Establishment of a new method to induce the differentiation of embryonic pancreatic cells into mature endocrine cells.

OBJECTIVE: To establish a new culture method to induce the differentiation of embryonic pancreatic cells into mature endocrine cells. METHODS: 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. RESULTS: 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. CONCLUSION: We successfully established a new culture method, with which embryonic pancreatic cells can efficiently differentiate into mature endocrine cell.

CD72 gene expression in immune thrombocytopenia.

To explore the role of CD72 in the pathogenesis of immune thrombocytopenia (ITP), we detected CD72, Sema4D, IL-2, IL-4, and IFN-γ mRNA expressions and the levels of plasma Sema4D, IL-2, IL-4, IL-6, and IFN-γ in ITP patients (n = 39) and controls (n = 23). The levels of plasma IL-2, IL-4, and IL-6 were assayed by radioimmunoassay, and the levels of plasma IFN-γ and Sema4D were analyzed by enzyme-linked immunosorbent assay. Sema4D, CD72, IL-2, IFN-γ, and IL-4mRNA expressions were analyzed by real-time quantitative reverse-transcription polymerase chain reaction. The expression of CD72 mRNA in ITP patients (n = 23) with active disease was significantly lower than that in patients in remission (p = 0.029) (n = 16) and controls (p = 0.0296) (n = 23). The IFN-γ/IL-4 mRNA (Th1/Th2) expression in ITP patients with active disease and in remission was significantly higher than that in controls (p = 0.0023, p = 0.0125, respectively). The expression of IL-2 mRNA in ITP patients with active disease was significantly lower than that in patients in remission (p = 0.0418) and controls (p = 0.004). The level of plasma IL-2 in ITP patients with active disease was significantly lower than that in patients in remission (p = 0.0029) and controls (p = 0.0101). The levels of plasma IL-4 in ITP patients with active disease and in remission were significantly higher than that of controls (p = 0.0093, p = 0.0053, respectively). CD72 mRNA expression level might correlate with Sema4D mRNA expression in peripheral blood mononuclear cells and level of plasma IL-2 in active ITP patients (p = 0.024 and p = 0.036). Our findings suggest that CD72 might be involved in the pathophysiological process of the ITP disease by increasing B-cell receptor signals.

Comparison of hematopoietic supportive capacity between human fetal and adult bone marrow mesenchymal stem cells in vitro.

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.

[Umbilical cord mesenchymal stem cell transplantation for treatment of experimental autoimmune myasthenia gravis in rats].

Umbilical cord mesenchymal stem cell (UCMSC) transplantation has been widely used in the treatment of a variety of diseases due to their advantages such as abundant resources, low immunogenicity and large ex vivo expansion capacity. This study was aimed to investigate the effects of UCMSC on experimental autoimmune myasthenia gravis (EAMG) rats. The distribution of human-derived cells was observed by immunofluorescence method, the effect of MSC on B-cell in situ-secreted antibodies was assayed by ELISPOT, the secreted IFN-γ level was detected by using Transwell test. The results showed that UCMSC were able to migrate to inflammation region and lymph nudes, moreover human-derived cells could be detected in medulla zone of lymph nudes. In vitro in situ detection of AchR specific antibody secretion revealed that the full contact of MSC with lymphnode-derived lymphocytes could effectively inhibit production of AchR antibody. Transwell test indicated that the direct contact of UCMSC with CD4 T cells could effectively decrease production of IFN-γ, which modulated the unbalance between Th1/Th2 to a certain extent. It is concluded that UCMSC can regulate the immune system by direct cell-cell contact or/and release of cytokines, which bring a new insight into knowledge about MSC-based therapy for EAMG.

[Isolation of mesenchymal stem cells from bone marrow filters by primary explant culture].

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.

More mesenchymal stem cells enriched from bone marrow aspirates by culturing bone marrow particles and mononuclear cells separately.

