiment group when TPO has stimulated CD41⁺ and UT7 cells or serum has stimulated U973 cells. This indicates that is a dominant negative effect of mutation MEK gene. EC1-4 gene transduced MDA-MB-435 cells have expressed EC1-4. CONCLUSION:The recombined murine stem cell retrovirus can effectively mediate gene transduction of CD41⁺,UT7,U937 and MDA-MB-435 cells,and transduced genes can be stably expressed.     

Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.     

Biological characteristics of JAK2 transduced CD34+ cells from cord blood during ex vivo expansion

AIM: To explore the feasibility and biological characterization of long-term regulated expansion of JAK2 transduced human CD34⁺ cord blood cells in vitro.METHODS: A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducer,dimerization (AP20187),was cloned (designated MGI-F₂JAK2).CD34⁺cells were enriched from cord blood with a MiniMACS system.The purified CD34⁺ cells were transfected with supernatant from the retrovirus packaging cell line that expressed JAK2.Following transduction,cells were expanded into four groups: AP20187 alone,FL alone,TPO,alone,AP20187+FL+TPO,respectively.The expanded cells were monitored by GFP expression,immunophenotyping,progenitor colony assay,karyotype analysis as well as tumorigenesis in nude mice.RESULTS: The purity of selected CD34⁺ cells was over 91% and gene transfer rate was 49.32%±6.21%.Only the group of AP20187 +FL+ TPO was obtained a significant sustained outgrowth of the transduced CD34⁺ cord blood cells.The percentage of GFP+ cells consistently produced a rise to the 90% peak level by the end of 8th week of culture.Flow cytometry analysis showed that the phenotype of the expanded cells was CD33⁺,CD61⁺ and Gly-A⁺ partial positive;CD38⁺ and HLA-DR⁺ strong positive,while CD2,CD7 and CD19 were almost negative.Colony assays performed in methycelluos,which can give rise to BFU-E,CFU-GM and CFU-Mix,the CFU-GM was predominantly in all colonies.The tumor was not observed in nude mice and the karyotype analysis was normal from expanded cells.CONCLUSION: The results demonstrate that AP20187-mediated activation of JAK2 signaling is capable of stimulating expansion JAK2 transduced CB CD34⁺ cells in combination with FL and TPO.This system may have applications for studies in signaling transduction,hematopoiesis,and for gene and cell therapy.     

The transdifferentiation of Sca-1+ cells from murine fetal liver

AIM:To explore transdifferentiation potential of Sca-1⁺ cells from murine fetal liver. METHODS:2×10³ of Sca-1⁺ cells from male murine fetal liver were transfused into female mouse irradiated lethally with γ ray from ⁶⁰ Co source (10 Gy) via tail vein. Two months later, FISH and immunohistochemistry were used to detect the situation for transdifferentiating of the donor cells (male cells) in tissues of female recipient mouse. RESULTS:The renal tubular epitheliocyte-like and neurocyte-like cells with Y chromosome were found on the sections of renal and brain tissues from female recipient mice. These cells have phenotype characteristics of RCA⁺/CD₄₅^-F_4/80^- and NueN⁺/CD₄₅^-F_4/80^-, respectively. CONCLUSION:The evidence is provided for Sca-1⁺ cells from murine fetal liver to transdifferentiate into both renal and brain tissue cells.     

Adult human mesenchymal stem cell differentiates into adipocytes

AIM:To investigate the differentiation from human mesenchymal stem cells (hMSC) to adipocytes.METHODS: hMSC were separated from rib marrow and expanded in culture medium. To detect the surface antigens, the labeled cells were analysed on a FACScan flow cytometer. hMSC were induced with dexamethasone, insulin, 1-methy1-3-isobutylxanthine and indomethacin which acted as adipocyte differentiation inducer. The cells were stained with Oil Red O. The number of adipocytes were counted on a phase-contrast microscope.RESULTS: hMSC were expanded as undifferentiated cells in culture for more than 5 passages. The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. These expanded, attached MSC were uniformly positive for CD29, CD44, CD90, CD105, CD166 and didn't express CD14, CD34, CD45, CD11a. After induced with induction medium, lipid vacuoles were first detectable within the cells at 48 hours. Two weeks later, more than 85% MSC differentiated into adipocytes which displayed a perinuclear accumlation of lipid vacuoles, as detected by Oil Red O. CONCLUSION:hMSC can be induced to differentiate into adipocytes.     

