Comparative Study of the Two High-Efficient Strategies for in vitro Generation of Human Umbilical Cord Blood-derived Natural Killer Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies.@*METHODS@#Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy.@*RESULTS@#After a 14-day's culture, the contents of CD3-CD56+ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3+CD4+ T cells and CD3+CD56+ NKT cells in M-NK group decreased significantly. The percentages of CD16+, NKG2D+, NKp44+, CD25+ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a+ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05).@*CONCLUSION@#The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.

Comparison of the Biological Functions between Human Bone Marrow Derived CD106 Mesenchymal Stem Cells and CD106 Subgroup / 中国医学科学院学报

Objective To analyze the differences in biological functions between bone marrow(BM)-derived CD106 mesenchymal stem cells(MSCs)and the CD106 subgroup. Methods The MSCs from normal BM were isolated and expanded.The subgroups of CD106 and CD106 MSCs were sorted.The cell proliferation and adhesion functions,chemotactic activities,adipogenic and osteogenic potentials,senescence,and senescence protein 21(p21)were detected.The capacity of translocation into nucleus of nuclear factor-kappa B(NF-κB)when stimulated by tumor necrosis factor(TNF-α)was measured. Results The proliferative ability was higher in CD106 MSCs than that in CD106 MSCs.In 48 hours,the value of optical density(OD)was significantly higher in CD106 MSCs than that in CD106 subgroup(1.004±0.028 0.659±0.023,=3.946,=0.0225).In 72 hours,this phenomenon was even more pronounced(2.574±0.089 1.590±0.074,=11.240,=0.0000).The adhesive capacity of CD106 MSCs was significantly stronger than that of CD106 subgroup(0.648±0.018 0.418±0.023,=7.869,=0.0002).Besides,the metastasis ability of CD106 MSCs were significantly stronger than that of CD106 subgroup(114.500±4.481 71.000±4.435,=6.900,=0.0005).The CD106 MSCs had signifcnatly lower proportions of senescent cells.The expression of aging protein p21 in CD106 MSCs was significantly lower than that in CD106 MSCs [(17.560±1.421)% (45.800±2.569)%,=9.618,=0.0000].Furthermore,there were no visible pigmenting cells after β-galactosidase staining in CD106 MSCs subgroup.However,in CD106 MSCs,some colored green cells were detected.The rate of NF-κB translocation into nucleus after stimulated by TNF-α was significantly higher in CD106 MSCs than CD106 MSCs [(37.780±3.268)% (7.30±1.25)%,=8.713,=0.0001]. Conclusion Bone marrow-derived CD106 MSCs possess more powerful biological functions than CD106 MSCs.

Effect of CD106 Mesenchymal Stem Cell on Bone Marrow Vascular Failure in Patients with Aplastic Anemia / 中国医学科学院学报

Objective To investigate the vascularization ability of mesenchymal stem cells(MSCs)and explore its influencing factors in aplastic anemia(AA) patients. Methods MSCs were isolated from the bone marrow of AA patients(AA MSCs) and normal controls(N MSCs) were cultured and then evaluated by flow cytometry and immunofluorescene staining technique.The expression level of vascular cell adhesion molecule-1(CD106) was detected by gene sequencing,and the content and fluorescene intensity of CD106MSCs was determined by fluorescence-activated cell sorting.The content of CD105CD106MSCs in fresh AA bone marrow was measured,followed by the determination of the capability of endothelial differentiation from AA MSCs and N MSCs with immunofluorescene analysis;finally,the capability of CD31cell differentiation from CD106-blocking N MSCs and its tubular structures formation in matrigel were tested.Results The expression of CD106 in AA patients was defective(decreased by 12.13 times when compared with N MSCs) and the concentration and fluorescene degree of CD106MSCs was also decreased in AA patients [(28.03±17.71)% vs.(59.61±12.26)%,P=0.000].The content of CD105CD106MSCs decreased significantly in the fresh bone marrow [(0.33±0.10)% vs.(2.98±0.46)%,P=0.0005].Besides, the capability of CD31cell differentiation from AA MSCs was significantly delayed [(13.67±1.50)% vs.(43.24±0.96)%,P=0.0004].Also,the capability of CD31cell differentiation and tubular structures formation of CD106-blocking N MSCs was also obviously decreased [(26.00±2.65)% vs.(91.78±2.44)%,P=0.000;(13.81±1.98)mm vs.(68.12±6.78)mm,P=0.0015].Conclusion The deficient or decreased expression of CD106MSCs accelerate the bone marrow vascularization failure in AA patients.