Bone marrow (BM) is the major source of mesenchymal stem cells (MSC). In most experiments, MSC were classically cultured from mononuclear cells (MNC) isolated by density gradient centrifugation method. However, several studies have demonstrated that this method was less efficient for MSC recovery. This study was aimed to investigate whether BM particles were the cause resulting in less efficiency of this method and how to isolate them. A total of 20 patients were enrolled in this study. MNC were cultured by standard adherence and BM particles were cultivated by primary explant culture. For BM from patients 1-10, MNC were first isolated and BM particles were then filtered out. The morphology and the fibroblastic colony number were compared between cultures of MNC and BM particles. For BM from patients 11-20, MNC isolation and BM particle filtration were processed in opposite order, then the immunophenotype and function between adherent cells expanded from MNC and BM particles were compared. In addition, for patients 11-20, the left BM aspirates were cultured too after BM particles and MNC were isolated separately. The results showed that adherent cells from BM particles were MSC. After BM particles were filtered out and cultured separately, MSC could be recovered completely from MNC isolated by density gradient centrifugation and no MSC were left in the residual BM aspirates. BM particles, which have been mostly discarded by the method of density gradient centrifugation, are another important source of MSC and they can be cultivated reliably by primary explant culture. It is concluded that more MSC are recovered from a single BM sample by culturing BM particles and MNC separately.

[Enhancement of all-trans retinoic acid induced HL-60 leukemia cell differentiation by human umbilical cord mesenchymal stem cells].

This study was aimed to investigate the enhancement of all-trans retinoic acid-induced HL-60 leukemia cell differentiation by human umbilical cord mesenchymal stem cells (hucMSC). The HL-60 cells were divided into 4 groups: control group (HL-60 cells treated without ATRA), hucMSC group (HL-60 cells co-cultured with hucMSCs), ATRA group (HL-60 cells treated with ATRA) and ATRA + hucMSC group (HL-60 cells treated with ATRA and co-cultured with hucMSCs). The proliferations of control group and hucMSC group were compared by Cell Counting Kit-8 (CCK8). The morphology of HL-60 cells and NBT positive rate in 4 groups were observed and compared by means of microscopy, the c-myc expression of HL-60 cells in different groups was evaluated by real-time PCR, and the CD11b expression on HL-60 cells in different groups were detected by flow cytometry. The results showed that in the co-culturing system, hucMSCs could inhibit the proliferation of HL-60 (hucMSC:HL-60 is 1:1, 48 hours p < 0.05, 72 hours p < 0.01; hucMSC:HL-60 is 1:5, 72 hours p < 0.05). In condition of stimulation with 2 micromol/L ATRA, the neutrophil like HL-60 cells and NBT positive rate in ATRA + hucMSC group were higher than those in ATRA group (p < 0.05). The c-myc expression of HL-60 cells in ATRA + hucMSC group was lower than that in ATRA group (p < 0.05). Furthermore, HL-60 cells in ATRA + hucMSC group had stronger CD11b expression than ATRA group (48 hours p < 0.05, 72 hours p < 0.01). It is concluded that hucMSC not only can inhibit the proliferation of HL-60 cells, but also can enhance the differentiation effect of HL-60 cells induced by ATRA.

[Differentiation of human umbilical cord derived mesenchymal stem cells into low immunogenic and functional hepatocyte-like cells in vitro].

OBJECTIVE: To investigate the biological function of hepatocyte-like cells derived from mesenchymal stem cells that isolated from human umbilical cord UC-MSCs in vitro, and to detect the changes in the immunogenicity of the differentiated hepatocyte-like cells (DHC). METHODS: Transdifferentiation of UC-MSCs into hepatic lineage in vitro was induced in modified two-step induction medium. The expressions of hepatic specific markers were detected by RT-PCR analysis and immunofluorescence staining at different time points after induction. The levels of albumin and urea in the supernatants of cultures were measured by enzyme-linked immunosorbent assay. Furthermore, the immunosuppressive property of DHC was detected by one-way mixed lymphocyte culture. RESULTS: The mRNA and proteins of alpha fetoprotein (AFP), albumin (ALB),and cytokeratin-19 (CK-19) were expressed in naive UC-MSCs at low levels. DHC highly expressed hepatic markers AFP, ALB, CK-19, and tryptophan 2, 3-dioxygenase 14 and 28 days after hepatic differentiation and were accompanied by an increased production of ALB and urea in supernatant in a time-dependent manner. DHC did not express human leukocyte antigen DR antigen and significantly decreased the lymphocyte proliferation. CONCLUSION: UC-MSCs are able to differentiate into functional hepatocyte-like cells in vitro, while the immunogenicity of DHC remains low.