Protective effect of nicotinic acid amide on human umbilical cord mesenchymal stem cells

AIM: To investigate the effect of nicotinic acid amide (NAA) on the infusion damage of human umbilical cord mesenchymal stem cells (hUC-MSCs) under the condition of instant blood-mediated inflammatory reaction (IBMIR). METHODS: Normal peripheral blood without anticoagulant at volume of 2.7 mL was mixed with 0.3 mL physiological saline (as blank group), CFSE labeled hUC-MSCs (1×10⁶ cells in 0.3 mL as MSC group) and CFSE labeled hUC-MSCs (1×10⁶ cells in 0.3 mL) preprocessed with NAA at concentration of 10 mmol/L for 24 h (as MSC+NAA group), respectively. The mixture was immediately injected into the improved Chandler Loop model, placed in 37 ℃ water bath, and then started the peristaltic pump at the speed of 20 mL/min for 1 h. The number of CFSE labeled hUC-MSCs, platelets, white blood cells were counted and the concentration of complement C3a was measured before and after cycling, respectively. RESULTS: After 1 h circulation, the platelet dissipation rate were (29.96±10.88)% in blank group, (77.76±19.29)% in MSC group all and (50.13±18.10)% in MSC+NAA group; and the leukocyte counts were (37.82±13.81)% in blank group, (64.57±17.08)% in MSC group and (41.52±17.26)% in MSC+NAA group. Compared with blank group, the differences of the dissipation rates in MSC group and MSC+NAA group all had statistical significance. The hUC-MSCs relative survival rate in MSC+NAA group was higher than that in MSC group. C3a concentrations in blank group, MSC group and MSC+NAA group were (206.27±58.10), (230.47±39.61) and (208.37±40.66) μg/L, respectively. CONCLUSION: Co-circulating the mixture of hUC-MSCs with normal peripheral blood without anticoagulant in the improved Chandler Loop for 1 h depletes a large number of hUC-MSCs and blood components, and increases C3a, suggesting that this model can induce IBMIR. NAA has a protective effect on the hUC-MSCs in the infusion damage by inhibiting IBMIR, reducing the wastage of the blood components and enhancing the survival rate of the hUC-MSCs.     

Percentage change on natural killer subsets of peripheral blood and decidua in between normal early pregnancy and spontaneous abortion

AIM: To determinate the percentage change on natural killer(NK) subsets of peripheral blood and decidua in between normal early pregnancy and spontaneous abortion. METHODS: Flow cytometry was used to detect the expression of CD56 and CD16 on lymphocytes of decidua and peripheral blood in normal early pregnancy and spontaneous abortion. RESULTS: In peripheral blood, the percentage of CD56⁺ of spontaneous abortion tended to decrease in comparison with normal early pregnancy, and the percentage of CD56⁺CD16⁺ in spontaneous abortion was significantly less than that in normal early pregnancy. The percentages of different natural killer subsets in decidua of spontaneous abortion were significantly lower than those in decidua of normal early pregnancy. CONCLUSION: The decrease of CD56⁺NK cells in decidua may be one of causes for abortion, the loss of CD56⁺ and CD56⁺CD16^+NKcells in peripheral blood could have a diagnostic value for spontaneous abortion.     

Mesenchymal stem cell-induced CD8α+ Jagged2high CD11bhigh regulatory dendritic cells prevent and treat mouse aGVHD