Human Umbilical Cord-derived Mesenchymal Stem Cells Secrete Interleukin-6 to Influence Differentiation of Leukemic Cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effect of human umbilical cord-derived mesenchymal stem cells (UC-MSC) on the differentiation of leukemic cells.</p><p><b>METHODS</b>The co-culture system of UC-MSC with acute promyelocytic leukemic cell line NB4 cells was constructed in vitro,and the differentiation status of the leukemic cells was assessed by cell morphology,nitroblue tetrazolium reduction test,and cell surface differentiation marker CD11b.</p><p><b>RESULTS</b>UC-MSC induced the granulocytic differentiation of NB4 cells. When UC-MSC and a small dose of all-trans retinoic acid were applied together,the differentiation-inducing effect was enhanced in an additive manner. Interleukin (IL)-6Ra neutralization attenuated differentiation and exogenous IL-6-induced differentiation of leukemic cells.</p><p><b>CONCLUSION</b>UC-MSC can promotd granulocytic differentiation of acute promyelocytic leukemia cells by way of IL-6 and presented additive effect when combined with a small dose of all-trans retinoic acid.</p>

Effects of rapamycin on biological characteristics of bone marrow mesenchymal stem cells from patients with aplastic anemia / 中国实验血液学杂志

This study was aimed to investigate the effects of rapamycin on biological function and autophagy of bone marrow mesenchymal stem cells (BM-MSC) from patients with aplastic anemia so as to provide experimental basis for the clinical treatment of aplastic anemia (AA) with rapamycin. BM-MSC were treated with different concentrations of rapamycin (0, 10, 50, 100 nmol/L) for 48 h, the expression of LC3B protein was detected by Western blot to observe the effect of rapamycin on cell autophagy; cell apoptosis and cell cycles were detected by flow cytometry; the proliferation of BM-MSC of AA patients was measured by cell counting kit-8; the adipogenic differentiation of BM-MSC were tested by oil red O staining after adipogenic induction for 2 weeks; the adipogenic related genes (LPL, CFD, PPARγ) were detected by real-time PCR. The results showed that the proliferation and adipogenesis of BM-MSC of AA patients were inhibited by rapamycin. Moreover, the autophagy and apoptosis of BM-MSC were increased by rapamycin in a dose-dependent way.Rapamycin arrested the BM-MSC in G0/G1 phase and prevented them into S phase (P < 0.05). It is concluded that rapamycin plays an critical role in inhibiting cell proliferation, cell cycles, and adipogenesis, these effects may be related with the autophagy activation and mTOR inhibition resulting from rapamycin.

Biological characteristics of exosomes secreted by human bone marrow mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to explore the immunoregulatory function and capability supporting the angiogenesis of exosomes secreted by bone marrow mesenchymal stem cells (BMMSC) from healthy persons. Supernatant of BMMSC (P4-P6) was collected for exosome purification. Transmission electron microscopy (TEM) and Western blot were used to identify the quality of isolated exosomes. The amount of exosomes was quantified through bicinchoninic acid (BCA) protein assay. Human peripheral blood mononuclear cells (PBMNC) were isolated from healthy donor and added with isolating exosomes. After co-cultured for 72 h, IFN-γ from the co-culture system was detected by ELISA. The expression of miRNA-associated with immunity were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). The interactions between exosomes and human umbilical vein endothelial cells (HUVEC) were observed with confocal microscopy. Subconfluent HUVEC were harvested and treated with the indicated concentration of exosomes. Nude mice were injected subcutaneously with exosomes or PBS as control to verify the ability of angiogenesis. The results showed that diameter range of exosomes was range from 40 to 160 nm. The isolated exosomes expressed the CD9. There was approximately linear relation between the secretion of exosomes and cell density. The exosomes suppressed the production of IFN-γ from PBMNC, and contained miRNA associated with immune regulation such as miR301, miR22 and miR-let-7a. Exosomes induced vascular tube formation in vitro and vascularization of Matrigel plugs in vivo. It is concluded that the BMMSC-derived exosomes can regulate immunity and support vascularization.