[IL-27 regulates the expression of Mac-1, fMLP-R and IL-1beta in human neutrophils through p38 MAPK and PI3K signal pathways].

The present study was aimed to investigate the pathways, by which IL-27 regulates the expression of adherent molecule Mac-1, chemotactic factor receptor fMLP-R and pro-inflammatory cytokine IL-1beta in human neutrophils. Highly purified human neutrophils were isolated from peripheral blood using Ficoll-Hypaque gradients centrifugation and erythrocyte lysis. The mRNA expression of IL-27 receptor components (WSX-1/TCCR and gp130) in human neutrophils was detected by reverse transcription polymerase chain reaction (RT-PCR). After incubation with IL-27 and specific inhibitors (p38 MAPK inhibitor SB203580, PI3K inhibitor LY294002 and ERK inhibitor U0126), the mRNA levels of fMLP-R and IL-1beta were determined by real time RT-PCR, and the adherent molecule Mac-1 expression in human neutrophils was determined by flow cytometry. The IL-1beta level in culture supernatant of human neutrophils was assayed by radioimmunoassay. The results showed that IL-27 receptor components (WSX-1/TCCR and gp130) were constitutively expressed in human neutrophils. IL-27 down-regulated Mac-1 expression in human neutrophils (p<0.05). After incubation with specific inhibitors, SB203580, not LY294002 and U0126, inhibited the down-regulation of Mac-1 expression by IL-27. However, IL-27 up-regulated the mRNA expression of fMLP-R and IL-1beta, and increased the release of IL-1beta (p<0.05). Interestingly, LY294002, not SB203580 and U0126, inhibited the up-regulation of fMLP-R and IL-1beta by IL-27. It is concluded that the IL-27 may regulate the expression of Mac-1, fMLP-R and IL-1beta in human neutrophils through p38 MAPK and PI3K signal pathways.

[Effect of fetal lung mesenchymal stem cells on differentiation of umbilical cord blood mononuclear cells into megakaryocytes].

In order to analysis the effect of fetal lung mesenchymal stem cell (FL-MSC) on differentiation of umbilical cord blood mononuclear cells (MNC) into megakaryocytes, the fresh umbilical cord blood MNC were isolated and divided into 2 groups in the culture added with TPO, IL-11 and heparin. In the first group MNC were cultured alone and in the second group MNC were cocultured with FL-MSC. The cells were collected at day 7, 10, 14 for cell counting and detection of CD41a and CD61 by flow cytometry. The morphology and ultrastructure of megakaryocytes were observed by immunohistochemistry method and transmission electron microscopy at day 14. The content of DNA was analyzed by flow cytometry at day 14 too. The results indicated that the of CD41a+ and CD61+ cells were obtained mostly in the second group at day 10 and were in 4.5 and 4.7 fold as much as the MNC cultured alone. The morphology and ultrastructure of megakaryocytes showed immature of nuclei in both of two groups. It is concluded that the FL-MSC could effectively enhance the production of CD41a+ and CD61+ cells, where the effect on nucleus development of the young megakaryocyte was not obviously shown.

[Effect of silencing bmi-1 by RNA interference on function of K562 cell line].