AIM: To investigate the role of mesenchymal stem cell-induced regulatory dendritic cells (MSC-DCregs) in mouse acute graft-versus-host disease(aGVHD) model. METHODS: Bone marrow cells from BALB/c (H-2^d) mice were isolated and were induced to differentiate into DCs. The DCs were selected by flow cytometry, and after 10 d co-culture with MSCs, they were induced to be MSC-DCregs. Male 8-week-old C57BL/6 (H-2^b) mice were used as donor mice. The female 8-week-old BALB/c (H-2^d) mice, who had received 100 cm source-skin distance, 30 cm×30 cm radiation field, 700 cGy total body irradiation (TBI) pretreatment were used as recipient mice. The recipients were divided into 5 groups: control group, TBI group (injected with medium only), bone marrow transplantation group (injected with 1×10⁷ bone marrow cells), aGVHD group (injected with 1×10⁷ bone marrow cells and 1×10⁷ spleen cells), and MSC-DCregs group (injected with 1×10⁷ bone marrow cells, 1×10⁷ spleen cells and 1×10⁶ MSC-DCregs). The white blood cell count, recipients' chimerism, clinical evaluation of aGVHD, survival analysis and pathological changes were determined. RESULTS: Hematopoieic recovery was seen at 10 d after transplantation. The recipients' chimerism was parallel to the donors' at 30 d. The median survival time of the mice in aGVHD group and MSC-DCregs group was 27 d and 33 d, and the survival rates at 30 d were 20% and 100% (P<0.01), respectively. The clinical scores of the mice in MSC-DCregs group were lower than those in aGVHD group (P<0.01). Moreover, the pathological changes in the skin and liver of the mice in MSC-DCregs group were less serious than those in aGVHD group. CONCLUSION: The MSC-DCregs induce an aGVHD tolerance in vivo, and further research of its mechanism is still in great necessary.     

Human mesenchymal stem cells differentiate into neuron-like cells with Tanshinone II A

AIM:To investigate the differentiation from human mesenchymal stem cells(hMSC) into neuron-like cells with Tanshinone II A.METHODS:hMSC were separated from rib marrow with Ficoll-Paque reagent and expanded in culture medium. To detect the surface antigens, the labeled cells were analysed on a FACScan flow cytometer to determine the effect of the capacity of proliferation and differentiation of the mesenchymal stem cells with FGF-2. hMSC were induced to differentiate into neurons with DMEM Tanshinone II A. Neuron-specific enolase(NSE), neurofilament(NF), Nestin, glial fibrillary acaidic protein(GFAP) were detected by immunohistochemistry.RESULTS:hMSC were expanded as undifferentiated cells in culture for more than 15 passages. The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. These expanded attached MSC were uniformly positive for CD29, CD44, CD90, CD105, CD166 and didn't express CD11a, CD14, CD34, CD38, CD45, CD80, CD86. FGF-2 have special effect on low denisity MSCs. Simple methods with Tanshinone II A induced hMSC to exhibit a neuronal phenotype, expressing NSE, NF-M, Nestin at 5 hours. But the neuron-like cells didn't express the glial astrocyte marker GFAP.CONCLUSION:hMSC can be induced to differentiate into neurons with Tanshinone II A.     

Adipose-derived stem cells mediate immunosuppression of Th17 in multiple sclerosis

AIM: To investigate how human adipose-derived stem cells (hASCs) regulates the differentiation of Th17 cells in multiple sclerosis. METHODS: hASCs were isolated from the adipose tissues. Magnetic-activated cell sorting (MACS) kit was used to isolate CD4⁺ T cells from peripheral blood mononuclear cells (PBMCs) which were isolated by density gradient centrifugation. The percentage of CD4⁺ T cells was detected by flow cytometry. The activated CD4⁺ T cells were co-cultured with hASCs for about 4 d at different ratios of hASCs to CD4⁺ T cells (1:4 and 1:10) in a Th17 polarised condition. Another group adding anti-leukemia inhibitory factor (LIF) antibody was set up. Th17 cell proportion of the CD4⁺ T cells was determined by flow cytometry. The level of LIF in the supernatant of co-cultured system was measured by ELISA. The mRNA expression of retinoid-related orphan receptor γt (RORγt), interleukin-6 receptor (IL-6R), interleukin-23 receptor (IL-23R), LIF and leukemia inhibitory factor receptor (LIFR) was detected by real-time PCR. RESULTS: The result of flow cytometry suggested there were mainly hASCs, and the percentage of CD4⁺ T cells in the PBMCs were above 90% after MACS. The Th17 cell proportion decreased in 1:4 and 1:10 co-cultured groups in a dose-dependent manner. The mRNA expression of IL-6R, IL-23R and RORγt was downregulated and the expression of LIFR and LIF was up-regulated. When the anti-LIF was added into the co-cultured system, the ratio of Th17 cells increased and reached to the control level. The protein level of LIF obviously increased after co-cultured. After anti-LIF added, the mRNA expression of RORγt and IL-6R was up-regulated. CONCLUSION: hASCs inhibits the differentiation of Th17 cells from multiple sclerosis patients through the competitive inhibition of LIF/IL-6 by secreting LIF.     