Adipogenic potentials of mesenchymal stem cells from human bone marrow, umbilical cord and adipose tissue are different / 中国实验血液学杂志

Mesenchymal stem cells (MSCs) could be obtained from many sources, and there are differences between them. This study was purposed to compare and analyze the basic biological characteristics of umbilical cord, adipose tissue-and bone marrow-derived MSC (UC-MSCs, AD-MSCs and BM-MSCs). The MSCs were isolated from umbilical cord, adipose tissue and bone marrow were cultured; the morphology of UC-MSCs, AD-MSCs and BM-MSCs was observed by using microscopy; the immunophenotype, differentiation potential and expression of peroxisome proliferation-activated receptor-γ (PPAR-γ) mRNA were detected by using flow cytometry, differentiation test (von kossais and 0:1 red O staining) and quantitative fluorescent PCR, respectively. The results showed that the UC-MSCs, AD-MSCs and BM-MSCs displayed similar morphology under confocal microscope after being stained with rhodamine phalloidin and DAPL. The immunophenotypes of these three originated cells conform to coincide with identification criterion for MSCs, and showed similar expression level. During adipogenic induction the adipogenic potential of these MSCs was different, AD-MSCs exhibited the highest adipogenic potential, UC-MSCs displayed the lowest, while potential of BM-MSCs get between; however, the osteogenic differentiation potential of UC-MSCs, AD-MSCs and BM-MSCs was similar. The PCR detection showed that the expression level of PPAR-γ mRNA was the highest in AD-MSCs and the lowest in UC-MSCs, while expression level in BM-MSCs get between, these results were identical with the adipogenic potential, suggest that the difference of adipogenic potential in 3 kinds of MSCs was associated with basic expression level of PPAR-γ mRNA. It is concluded that UC-MSCs, AD-MSCs and BM-MSCs exhibit similar morphology, the immunophenotypes of these MSCs coincide with identification criterion for MSCs, the osteogenic potential of these MSCs is similar, while the adipogenic potential and the expression level of PPAR-γ mRNA are different. The difference-associated mechanisms need to further study.

Effect of 15-deoxy-Δ(12), 14-prostaglandin J2 on the cytokines in the culture supernatant of bone marrow mesenchymal stem cells / 中国实验血液学杂志

15-Deoxy-Δ(12), 14-prostaglandin J2 (15d-PGJ2), a well known peroxisome proliferator activated receptor (PPAR) γ ligand, has been shown to inhibit cellular proliferation and induce apoptosis and differentiation. However, whether 15d-PGJ2 influences the cytokines in the culture supernatant of bone marrow mesenchymal stem cells (BM-MSC) is unknown. This study was purposed to investigate the influence of 15d-PGJ2 on cytokines in the culture supernatant of BM-MSC. The fibroblast-like cells attached to the culture dish from bone marrow of healthy donors were isolated. The immunophenotype and differentiation potential of the obtained cells were detected by flow cytometry and oil red O and von kassa staining respectively to confirm that these cells were BM-MSC. Thereafter, the BM-MSC were cultured with complete medium supplemented with 10, 20, 40 and 60 µmol/L 15d-PGJ2 for 24 hours respectively. The real-time PCR was used to assay the PPARγ mRNA level, the confocal immuno fluorescence technique was used to detect the expression level of PPARγ. The results showed that the BM-MSC underwent apoptosis and got detached from the culture dish when the concentration of 15d-PGJ2 was no less than 20 µmol/L. The PPARγ mRNA level of BM-MSCs cultured with medium containing 10 µmol/L 15d-PGJ2 was higher than that cultured without 15d-PGJ2, and the difference was statistically significant (P < 0.05). The enhancement of PPARγ expression was observed after stimulated by 15d-PGJ2. The protein chip detecting the culture supernatants of BM-MSC cultured with 10 µmol/L 15d-PGJ2 or without 15d-PGJ2 for 24 hours demonstrated that expression levels of some of the cytokines varied. It is concluded that the down-regulation of TIMP-2 exists after treatment of 15d-PGJ2, which is statistical significant.