Bmi-1 is a transcriptional repressor, which belongs to the polycomb group family. It has been demon- started that over-expression of Bmi-1 occurs in a variety of cancers, including several types of leukemia. Bmi-1 gene plays a key role in regulation of self-renewal in normal and leukemic stem cells. Acute myeloid leukemic cells lacking Bmi-1 undergo proliferation arrest and show signs of differentiation and apoptosis, which leads to the proposal of Bmi-1 as a potential target for therapeutic intervention in leukemia. The purpose of this study was to investigate the effect of short hairpin RNA (shRNA) targeting Bmi-1 on functions of K562 cell line. The shRNA eukaryotic expression vector targeting Bmi-1 was constructed and transfected into K562 cells through lipofectamine 2000. The mRNA and protein levels of Bmi-1 were detected by PCR and Western blot respectively. The proliferation of K562 after Bmi-1 silencing was measured by using MTT assay and clone formation assay. The cell cycle was detected by flow cytometry. The results indicated that among the four shRNA designed, there was a shRNA which efficiently interfered with the expression of Bmi-1. The results of PCR and Western blot validated that the Bmi-1 gene of K562 cells transfected with such a Bmi-1 shRNA was suppressed successfully. Although levels of Bmi-1 mRNA and protein were significantly reduced, delivery of this siRNAs had no effect on cell viability or growth. Flow cytometry analysis suggested that Bmi-1 inhibition did not affect the cell cycle. It is concluded that the suppression of Bmi-1 expression is not able to reduce proliferation of K562 cells, suggesting existence of some other parallel signaling pathways, which are fundamental for leukemic transformation and are independent of Bmi-1 over-expression. Bmi-1 over-expression may play a secondary role in chronic myeloid leukemia transformation.

[Expression of human factor IX in retrovirus-transfected human umbilical cord tissue derived mesenchymal stem cells].

The purpose of this study was to investigate the expression of human Factor IX (hFIX) in retrovirus-transfected human umbilical cord tissue derived mesenchymal stem cells (hUCT-MSCs). The pLEGFP-N1-hFIX vector was generated by cloning a 3.0 kb Bgl II-BamH I fragment from the pIRES2-EGFP-hFIX plasmid containing the hFIX cDNA and part of intron 1 of hFIX in pLEGFP-N1 vector. The retroviral supernatants were produced from the Phoenix packaging cell line and then infected the hUCT-MSCs. After selection with G418 for 10 day, the expression of FIX was detected by ELISA and Western blot. The biological activity of FIX was determined by the clotting assay employing human Factor IX-deficient plasma. The results showed that compared with the activity of pooled human normal plasma (100%), transduced cells produced biologically active hFIX with 100-130% activity in two-day culture supernatant and expressed hFIX at levels of 2.68 +/- 0.36 microg/10(6) cells/24 hours after G418 selection for 10 days. The secretion of hFIX into culture supernatant was also confirmed by Western blot analysis. It is concluded that genetically modified hUCT-MSCs can express biologically active hFIX and thus serve as an efficient drug delivery vehicle carrying hFIX used as a way of somatic gene therapy for hemophilia B.

[Mesenchymal stem cells derived from human umbilical cord tissue modulate the secretion of antiplatelet antibody from splenocytes of ITP patients in vitro].

The study was aimed to investigate the potential immunotherapeutical values of umbilical cord tissue-derived mesenchymal stem cells (UC-MSC) on patients with chronic idiopathic thrombocytopenic purpura (ITP). UC-MSC was cocultured in vitro with splenocytes isolated from ITP patients who experienced splenectomy. The level of IgG antiplatelet antibody (PAIgG) was determined by a competitive micro-enzyme-linked immunosorbent assay (ELISA) method. The proliferation of platelet-reactive CD4+ T lymphocytes was also measured in the presence of UC-MSCs. The results showed that UC-MSCs could stimulate the spontaneous secretion of PAIgG in supernatants; In the platelet-inducing condition, UC-MSC inhibited the production of PAIgG at a low ratio of 1 UC-MSC to 100 splenocytes, but promoted at a high proportion of 1 UC-MSC to 10 splenocytes. Moreover, UC-MSC exerted a suppressive effect on proliferation of platelet-reactive T helper cells in a dose-dependent manner. It is concluded that the UC-MSCs can regulate secretion of antiplatelet antibodies in vitro. Its concrete regulation mechanism and potential immunotherapeutical value are need to further study.

Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials.