Changes of CD28, CD56 and CD57 expression on CD8+T cells in the peripheral blood of healthy elderly individuals

AIM:The expression of CD28, CD56 and CD57 on CD8⁺T cells in the peripheral blood of young (age range 20-35) and elderly(age range 60-75) healthy donors were compared to explore the change of the cellular immune function with aging.METHODS:Three-color fluorescent flow cytometry was performed to analyze the differences in percentage of CD8⁺CD28⁺, CD8⁺CD56⁺ and CD8⁺CD57⁺T cells in the peripheral blood between the young and elderly groups.RESULTS:CD8⁺CD28⁺T cells in the peripheral blood of the elderly group was significantly lower than those in the young group, with percentage of 34.07±5.28 and 49.84±7.43,respectively (P＜0.05). Conversely, CD8⁺CD56⁺T cells and CD8⁺CD57⁺T cells in the peripheral blood of the elderly group were significantly higher than those in the young group, and the percentage was 6.60±2.40 vs 2.10±0.35,41.82±6.01 vs 22.89±2.80, respectively(P＜0.05).CONCLUSION:The expression of CD28, CD56 and CD57 on the CD8⁺T cells in the peripheral blood are changed significantly with aging. The decrease in CD28 expression may play an important role in the immunosenescence, while the increase in CD56 and CD57 expression seems to be a compensatory adaptation for the immune dysfunction.     

Sca-1+ cells from mouse fetal liver can differentiate into neurons in vitro

AIM: To study whether Sca-1⁺ cells from fetal liver can be induced to differentiate into neuronal cells in vitro. METHODS:Sca-1⁺cells from 14 5-days-old murine fetal liver were isolated with a magnetic cell sorting kit, and were cultured in Dulbecco s modif ied Eagle s medium(DMEM)/F12 supplemented with 10%fetal bovine serum(FBS), and passaged at a rat io of 1 3 when cells reached more than 80%confluence.The 5 passage cells were induced by 10^-3mol/Lβ-mercaptoethanol(β-ME)and 5×10^-7 mol/L all-trans-retinoic acid(RA)for 24 hours, and then incubated in serum-free medium for 5 hours to 5 days.The characteristics of treated cel s were assayed by immunocytochemistry staining analysis at 5 hours, or 5 days.RESULTS: Cells treated with β-ME and RA exhibited neuronal phenotype and expressed neuron-specific protein such as neuron-specific nuclear protein (NeuN), neuronfilament-M, and neuron-specific tubulin-1 (TuJ-1) but not tau, MAP-2, or the astrocyte-specific marker glial fibrillary acidic protein (GFAP).CONCLUSION: Sca-1⁺ cells from fetal liver, of which most are regarded as hematopoietic stem cells, could differentiate into early immature neuronal cells in vitro. These findings suggest that Sca-1⁺ cells from fetal liver may be an alternative source in cell therapy and gene therapy of neural dysfunction.     

Study on adherent precursors from human cord blood

AIM: To confirm the existence of the endothelial progenitor cells in human cord blood and to study its differentiation and development process. METHODS: The mononuclear cells in human cord blood were isolated using lymphocyte separation solution. Then the mononuclear cells were cltured in MCDB131 containing 20% fetal bovine serum. The effects of 5 μmol/L dexamethasone, the extract from bovine brain, insulin and hypoxanine on the proliferation and differentiation of the adherent cells were observed. The morphology of the adherent cells were examined twice daily by inverted phase contrast microscope. CD34 and CD14 expression were determined by FACS. Immunohistochemistry was used to confirm the expression of factor Ⅷ. RESULTS: The proliferative endothelial progenitor cells existed within the CD34^-adherent mononuclear cells of human cord blood. Dexamethasone and hypoxanine decreased the number of spindle-shaped cells and caudated cells. Bovine brain extract, insulin and FCS enhanced the number of spindle-shaped cells and caudated cells. CONCLUSION: The existence of endothelial progenitor cells within the CD34 - adherent monouclear cells of the human cord blood was observed and these cells were able to differentiate into endothelial-like cells in vitro.     