Long term in-vitro expansion reduces immune modulation function of placental chorionic villi mesenchymal stem cells / 中国实验血液学杂志

The main aim of this study was to investigate the biological activities and immune modulation changes of chorionic villi mesenchymal stem cells (CV-MSC) after long term culture. The morphology of the CV-MSC of passage 3 and passage 9 were observed by microscopy, and their phenotypes were detected by flow cytometry. CV-MSC of passage 3 and 9 were co-cultured with PHA-stimulated PBMNC, and IFN-γ concentration in culture medium was detected by ELISA. The mRNA expression of COX-2, HGF and HLA-G in CV-MSC were detected by real-time PCR. The results showed that after long term culture, the CV-MSC kept the MSC morphology and most of the phenotypes including CD31, CD34, CD44, CD45, CD62L, CD73, CD90, CD105, CD117, CD151, CD235a, CD271 and HLA-DR, while the CD49d was significantly up-regulated. Immune modulation ability of CV-MSC was reduced and the mRNA expression of COX-2 and HGF was down regulated after long term culture, but the expression of HLA-G did not found to be obvious change. It is concluded that the long term in vitro expansion changes the expression of CD49d and reduces immune modulation of CV-MSC.

Culture of pancreatic progenitor cells in hanging drop and on floating filter / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct a method to culture pancreatic progenitor cells in hanging drop and on floating filter,and to examine if pancreatic progenitor cells can differentiate into mature endocrine cells with this method.</p><p><b>METHODS</b>Murine embryos at day 12.5 were isolated and digested into single cells,which were then cultured in hanging drop for 24h and formed spheres.Spheres were cultured on the filter for 6 days,which floated in the dish containing medium.During culture,the expressions of pancreas duodenum homeobox-1(PDX-1)and neurogenin3(Ngn3)were determined.The expressions of endocrine and exocrine markers,insulin,glucagon,and carboxypeptidase(CPA)were determined on day 7 by immunohistochemistry.Insulin secretion of spheres stimulated by glucose was detected by ELISA.The changes of pancreatic marker expressions during culture were monitored by real-time polymerase chain reaction(PCR).</p><p><b>RESULTS</b>One day after the culture,there were still a large amount of PDX-1 positive cells in pancreatic spheres,and these cells proliferated.On day 3,high expression of Ngn3 was detected,and the Ngn3-positive cells did not proliferate.On day 7,The expressions of endocrine and exocrine markers in the differentiated pancreatic progenitor cells were detected,which were consistent with that in vivo.Insulin was secreted by spheres upon the stimulation of glucose.</p><p><b>CONCLUSION</b>In hanging drop and on floating filter,pancreatic progenitor cells can differentiate into mature endocrine cells.</p>

IL-1β promotes the hematopoietic support of human umbilical cord mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to investigate the effect of IL-1β on hematopoietic support of human umbilical cord mesenchymal stem cells (hUC-MSC). 2×10(6) hUC-MSC were seeded in 75 cm(2) flasks, after adherence to wall for 2 h, 10 ng/ml IL-1β was added in hUC-MSC supernatant and cultured for 36 h, then the culture supernatants and cells were harvested. The effect of conditioned medium with/without IL-1β on CD34(+) cell hematopoietic support was observed, mRNA expression changes of hUC-MSC cultured in medium with/without IL-1β were monitored by real time PCR, the differences in hematopoiesis-related factors were detected by ELISA. The results showed that the conditioned culture medium of hUC-MSC with IL-1β enhanced the ability to form colony of CD34(+) cells, especially CFU-G and CFU-GM in vitro; IL-1β promoted the mRNA expression of GM-CSF, G-CSF, IL-6 on MSC; IL-1β also promoted the secretion of GM-CSF, G-CSF, and IL-6 protein from hUC-MSC. It is concluded that IL-1β enhances hematopoietic support capacity especially, capability of MSC to myeloid differentiation.

Human umbilical cord mesenchymal stem cells reduce the sensitivity of HL-60 cells to cytarabine / 中国实验血液学杂志

This study was purposed to investigate the impact of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) on the sensitivity of HL-60 cells to therapeutic drugs so as to provide more information for exploring the regulatory effect of hUC-MSC on leukemia cells. Transwell and direct co-culture systems of HL-60 and hUC-MSC were established. The apoptosis and cell cycle of HL-60 cells were detected by flow cytometry. RT-PCR and Western blot were used to detect the mRNA and protein levels of Caspase 3, respectively. The results showed that the apoptosis of HL-60 induced by cytarabine (Ara-C) decreased significantly after direct co-cultured with hUC-MSC cycle mRNA (P < 0.05). The similar phenomenon was observed in transwell co-culture system. Cell cycle of HL-60 cells were arrested at G0/G1 phase and did not enter into S phase (P < 0.05) and the expression of Caspase-3 mRNA and protein in HL-60 cells were reduced (P < 0.05). It is concluded that hUC-MSC protected HL-60 from Arc-C induced apoptosis through regulating the cell cycle and down-regulating expression of Caspase 3 in HL-60 cells. In addition, this effect is caused by the soluble factors from hUC-MSC.