BACKGROUND AND OBJECTIVES: Adult bone marrow (BM) is the major source of mesenchymal stem cells (MSC) for cell therapy. However, aspiration of BM involves invasive procedures. We isolated MSC from human full term umbilical cord tissues (UC). The biological characteristics of MSC derived from UC (UC-MSC) were further determined and compared with normal adult bone marrow-derived MSC (BM-MSC). DESIGN AND METHODS: MSC were isolated from UC by enzyme digestion and cultured in appropriate growth medium. The isolation efficiency, cell yield, colony-forming unit-fibroblast (CFU-F) frequency, growth kinetics, phenotypic characteristics, multi-lineage differentiation capacity, cytokine spectrum as well as hematopoiesis-supportive function of UC-MSC were determined and compared with those of BM-MSC. RESULTS: MSC were successfully isolated from all 36 UC and six BM samples we collected for this study. The mean number of nucleated cells isolated from UC was 1yen106/cm and the yield of adherent cells was 8.6yen105/cm. UC-MSC shared most of the characteristic of BM-MSC, including fibroblastic-like morphology, immunophenotype, cell cycle status, adipogenic and osteogenic differentiation potentials, and hematopoiesis-supportive function. The CFU-F frequency was higher in UC nucleated cells (1:1609 +/- 0.18) than in BM nucleated cells (1:35700 +/- 0.01) (p < 0.05). Furthermore, in comparison with BM-MSC, the UC-MSC had a higher proliferation capacity and lower levels of expression of CD106 and HLA-ABC (p < 0.05). Immunofluoresent and western blot assays revealed that UC-MSC had a higher percentage of neuron specific enolase-positive cells than had BM-MSC after neuronal induction. Finally, reverse transcriptase polymerase chain reaction analysis showed that UC-MSC had a cytokine spectrum very similar to that of BM-MSC, including expression of the mRNA of stem cell factor, leukemia inhibitor factor, macrophage-colony stimulating factor, Flt3-ligand, interleukin-6, vascular endothelial growth factor and stromal-derived factor-1, but UC-MCS additionally expressed mRNA of granulocyte macrophage and granulocyte colony-stimulating factors. After co-culture with CD34+ cord blood cells for 5 weeks, no significant difference in colony-forming cells was observed between the CD34+ cells/UC-MSC and CD34+ cells/BM-MSC co-cultures (p > 0.05). INTERPRETATION AND CONCLUSIONS: We have established a protocol to isolate abundant MSC from human umbilical cords with a 100% success rate. The comparative study indicates that UC is an excellent alternative to BM as a source of MSC for cell therapies.

[In vitro vasculogenesis and angiogenesis of mouse embryonic stem cells].

OBJECTIVE: To explore an optional condition to induce mouse embryonic stem (ES) cells to differentiate into endothelial cells and to establish in vitro models of vasculogenesis and angiogenesis. METHODS: Mouse ES cells were cultured in differentiation medium containing a cocktail of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-6 (IL-6) and erythropoietin (EPO) in 1% methylcellulose to induce formation of embryoid bodies (EBs). At day 11, EBs were harvested and suspended in rat-tail collagen type I with the same cocktail of cytokines cultured for three additional days. The differentiation of ES cells into endothelial cells, processes of vasculogenesis and angiogenesis were examined using immunostaining of EBs slices and whole-mount immunocytochemistry of EBs with monoclonal antibodies (mAbs) against platelet endothelial cell adhesion molecule-1 (PECAM-1) and alpha-smooth muscle actin (SMA). RESULTS: Under appropriate culture conditions; ES cells spontaneously differentiated and formed EBs containing vascular structures and tubular channels, which were positive for PECAM-1 co-differentiated with smooth muscle. When not treated with angiogenic growth factors, PECAM-1-positive cells could not organize into vascular structures of 11-day-old EBs. In the presence of angiogenic factors 11-day old EBs embedded into type I collagen, and rapidly developed an endothelial networks. Whole-mount immunocytochemistry of collagen gel with anti-PECAM-1 antibody showed the formation of primary vascular structures sprouting from EBs. Quantitative analysis revealed that 100 microg/ml thalidomide significantly reduced the number and length of EBs endothelial sprouting. CONCLUSIONS: Mouse ES cells can differentiate into endothelial cells combined with smooth muscle differentiation during EBs formation and further develop endothelial outgrowths after EBs embedded into collagen, which respectively recapitulate vasculogenesis, angiogenesis, and arteriogenesis processes in vivo. The models provide a useful tool to investigate vasculogenesis, angiogenesis, and arteriogenesis mechanisms and evaluate the effects of angiogenic and angiostatic agents.