Transplantation of mouse fetus-derived MSCs alleviates physical fatigue in mouse

AIM:To observe the effect of mouse fetus-derived mesenchymal stem cell(MSC) transplantation on alleviating fatigue in mice. METHODS:MSCs were derived from the mouse fetus at 13.5 d. The 6-month-old BALB/c mice were randomly divided into non-swimming group, swimming control group and swimming+MSCs group. The mice in swimming+MSCs group were injected with fetus-derived MSCs through the tail vein, while the mice in non-swimming group and swimming control group were injected with equal volume of normal saline. The anti-fatigue activity was evaluated using a weight-loaded swimming test, along with the determination of blood urea nitrogen(BUN), blood lactic acid(BLA),and glycogen in the hepatic and muscular tissues 24 h later. The antioxidant activity was evaluated by determining the activity of superoxide dismutase(SOD) and the content of malondialdehyde(MDA). The cardiac functions were measured by echocardiography. RESULTS:Primary cultured cells were spindle-shaped in scattered colony. The cells in fifth passage grew parallelly or swirlingly. The cells expressed CD44 and CD105, but not CD34 or CD45. Inducing experiments showed that the cells differentiated into osteoblasts or adipocytes. Transplantation of MSCs alleviated fatigue in mice. In swimming+MSCs group, weight-loaded swimming time was longer, the levels of BUN, BLA, hepatic and muscular MDA were lower, and the levels of glycogen and SOD in the hepatic and muscular tissues were higher than those in control group. Stroke volume, cardiac output and left ventricular diastolic volume in swimming+MSCs group increased, indicating that the cardiac functions were enhanced. CONCLUSION: Transplantation of mouse fetus-derived MSCs alleviates physical fatigue.     

Characters of the fibroblast-like cells cultured from the mobilized peripheral blood cells

AIM: To explore the characters of fibroblast-like (F-L) cells cultured from granunocyte clony stimunating factor (G-CSF)-mobilized peripheral blood cell (PBC) harvests. METHODS: The adherent cells in the PBC harvests were cultured for 2 week in the mediums of RPMI-1640/L-DMEM/G-CSF or interleukin-3 (IL-3) plus RPMI-1640, the cultured F-L cells were analyzed by flow cytometry (FC). RESULTS: The adherent non-confluent F-L cells obtained from the four groups were similar in their phenotypes: CD33+, CD11c+, CD64+, CD14+, CD45+, HLA-DR+, CD86+, CD34-, CD38-, CD3-, CD19-, CD56-, CD29-, CD44-, CD105-. The F-L cells are similar to monocytes except CD38- and were distinct from dendritic cells (DC) or mesenchymal stem cells (MSC). CONCLUSION: The cultured F-L cells are macrophages rather than DC or MSC. G-CSF, rhIL-3 enhances their numbers.     

Effect of forsythiaside A on immune function in rats with ulcerative colitis

AIM To investigate the effect of forsythiaside A (FA) on immune function in rats with ulcerative colitis and its related mechanism. METHODS Healthy SD rats were randomly divided into 5 groups: control group (no treatment, normal feeding), model group (establishment of rat ulcerative colitis model), and low, medium and high doses of FA groups (treatment of the model rats with FA at 5 mg/kg, 20 mg/kg and 80 mg/kg, respectively). The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in rat colon tissues were measured by colorimetry, and the serum levels of tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2) and IL-4 were detected by ELISA. The spleen index and thymus index, the percentages of CD3⁺, CD4⁺ and CD8⁺ T-lymphocytes in peripheral blood mononuclear cells （PBMC）, the serum IgA and IgG levels, and the serum complement C3 and C4 levels were also determined. RESULTS The colon tissues of the rats in model group showed obvious inflammation and ulceration, indicating that the animal model was successfully established. Compared with model group, the colonic inflammation and ulceration were significantly attenuated in FA groups, among which the high dose had the best effect. Compared with control group, the spleen index and thymus index of the rars in model group were decreased (P<0.05), MDA content in colon tissues was increased (P<0.05), and SOD activity in colon tissues was decreased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were decreased (P<0.05), while the serum levels of IgA, IgG, TNF-α, and IL-2 were increased in model group as compared with control group. Furthermore, the spleen index and thymus index of the rats in FA groups were increased (P<0.05), the MDA content in the colon tissues was decreased (P<0.05), and the SOD activity in the colon tissues was increased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were increased (P<0.05), while serum IgA, IgG, TNF-α and IL-2 levels were decreased in FA groups as compared with model group (P<0.05). CONCLUSION Forsythiaside A effectively attenuates the colonic lesions in rats with ulcerative colitis, and its mechanism may be related to reinforcement of oxygen free radical scavenging power, alleviation of inflammatory response, and enhancement of immune function.     