Isolation and biological characteristics of mesenchymal stem cells derived from human placenta decidua basalis / 中国实验血液学杂志

Comparing to bone marrow mesenchymal stem cells (MSCs), placenta-derived MSCs have the advantages of adequate sources, low immunogenicity, little risk of viral contamination, and no ethical controversy, and thus possess a better prospect for clinical application. Placental tissue not only includes chorionic and amniotic, but also contains decidua basalis which locate in the maternal placenta surface. The biological characteristics of MSCs isolated from decidua basalis have not been well studied. This study was aimed to investigate the biologic characteristics of placenta decidua basalis-derived MSC from placenta decidua basalis (DB) by enzymatic digestion. Short tandem repeats (STR) test was used to identify the cells derived from the maternal placenta surface. Growth rate of decidua basalis mesenchymal stem cells (DB-MSC) was measured by MTT. Cell cycle and cell phenotype were detected by flow cytometry. Inducing differentiation was used to evaluate multipotency of DB-MSC. For testing the immunosuppression of DB-MSC, they were co-cultured with peripheral blood mononuclear cells (PBMNC) stimulated by phytohemagglutinin (PHA) and then IFN-γ in the co-cultured media was quantified by ELISA. The results showed that the cells were derived from the maternal placenta by STR analysis. DB-MSC showed typical fibroblast morphology in the culture and were positive for the MSC surface markers: CD90, CD73, CD105, CD44 and negative for CD45, CD11b, and CD34. DB-MSC underwent osteogenic, adipogenic and chondrogenic differentiation in inducing medium. DB-MSC could inhibit the secretion of IFN-γ by PBMNC. It is concluded that the cells are isolated from placenta decidua basalis and possess the basic characteristics of MSC. DB-MSC can be an important maternal autologous MSC and may be a safe and effective treatment for immune system diseases, which makes the DB-MSC as an important source of autologous MSC from mother. DB-MSC can be safely for the treatment of the mother's immune system diseases.

Supportive effects of conditioned culture media of human umbilical cord mesenchymal stem cells on hematopoiesis in vitro / 中国实验血液学杂志

This study was aimed to explore whether the conditioned culture medium of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) has supportive effects on hematopoiesis in vitro. hUC-MSC were cultured in 75 cm(2) culture flasks at a concentration of 2×10(6) cells per flask. After 48 h, the conditioned culture medium was harvested. CD34(+) cells were isolated with the human cord blood CD34 positive selection kit. The CD34(+) cells were plated in three different culture systems: the culture supernatant from hUC-MSC added into incomplete methylcellulose without recombinant human cytokines as conditioned culture medium; the complete methylcellulose medium with recombinant human cytokines as positive control medium; incomplete methylcellulose adding DMEM/F12 with 10% FBS instead of conditioned culture medium as the negative control medium. After 14 days of culture, colonies containing ≥ 50 cells were scored and types of colonies were classified under inverted microscope. The immunophenotypes of cells which were collected from the colonies were detected by flow cytometry. The results showed that conditioned culture medium of hUC-MSC supported the differentiation of CD34(+) cells into CFU-G (47.67 ± 0.58), CFU-GM (48.67 ± 4.73) and CFU-M (3.00 ± 2.00) in vitro, while the CFU-E, BFU-E or CFU-GEMM were absent. Comparatively, in the positive control medium all kinds of CFU were observed. Interestingly, the percentage of CD45(+)cells of CFU in conditioned culture medium (97.43 ± 2.15)% was more than CD45(+)cells in positive control medium (39.69 ± 0.96)% (P < 0.05). It is concluded that the conditioned culture medium of hUC-MSC has been confirmed to have ability to support hematopoiesis separately in vitro. Besides, it enhances the differentiation of CD34(+) cells into myeloid cells except cells of erythroid lineage.

Establishment of a new method to induce the differentiation of embryonic pancreatic cells into mature endocrine cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a new culture method to induce the differentiation of embryonic pancreatic cells into mature endocrine cells.</p><p><b>METHODS</b>Mouse embryos at day 12.5 were used and embryonic pancreata were isolated. The isolated embryonic pancreata were cultured on the filter for 7 days, which floated in the dish containing medium. During culture, the expression of pancreas duodenum homeobox-1 (PDX-1), a pancreatic stem cell marker, was examined at day 1. The expression of neurogenin 3 (Ngn3), a pancreatic progenitor cell marker, was examined at day 3. The expressions of endocrine and exocrine markers, insulin, glucagon, and carboxypeptidase (CPA) were examined at day 7 by immunohistochemistry. The kinetics of pancreatic marker expression during culture was assayed by real-time PCR.</p><p><b>RESULTS</b>Many pancreatic stem cells still existed in embryonic pancreata cultured for 1 day; meanwhile, these pancreatic stem cells proliferated in high rate. A large amount of pancreatic progenitor cells were found in embryonic pancreata cultured for 3 days.Pancreatic stem/progenitor cells differentiated into mature endocrine and exocrine cells in embryonic pancreata after having been cultured for 7 days. Furthermore, the expression pattern of pancreatic marker is consistent with that in vivo.</p><p><b>CONCLUSION</b>We successfully established a new culture method, with which embryonic pancreatic cells can efficiently differentiate into mature endocrine cell.</p>

Comparative study of biological characteristics of human umbilical cord and placental chorionic villous mesenchymal stem cells / 中国实验血液学杂志

Because advantage of tissue origin and proliferation potential, the umbilical cord-derived mesenchymal stem cells (UC-MSC) and placental chorionic villous-derived mesenchymal stem cells (CV-MSC) have clinical application potential, as compared with bone marrow MSC. But whether the differences of biological characteristics exist between UC-MSC and CV-MSC, which deserve to be further explored. This study was purposed to compare the biological characteristics of UC-MSC and CV-MSC. The placental and umbilical cord were cleaned by using the sterile physiological salt, the UC-MSC and CV-MSC were separated by enzyme digestion. Short tandem repeat (STR) analysis was used to detect whether the MSC obtained from fetal tissue. MTT method was used to detect proliferation of MSC. Flow cytometry was applied to analyze cell phenotype. The different differential medium was used to detect their multi-directional differentiation capacity. After the MSC and PHA-stimulated peripheral blood mononuclear cells were co-cultured, the γ-interferon (IFN-γ) levels of the co-culture supernatant were detected using the ELISA. The results showed that these MSC were derived from fetal tissue by STR analysis. They were adherent cells with typical fibroblast morphology. Cells expressed the MSC surface markers CD90, CD73 and CD105 and CD44, not expressed CD45 and of CD11b and CD34.These cells could differentiate into osteoblasts and adipoblasts under culture with different conditioned medium, but in the adipogenic differentiation of CV-MSC, the larger lipid droplets appeared. It is concluded that these cells are obtained MSC. These MSC can inhibit peripheral blood mononuclear cells stimulated by PHA to secrete IFN-γ, and the the CV-MSC have a stronger suppression capacity, which makes the CV-MSC to have a greater advantage in the treatment of autoimmune diseases.

Effects of interferon-γ on biological characteristics and immunomodulatory property of human umbilical cord-derived mesenchymal stem cells / 中国实验血液学杂志

The aim of this study was to investigate the effects of interferon (IFN)-γ on biological characteristics and immunomodulatory property of human umbilical cord-derived mesenchymal stem cells (hUC-MSC). hUC-MSC were treated with IFN-γ 10 ng/ml (IFN-γ group) or without IFN-γ (control group). The phenotype of hUC-MSC was detected by flow cytometry. The proliferation status was detected by CCK-8 method, and its differentiation ability was assessed by oil red O and von Kossa staining. The production of PGE-2 was measured by ELISA, and the mRNA expression levels of COX-2, IDO-1 and IDO-2 in hUC-MSC were detected by real-time quantitative PCR. Furthermore, the proliferation of human peripheral blood mononuclear cells (hPBMNC) was evaluated after co-culture with hUC-MSC, IFN-γ pretreatment or not. The results showed that after IFN-γ stimulation, the expression of SSEA-4 on hUC-MSC decreased significantly [(8.15 ± 2.94) vs (16.42 ± 8.5), P < 0.05], and the expression of CD54 increased [(96.64 ± 3.29) vs (84.12 ± 10.73), P = 0.051]. The immunomodulatory property of hUC-MSC on the proliferation of hPBMNC was enhanced (P < 0.05). All the above mentioned effects were IFN-γ concentration-dependent. When hUC-MSC were stimulated by IFN-γ for 24 h, the production of PGE-2 secreted by hUC-MSC decreased significantly (P < 0.01). The mRNA expression level of COX-2 also decreased though the difference did not reach to statistically significant level. Compared with control group, IDO-1 expression level in IFN-γ group increased significantly (P < 0.01), and the mRNA expression level of IDO-2 remained unchanged. It is concluded that IFN-γ can influence the phenotype of hUC-MSC and enhance the immunomodulatory property of hUC-MSC.