Osteogenic and neurogenic differentiation of human yolk sac mesenchymal stem cells

AIM: To purify human yolk sac mesenchymal stem cells (hYS-MSC) and investigate its osteogenic and neurogenic differentiation potentials. METHODS: hYS-MSC were separated from yolk sac and purified via passage culture. The karyotype of hYS-MSCs was analyzed via G-banded characteristics. Flow cytometric analysis was used to determine the cell cycle and phenotype of hYS-MSC. The AKP expression of hYS-MSC was also tested. Osteogenic differentiation of hYS-MSCs was induced by 10-8mol/L dexamethasone, 10 mmol/L β-glycerophosphate and 50 mg/L vitamin C. Alizarin red S stain was used for identification of mineralization. β-mecaptoethanol or salviae miltiorrhizae were used to induce neurogenic differentiation of hYS-MSCs. The expressions of NSE, NF and GFAP were identified by immunohistochemical method. RESULTS: hYS-MSCs could be purified at passages 2 or 3. The cell cycle analysis suggested that hYS-MSCs showed strong proliferational potentials by which the cells kept normal diploid karyotype during the in vitro culture. Flow cytometry showed the phenotype of purified hYS-MSCs was uniformly positive for CD29, CD44, CD105, and CD166, and negative for reactivity to antigens CD34, CD45, or CD86. hYS-MSCs were weakly but clearly positive in AKP. Osteogenic differentiation was appeared after induction of osteogenic differentiation. hYS-MSCs, which were of spindle shape, uniform in size, were induced to pleomorphism osteoblast-like cells which expressed high level of AKP. Aggregates or nodules were formed at day 7 and calcium accumulation was detected by alizarin red S staining on day 10 or day 14. Neurogenic differentiation of hYS-MSCs was induced by β-mecaptoethanol or salviae miltiorrhizae. NSE, NF or GFAP positive cells were detected by immunohistochemical staining. CONCLUSIONS: hYS-MSCs have strong proliferation potential and the normal diploid karyotype is kept during the in vitro culture. The phenotype of hYS-MSCs is coincident with adult hMSCs. hYS-MSCs could be induced to differentiate into osteogenic or neurogenic cells.     

Isolation and multilineage differentiation of mesenchymal stem cells from human umbilical cord vein in vitro

AIM: To investigate the isolation, purification, expansion and multilineage differentiation of mesenchymal stem cells (MSCs) derived from human umbilical cord vein in vitro.METHODS: By 1% collagenase Ⅱ digestion, endothelial cells were isolated from human umbilical cord vein and cultured in IMDM medium.The morphology of the cells was observed by Wright’s staining and electron microscope.Cell cycle and immunophenotype were investigated by flow cytometry.Assays of adipogenic and osteogenic differentiation were performed in vitro.von Kossa staining, Oil Red O staining and mRNA expression of osteopontin and lipoprotein lipase were studied in the induced cells.RESULTS: The cells from the cord vein displayed a fibroblast-like morphology adhering to the culture plate.FACS showed that the cells expressed several MSCs-related antigens such as CD29, CD44 and CD105, while CD13, CD31, CD45, CD34, and HLA-DR were negative.Adipocyte and osteocyte differentiation were induced successfully.CONCLUSION: The morphology, growth characteristics, immunophenotype and pluripotentiality of the MSCs from human umbilical cord vein are similar to the MSCs from bone marrow (BM).They could potentially be an excellent source of MSCs for experiments and clinics.