Role of microenvironment in the process of expansion of late endothelial progenitor cell in vitro / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the role of the feeder layer cells as niche in the process of expansion of late endothelial progenitor cell in vitro.</p><p><b>METHODS</b>We cultured mononuclear cells (MNC)from human peripheral blood (PB)on the plate with the feeder layer cells which were irradiated late endothelial progenitor cells(EPC)or human umbilical vein endothelial cells (HUVEC) by EGM-2. After 21 days, the numbers of obtained late EPC colonies were counted separately, and their surface antigen of the late EPC was verified by fluorescence-activated cell sorter (FACS) analysis, and their ability of forming vessel structure with Matrigel in vitro. The differentiation of single stem cell on the feeder layer cell was traced by video-microscopy.</p><p><b>RESULTS</b>After 21 days of culture,(40.0±3.9)and(39.3±3.1)late EPC colonies that MNC of a hundred milliliter PB were cultured, respectively, on the feeder layer cells of EPC and HUVEC were much more than (2.0±1.3) colonies cultured on without the feeder layer cells (all P <0.05). These cells also expressed CD31,CD34,eNOS,FLt-1,P1H12,Sendo,VE cadherin,and CD117, as shown by FACS analysis. Furthermore, they formed vessel structure with Matrigel in vitro. The video-microscopy showed the asymmetric cell division was participated by the feeder layer cell during the expansion of single stem cell.</p><p><b>CONCLUSION</b>The massive expansion of late EPC can be achieved by the provision of the feeder layer cells, which may be involved in the stem cell asymmetric cell division.</p>

Comparison of hematopoietic supportive capacity between human fetal and adult bone marrow mesenchymal stem cells in vitro / 中国实验血液学杂志

Hematopoietic stem cells (HSC) shift from fetal liver and spleen to bone marrow at neonatal stages and this movement may be due to inductive signals from different microenvironments. Mesenchymal stem cells (MSC) are the precursors of stromal cells in bone marrow microenvironments such as osteoblasts and endothelial cells. Some researchers speculated that fetal bone marrow before birth might be not perfectly suit HSC growth. However, it is still lack of direct evidence to prove this hypothesis. This study was aimed to compare the hematopoietic supportive capacity between human fetal and adult bone marrow MSC in vitro. Adult bone marrow MSC (ABM-MSC) were isolated from three healthy donors and fetal bone marrow MSC (FBM-MSC) were isolated from three fetuses between gestations of 19 to 20 weeks. After irradiation, MSC were co-cultured with CD34(+) cells isolated from umbilical cord blood in long-term culture-initiating cell (LTC-IC) assay. The colony number of colony forming cells (CFC) was counted and the phenotypic changes of co-cultured CD34(+) cells were analyzed by flow cytometry. Cytokine expressions in both kinds of MSC were detected by reverse transcription polymerase chain reaction (RT-PCR). The results showed that ABM-MSC had a stronger hematopoietic supportive capacity than FBM-MSC. Both of them enhanced the differentiation of CD34(+) cells into myeloid lineages. Cytokines were expressed differently in ABM-MSC and FBM-MSC. It is concluded that ABM-MSC possess more potential application in some treatments than FBM-MSC, especially in hematopoietic reconstitution.

Isolation of mesenchymal stem cells from bone marrow filters by primary explant culture / 中国实验血液学杂志

This study was aimed to investigate whether mesenchymal stem cells (MSC) can be isolated from bone marrow filters which have always been discarded. The bone marrow (BM) particles from BM filters of 2 healthy donors were cultivated by primary explant culture. After expansion, the number of MSC was counted and their immunophenotype and differentiation potential were detected. The results indicated that many MSC were found in bone marrow particles from filters, and nearly 10(7) MSC were obtained at 3 passages of expansion. They not only possessed the characteristics of morphology and immunophenotype of MSC, but also could differentiate into osteoblasts, chondrocytes and adipocytes. In conclusion, a large amount of MSC can be obtained from BM filters if the BM particles were cultivated by primary explant culture.